Faculty of Biological and Environmental Sciences

 

Recent Submissions

  • Soininen, Laura (Helsingin yliopisto, 2022)
    Residents of modern cities have an elevated risk of developing immune mediated diseases due to lower odds of encountering rich microbial communities that help the formation of a healthy immune system. Due to this elevated risk, we are in search of prophylactic methods in order to reinforce urban microbial communities outdoors, and indoors, where city residents spend most of their time. In the light of previous studies regarding the biodiversity hypothesis, we should increase microbial diversity and occurrence of health-associated environmental microbes in cities. Such bacterial groups are lactobacilli, mycobacteria and proteobacterial classes including saprophytic taxa. Vegetation and soil are among the main sources of environmental microbes and their type and amount in the surrounding environment affect the local microbial communities. Bacterial community compositions, in turn, are reflected in the microbiomes of the residents. Several recent intervention studies support the biodiversity hypothesis and show that the immune system can be stimulated with increased exposure to rich microbial communities residing in soil and vegetation. The studies in this thesis focused on the immunomodulatory potential of plant and soil products in order to increase exposure to rich microbial communities in cities via plant substrates and vegetation. Most soil studies focus on natural soil but soil products (used in landscaping and as plant substrates), by which city dwellers are surrounded, are seldom studied. The first study of this thesis showed that long-term storage may lower the immunomodulatory potential of soil products even though their use may still be safe. To promote efficient and safe immunomodulatory use of soil products, I investigated the effect of long-term storage on the diversity and abundance of health-associated bacteria, as well as bacterial functions, in eight soil products. After the three-year storage, the diversity and relative abundance of most health-associated (potentially beneficial or harmful) bacterial taxa reduced. Indications of pathogenic functions did not increase during storage. Based on the results, it would be recommendable to determine an immunomodulatory shelf life for soil products and to keep storage times short. The second study recognized the potential of soilless air-circulating green walls as sources of immunomodulatory exposure at work spaces. A two-week exposure to the green walls during work days induced shifts in the skin bacterial communities of the study participants and in the immunomodulatory cytokines in the blood. The relative abundance of Lactobacillus spp. increased in connection to the green wall exposure. Additionally, the green walls induced an increase in the diversity of phylum Proteobacteria and class Gammaproteobacteria, which were connected to the reduction of pro-inflammatory cytokine IL-17A. The exposure also increased the concentration of anti-inflammatory cytokine TGF-β1. The third study demonstrated boosting the immunomodulatory potential of a standard plant substrate by supplementing it with side streams of forestry and agriculture. In yearlong growing experiments with grass vegetation, the bacterial community compositions in experimental substrates, which contained side streams, were compared to a commonly used grass substrate. The relative abundances of several proteobacterial taxa and Mycobacterium spp. increased in substrates containing moss, conifer needles, alnus litter and/or reed. Mixing commercial substrates containing side streams increased richness of the bacterial communities and biochar supplementation increased richness of phylum Proteobacteria and class Alphaproteobacteria. In the light of the results in this thesis, it seems that the immunomodulatory potential of soil products is higher when recently produced and supplemented with plant-based side streams that allow versatile habitats for microbes with varying preferences. The beneficial impact of green walls on the office workers indicates the immunomodulatory potential of indoor greening.
  • Chichorro de Carvalho, Filipe (Helsingin yliopisto, 2022)
    Species are declining and going extinct at unprecedented rates due to human activity. Understanding the drivers of extinction may help conservationists to proactively protect the species most in need. Species’ vulnerability to extinction has been hypothesised to be related to both their intrinsic biological traits and external human factors, often in synergy. In the past twenty years, many studies have compared the traits of threatened species to those of non-threatened species, within taxonomic groups. However, the literature in the topic has often focussed on few taxa or geographical realms, which has limited our capacity to find and understand general predictors and mechanisms of extinction. The primary aim of this dissertation is to identify which traits are related with extinction risk across the generality of life forms and geographical realms. To do so, I have applied a set of three distinct approaches: in Chapter I, I have identified gaps in research and synthesised past literature using a meta-analytical approach. Given the identified gaps and biases, in Chapter II I compiled a dataset including species from all realms and 15 taxonomic groups, to find predictors of extinction risk across taxa. Because traits and human threats are likely to interact, in Chapter III, I have implemented, tested, and analysed a virtual conceptual model to understand how different traits interact with the different threats to increase the extinction risk of species. The literature review of Chapter included 173 publications with most studies focussing on either mammal or bird taxa. The spatial extent of studies has also been narrow, with a very strong focus on the Palaearctic realm, particularly for invertebrate and plant groups. Body size was the most studied trait, followed by geographical range and fecundity. Among the top 10% studied traits, geographical range size had the greatest percentage of significant relationships with extinction risk. Meta-analyses were possible for geographical range size, habitat breadth and body size, with the first two being negatively related to extinction risk, and the third showing no consistent effect. In Chapter II, I compiled and analysed trait-extinction risk relationships for 10 traits, with a dataset consisting of 874 species evenly sampled from 15 taxonomic groups ranging from vertebrates, invertebrates, and plants. I identified narrow habitat breadth as a universal predictor of extinction risk across vertebrates, invertebrates, and plants. Long generation length, low fecundity, and large offspring size - characteristic of species with slow life cycles - and small climatic breadth were also identified as general predictors, but available data was limited and therefore results are not conclusive for these traits. Poor dispersal ability is a general predictor among invertebrates and plants, but not among vertebrates. The remaining traits (body size, microhabitat verticality, trophic level, and diet breadth) showed contrasting responses depending on the taxa. In the third Chapter, my aim was to understand how different anthropogenic drivers may impact species with different traits. I built a conceptual agent-based model with virtual organisms competing in a virtual environment for resources, upon which I simulated human threats. My model simulations showed that traits and threats interact in predictable ways. Namely, threats that directly increase the mortality of individuals - direct killing- have a higher impact on organisms with slow life cycles and poor dispersal ability. Threats decreasing the amount of habitat - habitat loss and fragmentation- negatively affect large sized, poorly fecund organisms. The effect on dispersal ability depends on the spatial configuration of the threat. Finally, threats decreasing habitat quality or the amount of resources available to organisms - habitat degradation and invaders - severely affect large, fast-lived, and highly fecund organisms. For a clearer picture on which traits predispose species to extinction, more data should be collected on underrepresented groups and regions, such as invertebrates, plants, and fungi in the tropics. Despite this shortcoming, in this thesis I demonstrate that some traits predispose species to and can be used as predictors of extinction. Species with narrow geographical and habitat breadths, two of the classical dimensions of rarity, are more at risk. Slow life cycles and narrow climatic breadths should also drive greater extinction risk. Other traits do not seem to have a general response but are taxon-dependent. Finally, intrinsic traits must in the future be studied considering anthropogenic threat types, as these two dimensions interact to drive species extinctions.
  • Pulkkinen, Lauri (Helsingin yliopisto, 2022)
    Tick-borne encephalitis virus (TBEV) is a dangerous viral pathogen that causes a severe neurological disease. Like other members of the Flavivirus family, the virus has a positive-strand RNA genome that forms a nucleocapsid (NC) with multiple copies of the capsid (C) protein. The NC is surrounded by a lipid envelope derived from host membranes. The viral envelope (E) and membrane (M) proteins are embedded into the bilayer, and form an icosahedrally symmetric lattice on the virion surface. The aim of my thesis work was to understand what sets TBEV apart from other flaviviruses, as well as to increase our knowledge of TBEV particle assembly. To identify open questions in the field, I performed a literature review on TBEV structural biology. My review highlighted that despite the danger it poses, TBEV has been under-studied, especially in comparison to other flaviviruses. Consequently, our understanding of TBEV biology relies heavily on extrapolation from other species, and many aspects of the virus’ lifecycle remain unknown. To better understand the unique features of the TBEV virion, I purified the virus, and used cryogenic electron microscopy (cryo-EM) combined with single-particle reconstruction to build a 3.3 Ångström-resolution three-dimensional model of the virus. The reconstruction revealed a novel M protein conformation that may be unique to TBEV based on sequence alignment. Additionally, the cryo-EM reconstruction allowed me to characterize the protein-protein and protein-lipid contacts that affect TBEV virion stability. I discovered that the M proteins form lateral contacts on the virion surface, stabilizing the virus. These interactions compensate for the lack of E-E contacts in TBEV compared to other flaviviruses. Additionally, I showed that the E and M proteins coordinate ordered phosphatidylcholine molecules that increase the stability of the virion. Despite the central role the envelope plays in particle stability and virus entry, the contribution of lipid interactions in virion assembly has received little attention. To understand the role of lipids in NC formation, I investigated the C protein interactions with membranes. I used liposome sedimentation assays, quartz-crystal microbalance with dissipation monitoring measurements with supported lipid bilayers, and Langmuir-Blodgett trough-based tensiometry to characterize the interactions of the C protein with membranes. I showed that the C protein can bind and insert into membranes only in the presence of lipids with negatively-charged headgroups and that it can recruit RNA while membrane-bound. I characterized the TBEV lipidome and observed that the lipids the C protein needs for membrane binding to are also incorporated into the virion envelope confirming the physiological relevance of the biophysical findings. My results show that while there are commonalities between flaviviruses, generalising from one taxon to the others is not always applicable, and thorough investigation is needed to establish pan-flaviviral characteristics. Additionally, it is evident that both protein-protein and protein-lipid interactions play important roles in TBEV biogenesis and particle stability, and merit further study.
  • Johansson, Tiira (Helsingin yliopisto, 2022)
    The advent of next generation sequencing (NGS) technologies has changed the nature of human leukocyte antigen (HLA) research. Thanks to its increased sequencing throughput, NGS empowers high-accuracy HLA genotyping in clinical settings, disease association studies, and the development of potential future immunotherapeutics. Current NGS can be divided into two different approaches. Illumina’s technology with massively parallel sequencing produces a high number of short reads. Illumina provides highly accurate data with a minimal number of sequencing errors; however, the short reads can cause issues with alignment and phasing in HLA genotyping. In contrast, the long-read technologies, Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), offer a single-molecule sequencing approach enabling sequencing of ultra-long reads. However, these two suffer from higher error rates, making HLA genotyping potentially less accurate. Concurrently with the development of NGS applications, several bioinformatics software have been developed for assigning HLA alleles based on existing genomic and RNA sequencing (RNA-seq) data and for imputing HLA alleles using single-nucleotide polymorphism (SNP) markers. In addition to HLA genotyping, NGS provides a powerful tool for studying the expression of several HLA genes and alleles in multiple samples simultaneously, replacing more conventional methods such as quantitative PCR (qPCR) and microarray. At the beginning of this thesis, earlier studies had already identified associations between differential HLA gene- and allele-level expression and human diseases. However, an RNA-seq method providing accurate and multiplexed way to study HLA gene- and allele-specific expression was lacking. To study comprehensively HLA gene- and allele-specific expression in normal peripheral blood mononuclear cells (PBMCs), a highly multiplexed RNA-seq method for Illumina using unique molecular identifiers (UMIs) in expression quantification was developed in study I. The combination of a personalized HLA reference and an in-house pipeline, written in R, allowed an extensive comparison of HLA gene and allotype expression in PBMC samples of 50 individuals. The results showed that although the expression in HLA was clearly gene- and allele-specific, there was also variation within genes and alleles representing the differential expression between individuals. Additionally, study I revealed haplotype-specific expression of six common Finnish HLA haplotypes. Interestingly, two autoimmune haplotypes, which have been associated with e.g. celiac disease and type I diabetes, had very distinct expression levels suggesting that the level of haplotype expression alone is not the primary predisposing factor. In study II, a targeted RNA-based method was developed for HLA ONT sequencing. The method employed PCR-based enrichment and barcoding, enabling 10 samples and several HLA genes to be multiplexed and sequenced in a single sequencing run. By using the MinION sequencer together with SpotOn flow cells, a sufficient number of reads per sample for HLA genotyping was generated. To achieve the best possible genotyping accuracy, only the higher quality 2D reads were included in the analysis. Despite the sequencing errors that ONT introduces during sequencing, the HLA genotyping results were obtained in 80% of HLA class I alleles and 95% of HLA class II alleles. Since HLA has a crucial role in immune surveillance and in the initiation of antitumor immune responses, the aim of study III was to investigate HLA expression in tumor samples acquired from a longitudinal high-grade serous ovarian cancer (HGSC) cohort. The sample material consisted of ovarian tumors and various intra-abdominal anatomical sites collected prior and after chemotherapy. In the inter-tissue analysis, differential expression levels in mainly non-classical HLA genes were found between distinct anatomical sites, indicating tissue-specific HLA expression levels. Additionally, the results in study III showed that in one of the anatomical sites, omentum, chemotherapy altered the expression of class II. Interestingly, the intra-patient analysis revealed that the allelic imbalance between two heterozygous alleles changed in the samples acquired from different tissues and treatment phases. To conclude, this thesis provides novel insights into gene- and allele-level HLA expression in different tissues. Additionally, it introduces new RNA-based methods for HLA genotyping and HLA expression quantification, which can be applied in future studies. Finally, it provides a comprehensive review of methods and bioinformatics tools designed for HLA allele-specific expression and the diseases associated with differential HLA allele expression.
  • Sihvonen, Reetta (Helsingin yliopisto, 2022)
    Streptococcus pyogenes and Streptococcus pneumoniae are important human pathogens capable of causing invasive infections which need antimicrobial treatment. Aerococcus urinae is a rare and relatively unknown causative agent in invasive infections but its clinical importance is emerging. Antimicrobial susceptibility testing (AST) is a crucial method to study the usefulness of the clinically used antimicrobial drugs and to observe trends in the prevalence of the resistance or its emergence. This thesis project aimed to analyse the antimicrobial susceptibilities of S. pyogenes and S. pneumoniae towards commonly used antimicrobial drugs and to characterize both species to gain more understanding related to their resistance. The aim was also to study bacteraemic infections caused by A. urinae and to estimate the effectiveness of the given empiric cefuroxime treatment. In addition, the objective was to determine the in vitro susceptibility of A. urinae to benzylpenicillin, cefuroxime and ceftriaxone. Species identification was performed by latex agglutination (S. pyogenes), optochin susceptibility (S. pneumoniae) and/or MALDI-TOF MS (S. pneumoniae and A. urinae). Antimicrobial susceptibilities were determined by disk diffusion, gradient diffusion (Etest) and/or broth microdilution test (Sensititre) according to the EUCAST standards. S. pyogenes isolates were characterized by emm typing and resistance gene analysis was performed by multiplex PCR to detect ermB, ermTR, and/or mefA. S. pneumoniae isolates were serotyped. Clinical bacterial isolates were found from the laboratory database using WHONET or collected particularly for the study project. Clinical data from the patients were collected retrospectively. The patient catchment area was the Helsinki metropolitan area in southern Finland representing nearly a third of the Finnish population. Susceptibility and patient data were analysed statistically by SPSS. In this thesis project we described a skin and soft tissue infection epidemic in the adult population caused by macrolide and clindamycin resistant S. pyogenes emm33 in 2012–2013. Emm33 is a rare and previously unknown emm type in Finland. Epidemic S. pyogenes emm33, a putative clone, harboured the ermTR and expressed a constitutive MLSB phenotype but was susceptible to telithromycin. S. pyogenes emm33 was also susceptible to tetracycline, doxycycline, levofloxacin, moxifloxacin, and vancomycin. Studied patients were mainly 41–60 years old and bacteraemic patients often had alcoholism and/or intravenous drug abuse. The clinicians in the HUS district were informed about the epidemic and advised in empirical treatment choices. We studied the susceptibility patterns of invasive and non-invasive S. pneumoniae isolates in 2009–2014 and showed that S. pneumoniae non-susceptibility to clinically important antimicrobial agents decreased significantly in the Helsinki metropolitan area during the four years after the 10-valent pneumococcal conjugate vaccinate (PCV10) implementation in 2010. This trend was seen especially in non-invasive isolates from <5 years old patients, where non-susceptibility decreased towards penicillin, erythromycin, clindamycin, tetracycline, and trimethoprim–sulfamethoxazole. The proportion of multidrug resistant (MDR) isolates decreased also significantly from 22% in 2009 to 6% in 2014 among these patients. S. pneumoniae serotype distributions showed that the number of the PCV10 serotypes decreased among invasive isolates. In contrast, the non-PCV10 serotypes increased among invasive isolates from ≥5 years old patients and of these, serotypes 19A (38%), 3 (13%), and 6A (13%) were the most frequent. Especially 19A is associated with high prevalence of resistance therefore making the continuous surveillance of pneumococcus essential. We retrospectively studied 141 clinical A. urinae isolates and the clinical data of 77 patients with bacteraemic A. urinae infection from 2013–2018. All A. urinae isolates were susceptible to benzylpenicillin (S ≤0.125, EUCAST breakpoints for aerococci) and cefuroxime (S ≤4, EUCAST PK/PD breakpoints) but 2–3% were resistant to ceftriaxone (R >2, EUCAST PK/PD breakpoints). Bacteraemic A. urinae patients were primarily elderly men harbouring several comorbidities. All but one patient was given antimicrobial therapy and the response was mainly good based on decreased fever and CRP. Thirty-day mortality was 17% and patients who died were on average older compared to survivors. Cefuroxime was the first empiric antimicrobial agent given to 87% of the patients and it was given equally often to patients who survived and did not survive. There were no differences between the cefuroxime MIC values of the survivors versus non-survivors. Based on these findings, A. urinae was clinically susceptible in vitro to cefuroxime and furthermore, empiric cefuroxime treatment likely covers bacteraemic infections caused by A. urinae. The results of this study made A. urinae more familiar to HUS district physicians and have improved the treatment of bacteraemic A. urinae patients.
  • Mahato, Arun Kumar (Helsingin yliopisto, 2022)
    Motor symptoms of Parkinson’s disease (PD) are caused by the degeneration of dopamine (DA) neurons. Currently, no disease-modifying therapies against PD are available. Therefore, the major challenge is developing a drug to protect and restore DA neurons. Glial cell line-derived neurotrophic factor (GDNF) has been identified and characterized for its ability to promote the survival of cultured nigrostriatal dopamine neurons and protect and repair them in animal models of PD. However, the outcomes of the clinical trials with GDNF in PD patients remain inconclusive. GDNF is a promising protein that halts neurodegeneration. GDNF does not cross through the blood-brain barrier (BBB) and has poor diffusion properties, complicating clinical use in PD patients. This thesis aimed to characterize the biological activity of GDNF family ligand (GFL) receptor agonists in both in vitro and in vivo preclinical models of PD. We have developed small molecular weight compounds acting similarly to GDNF in the dopamine system (GFL receptor agonist) with improved pharmacological characteristics. The first-generation GFL receptor agonist, a compound named BT13, potently activated the GDNF receptor REarranged during transfection (RET) and its downstream targets. It protected cultured dopamine neurons from neurotoxin-induced cell death and stimulated dopamine release in the mouse striatum. We further optimized BT13 and developed the second-generation GFL receptor agonist BT44 with ten-fold improved potency. BT44 activated RET, supported cultured DA neuron's survival and protected them from neurotoxin lesions, induced functional recovery, and protected striatal dopaminergic fibers in rats with experimentally induced PD. We also studied the selectivity of both BT13 and BT44 using different functional assays. Our results demonstrate that both BT13 and BT44 are selective towards RET. The compounds promote survival and protect the cultured dopamine neurons only in the presence of RET. BT44 is a promising lead compound that could be potentially developed as a drug for PD treatment. However, BT44 suffers from intrinsic poor aqueous solubility like many other hydrophobic drugs, making its preclinical development and clinical translation difficult. Therefore, to improve BT44 solubility, we developed nanoformulation of BT44 with Pox-based ABA triblock copolymers. We found that nanoformulated BT44 retains its biological activity and has improved absorption and blood-brain barrier (BBB) penetration compared to the pristine BT44. We also demonstrate that nanoformulated BT44 binds to the GFL receptors and competes with GDNF for binding to the RET receptor. The receptor binding studies were not possible with pristine BT44 due to its poor solubility. Taken together, our results indicate that both BT13 and BT44 can activate the RET receptor and its downstream signaling cascade necessary for neuronal survival and protection from neurotoxin insults. Further, our results show that in vitro BT44 is active in 10 nM concentrations, which is a realistic target concentration for actual drugs. Moreover, we developed a nanoformulation of BT44 to improve the solubility of the pristine compound and showed that this nanoformulation retains biological activity. With further optimization and detailed studies, BT44 could be developed as a drug-like candidate for disease-modifying treatment for PD.
  • Luoma, Emilia (Helsingin yliopisto, 2022)
    Sustainability and sustainable development have reached global attention and sustainable development has become a common objective in various environmental programs and projects. Although sustainable development and sustainability have usually been acknowledged to consist of three aspects: environment, economic and social, their exact definitions have remained hazy allowing people to define them in ways that best suit them. Socio-ecological problems threatening sustainability are both complicated and multiscale, and their environmental management requires multidisciplinary cooperation, trade-offs and systems understanding. This thesis consists of four Articles [I-IV], which aim at deepening the systems understanding related to marine environmental management issues and searching for the methods and practices supporting sustainable development. I apply different causal network modeling approaches (CNMAs) in the context of two management-oriented themes: ship biofouling management and sustainable development of marinas. In this summary, I study and integrate the combined results of the articles through two sub-questions to aim to understand the sustainability and sustainable development related issues on a broader scale. They are: 1) How can causal network modeling approaches support sustainable development in environmental management?; 2) What prevents or promotes sustainable development in environmental management? In Article [I] we developed a conceptual influence diagram (CID) to visualize the multi-disciplinary nature of biofouling management and to analyze the ships’ biofouling management issue in the Baltic Sea. The developed CID demonstrates the causal interlinkage between the main factors and presents the multiple aspects of the issue. Further, the CID can advance communication between different parties, providing a starting point for conversations. Article [II] presents a decision analysis model where the CID of the Article [I] was used as a starting point in developing a multi-criteria decision support model (MCDAM) using Bayesian networks. The developed MCDAM advances holistic understanding about the biofouling management in the Baltic Sea and compares the case-specific sustainability (the combination of risks and utilities) of alternative management strategies (the combination of multiple decisions). Article [II] demonstrates the optimal biofouling management method is case-specific but with careful considerations, both economic and environmental savings can be reached. In Article [III] we study how service providers and users define sustainable development and its goals in the marina context. The constructed CIDs were structured based on the interview data and used as a method to analyze the issue. The results present the differences and similarities in stakeholders' thinking. In addition, the study indicates stakeholders consider sustainable development from a more multi-dimensional perspective when setting the goals than defining the term. In Article [IV] actor-network theory (ANT) provides a philosophical framework for a conceptual systems analysis, presented as a concept map, on boat-sourced sewage management (BSSM). Based on literature, questionnaires and interviews, a multi-material collective of human and non- human entities was developed to realize how and under what conditions BSSM facilities support the sustainable development of marinas. Since the BSSM facilities are both core marina services and governance artefacts if managed optimally, they can support both sustainable boating and marina operation. Based on the results, I state that a systems approach is needed in studying environmental management problems and advancing sustainable development. By revealing the multi-dimensionality and the interconnections between different elements, CNMAs can promote the identification and involvement of the key components, the explanation of the mechanisms and principles of operation, and this way increase the comprehensive understanding concerning the management questions or problems. Based on this, I argue that CNMAs have potential to enhance the understanding about what knowledge is needed and what aspects should be considered to make decisions that advance sustainable development. Further, I argue that CNMAs can promote reciprocal stakeholder participation and open communication and thus aid pay attention to the factors and actors that prevent sustainable development.
  • Karjalainen, Linda (Helsingin yliopisto, 2022)
    Sustainability is a globally prominent guideline in policy-making and planning, with cities currently leading the way by pursuing sustainability transitions locally. The urban transportation sector is no exception in this regard. Urban transportation sustainability (UTS) pursues accessible, affordable, safe, and just mobility, efficient operations and investments, and lower levels of emissions, pollution, resource use, and other environmental impacts. Such a system requires long-term commitment to policies alongside wide and cooperative collaboration between diverse actors within municipalities, as well as between their surrounding regions and the national level. While sustainability promotes a tri-dimensional approach that addresses environmental, social, and economic aspects together, local applications have been found to mostly focus on environmental and efficiency-related issues. The simultaneously marginalised social aspects of sustainability tend to be the most relevant to local citizens, as they touch upon the diverse perceptions, experiences, needs, and vulnerabilities of the population. If sustainability is narrowly conceptualised, it likely leads to a narrower set of measures selected and implemented as a solution, as well as potentially limited indicator use when monitoring these measures locally. Together, they affect the urban context that is created as a result of policy-making and planning. Thus, critical exploration of the links between conceptualisation, operationalisation, and realisation of UTS is needed. This thesis directly addresses the space between concept and practice that has been overlooked in transportation policy research and is in need of connecting. Specifically, the following two-fold research question is posed: RQ1 (A) How is UTS conceptualised and operationalised in municipal policy-making and planning, and (B) in what ways does (A) affect the realisation of sustainability in cities? An integrative analytical framework is applied to analyse the journey of UTS from an idea to a reality through the stages of the policy cycle. This thesis presents an interdisciplinary policy analysis that is rooted in empiricism and realism. It draws from environmental social sciences, geography, policy studies, and urban planning to cover each step of the policy cycle, to utilise diverse datasets and mixed methods, as well as to incorporate viewpoints from academia, municipal policy-makers and planners, and citizens. The viewpoints are synthesised as integrated perspectives to answer the research question reliably and broadly. The data consists of academic research articles that focus on UTS assessments (Paper I), municipal policy-maker and planner interviews that focus on political agenda formation (Paper II), and household car ownership statistics that focus on different types of carlessness (Paper III). Mixed methods are applied to both data collection and analysis. The methods consist of a systematic review, semi-structured interviews, thematic analysis, and statistical analysis. Local cases representative of the Nordics (Helsinki, Finland; Oslo, Norway; Stockholm, Sweden) are utilised. The key findings reveal that UTS in practice is most prominently framed through climate change and carbon emissions. Despite this politically established direction, car travel presents a significant challenge to UTS realities even though current policies strongly address emissions. Furthermore, the UTS focus throughout the policy cycle is on the quantitative and easily measured emissions, traffic volumes, and accidents. The limited UTS coverage is guided by politics and begins in the agenda setting phase, carries onto the measure selection and implementation phase, and finally onto the monitoring phase, producing an application of UTS that marginalises many social and qualitative aspects. The narrow monitoring of UTS then loops back to problem definition and agenda setting. Academic assessments display the same disregard towards diverse and comprehensive indicator and data use, and display minimal efforts to broaden their approaches to sustainability to produce more conclusive results. A vicious circle that does not fully account for sustainability as a concept nor as a reality is thus created. The notion of accounting for social equity and planning for the most vulnerable is largely missing from current practices, yet it presents a reality to many citizens as empirically detected in Paper III. UTS ought to be broadly framed throughout the policy cycle to diversely understand and sensitively include different citizen realities, vulnerability, and other aspects of sustainability into the policy-making and planning processes. This thesis empirically demonstrates that while on the surface UTS appears to be advancing – regarding political agendas, solution implementation, and evaluations – a critical in-depth analysis of its conceptualisation, operationalisation, and realisation reveals major issues and further implications in sustainability coverage and sensitivity. Currently, UTS is mainly advanced within the existing system and practices, and no significant changes in decision-making and planning (including assessments) are visible. The narrow view on sustainability is applied in practice throughout the policy cycle and round again, disregarding the purpose and comprehensiveness of the UTS paradigm, as well as the complex citizen realities.
  • Piilo, Sanna (Helsingin yliopisto, 2022)
    The high-latitudes are warming at more than twice the rate of the global average. Warming and the consequent changes in hydrology affect peatland functioning, especially through changes in vegetation and carbon dynamics. The majority of the world’s peatlands are found in the Northern Hemisphere, forming a globally significant carbon storage and they are in constant interaction with the atmosphere through carbon uptake and release. The global importance of peatlands is widely recognised; however, the role played by high-latitude peatlands in changing climates is still unclear. It is not thoroughly understood how warmer future climates and hydrological changes will affect peatland vegetation and carbon processes. These uncertainties result from the complexity of peatlands and from the manifold future trajectories that are affected by different forcing factors from climate to local conditions. In this dissertation, I aim to increase our knowledge of high-latitude peatland vegetation and carbon dynamics under changing climatic conditions. My approach is palaeoecological, because I use peat records as an archive to reconstruct the response of high-latitude peatlands to known changes in climate. Peatlands function as important archives, since under anoxic and acidic conditions, peat-forming plant remains are well preserved. By identifying these plant remains, we can reconstruct past vegetation compositions. The various peat-forming plants have their own ecological niche, since they prefer and require specific hydrological or nutritional conditions and thus are good indicators for past hydrological changes and conditions. For this dissertation, I collected, in total, 47 peat records from eastern Canada, northern Sweden and Finland, the Kola Peninsula and the northeast of European Russia. I investigated how peatland habitats, carbon accumulation and cycling of our study sites have changed in response to changes in climate. For this, I used plant macrofossils, peat geochemical measurements and dating methods. In addition, I statistically inspected the change in vegetation compositions over time and used a model of carbon accumulation that considered peat decay aspects to determine whether carbon accumulation has been higher or lower than what we would otherwise predict, based on carbon accumulation models. My results show that during recent centuries, the vegetation compositions of the microhabitats examined have mostly changed from typical wet sedge fen vegetation to Sphagnum moss-dominated intermediate surfaces and dry moss- and dwarf shrub-dominated surfaces. During recent decades, these vegetation compositions have remained rather stable, with no major changes in vegetation. However, the spatiotemporal variation within and between the study sites was prominent, and thus to detect any large-scale signals from the data it was essential to use multiple samples and sampling points. Based on my data, it was plausible to consider that if high-latitude peatland vegetation changes from sedge fen vegetation to more hummocky vegetation types, high-latitude peatland carbon accumulation and sink capacity may remain significant or even increase. To better predict the role of peatlands under changing climates, it is crucial that peatland vegetation responses, carbon dynamics and their linkages with climate are more thoroughly understood. My data also support the prevailing understanding that peatlands are important carbon sinks and storages and thus preserving ecosystems that form a nature-based solution to the problem of mitigating the effects of climate warming is highly important.
  • Milocco, Lisandro (Helsingin yliopisto, 2022)
    Understanding the genotype-phenotype map (GPM), or how specific genetic variation relates to specific phenotypic variation, is a fundamental objective of biology. Particularly for the study of evolution, the GPM is important since it bridges genes, the heritable units, with the traits that interact with the environment and determine fitness. The applied field of quantitative genetics approximates the GPM using linear statistical models. This approximation is the basis of many important applications, including methods to predict the response to selection in a population. The other major field concerned with the GPM is evolutionary developmental biology or evo-devo, which studies the gene and cell interactions by which the phenotype is built through the process of development. A main result from this field is that the GPM is notoriously complex and nonlinear, with genetic effects being highly dependent on genetic background, biophysical factors and the environment. This can be in conflict with the linear approximation of quantitative genetics. Because evo-devo and quantitative genetics have developed independently, there is a gap in the understanding of how these conceptualizations of the GPM fit with each other, what are their limitations, and if there is a potential to improve our ability to make prediction about evolutionary systems by combining insights from both approaches. This thesis deals with these issues. The thesis can be divided in two parts. The first part consists of theoretical work studying in detail how quantitative genetics predictions and models behave under complex and nonlinear GPMs that arise from development. For this, I performed large-scale evolutionary simulations using a realistic representation of the GPM given by a computational model of tooth development. Using this set of simulations, I first studied how well the linear approximation of quantitative genetics is able to predict the response to selection. I found that predictions using the linear approximation can be biased when the GPM is nonlinear. In other words, there can be a significant part of the response to selection that is missed by the linear approximation. Using the same set of simulations, I studied how the linear approximation evolves in a complex GPM. I found that the evolutionary dynamics of the linear approximation are highly dependent on the GPM, and differ substantially from what is expected for a linear GPM. These dynamics are not purely stochastic, but rather deterministic ways in which the linear approximation changes as a reflection of the curvature of the GPM. In the second part of my thesis, I use the insight obtained in the first part to develop an applied method that uses techniques of quantitative genetics, combined with insight from evo-devo, to provide better predictions of evolutionary response to selection. The method uses a Kalman filter to combine information from selection acting on each generation, with information from the evolutionary time-series. I test the method with the simulated dataset using the model of tooth development, and also with an artificial selection experiment on the wing of the fruit fly, where 16000 flies were measured. The new method is able to improve predictions and is a promising path to combine knowledge from both fields studying the GPM. In this way, the work in this thesis shows that rather than being a nuisance, the nonlinear nature of the GPM contains information that can improve our understanding of evolutionary processes.
  • Mogollon, Isabel (Helsingin yliopisto, 2022)
    Specialized signaling centers act as organizers of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called the enamel knots (EKs) determine tooth number, size, and shape. The tooth initiating placode and later primary EK signaling center share molecular markers, but it was not clear what the relationship between these two is and what cellular behaviors are contributing to early molar morphogenesis. In this thesis work, I used molecular tools and fluorescent reporter mouse models to visualize cellular behaviors with confocal microscopy of intact live and fixed tissues on a single cell level. I uncovered that the molar is regulated by an initiation knot (IK) signaling center prior to the EKs. The molar IK arises in the placode: cells exit the cell cycle, condense via migration and drive proliferation in the bud. The IK is apoptotically silenced as the primary EK arises independently. I verified that the IK is an early signaling center belonging to the molar itself and argue that it plays a fundamental role in establishing the tooth fields and defining tooth shape. I also explored the mechanisms of signaling center dysregulation in tooth dysplasia models lacking the transcription factor Forkhead box i3 (Foxi3). Foxi3 placode and bud stage mutants revealed abnormal cell behaviors including signaling center fate definition, cell cycle regulation abnormalities and signaling center longevity. These changes result in arrest of tooth morphogenesis and abnormal tooth shape. Lastly, I reviewed the different experimental setups used to study tooth development with imaging approaches. I concluded that the continual advancement of imaging technologies is shedding light on the fundamental mechanisms of tooth development, especially during the earliest stages.
  • Kahma, Tuomas I. (Helsingin yliopisto, 2022)
    Coastal zones are vulnerable to human activities, and eutrophication is one of the most severe anthropogenic stressors of coastal ecosystems. The decline of Fucus vesiculosus in the Baltic Sea has been of major concern in the past decades. The present thesis highlights the role of this perennial macroalgae not only as a habitat-forming key species maintaining biodiversity in rocky seafloor communities but also as a cross-habitat provider of energy subsidy to adjacent soft-sediment ecosystems and associated macrofauna communities. In coastal ecosystems, the flow of detrital matter connects spatially distinct habitats, and the provision of such cross-habitat energy subsidies is fundamental for many ecological processes. The local primary production and carbon export potential of large macrophytes can be very high. However, the overall importance of macrophyte-originated detritus for coastal food webs is not fully understood. In oceanic systems, the role of large kelps as an organic matter subsidy has been studied previously. There is growing evidence that macroalgae-derived detritus are significant spatial subsidies from shallow rocky vegetated habitats to nearby soft-sediment shallow areas and to deeper offshore areas, where it fuels benthic macrofauna food webs. In shallow habitats of the Baltic Sea, habitat-forming rockweed species Fucus vesiculosus forms abundant and highly productive underwater forests, which host a high diversity of associated macrofauna. The carbon export potential of F. vesiculosus to nearby shallow bare sediment habitats has been recently recognized, but we know little about the role of this perennial macroalgal species in the coastal food webs and the dynamics of associated benthic macrofauna communities. The general aim of this thesis was to examine the dietary contribution of macrophyte-originated detrital matter, especially drifting F. vesiculosus, to the associated benthic macrofauna communities in coastal areas of the Baltic Sea influenced by large external inputs of macrophyte detritus. Additional aims were to investigate how this role may change spatially and seasonally and to provide insights into the fate of primary production in coastal systems dominated by large macrophytes and affected by increasing ephemeral algal accumulations. A stable isotope approach and Bayesian modelling were applied to assess the contributions of macrophyte-originated components to the adjacent basal food webs potentially influenced by drifting detrital macrophyte matter. The study took place in Hanko Peninsula, SW Finland. There is a high diversity of potential macrophyte food sources in the shallow vegetated habitats of the Baltic Sea since both marine and limnic species are present. The results indicated that the dominating habitat-forming macrovegetation of a habitat (F. vesiculosus or angiosperm plants) does not necessarily affect the contribution of the macrophyte component in the diets of detritivorous benthic infauna. The stable carbon and nitrogen isotopic niche structures of a particular habitat, however, are strongly affected by the dominant macrovegetation assemblage. In the Storfjärden Bay, most of the sedimentary carbon on the seafloor seemed to be of autochthonous origin, despite the proximity of riverine runoff. In offshore areas, pelagic primary production dominated as the main energy source of the benthos, but the results showed that the carbon export from F. vesiculosus can have a large contribution to the adjacent soft-sediment food webs also here. Depth was an important factor regulating energy source dynamics. The results from different depths (from 3.5 to 45 m) indicated that the rockweed F. vesiculosus fuels soft-sediment benthic macrofauna communities in the direct proximity of shallow vegetated habitats, but it can also have a significant contribution in deeper soft-sediment areas of the coastal ecosystem. In shallow (< 5 m) vegetated habitats, F. vesiculosus is one of the main energy sources of the clam Macoma balthica, but also obligate suspension-feeding mussels (Mytilus trossulus) can incorporate some of the fine-sized detrital matter exported from F. vesiculosus rocky bed communities. In deeper areas (> 15 m), the amount of this cross-habitat energy subsidy might depend on the magnitude of physical transport processes. A temporal field study, examining species-specific dietary responses across three sampling dates, showed that the preferred feeding strategy, but not the season, mainly shapes the dietary compositions of the abundant benthic macroinvertebrate consumers. The results show that accumulations of drifting filamentous algae are rapidly incorporated into the food web through epigrazers. An in situ field experiment simulating a natural accumulation event and subsequent decaying process of F. vesiculosus at different depths showed that algal decomposition progressed relatively slowly. Detectable incorporation of Fucus-derived matter to epigrazers and detritivorous bivalves occurred after a few weeks, while simultaneously the incorporation of filamentous algae decreased over time. Hence, it can be suggested that the ecological role of decomposing F. vesiculosus is more important in areas where the algal matter can accumulate for several months. The effect of depth influenced the food incorporation of typical epigrazers. As a rough quantitative estimate, the effect of depth (3 m vs. 6 m) on the proportion incorporated was approximately 10 % units between the two depths, which was roughly similar to the observed temporal dietary variations in the studied area.
  • Kotila, Tommi (Helsingin yliopisto, 2022)
    Cells are the minimal compartments of life that constantly respond to cues coming from both inside and outside of their environment. In response, cells migrate, transport different molecules and undergo sudden shape changes to meet their environmental demands. These processes are regulated by the actin cytoskeleton, a ubiquitous component of eukaryotic organisms that dynamically cycles between monomeric and polymeric form at the sites of cellular rearrangements. Polymerization of actin against plasma membranes and organelles provides force to the constant remodeling of cells. Consequently, the actin cytoskeleton is critical for many fundamental cellular processes, including cell division, cell migration, cellular morphogenesis, endo- and exocytosis. Actin molecules bind ATP and polymerize to polar filaments with structurally two different ends. Actin filaments grow from the ‘plus’ (+) ends by the incorporation of ATP-actin monomers, after which they convert through ATP-hydrolysis to ADP-state that populates the ‘minus’ (-) end of the filaments. ADP-state renders actin molecules to a state of depolymerization, and thus actin filaments slowly shrink from the (-) ends in steady-state conditions. This is a fundamental property of actin filaments that drives their directional growth in the force production. Thus, cells require a constant supply of actin monomers from depolymerization of ‘old’ filaments and conversion of these actin monomers from ADP- to ATP-state. Several proteins, including evolutionary conserved ADF/cofilin and cyclase-associated protein (CAP), are involved in the regulation of these steps in cells, but the exact molecular mechanisms have remained elusive. In this work, I studied the molecular principles of how actin filaments are dynamically converted between monomeric and filamentous forms. CAP is a multidomain protein and earlier shown to be critical for actin dynamics in various model organisms. However, the molecular mechanisms by which CAP regulates actin dynamics have remained unknown. In the first and second parts of my thesis, I utilized X-ray crystallography, mutagenesis and biochemical assays to reveal how CAP interacts with both actin monomers and filaments. I show that the N-terminal domains of CAP are responsible for binding to the (-) ends of actin filaments and catalysis of rapid filament depolymerization in synergy with ADF/cofilin. The C-terminal domains of CAP scavenge the depolymerized ADP-actin molecules from ADF/cofilin and catalyze their conversion to ATP-state. These data establish CAP as a recycling machine for actin molecules and explain the earlier in vivo observations why different domains of CAP are vital for rapid actin dynamics in cells. In the fourth part of my thesis, I show how rapid actin dynamics are achieved in an evolutionary distant organism, pathological Leishmania parasite that contains only a very simple actin-regulatory machinery. By utilizing the molecular tools developed in the third study, combined with cryo-electron microscopy, mutagenesis and biochemical assays, I show that Leishmania actin filaments are inherently dynamic and susceptible to disassembly by ADF/cofilin, and reveal the atomic mechanisms behind these attributes. These data help to understand the mechanisms that regulate the actin cytoskeleton evolved in eukaryotic organisms and could provide a molecular rationale for development of inhibitory compounds against Leishmania parasite actin in future. Together, this work provides a detailed molecular level understanding of new mechanism by which the dynamics of actin cytoskeleton are regulated in eukaryotic organisms. This work also provides insight into the evolution of the actin cytoskeleton and its regulatory mechanisms. Finally, the findings presented here help understanding the basic mechanisms of biological processes, and also provide molecular level understanding of the mechanisms of human diseases.
  • Chen, Chi-Chuan (Helsingin yliopisto, 2022)
    Microsoroid ferns (Microsoroideae) are the third largest subfamily of the Polypodiaceae, containing ca. 180 species (PPG I, 2016) and found mainly in tropical and subtropical Asia, Australasia, the Pacific Islands, Madagascar, and tropical Africa. The relationships between groups of this subfamily have long been controversial, especially the delimitation of the genus Microsorum, which, based on current understanding, is not a monophyletic entity. By using mostly DNA sequence data, as well as the micro-morphological information (spore ornamentation), the focus of this thesis is to clarify the delimitation of each genus and subgroup within Microsoroideae, in order to improve our understanding of the diversity of the subfamily. In the first and second study, molecular data was used to infer the phylogenetic relationships of microsoroid ferns. The results of the first study confirmed the placement of the Java Fern (Leptochilus pteropus), the common plant used in aquaria, in the monophyletic Leptochilus, with L. macrophyllus as the most basal branch. The second study shows that five tribes should be recognized within Microsoroideae, containing ca. 11 genera/groups with 205-225 species total. The previous Microsorum s.l. is divided into six clades in two tribes, Microsoreae and Lecanoptereae. By integrating molecular and micro-morphological data in the third study, more information is provided regarding possible classification, especially in tribe Microsoreae, indicating that there are two potentially new genera that need to be further confirmed. Most species of the six subfamilies of Polypodiaceae are epiphytic. Microsoreae is, in this respect, exceptional with a relatively low percentage of epiphytes. Therefore, in the fourth study, I explored the evolution of the substrate preference of the microsoroid ferns by combining the data of divergence time estimation based on fossils, trying to answer the questions “why” and “when”. The results show that the evolution of substrate preference has possibly been influenced by climate fluctuations and the colonization of new geographic regions during the Oligocene-Miocene periods.
  • Safronov, Omid (Helsingin yliopisto, 2022)
    Inonotus obliquuse, commonly called chaga mushroom (Pakurikääpä), is known and used by many ethnic societies for its medicinal properties, to the degree that they believed to its magical and supernatural powers. Chaga mushroom mainly grows on Betula species, and easily identifiable when the sterile black conks burst through the bark of a host like Betula pendula (silver birch). The infected host tree might not even show any symptoms for several years, while the white-rot disease is progressing in the heart of the woody stem of the infected tree. However, very little known about the evolutionary history of chaga mushroom, and the molecular dynamics involves in pathogen-host interaction of chaga. In contrast, there is no lack of research over the study of secondary metabolites, extracted from chaga, and their potential for biotechnological, pharmaceutical, and industrial applications. Among the compounds are triterpenoids, such as betulin and betulinic acid, which found to be produced naturally by both chaga and its hosts Betula species. This begs a question what is the molecular mechanism which drive the production of the same compounds in host and the pathogen? To this end, we sequenced, assembled, and annotated the genome of B. pendula to predict high-quality gene models. We also studied the transcriptomic profiling of silver birch bark to understand the molecular pathways which contribute to the triterpenoid’s biosynthesis in different tissues of silver birch bark. In addition to the host genome, we sequenced, assembled, and annotated a high-quality genome of the pathogen (chaga mushroom), which provides an important foundation for our evolutionary analysis. Furthermore, we also identified and characterized the candidate CYP450 monooxygenase genes from silver birch and chaga mushroom, which then for the functional analysis, they subjected to heterologous gene expression analysis.
  • Barrass, Sarah (Helsingin yliopisto, 2022)
    Tick-borne encephalitis virus (TBEV) is an important flavivirus that affects the central nervous system, and is endemic in many parts of Europe, Russia and China. Despite its importance, TBEV is less extensively studied in comparison to its mosquito-borne relatives and much of the knowledge about TBEV is extrapolated from these. In this thesis work, I use an integrative structural biology approach, combining cryo-electron microscopy and cross-linking mass spectrometry to study the structure of TBEV and its interaction with host cells. Thus, identifying key differences and similarities between TBEV and its mosquito-borne relatives. The TBEV virion structure of the Kuutsalo-14 isolate was solved using cryo-electron microscopy to a resolution of 3.3 Å. The high-resolution structure allowed the visualisation of structural- and sequence-based differences among flaviviruses, which may influence their interaction with host proteins. In addition, the structure reveals conformational switching of the peripheral and transmembrane helices of the membrane protein (M), resulting in a previously unobserved M-M interaction. The residues responsible for the interactions are highly conserved in TBEV, but not mosquito-borne flaviviruses. Furthermore, two lipid interaction sites were resolved in the envelope protein (E), corroborating the presumed role of lipid pockets in stabilising TBEV virions. I examined the methodologies in the field of receptor identification and recognised a lack of cell-based proteomic approaches. Therefore, a novel method was developed to identify virus-host interactions on the surface of cells using cross-linking mass spectrometry. During this process, it became evident that cross-link validation methods for proteome-wide studies were poorly established. Using the intraviral protein cross-links as an internal control, I assessed the cross-link confidence level, and filtered the dataset to remove false positives arising due to poor cross-linked peptide spectrum matches. In total, 59 virus-host interactions were detected using cross-linking mass spectrometry. Notably, I identified an interaction between the surface of TBEV and the substrate-binding domains of HSP74 and BiP, two entry receptors for multiple flaviviruses. I also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located within the cytoskeleton, suggesting that both clathrin-mediated endocytosis and macropinocytosis may be used as entry mechanisms for TBEV.
  • Pakarinen, Emmi (Helsingin yliopisto, 2022)
    Protein homeostasis is essential for the whole cell function. Therefore, cells monitor protein synthesis, folding, and degradation closely. Disturbances in secretory and membrane protein folding can cause accumulation of misfolded and unfolded proteins in the endoplasmic reticulum (ER). This condition, called ER stress, can predispose for manifestation of various diseases including neurodegenerative diseases such as Parkinson’s disease. Cells activate the unfolded protein response (UPR) to alleviate ER stress and restore homeostasis. Members of a novel neurotrophic factor family, cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF), are components of the UPR machinery. They are small proteins widely expressed in mammalian tissues and mostly localized to the ER lumen. CDNF and MANF can reduce ER stress with a mechanism that is not fully understood. In addition, they also promote survival of neuronal and non-neuronal cells in different animal disease models. In animal models of Parkinson’s disease, they both protect and even restore the function of midbrain dopamine neurons. However, the involvement of endogenous CDNF and MANF in the maintenance and regulation of dopamine neurons in mice is not known. The aim of this thesis was to examine the role of endogenous CDNF and MANF deletion in UPR regulation in vivo. We also aimed to investigate the role of their deficiency in the maintenance and function of midbrain dopamine neurons as knockdown of MANF results in changes in the dopamine system in fruit fly and zebrafish. In this thesis, we characterized CDNF and MANF knockout mice and analyzed their UPR gene expression and the midbrain dopamine system by gene expression analyses, immunohistochemical methods, and behavioral tests. Compensation between CDNF and MANF was investigated in the third study where we developed and characterized mice lacking both CDNF and MANF. This study demonstrates that MANF ablation causes an increase in UPR gene expression in the developing, adult, and aged brain in vivo and in cortical neurons in vitro. In particular, the inositol-requiring enzyme 1 α (IRE1α) pathway of UPR is persistently activated in the MANF-deficient brain. CDNF ablation, instead, induces UPR gene upregulation in the skeletal muscle, which is aggravated by MANF ablation. This indicates that MANF can functionally compensate for CDNF. We show that vice versa, CDNF cannot compensate for MANF function in the tissues studied. Furthermore, this thesis addresses the role of CDNF and MANF in the function and maintenance of the midbrain dopamine system. We investigated the integrity of dopamine fibers and dopamine neurons in aged mice lacking CDNF, MANF, or both. Furthermore, motor behavior of these mice was investigated to measure the function of the dopamine system. Our data indicated that these mice did not show degeneration of the midbrain dopamine system upon aging although CDNF-deficient mice display functional changes in dopamine neurons. In conclusion, this thesis work elucidates the importance of CDNF and MANF in regulating the UPR with tissue specificity in vivo. In addition, we provide data about the role of endogenous CDNF and MANF in the maturation and maintenance of the midbrain dopamine system.  
  • Sagath, Lydia (Helsingin yliopisto, 2022)
    The aim of this thesis project was to improve the methods of molecular genetic mutation analysis in neuromuscular disorders, mainly focusing on challenging repetitive intragenic regions and making these methods more accessible. The large skeletal muscle genes nebulin and titin contain large repetitive, i.e., segmental duplication regions. These regions cause hurdles for standard copy number variation detection methods and are therefore often omitted from analysis. The segmental duplication region of nebulin has been previously shown to harbor pathogenic copy number variants; when the number of duplicated blocks deviates by two or more blocks per allele from the normal number, the alteration is considered a pathogenic recessive mutation. We developed a custom comparative genomic hybridization array for the detection of copy number variants in neuromuscular disorder-causing genes. The array covers all 11 known nemaline myopathy genes and 176 additional known and potential neuromuscular disorder genes. The genes are divided by coverage into three groups, all of which allow copy number variant detection at the exon level. Using the array, we detected the largest nebulin deletion (loss of genetic material) hitherto in a patient presenting with an asymmetric distal myopathy. We were also able to determine that the deletion was present in the patient in a mosaic state, which may contribute to the asymmetric phenotype of the patient. The deletion is the first causative dominant de novo deletion described in nebulin. We have previously used the comparative genomic hybridization array for routine screening of samples from patients in whom a copy number variant in the nebulin segmental duplication region is suspected to be a causative mutation. Moreover, the array detected previously unrecorded recurrent copy number variation in the segmental duplication region of titin. The array, however, is both relatively costly and laborious to run and yields far more data than necessary. In addition, the repetitive nature of the titin segmental duplication region does not allow for exact copy number determination by the comparative genomic hybridization array alone. We, therefore, set out to develop custom droplet digital PCR assays for routine screening of the nebulin segmental duplication region and assays to verify the copy number variation of the titin segmental duplication region. Using the droplet digital PCR assays targeting the nebulin segmental duplication region, we were able to determine the copy number of the region adequately. The droplet digital PCR assays targeting the titin segmental duplication region confirmed that the region is subject to copy number variation, revealing a novel putative pathogenic or modifying mechanism in the gene.
  • Koskenpato, Katja (Helsingin yliopisto, 2022)
    Climate change has become a major challenge which organisms need to cope with to survive. However, it is challenging to measure how and to what extent climate change affects organisms and what characteristics make an organism vulnerable to these changes. An option to study the effects of climate change on animals is to do research in colour polymorphic species. Species that display two or more genetically determined discrete colour morphs within a population are considered colour polymorphic. Different colour morphs are often documented to have different characteristics considering behavioural-, physiological- and life history traits. Thus, by observing colouration shifts in space and time, it is possible to study the environment-specific adaptive values of different traits in addition to colouration itself. In this thesis, I use the colour polymorphic tawny owl (Strix aluco) as a study organism to investigate colour-associated adaptation to climate change. The tawny owl has two colour morphs: pale grey (grey) and reddish-brown (brown). In the species northernmost range margin in southern Finland, the brown morph suffers from higher mortality during cold and snowy winters compared to the grey morph. However, the selection pressure against the brown morph is absent during milder winters and the frequency of the brown morph has increased in the area over the decades. To gain a more comprehensive view on the associations between colouration and climatic variables, I conduct a large spatiotemporal scale study on the occurrence of the morphs across the species range in Europe. I also aim to find out the reasons behind the differential winter survival of the tawny owl colour morphs in the north by investigating potential adaptive traits. In particular, I study if the colour morphs differ in their plumage insulation capacity, camouflage or foraging strategy. Regarding the spatiotemporal occurrence of tawny owl colour morphs, we found in chapter I that in general the colouration seems to follow Gloger’s rule, i.e. the brown morph dominates in warm and humid environments and the grey morph in cold and dry environments. We also discovered that different climate zones have different temporal trends in colour morph frequencies in line with climate trends, and that in some cases regional weather affects these patterns. In chapter II we found that the brown morph has poorer plumage insulation capacity than the grey morph, suggesting that the grey morph is more resistant to cold temperatures than the brown morph. In chapter III we found that plumage colour affects detectability depending on the background as the brown morph was more conspicuous than the grey one in snowy landscapes, but less so in snowless landscapes. In chapter IV we used diet data to show that albeit the tawny owl is a generalist predator, the grey morph is more specialised in small mammal prey than the more generalistic brown morph. These results indicate that the higher mortality of the brown morph during harsh winters results from its poorer plumage insulation capacity together with poorer camouflage in a snowy landscape compared to the grey morph. In addition, the specialised foraging strategy on small mammals of the grey morph might be more beneficial than a broader foraging strategy of the brown morphs in years when winters are harsh. The mammalian prey is available all year round, whereas most of the alternative prey that the brown morph is more prone to use is unavailable as most birds migrate and amphibians hibernate during winter. The spatiotemporal patterns further indicate differential adaptive abilities of the morphs on a broader scale. The differential lifestyles and adaptive strategies of the colour morphs may buffer the tawny owl against climate change induced extinction risk.
  • Salomies, Lotta (Helsingin yliopisto, 2022)
    Teeth are essential organs for the feeding process of the vast majority of vertebrates. Parameters such as tooth shape, size, identity, and number can have substantial implications on the ecology of a given species. However, understanding the development of many of these parameters has been limited by the lack of available model organisms. Indeed, most dental research has been so far performed on a single mammalian species, the house mouse, which has a highly derived and reduced dentition. Furthermore, one of the most characterizing features of many vertebrate dentitions, continuous tooth renewal, is not present in the mouse. Reptiles are one of the most diversified vertebrate clades, with over 10,000 species representing numerous ecologies and habitats. In particular, lizards in the clade Acrodonta possess many of these understudied dental features, including tooth renewal (polyphyodonty) and dentitions that vary along the length of the jaws (heterodonty), and thus offer valuable models for studying these fundamental properties. This thesis explores tooth development and replacement focusing on one Acrodontan species, the bearded dragon, which possesses a heterodont dentition with replacing anterior teeth and non-replacing posterior teeth. In this thesis, I show that in bearded dragon, tooth replacement is dependent on two putative dental stem cell populations. Unlike in other species with no tooth replacement, non-replacing teeth in bearded dragon do not show signs of degradation of the dental lamina, a key structure for tooth renewal. Instead, they lack one of the stem cell populations and show decreased expression of previously uncharacterized dental genes, thus losing the ability to produce replacement teeth from the dental lamina. In addition, I characterize the role of EDA signalling in the bearded dragon and show how changes in the activity of this pathway may alter dental formula through changes in tooth size. Finally, I show a universal rule guiding the correlation between tooth formula and size in Acrodonta, suggesting a previously undiscovered mechanism underlying the evolution of dentitions. Altogether, this thesis elucidates multiple central phenomena in vertebrate dentitions, and bridges our understanding of the relationship between the development and evolution of teeth in reptiles.

View more