Faculty of Biological and Environmental Sciences

 

Recent Submissions

  • Oliveira da Silva, Filipe (Helsingin yliopisto, 2021)
    Snakes are intriguing organisms, yet their ancestral ecology and evolutionary history remained uncertain for centuries. This debate received renewed attention due to controversies about the interpretations of fossils, ecologies, and evolutionary relationships. Thus, new approaches were needed to investigate the early evolution of snakes. It is well-known that biological shape can be linked with certain functions and that the skull is an important ecological structure. So, it is relevant to investigate the skull shape to understand more details about the evolution and ecology of early snakes. In this dissertation, I investigated hundreds of skulls of lizards, snakes, and tuatara to address the ecological origin and radiation of snakes from lizards. I used cutting-edge geometric morphometrics (investigates the form of biological structures), comparative (uses phylogenetic trees), and multivariate analyses (takes higher biological complexity into consideration). I evaluated four hypotheses. i: skulls of lizards and snakes that dig in the soil and mostly live underground (fossorial) are more similar than expected under a neutral model of evolution; ii: skull shapes and ecologies are correlated, meaning that certain shapes are commonly associated with certain ecologies, which also allows us to predict the ecology of fossils and estimated ancestors based on the skull shape; iii: snakes evolved from either a fossorial, marine, or terrestrial ancestor; and iv: heterochrony - evolutionary changes due to changes in the rate and length of development - underlies the skull shape development and morphological innovation within and among snakes. I found that the skull shape of fossorial lizards and snakes are convergent, indicating that natural selection played an important role. I found a significant correlation between form and function or skull shape and ecology. In an analogy, this is like thinking about the different kinds of pans you have in your kitchen. The frying pan has a different shape in comparison to the boiling pot and you recognize their use based on their shape. I then estimate the shape of the ancestors and with high statistical confidence the ancestral ecology of snakes. Surprisingly, snakes most likely evolved from a terrestrial lizard-like ancestor. The earliest snake ancestor was most likely small and fossorial. Finally, heterochrony through acceleration in the skull shape development was detected in alethinophidian snakes, likely underlying the rise of evolutionary innovation and ecological radiation. All that said, this dissertation opened new avenues and approaches to investigate snake and vertebrate evolution in innovative ways that led to intelligible, plausible, and fruitful outcomes.
  • Kyrö, Kukka (Helsingin yliopisto, 2021)
    Urbanisation drives drastic changes in land cover and land use with multiple environmental impacts. A compact city structure, e.g., helps to reduce energy consumption and fossil fuel emissions. Yet, the increase in urbanized land cover has become a major cause of habitat loss and fragmentation – factors that are rated among the greatest threats to the variety of life on earth. Thus, there is an urgent call to search for tools that mitigate and, eventually, halt the decline of wildlife and plants with urbanization. In this thesis, I study vegetated roofs as tools for arthropod conservation in urban environments. Vegetated roofs, also known as green roofs, are purposely covered with substrate or soil and plants. They avert a multitude of environmental problems caused by the replacement of natural habitats with anthropogenic land use. They can be used, e.g., for stormwater retention and carbon sequestration. In addition, they turn building tops into habitat for plants and mobile animals. Thus, they are assumed to mitigate the loss of habitat and biodiversity due to urban development, but knowledge from vegetated roofs as habitat patches has not been sufficient to evaluate this assumption. I use arthropod data from vegetated roofs with low-growing, drought tolerant vegetation dominated either by forbs and/or grasses or succulents and mosses. I describe arthropod assemblages (beetles in Chapter I and multi taxa assemblages in Chapters II & III) using taxonomic and trait data and apply island biogeography theory and community ecology as frameworks to study the effects of biophysical roof characteristics, roof age and the landscape on arthropod abundance, richness and community composition. I found vegetated roofs to host arthropods with active and passive aerial dispersal strategies and either tolerating a wide range of habitats or associated with dry habitats. Most species were common generalists, but a few rare and endangered species also occurred on roofs. In addition, I found indication that vegetated roofs may sometimes serve as platforms for introductions of exotic arthropod species. Both local roof characteristics and the landscape shaped arthropod community composition on vegetated roofs. Roof characteristics, particularly vegetation, but also roof height and age, shaped arthropod abundance with taxon-specific effects. Most taxa responded positively to forb cover or to a combined cover of forbs and grasses, and some phytophagous groups were rare on roofs that had vegetation consisting almost exclusively of succulents and mosses. The vertical isolation of roof habitats is an effective filter that excludes less mobile species, but species that were able to colonize the roofs responded even positively to roof height, possibly because of decreased competition and/or predation. Roof age had a variable effect on arthropod abundance and richness, which are likely connected to variation in the vegetation and changes in biotic interactions. In this thesis, I have shown that urban vegetated roofs with shallow substrate benefit native arthropods associated with dry habitats and open vegetation, but do not automatically provide high biodiversity values and may sometimes serve as agents for exotic species. The ecological value of roofs can be improved by designing them from a habitat provision perspective and as part of the habitat network existing at ground level. My results point to the benefits of planting roofs with diverse vegetation instead of using only a few succulent species, when designing vegetated roofs to support a rich arthropod fauna.
  • Pospelov, Alexey (Helsingin yliopisto, 2021)
    Treatment of birth asphyxia (BA) is a challenging problem – this condition is common and often leads to severe life-long neurological dysfunction. The pathophysiology of BA is complex and not fully understood, and the existing therapeutic approaches are not effective. Animal models are the main source of knowledge about the pathophysiology of BA, but many of the existing models have little relevance to the defining features of BA – the co-occurring hypoxia and hypercapnia – making the results obtained from such models, which concentrate on hypoxia in isolation, difficult to apply in clinical practice. This Dissertation consists of three studies (Studies I–III), which address the above shortcomings and suggest alternative approaches. Study I characterizes the pH-dependent vasomotor responses in a novel, physiologically-validated model of BA. The key advantage of this model is that it reproduces not only the hypoxic but also the hypercapnic component of asphyxia. The importance of the co-occurrence of these two components is directly demonstrated in experiments showing that in the absence of hypercapnia, shifts in brain pH and in partial pressure of oxygen (Po2) during and after hypoxia are qualitatively different from those during asphyxia proper. The respiratory acidosis, associated with the latter, triggers protective mechanisms that have emerged during mammalian evolution to ameliorate brain damage during asphyxia. In Study II, we investigated the therapeutic potential of carbon dioxide (CO2) supplemented in ambient air for the treatment of BA-seizures. Seizures are a common acute consequence of BA and they exacerbate the brain damage caused by asphyxia itself. We found that supplementing the inhaled air with 5% CO2 immediately after the asphyxia period prevents seizures, presumably by slowing down the brain pH recovery from acidosis caused by asphyxia. Study III focuses on carbonic anhydrase inhibitors (CAIs), drugs that cause respiratory acidosis by retention of metabolically-generated CO2. These drugs produced brain pH and Po2 responses similar to the responses to CO2 supplementation and similarly suppressed seizures triggered by BA. As first shown in this study, the most widely used CAI, acetazolamide (AZA) is therefore a candidate drug for the treatment of BA seizures. In sum, this doctoral thesis work developed and characterized a novel rodent model of BA and used this model for testing a novel treatment strategy for BA and post-asphyxia seizures.
  • Tavakoli, Shirin (Helsingin yliopisto, 2021)
    The prevalence of vision-threatening diseases of the posterior eye segment, such as age-related macular degeneration (AMD), diabetic retinopathy, diabetic macular edema and glaucoma, is increasing worldwide. This is a major burden to patients and health care systems. Current therapy includes intravitreal injections of anti-angiogenic agents against vascular endothelial growth factor (VEGF), because drug delivery to the back of the eye is hampered by anatomical and physiological barriers. Intravitreal injections are uncomfortable, may cause adverse reactions and reduced compliance leading to suboptimal treatment outcomes. Furthermore, current anti-VEGF drugs are not effective in all patients. Therefore, new drugs and delivery systems for targeted and prolonged action are needed for posterior segment eye treatment. Nanoparticles have been investigated as a long-acting and targeted ocular drug delivery systems that may prolong actions of small molecule drugs (e.g. corticosteroids and tyrosine kinase inhibitors) with short vitreal half-lives. Nanoparticles are also important for delivery of labile therapeutics with intracellular targets, including some proteins, neuroprotective peptides and nucleic acids (RNA, DNA). However, several barriers hamper retinal delivery of intravitreal nanoparticles and interspecies differences may lead to poor clinical translation. Hence, the overall objective of this study was to generate improved understanding of ocular barriers to retinal delivery of nanoparticles by using translationally valid preclinical models. We systematically studied vitreal diffusion of various liposomes and other lipid-based nanoparticles by analyzing the mobility of the nanoparticles with single particle tracking in intact porcine central vitreous with similar structure to human vitreous. We evaluated the physicochemical features of nanoparticles affecting their vitreal mobility (e.g. particle size, surface change, surface coating with polyethylene glycol or hyaluronic acid). Neutral and anionic liposomes showed faster diffusion than cationic liposomes. Small size and polymer coating modestly facilitated vitreal mobility of liposomes. Kinetic analysis demonstrated that nanoparticles’ distribution in the human vitreous is controlled by convection rather than diffusion, while vitreous liquefaction may increase the role of nanoparticle diffusion. We also studied protein corona formation on liposomes’ surface, since it may affect the hydrodynamic diameter and cellular interactions. In this regard, surface plasmon resonance analysis was performed to monitor the protein corona formation in the presence of porcine vitreous. Insignificant size change was seen indicating that vitreal diffusion is not influenced by protein corona. In addition, high-resolution proteomics confirmed identity of the proteins on liposomal surface that may change the biological interactions of the liposomes. Next, retinal permeation of liposomes was studied systematically using ex vivo analyses and bovine retinal explants. Neutral and anionic liposomes with high vitreal mobility were studied for their potential in overcoming the ILM barrier. Liposomes with diameters over 100 nm fail in retinal entry irrespective of their surface charge, while small anionic PEG-coated liposomes (<50 nm) distributed into the retina. Lastly, a liposomal formulation was developed to encapsulate sunitinib, a small molecule anti-neovascular drug for VEGF suppression. Unlike sunitinib solution, the liposomal formulation showed anti-neovascular effect in laser induced mouse choroidal neovascularization model. In summary, this study extended understanding of the retinal drug delivery barriers related to the intravitreal injections. It also informed about the role of nanoparticles’ characteristics on their interactions with ocular barriers. These findings can be leveraged in understanding pharmacokinetics and design of retinal drug delivery systems.
  • Hagolani, Pascal (Helsingin yliopisto, 2021)
    The complexity of organisms has astonished biologists for centuries. How complexity has evolved, has given rise to much debate. Many have claimed that natural selection is the main factor that made it possible to achieve high degrees of complexity. On the contrary, others argue that this is far from the truth, as complexity can increase passively, without the need of natural selection. Either way, the exact mechanisms by which complexity has increased in some groups of organisms remains largely unknown. For morphology, to understand which mechanisms have enabled an increase in complexity, requires to study development. As development is the process that establishes morphology, any evolutionary change in morphology is preceded by a change in its development. Additionally, to understand how morphological complexity evolves, it is necessary to comprehend the phenotypic variation that different developmental mechanisms can produce. The two main questions that I am interested to answer in this dissertation are:  1. Are there some logical requirements that developmental mechanisms should fulfill in order to lead to complex morphologies? 2. How does morphological complexity affect evolution?   To tackle these questions, I used a general computational model of development, EmbryoMaker. EmbryoMaker is a general model that allows to simulate the development of 3D morphologies of any type. It is precisely this generality of EmbryoMaker which was vital for this dissertation, since it allowed an unconstrained exploration of developmental mechanisms, without being limited to certain organisms or systems. This allowed me to tackle the questions I posed in a general way. The general results obtained are: 1. The development of complex morphologies does not require cell signaling or complex gene networks. 2. Extracellular signaling enhances robustness through the compartmentalization of the embryo into different regions of gene expression 3. Complex morphologies are rare 4. The more complex a morphology is, the more finely tuned its developmental parameters need to be 5. The more complex the morphology, the larger the mutational asymmetry towards simplicity 6. The more complex morphology, the more complex the GPM These results indicate that there are qualitative differences in the way complex and simpler morphologies evolve. Complex morphologies evolve under a complex GPM and higher developmental instability. Additionally, complex morphologies produce a higher morphological diversity than simpler morphologies for the same amount of genetic variation, therefore offspring of complex individuals spread across large regions of the morphospace. Finally, these results also indicate that the evolution of morphological complexity becomes progressively slower as complexity increases, until possibly arriving at a complexity trap, where it cannot effectively increase.
  • 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.

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