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  • Mäki, Ilona (2022)
    Biochar is a porous, carbon-rich material, made from organic material by pyrolysis in low oxygen conditions, and it can be used to sequester carbon into the soil. This review aspires to give an overview of the economic dimensions of using biochar in Finnish (Boreal and sub-boreal) forests. A literature review was conducted to collect and summarize the information about studies and applications elsewhere, and how we could possibly apply them into Finnish forest ecosystems. This thesis is done as part of Helsus Co-Creation Lab -project, where our group was tasked with looking into how biochar could enhance biodiversity in soil and accelerate transformation to low carbon economy. From this larger topic, this paper is looking into the economic side, and whether it is economically viable to use biochar to enhance and uphold biodiversity. This is evaluated by reviewing and categorizing 164 papers and conducting a literature review. My conclusions are that the current biochar applications show lower economic efficiency than other carbon dioxide abatement technologies. The stability of biochar in soil is a key factor, as the half-lives of biochars may not be as long as commonly suggested. Furthermore, competition for biomass resource use can restrict the availability of feedstock, and make it more expensive. Subsidies for biochar application are required if biochar is to be- come a significant part of the national or global climate mitigation policy. The results in different articles are quite variable and there is currently no standard approach to them. There is a need for specific research on what kind of biochar benefits what soil and vegetation, which is expensive. A primary goal is to incorporate a consistent and standardized testing or analysis method for biochar stability into the certification programs run and administered by the International and the European Biochar Initiatives. In the foreseeable future, biochar by itself is unlikely to play a significant role in climate mitigation strategies. Biochar might be just one of several alternatives in a bundle strategy to re- duce carbon emissions. However, its potential use must still be researched more.
  • Björn, Marko (2021)
    Abstract: The EEG measurement protocol is standardized and in use globally. The skull is measured to ensure that the electrodes are placed in the correct position. Measurements are necessary because skull sizes and shapes are different. Studies for placing electroencephalograph (EEG) electrodes on a human head are typically introduced theoretically before students are granted the opportunity to practice. Due to the limited availability of EEG equipment and supervisory staff, students encounter shortened practical training sessions and lengthy waiting periods transitioning from theory to practical components. The main aim of this project was to create a learning environment with game technologies to help students study electrode placement during the idle time between theory lessons and practical training. We set out to determine whether students experienced some learning gain and if they had a positive experience with the learning environment. We simultaneously assessed if fuzzy feedback is preferred over exact feedback. Additionally, the aim was to make use of a design-based approach with the information from a User Experience Questionnaire (UEQ) the EEG-simulator. Our group developed and tested a digital learning application that provides a 3D model of a human head, on which learners can practice placing EEG electrodes. We followed a user-centric design science approach to ensure our application appeals to our target audience. We used an observational post-test only design with two experimental groups and a control group. We applied a widely accepted user experience questionnaire to ascertain which of our two feedback systems elicited the best user experience. We also qualitatively analyzed diaries the students kept, as they worked with the learning environment, to better understand future development options for further maximizing the environment’s learning benefit. The overall application was well-received, and students opined that the application significantly enhanced their practical session experience. Although the post-test evaluation showed no difference between the two experimental groups, the user experience questionnaire showed that the fuzzy feedback system was preferred over the exact feedback. Furthermore, it was evident that students who had not used the learning environment struggled more to come to terms with the practical session. The personal experience recording by the students revealed several suggested improvements to the learning environment. We conclude that, with further development, this EEG placement learning application could address the idle period between demonstration lessons and practical training. We also venture to state that fuzzy feedback is preferred because of the high-fidelity mimicry of real teacher feedback. The last part of the research was to develop the EEG simulator so that it will increase theory learning with a simulator, that works, and this is ongoing. We have developed the last EEG simulator version with AR (augmented reality) mobile version that can be used with any smart devices. The future work is to test EEG application and does application influence student's theory learning process.
  • Partanen, Reeta-Maria (2020)
    There is a naturally reproducing Atlantic salmon population in the River Teno in northern Norway and Finland. The Teno population has a strong population structure and up to 28 subpopulations have been recognized. Estimation of effective population size is important in conservation of the subpopulations. Effective population size tells about genetic variation of a population and is among the most important concepts in conservation genetics. In this study, current and past effective population sizes of 28 subpopulations were estimated from high density SNP-data for 1137 individuals in total. The estimation was done with the linkage disequilibrium method and the effects of using different assumptions were studied. Current estimated effective population sizes in subpopulations were generally low and ranged from around nine to 272 individuals. Only four populations had a current effective population size bigger than 50 individuals. Past effective population sizes showed a clear declining trend from the most distant generations in all populations. The choice between physical and linkage map as well as female, male or average linkage map had an effect to estimates. Also, different sample size corrections resulted in different estimates. Furthermore, effective population size was estimated with temporal method in three populations. It was detected that the estimates from temporal and linkage disequilibrium method were different from each other. The results of this study suggest that Teno Atlantic salmon subpopulations have declined over the past 150 generations and are in risk of losing genetic variation due to current low effective population size. This should be taken into account in conservation plans.
  • Fabritius, Henna (2010)
    Genetic variation is vital for both contemporary and long-term wellbeing of populations. Whereas heterozygosity (Ho) and allelic richness (A) are commonly used to measure the level of genetic diversity in a population, effective population size (Ne) describes the speed of loss of genetic variation. Various effective population sizes are proposed as standards for safe retention of genetic variation in a Minimum Viable Population (MVP). Since the 1940s, several types of effective population size estimators have been developed. Earlier estimators relied on demographic parameters, whereas genetic estimators are based on the analysis of either one or two genetic samples from a population. All Ne estimators have their unique sensitivities and limiting assumptions, which complicate the choice of estimator, comparison of results of different studies and the assessment of the reliability of the results. Ne estimators have recently been used e.g. in the monitoring of many aquatic populations, but their reliability and comparability has not often been tested with extensive ecological and genetic data, and it is not well established how much added value they bring to the conservation of easily observable species. I tested this with an extensive dataset on the Siberian jays (Perisoreus infaustus) living in Suupohja, Finland (62°22'N, 21°30'E). The Suupohja Siberian jays form one of the few isolates of Siberian jays in Southern Finland. I utilised three demographic and three genetic Ne estimators to estimate the Ne and the Ne/N ratio in the Suupohja Siberian jays, and compared the findings to the Ho and A estimates calculated with the same data, and to various suggested MVP standards. The results showed that the ratio of effective and census population sizes (Ne/N) is close to 0.6 in the Suupohja Siberian jays. Uneven survival of offspring and population size fluctuations are the main factors in the formation of this ratio. The average genetic Ne estimate would, then, suggest a census population size of 44 % higher than the average N in the Suupohja study area. This result is probably connected to the high proportion of breeding immigrants in the data, which would cause the Ne estimates to reflect a larger genetic neighbourhood than the study area. The genetic Ne estimates also suggest that the Suupohja Siberian jays might not be able to maintain their genetic diversity in the long term if gene flow would cease due to further isolation, especially if isolation would also cause a faster demographic decline. Conservation attempts should aim at ensuring gene flow to the remaining Siberian jay isolates in Southern Finland, in order to protect them from increasing genetic uniformity and inbreeding. It is possible that while the average dispersal distances in the Siberian jay are short, occasional long-distance dispersal events have an important role in the pretention of genetic structuring in a Siberian jay population. Ne estimation based on demographic data was laborious in the case of the Suupohja Siberian jays, whereas the genetic Ne estimates showed large variation depending on year and estimation method used. Reliable estimation of Ne with genetic methods would have required information on the large-scale genetic structure of the population. In any case, Ne estimates gave a clearer picture on the genetic viability of the Suupohja Siberian jays than the Ho and A estimates, which did not indicate any decrease of genetic diversity during the study period.
  • Shrestha, Subhash (2019)
    SH3 domains are relatively short and most common of modular protein binding domain in eukaryotes. They are present in proteins that play critical role in various cell signaling and regulatory pathway. Human genome encoded 296 types of SH3 domains have been successfully displayed in phagemid using classical PelB signal sequence and used for finding novel binding partners. However, given its shorter length and tendency to fold rapidly it is useful to understand if signal sequence that directs SH3 translocation through Co translational pathway is much more efficient in displaying these domains than the one that translocate protein post translationally. For the study, PelB signal sequence of phagemid displayed human SH3 library was replaced with DsbA signal sequence using round the horn PCR method (Site directed mutagenesis) and verified with agarose gel electrophoresis. Subsequently, infective phages were prepared. The infective titer of newly generated DsbAss based library was found to be higher than that of PelBss based library. Both libraries normalized at 1 x1012cfu/ml were panned against known protein targets MC159(Molluscum contagiosum 159), NCF2(Neutrophil cytosolic factor 2) and NS1(Nonstructural protein 1). Enrichment with DsbAss library was moderately higher for each antigen. However sequencing results showed that results for proteins panned with PelBss library were congruent with previous finding whereas DsbAss library selected some potential weak binders and nonspecific ones along with strong binders. Panning results of DsbAss with NCF2 was striking as all clones selected were NCF1 SH3 domains. Although further functional study was not performed. Based on the study, we concluded that both libraries have its own advantage. PelBss based library can be used for finding strong binders while DsbAss based library can be used for studying weaker interaction and functional role of NCF2-NCF1 SH3 domain interaction is still an open question.
  • Päiviö, Elisa (2020)
    Behaviour is a key component in ecological interactions and studying its role in adaptation is central in our understanding of natural selection and phenotypic variation in the wild. Predation is an important driver shaping animal behaviour in the wild, since predators have been shown to select against particular behavioural phenotypes. However, it is not easy to demonstrate that specific behaviours are adaptive to certain levels of predation, since behaviours are often correlated with each other forming multivariate phenotypes and display notable phenotypic plasticity. I studied how predation shapes genetically determined behaviour of the nine-spined stickleback (Pungitius pungitius) through variation in historical predation pressures and by inducing phenotypic plasticity. This was achieved through rearing 65 full-sib families of nine-spined sticklebacks derived from four coastal marine (predator-sympatric) and four pond (predator-naïve) populations in a common garden experiment and quantifying their behaviour in the laboratory in the presence and absence of natural predators. Since the fish used were F1-generation offspring from artificial crosses, I was also able to estimate the heritability and genetic correlations of the studied behaviours. Pond sticklebacks tended to be more explorative and took more risks during foraging than marine sticklebacks regardless of predation risk. In all fish, predator presence decreased the propensity to take risks during foraging, but not exploration tendency. Since the fish were reared in a common garden setting, there is a genetic basis for these population differences. Both behaviours were heritable in all populations. In this study, I observed genetically based and heritable behavioural differences between pond and marine stickleback populations. Despite showing similar levels of behavioural plasticity as marine sticklebacks, pond sticklebacks were still inappropriately active in the presence of predators and would have a low survival probability in a predator-sympatric environment. In risk-taking during foraging, the behavioural trend caused by acute predation risk was directionally the same as that caused by evolutionary history of predation risk, implying that the behavioural differentiation between marine and pond populations in this behaviour is due to predation. These results provide evidence of local adaptation in behaviour to differing levels of predation in these populations, and that this adaptation comes about as differences in the overall level of behaviour rather than in phenotypic plasticity.
  • Wong, Carlton (2019)
    Meningeal lymphatics vessels (mLVs), the recently characterized lymphatics in the central nervous system (CNS), provide a link between the adaptive immune system and the CNS. mLVs could be important for the activation of T cell-mediated adaptive immune response, by draining antigens from the brain to the deep cervical lymph nodes, where they are presented to T cells. In traumatic brain injury (TBI), we hypothesized that the activation of self-reactive T cells (i.e., T cells able to recognize self, brain-derived antigens and promote an immune reaction), possibly underlies the pathogenesis of the disease. In order to test this hypothesis and to decipher the specific role of mLVs in the modulation of T cell-mediated neuro-immune response after TBI, we ablated the existing mLVs in adult male C57BL/6OlaJ mice (with the use of the AAV-mVEGFR3 1-4 Ig vector), induced TBI with controlled cortical impact, and examined the motor function of the mice and the activation of different T cell populations in the brain, as well as in the secondary lymphoid (spleen and lymph nodes – LNs) and non-lymphoid organs (meninges). Our data showed that the T cell-mediated adaptive neuro-immune response in TBI was unaffected by the depletion of mLVs. Our results, however, are preliminary, due to the limited sample size used in this study, which reduces the statistical power and restricts our ability to conclude for the effect of mLV depletion on TBI recovery.
  • Anastasiadou, Maria (2019)
    Tiivistelmä – Referat – Abstract Genetic variations within the MYO16 gene indicate a common predisposition to severe psychiatric, neurocognitive and neurodevelopmental disorders (NDD), as well as bipolar disorders (BD) and schizophrenia spectrum disorders (SSD). Myosin XVI’s ability to regulate actin and its involvement in cytoskeleton remodeling highlights the protein’s significance in neuronal circuitry development and signaling. Mutations in actin regulator protein-encoding genes like MYO16 have been found to shift cytoskeletal dynamics, as well as cause irregular dendritic spine and excitation/inhibition (E/I) synapse phenotypes. Interestingly, altered actin dynamics and E/I synapse dysregulation are two commonly detected molecular deficits associated with neuropathologies, namely autism spectrum disorders (ASD), SSD, and intellectual disability (ID). Therefore, synaptic E/I profiles are good candidates for investigating the neuropathologies they accompany, and also for revealing potential functional abnormalities. Hence, we determined that quantifying the levels of inhibitory synaptic proteins VGAT and gephyrin is the most suitable approach to investigate inhibitory synapse profiles and their relation to pathologies. Specifically, we investigated how microRNA (miRNA)-mediated myosin XVI protein knockdown (KD) affects pre- and postsynaptic inhibitory synapse density in rat primary hippocampal neurons. We achieved this by analyzing the density of VGAT and gephyrin puncta, signifying pre- and postsynaptic inhibitory synapses, respectively, and also by measuring their diameter to determine differences in inhibitory synapse size. Moreover, we quantified and assessed inhibitory synapse density and size differences between groups by comparing Myo16 KD-plasmid expressing hippocampal neurons to scrambled control cells. Common for both Myo16 KD plasmids was the active suppression of myosin XVI by 33%. However, Myo16 KD plasmids did not affect inhibitory synapse density and size to the same degree. Specifically, there was a significant reduction of inhibitory synapse density in the Myo16 KD3-plasmid expressing neurons, yet, no changes were observed in Myo16 KD5-plasmid expressing neurons. Finally, pre- and postsynaptic inhibitory synapse size differences were not significant between groups for either Myo16 KD plasmid when compared to scrambled control. Aberrant actin cytoskeleton remodeling, as well as altered E/I synapse ratios may lead to hyper/hypo-transmissive neuronal states or cause E/I imbalance, suggesting a complex relationship between actin regulator genes and inhibitory synapses. Our understanding behind their interplay is fairly limited, thus, gaining insight into the mechanisms associated with altered E/I balance remains the primary aim.
  • Sofieva, Svetlana (2019)
    Nemaline myopathy (NM) is a rare congenital disorder, the most common of congenital myopathies. It affects primarily the skeletal muscles and it is recognised by nemaline bodies in muscle tissue samples and muscle weakness. Mutation of eleven genes are known to lead to NM and the most frequent disease-causing variants are either recessive NEB variants or dominant ACTA1 variants. Variants in NEB are thought to be well tolerated and only 7% of them are hypothesized to be pathogenic. Over 200 pathogenic NEB-variants have been identified in Helsinki and the majority occurred in patients as a combination of two different variants. The missense variants were speculated to have a modifying effect on pathogenicity by affecting nebulin-actin or nebulin-tropomyosin interactions. Nebulin is a gigantic protein coded by NEB and is one of the largest proteins in vertebrates. It is located in the thin filament of the skeletal muscle sarcomere. Enclosed by terminal regions, nebulin has an extensive repetitive modular region that covers over 90% of the protein. The repetitive zone comprises of 26 modules called super repeats (SR). SRs consist of seven simple repeats. There are seven conserved SDXXYK actin-binding sites at each super repeat, one per simple repeat, and one conserved WLKGIGW tropomyosin-binding site. Due to its enormous size and highly repetitive sequence, nebulin is one of the least studied proteins in vivo, in vitro or in silico. In the NM patient database used for this study, there are 70 families with verified pathogenic mutations and in 30 of them, there were additional missense variants in NEB. These missense variants can be pathogenic modifying factors or have no impact on the phenotype. Seven missense variants were selected to study the effect of these mutations on actin-binding capacity compared to wild-type nebulin using the SR panel constructed previously by Laitila and Lehtonen. Also, due to the differences in actin-binding capacity of SRs compared to each other, one of the aims was to determine whether corresponding mutations in different SRs would have a similar or different effect on actin-binding capacity. For this aim, one missense mutation in the strongly actin-binding SR 1, and one in the weakly actin-binding SR 7 were selected from the NM database, and corresponding variants were created. Also, an in-frame deletion in SR7 found in the ExAC database and the corresponding mutation in SR1 were constructed for this study. The actin-binding strength was determined using actin co-sedimentation assay and actin affinity assay. The results for co-sedimentation assay indicate that missense variants can have an effect on nebulin-actin interactions and, therefore, can be a possible cause for NM. The corresponding mutations had no correlation in their effect on actin-binding strength, just the opposite. S1-m-2 decreased actin-binding strength of SR1 and S7-m-2 had no effect on SR7. Likewise, S7-m-1 and S7-del-1 decreased actin-binding strength of SR7 and corresponding mutations had no effect on SR1. The selected missense mutations found in NM patients in SRs 2 and 4 decreased actin-binding strength, if located at the actin-binding sites and in SR 10 increased the actin-binding strength, if located at the actin-binding site. The change in actin binding strength was defined as significant if the P-value was below 0.005. The more accurate affinity assay was performed as a trial only for S16 and S16-m-1, a variant at a tropomyosin-binding site close to an actin-binding site. It indicated a difference in actin-binding affinity missed by the actin co-sedimentation assay. The results are preliminary, but show big promise and should be optimized and implemented in the future missense mutation affinity studies. In an attempt to understand if the effect missense mutations have on nebulin-actin interaction is based on the change in nebulin structure, the 3D-structure of each produced fusion protein was predicted in silico. Considering that the variants were produced as GST-fusion proteins, the position and effect of GST in them is also a point of interest. In order to predict the structure of these large proteins, a combined approach was implemented using I-TASSER (Iterative Threading ASSEmbly Refinement) software. The software uses ab initio modeling, threading methods and atomic-level structure refinement to build an accurate 3D-model of a protein from sequence. According to the predicted 3D models of the fusion proteins, the GST-part of the proteins folds into a globular structure and acts as a core around which the nebulin fragments fold. The GST does not bind to actin and is positioned on the inside, which indicates minimal effect on nebulin-actin interaction, but may be a reason for an alternative nebulin fragment folding. The accuracy of the default set of programs in software does not give the definitive answer of the possible effect missense mutations can have on structural changes. However, I-TASSER approach for 3D-modeling is promising with further software optimization and can possibly serve as an effective bioinformatic tool in the future.
  • Pakarinen, Aku (2019)
    Modern agriculture uses great amounts of fertilizers. A large portion of these fertilizers leaches from the fields to the surrounding environment and causes eutrophication of water ecosystems. Fertilizers are an expense for the farmer. Sustainable agriculture aims to minimize the use of fertilizers by using natural processes and nutrient circulation. Crop rotation is an important tool in sustainable agriculture. In crop rotation different crop species are cultivated alternately in the same field. Arbuscular mycorrhizal (AM) symbiosis is one of the world’s most important symbioses — 80 % of land plants form it. AM symbiosis enhances host plant’s nutrient uptake and provides nutrients from low concentrations in the soil. The important role of AM symbiosis in agriculture is widely recognized. However, the effect of different crop sequences on soil AM fungi is poorly studied, especially in boreal climate. In this thesis, I study the effect of four different preceding green manure crops (white lupin Lupinus albus, french marigold Tagetes patula, crimson clover Trifolium incarnatum and common vetch/hairy vetch Vicia sativa/V. villosa) on the AM colonization rate and diversity in onion (Allium cepa) roots and rhizosphere. White lupin does not form a mycorrhiza, unlike the other three preceding crops in the experiment. Onion is one of the most cultivated vegetable crops in Finland and is particularly dependent on its AM symbiont. My thesis is based on a field experiment executed in Natural Resources Institute Finland’s study field, in Mikkeli, eastern Finland, from 2017 to 2018. One of the four preceding crops was sowed in each cultivated row for 2017 growing season. Preceding crops were ploughed into the soil in autumn 2017. Onion was cultivated in all rows in 2018 growing season. I studied the effect of different preceding crops on the AM colonization rate in onion roots with microscopic methods. I studied the effect of preceding crops on the AM diversity in onion roots and rhizosphere with molecular methods. The preceding crop did not have an effect on the AM colonization rate, nor did it affect the AM diversity in onion roots or rhizosphere. Unlike in previous studies in warmer climates, the non-mycorrhizal preceding crop white lupin did not have a negative impact on the soil AM community. My study suggests that non-mycorrhizal preceding crops can be used in crop rotations, in the climate conditions of eastern Finland, especially in combination with mycorrhizal plants. Future studies should investigate the effect of longer crop rotations on the soil AM fungal communities.
  • Puskarjov, Martin (2010)
    The Cl- and HCO3- electrochemical gradients across the plasma membrane dictate the electrical consequences of GABAA receptor (GABAAR) function and thereby play a significant role in neuronal GABA-mediated signalling. In adult pyramidal neurons, responses to GABA are maintained hyperpolarizing mainly by the action of K-Cl cotransporter isoform 2 (KCC2). KCC2 acts as a Cl- extrusion mechanism responsible for setting the intracellular Cl- concentration below the electrochemical equilibrium, a necessary condition for hyperpolarizing inhibition mediated by GABAARs. Recent evidence suggests that plasmalemmal KCC2 has a very high rate of turnover, pointing to a novel role for changes in KCC2 expression in diverse manifestations of neuronal plasticity. Some studies indicate that rapid down-regulation of KCC2 may be a general early response involved in various kinds of neuronal trauma. In this work, whole-cell patch-clamp was used to examine KCC2 function under a pharmacologically induced arrest of protein synthesis in living hippocampal brain slices from rat. The stability of KCC2 function was quantitatively assessed on the basis of the dendritic Cl- extrusion capacity in the presence of protein synthesis inhibitors cycloheximide and emetine. The parameter used for assessing extrusion capacity was a somato-dendritic Cl- gradient, which was imposed by a somatic Cl- load that resulted in a gradient of EGABA (ΔEGABA). The results of this study show that under general protein synthesis inhibitor-induced arrest of translation, KCC2 function persists unperturbed for at least 4 hours and hence that the cessation of mRNA translation cannot rapidly induce downregulation of KCC2-mediated Cl- extrusion. This finding precludes the use of protein synthesis inhibitors for rapid modulation of KCC2 function. Indirectly, the results presented here imply that the levels of KCC2 under pathophysiological conditions are primarily determined by the degradation rate and not by de novo synthesis.
  • Auvinen, Pauliina (2018)
    Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility.
  • Saarela, Sanna (2020)
    Ischemic heart failure is the leading cause of death in the world. The disease is caused by coronary heart disease, in which the narrowed coronary arteries limit oxygen- and nutrient-rich blood from reaching the myocardial tissue. Obstructed arterial blood flow can cause myocardial necrosis and scarring. Scar tissue is non-contractile and poorly elastic. It can thus compromise the pumping capacity of the heart. Current medical and interventional therapies have only very limited efficacy to reduce myocardial scarring. Preclinical and clinical research efforts are underway to generate myocardial scar-reducing and regenerative therapies. In the field of cardiac cellular therapies, the delivery of cells has conventionally been based on intramyocardial injections. However, epicardial patches have been demonstrated to reduce scarring and promote myocardial healing. In addition to merely being a carrier or cover for the cellular transplant, the biomembrane of the patch can also be considered as an active element for the patch’s therapeutic activity. Thus, the properties of the biomembrane can have a major impact on both the cellular and the therapeutic tissue response. The aim of this Master's thesis was to build a standardized test set up to study the properties of the biomembrane. Biomembrane permeability to small (glucose, lactate) molecules and different size proteins was investigated. In addition, the set up was modified to enable the investigation of biomembrane properties on the survival of the grafted cells. Finally, the test set up was evaluated by studying the properties of ProxiCorTM, the biomembrane currently used together with autologous atrial micrografts (AAMs) in epicardial patch. As a result, the set up was successfully constructed and characterized. The ProxiCorTM membrane demonstrated permeability to both small molecules and proteins, and a stable pH was maintained across the membrane. ProxiCorTM enabled traverse serum-induced proliferation of cells compared to the control impermeable membrane. Taken together, these results prove the functionality of the test set up and thus support its further development.
  • Singh, Abhishek (2019)
    Neurotrophic factors (NTFs) play an important role in regulating the survival, differentiation and maturation of developing neurons. Based on strong pre-clinical evidences, some of NTFs have been suggested to be efficient therapeutic agents for treatment of Parkinson’s disease (PD). PD is a neurodegenerative disorder characterized by loss of dopamine (DA) neurons from nigrostriatal pathway resulting in motor symptoms of the disease. A hallmark of the disease is the presence of Lewy bodies in the brain and they comprise majorly of aggregated alpha-synuclein (aSyn) protein. MANF, an unconventional NTF, was discovered over a decade ago and differs from traditional NTFs. Removal of MANF has been shown to trigger unfolded protein response in cells. Evidences indicate that increased endogenous level of aSyn may have a role in enhancing the process of aggregation of aSyn into Lewy body. Determining the initiation event of aSyn aggregation is an important step in Lewy body pathology and it is still under investigation. In the first part of this study, I aimed to elucidate if MANF knockout can trigger any change in endogenous level of aSyn. Transmission of Lewy bodies from cell to cell has been well studied by researchers and is suggested to spread across brain in a prion like fashion. CDNF has been neuroprotective and restorative for tyrosine hydroxylase (TH)-positive neurons in a toxin-based models of PD. However, presently exists no study which has evaluated the effects of CDNF on propagation of aSyn aggregates in vivo. In the second part of this study, I aimed at evaluating effects of long-term intrastriatal infusion of CDNF at two concentrations (1.5 μg/24h or 3 μg/24h) on propagation of endogenous phosphorylated aSyn inclusions in vivo. CRISPR/Cas9-mediated MANF knockout in SH-SY5Y cells did not yield any significant changes in the endogenous level of aSyn. Additionally, brain samples derived from MANF knockout mice yielded similar non-significant difference in level of aSyn compared to wild-type mice. MANF knockout primary DA neurons when inoculated either with only pre-formed fibrils (PFFs) or with a combination of PFFs and aSyn overexpression, showed no significant difference in the number of Lewy body like aggregates, suggesting no change in endogenous aSyn levels. Rats were injected with PFFs and then chronically infused with CDNF, 1 month and 2 months after PFFs at 2 different concentrations (1.5 μg/24h or 3 μg/24h). Immunohistochemical analysis of substantia nigra pars compacta (SNpc) derived from rats showed similar numbers of endogenous phosphorylated aSyn inclusions in animals treated chronically with either CDNF or PBS. In summary, only MANF knockout from cells or animals has no direct effect on endogenous level of aSyn. But external stressors may perhaps trigger upregulation of aSyn in MANF knockout cells. Furthermore, chronic infusion of CDNF either 1 month or 2 months after PFF injection doesn’t reduce the total number of phosphorylated aSyn inclusions in SNpc compared to control. Nevertheless, we need more data to corroborate this evidence.
  • Sagath, Lydia (2015)
    Welander Distal Myopathy (WDM) is caused by the p.E384K mutation in the TIA1 gene. The mutation supposedly causes the disease by a gain-of-function mechanism related to the formation of stress granules (Hackman et al. 2013). Also environmental factors have been proposed to affect the development of the disease: an increased number of stress granules has been observed in cells treated with cold shock compared to cells kept in 37 °C (Hofmann et al. 2012). When patients with WDM-like symptoms have been screened for changes in TIA1, an p.N357S-change has been found enriched in these patients. The p.N357S-change has earlier been reported as a polymorphism. The change in question is located in the same prion-like domain in exon 5, in which the p.E384K-mutation also lies. Therefore, the p.N357S-change could affect the predisposition to aggregation. The pro gradu project is divided into two parts: • The effect of the p.N357S polymorphism on stress granule formation in arsenite and possibly other stress treated cells • The effect of cold shock on stress granule formation on wild type and p.E384K TIA1 The results indicate, that the p.N357S change in TIA1 causes a change in the translated protein’s behavior. Similarly to the p.E384K change, the p.N357S change also induces an increased amount of stress granules in arsenite treated cells. However, the results also show that the stress granules recover faster in fluorescence recovery after photobleaching (FRAP) studies p.N357S transfected cells as compared to TIA1 p.E384K and wild type transfected cells. The cold shock experiment indicates that there is a difference in the stress granule formation between cells transfected with p.E384K and wild type TIA1. This supports previously published results of the effect of the p.E384K change on the stress response and stress granule formation, and also the use of cold shock as a stress inducing treatment. Used methods: PCR, transformation, DNA-extraction, cell culture, transfection, induction of stress granule formation by arsenite treatment and cold shock. The cells are cultivated on well plates, imaged and the data is analyzed with an automatized high content image analysis method (the CellInsight-platform). p.N357S cells were also analyzed with FRAP.
  • Liu, Zhenzhen (2022)
    Woodland strawberry (Fragaria vesca) is a perennial herb in the Rosaceae family with dimorphic leaves, summer and winter leaves, adapted to seasonal climate. Woodland strawberry produces a new set of leaves in spring that are photosynthetically active throughout the summer season (summer leaves), and the leaves senescence in autumn when they are replaced by a new set of leaves (winter leaves). The winter leaves retain photosynthetic capacity under the snow cover throughout the winter season, which prolong the photosynthetic period of the species. With the world-wide climate warming, the thickness of winter snow is decreasing, which can affect overwintering and spring phenology of plants. This thesis focuses on springtime ecophysiology and phenology of the senescing winter leaves and the formation of new summer leaves of woodland strawberry genotypes of different European origin. The 15 different genotypes of woodland strawberry are from Iceland, Italy and Norway, and they originate from different environments that are geographically separated from each other, so the populations are genetically distinct. In this study, these genotypes were kept at two different overwintering sites, coastal site at the Åland islands with mild temperatures, and continental site in Lammi with a persistent snow cover. According to the results all 15 genotypes showed earlier development of the summer leaves and earlier senescence of winter leaves in the group with Åland as overwintering site than in the group with Lammi. Another important finding is that the first summer leaves produced in spring begun to senesce shortly after they are fully developed and were replaced by later formed summer leaves. Specifically, the dates of summer leaf formation, flowering and stolon production were advanced, and the dates of winter leaf senescence were also advanced. The value of different leaf types to chlorophyll fluorescence was also lower at the Åland site. Therefore, it can be concluded that overwintering conditions have an effect on the subsequent phenological development in spring. In the context of global climate change, the spring development of woodland strawberry will be earlier, and the senescence of winter leaves will also be earlier.
  • Äikäs, Lauri (2021)
    Abstract Introduction: Atherosclerotic cardiovascular diseases (ASCVD) cause the biggest burden on our healthcare system and cause most premature deaths. Risk for ASCVD can be lowered by lifestyle choices and medication, as well as several therapeutics such as ethyl eicosapentaenoic acid (E-EPA) supplementation. Here we aimed to investigate the effect of EEPA intervention on known ASCVD risk factors including circulating lipoprotein levels as well as low-density lipoprotein (LDL) aggregation susceptibility, a new independent risk factor for ASCVD. Study design: A study group of 39 healthy men and women participated in a 4-week long dietary supplement trial with 3.9 g/day of E-EPA. A dose of 75 µg/day of vitamin D was included in the E-EPA capsules. Blood samples were drawn before the trial, at weeks 1 and 4 of the intervention and 1 week after the intervention. The study was an open design where participants’ own baseline measurements were used to measure changes. Outcomes: The mean plasma cholesterol concentration was reduced from 3.8 mmol/l to 3.6 mmol/l (p=0.0038 one-way ANOVA) after one week of E-EPA supplementation and remained the same until the end of study period. This change was followed by a change in plasma LDL (p=0.0028 one-way ANOVA) and triglyceride (p=0.0004 one-way ANOVA) concentrations after four week and one week of E-EPA supplementation, respectively. Vitamin D levels increased on average by 18%, showcasing a lower relative response than seen in other vitamin D trials, which can be attributed to high effective baseline concentrations of vitamin D in our study group and the related negative feedback system. LDL aggregation susceptibility did not significantly change in the entire group. However, we discovered that the change in LDL aggregation susceptibility correlated negatively ( = -0.451, p = 0.0039) with the baseline LDL aggregation susceptibility. Thus, LDL aggregation decreased in participants having aggregation-prone LDL at baseline. This finding highlights a possibility that participants with higher LDL aggregation susceptibility may benefit from addition of E-EPA to their diet.
  • Seiffert, Nina (2021)
    An increasing number of people are diagnosed with depression. One possible reason for the development of depression is faulty wiring and information processing in certain neural networks (network hypothesis) in the central nervous system. It has been shown that antidepressant drugs (ADs) can induce a juvenile-like plasticity state in the brain (iPlasticity) comparable to the plastic state of critical periods during development. iPlasticity enables the rewiring of neuronal networks in combination with environmental stimuli. At the molecular level, the binding of brain-derived neurotrophic factor (BDNF) to its high-affinity receptor tropomyosin kinase receptor B (TRKB) leads to TRKB dimerization and activation, triggering a downstream signalling cascade promoting brain plasticity. Activation of the TRKB signalling cascade is triggered by neuronal activity as well as AD treatment. Recent findings demonstrate that classical as well as rapid-onset ADs bind directly to the transmembrane domain of TRKB, leading to increased translocation of intracellularly stored TRKB to the plasma membrane and enhanced BDNF binding. Cholesterol, a sterol lipid known to regulate TRKB signalling, has been found to ensure optimal TRKB-BDNF signalling by changing the TRKB dimers’ relative orientation when altering the membrane thickness. A point mutation of TRKB tyrosine 433 to phenylalanine (TRKB.Y433F) has been found to hinder TRKB dimerization. Molecular dynamic simulations reveal that other membrane lipids are likely to participate in AD binding to TRKB. The aim of this thesis was to investigate whether lipid and drug compound treatments affect TRKB dimerization in Neuro2A cells expressing TRKB. Furthermore, we assessed whether the Y433F mutation modulates TRKB dimerization in such treatments. Protein fragment complementation assay (PCA) was used as in vitro protein-protein interaction assay to quantify dimerization of overexpressed TRKB carrying two split luciferase reporter proteins. Additionally, to avoid variability caused by transient transfection and be able to test large compound libraries, the establishment of a stably TRKB-expressing N2A cell line was initiated. The results show that lipid compounds, such as Allopregnanolone, as well as ADs, such as Imipramine and (2R,6R)-Hydroxynorketamine, increased TRKB dimerization in vitro in a dose-dependent manner within 40 minutes. The increase was more pronounced in the TRKB WT-expressing cells. This indicates that the compounds tested here may be directly interacting with TRKB, facilitating dimerization. Moreover, data seem to confirm previous research on the less effective TRKB.Y433F mutation. While stable expression of TRKB carrying one of the luciferase reporter proteins was successfully achieved in a monoclonal cell line, the amount of protein expressed seems to require further optimization before utilising it for PCA. In conclusion, lipid and AD treatments can induce an increase in TRKB dimerization in a dose-dependent fashion. Further investigations are needed to determine where the compounds bind and by which mechanisms they exert their effects on TRKB. Furthermore, the work on the stable cell line will be completed to avoid variability of transient transfection in the future.
  • Kuutti, Mirjami (2022)
    In recent years, psychedelics have shown promise in the treatment of conditions like depression and addiction. The therapeutic effects of psychedelics have been linked to their ability to increase plasticity in the brain, an effect that has also been seen for antidepressants. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which has an important role in the development of the nervous system, as well as promotion of neuronal survival and differentiation during adulthood. BDNF, through its receptor TrkB, has been implicated in antidepressant action, and BDNF-TrkB signalling is involved in many aspects of plasticity. Recently, antidepressants have been reported to bind directly to TrkB, and through this binding mediate their plasticity-enhancing, as well as behavioural effects. Psychedelics have been shown to increase structural and functional plasticity, but the mechanisms behind these effects are not fully understood. For example, the serotonergic receptor 5-HT2A is known to be behind the acute hallucinogenic effects of psychedelics, but its role in plasticity is still debated. The aim of this study was to investigate the mechanisms of LSD-induced plasticity. The dimerization of TrkB was examined after LSD treatment in the protein-fragment complementation assay (PCA). Phosphorylation of TrkB signalling markers mTOR and ERK, which have known effects on plasticity, was assessed in Western blot, and the total expression of BDNF was examined with the enzyme-linked immunosorbent assay (ELISA). The timeline of the effects was investigated, and the involvement of 5-HT2A in TrkB dimerization and the phosphorylation of ERK was assessed by combining LSD treatment with the 5-HT2A antagonist M100907. Dimerization was also assessed in a TrkB mutant (Y433F) that has previously been shown to disrupt antidepressant effects on plasticity. These experiments showed that LSD treatment increased TrkB dimerization as well as phosphorylation of mTOR and ERK. The Y433F mutation interfered with LSD-induced TrkB dimerization, but the effects of LSD on TrkB dimerization or ERK phosphorylation were not blocked by M100907. Together, these data suggest that 5-HT2A is not involved in LSD-induced promotion of TrkB dimerization or ERK phosphorylation. The increases in phosphorylation and dimerization were found to be most robust after a 1 h LSD treatment, however an increase in BDNF expression was seen in cortical neuron cultures only after a 24 h treatment with LSD. The results reported in this study support the view that 5-HT2A might not be needed for the plasticity-inducing effects of psychedelics. If this is true, the development of treatments that target plasticity without hallucinatory effects could be possible. Overall, this research provides insight into the mechanisms of LSD-induced plasticity and offers new and interesting directions for future research in the field.
  • Törrönen, Essi (2020)
    4-Methylmethcathinone (Mephedrone) is one of the the most prevalent synthetic cathinones that bears close structural similarity to amphetamines. Like other stimulants, mephedrone is often used with alcohol (ethanol). In animal studies ethanol has been observed to potentiate the neurotoxicity of amphetamine-type stimulants, and same has been observed when mephedrone and alcohol is combined. The long-term effects of mephedrone have still remained largely elusive. The aim of this thesis is to study the effects of mephedrone, methamphetamine, and ethanol on dendritic spine density and morphology in the hippocampus, nucleus accumbens and caudate putamen, and compare the spine densities with changes in brain activation observed in manganese-enhanced magnetic resonance imaging (MEMRI). Dendritic spines are small membranous protrusions on dendrites that act as the post-synaptic sites for most of the excitatory synapses. Amphetamine and methamphetamine have been shown to affect the density and morphology of the spines. The goal of this thesis was to investigate the long-term effect of binge-like (two times a day, four consecutive days) stimulant treatment on dendritic spines using Golgi-stained rat brain sections. The brains of 48 male Wistar rats were imaged using AxioImager Z2 microscope and the number and the size of the spines was analyzed using Reconstruct software. In this thesis no effect on dendritic spines was observed in the hippocampus and nucleus accumbens in animals treated with mephedrone, methamphetamine, ethanol or combination of them. In the caudate putamen significant increase in the total density of dendritic spines and in the density of filopodia-like spines was observed in mephedrone-treated animals. Other treatments showed no observable effect. These results were conflicting with previous studies where amphetamine-type stimulants have been shown to increase the spine density in the nucleus accumbens and the hippocampus and increase the density of branched spines. In the caudate putamen methamphetamine has been observed to decrease the spine density. There was no correlation between spine densities and brain activation observed in MEMRI. To my best knowledge this is the first time when the effect of mephedrone on dendritic spines has been studied. It is possible that the treatment regimen used here was not strong enough to produce marked long-term changes on dendritic spines. It is also possible, that mephedrone is not as neurotoxic as other amphetamine-type stimulants, which may explain why the effects remained limited and conflicting. More research is still required to establish the long-term structural effects of mephedrone.