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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.

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.

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.

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.

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.

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.

Poor quality of sleep and the following health problems affecting daily life are in many cases caused by cognitive and physiological arousal resulted from a stressful event. Such stress detrimental to sleep may originate from psychosocial factors such as feelings of shame and social rejection. Our goal was to elucidate the impact of acute psychosocial stress occurring before bedtime on sleep macrostructure and the early night non-rapid eye movement sleep (NREMS). In addition, virtual reality solutions are emerging as options to simulate social threats in laboratory environments. We studied whether a virtual reality variation of a public speaking scenario was sufficient in producing a physiological stress response evident in heart rate variability (HRV) parameters. We compared two experimental groups of healthy young adults (n=34), which differed in the scenario completed within the virtual reality. The stress condition involved a public speaking simulation in front of an attentive virtual audience whereas the control condition involved listening to a neutral presentation in the same but empty virtual seminar room. The participants’ physiological responses were measured with a HRV monitor for 38 hours and the quality of sleep during the laboratory night following stress induction with electroencephalography (EEG). The examined early sleep period was divided into two separate cycles of NREMS, whose results were juxtaposed. For analysing frequency band activity during sleep, we processed the data from EEG with Fourier transformation to yield power spectral density values i.e. frequency activity values. Comparing the two conditions, we observed a distinct effect of stress both during the virtual public speaking scenario and in the subsequent early sleep in the participants from the stress group. We found a significant increase in heart rate and rising fluctuations in the LF/HF (HRV power spectrum high frequency/low frequency) ratio around the stress task period contrasting the results of the control condition, reflecting increased sympathetic tone in the stress group. In the following night, the percentage of stage N3 sleep significantly increased at the cost of N2 sleep during the first NREMS cycle in the stress condition, but this effect resolved in the second NREMS cycle where group differences were absent. As a key finding, the stress group exhibited higher beta frequency activity in proportion to delta activity throughout both cycles and sleep stages. This effect was significantly magnified in N3 sleep where the delta/beta activity ratio decreased in the stress group from cycle 1 to 2, indicating worsening quality of sleep as the night progressed. We reflected our results through a homeostatic point of view, where the increased high frequency beta activity at sleep onset and early sleep in the stress group might explain their increased N3 sleep duration in the first NREMS cycle. A stronger affinity for the important N3 sleep may be a sleep protective mechanism to counter the stress induced abnormally high frequency EEG activity at sleep onset and early sleep to ensure the restorative benefits of slow-wave activity.

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.

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.

Early life stress (ELS) has been associated with the development of psychiatric disorders such as anxiety and depression later in life. The central hypothesis is that these disorders are caused by a malfunctioning of the serotonin system and serotonin (5-HT) produced in the dorsal raphe nucleus (DRN). The DRN is anatomically connected to the medial prefrontal cortex (mPFC), especially to the infra- and prelimbic cortex, where 5-HT modulates behaviors such as impulsivity and cognitive flexibility. The DRN and mPFC mediate with low-frequency network oscillations, which are indicative of the state of the network and its funtional connectivity, as disturbances in these network oscillations have been connected to neuropsychiatric disorders. The aim of the thesis is to investigate whether and how ELS can influence the local field potential (LFP) activity of the mPFC and DRN and the functional connectivity of the DRN and mPFC. This is researched by characterizing and comparing the LFP activity recorded in the DRN, where 5-HTergic neurons are located, and in layer 5 of the infralimbic area of the mPFC. To accomplish these aims, a well-established animal model of early-life stress, the limited bedding and nesting model (LBN), was used. The model causes fragmented maternal care due to the stress of the dam, which in turn leads to the stress of the pups. Simultaneous multi-site recordings of LFP and multi-unit activity (MUA) within DRN and mPFC were performed in vivo during postnatal days (PND) 10-11 from control and LBN pups to characterize the network activity of these two brain areas and then investigate possible changes in their functional connectivity. The efficacy of the LBN model was determined by the observed decreased weight gain of LBN animals compared to controls. From the data, the LFP activity of the DRN and mPFC were characterized. The activity was characterized as power spectrum, wavelet spectrum, and MUA with DRN showing discontinuous activity with low signal-to-noise ratio and low frequency theta oscillations (4-12 Hz), while mPFC showed almost continuous activity with higher signal-to-noise ratio and developing gamma oscillations (20-50 Hz). The power of LFP signal of the areas was not found to be affected by ELS. To investigate if the coupling by synchrony between DRN and mPFC networks is altered by ELS, I analyzed wavelet coherence by computing coherence values between LFP signals in DRN and mPFC in a control and ELS for frequencies from 1 to 50Hz. The functional connectivity was affected by ELS. Statistically significant changes were observed in wavelet coherence in the lower frequencies of 1-2.8 Hz between the control and LBN treatment, suggesting impaired synchronization between DRN and mPFC at 1-2.8Hz frequency range immediately after ELS exposure at PND 10-11 mice. Caveats of the study were low signal-to-noise ratio of the recordings, the small group size of LBN animals (n=5) as well as the uneven sex distribution (male n=11, female n=3) which prevented the sex-based comparison of the effects of ELS. The thesis examines postnatal LFP brain activity in the DRN and mPFC and the functional connectivity between these brain areas. The results of the thesis show that ELS exposure is able to influence the functional connectivity of these two brain regions. The results support previous findings, which have found alterations in the functional connectivity of the neural networks underlying neuropsychiatric disorders in adulthood. The findings of this thesis suggest that ELS could affect the functional connectivity of a developing network and thus increase the risk of the development of neuropsychiatric disorders. Further studies are needed with larger group size, even gender balance, and better signal-to-noise ratio of recordings.