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Tämä opinnäytetyö käsittelee uuden ympäristöseurantamenetelmän kehitystyötä. Hauruja (Fucus spp.) on seurattu Suomen rannikolla jo pitkään, mutta nykyisiin kartoitus- ja seurantamenetelmiin liittyy haasteita ja epätarkkuutta. Hauruvaltaiset elinympäristöt ovat uudessa uhanalaisuusarvioinnissa määritelty erittäin uhanalaisiksi. Tarvetta uusille seurantamenetelmille siis on. Drone-teknologia on kehittynyt ja halventunut viime vuosina, mikä on avannut uusia mahdollisuuksia hyödyntää kaukokartoitusta. Edulliset valokuvaamiseen tarkoitetut dronet mahdollistavat sekä ajallisesti että paikallisesti erittäin tarkkojen ilmakuvien tuottamisen. Näitä on maailmalla käytetty menestyksellisesti myös rannikon kartoituksessa ja seurannassa. Suomessa drone-kuvia ei kirjallisuuden perusteella ole käytetty makrolevien kartoitukseen tai seurantaan. Tämän tutkimuksen tarkoitus on selvittää, sopivatko tällaiset drone-kuvat hauruyhteisöjen kartoitukseen ja monitorointiin itäisellä Suomenlahdella sekä tuottaa tietoa menetelmän jatkokehitystä ajatellen. Kuvasin 22 merenranta-alueen reunaympäristön alkukesällä 2020. Kuvatuista alueista valitsin kahdeksan jatkotutkimuksiin, jotka kuvattiin uudelleen syksyllä 2020. Tällöin niiden alueelta kerättiin kuvatulkinnan tueksi 263 tukipistettä, joilta mitattiin veden syvyys ja arvioitiin haurujen esiintyvyys Drop-kuvausmenetelmällä. Rajasin ilmakuvilta näkyvät haurualueet ja vertasin niitä tukipisteaineistoon. Havaitsin, että 80 % havainnoista tieto luokittui oikein. Cohenin kappa -testin perusteella kohteiden välillä sekä eri syvyyksillä olevilla kasvustoilla oli suuria eroja havaintojen luotettavuudessa. Syvemmällä kuin 2,5 metrissä olevien haurukasvustojen erottaminen onnistui huomattavasti huonommin. Myös matalimman kasvuston (0 – 0,5 m) erottaminen oli hankalaa. Tulokset ovat lupaavat, mutta niiden perusteella menetelmää ei kuitenkaan vielä voi todeta toimivaksi, vaan lisätutkimuksia tarvitaan. Tämän tutkimuksen perusteella kehitystyötä kannattaa jatkaa käyttämällä tarkkaa paikkatietoa ilmakuvien ja tukipisteaineiston tuottamiseen sekä uusia kuvantamismenetelmiä, jotka kykenevät tuottamaan tavanomaista RGB-kameraa paremman erottelukyvyn. Drone- ja meritöiden turvallisuuden parantamiseksi tehtiin riskienhallintaprosessi, jossa tunnistin, analysoin sekä raportoin töihin liittyvät merkittävimmät riskit.

Upregulation of specific helpful proteins represents a possible method for preventing or treating human diseases. Endogenous upregulation (knockup) is the increase of a gene's expression only in cells in which it is already expressed, thus avoiding physiologically abnormal spatiotemporal patterning. A gene's three prime untranslated region (3′UTR) affects protein expression through stability regulation of RNA already transcribed, which suggests 3′UTR modification as a viable route for endogenous upregulation. Mammalian model organisms can be generated in order to test the effects of different 3′UTR modifications, but at great cost of time, effort, and money. If able to predict in advance with an in vitro assay whether an in vivo modification would cause a desirable or undesirable change, these costs could be substantially reduced. In this thesis project, an in vitro assay was used to compare the protein expression influence of twenty neurodegeneration-relevant mouse genes' 3′UTRs to that of a flip-excision cassette (flex-cassette) previously used for in vivo conditional knockup. The assay used was the Promega Dual-Luciferase Reporter Assay, in which plasmids expressing Renilla and Firefly luciferase as reporter and internal control are co-transfected into in vitro cells, then each luciferase's expression measured with its respective substrate and a luminometer. Transfections were carried out in three-well replicates and on multiple days. The aims of the project were the evaluation of the assay's ability to predict in vivo results, the suggestion of 3′UTRs which could be upregulated in vivo by the conditional knockup flex-cassette, and the identification of any trends in 3′UTR-based protein expression influence according to gene function. A number of gene 3′UTRs were identified which were either candidates for flex-cassette upregulation or candidates for use in the flex-cassette to upregulate other genes. However, the flex-cassette's in vitro results were only partially consistent with its previous in vivo results. Specifically, the lox sites in the flex-cassette was observed to lower expression level to a degree not observed in vivo. Additionally, in the course of the project a number of possible workflow improvements were identified, for which suggestions have been made in the text. As such, this in vitro approach requires further study in order to determine suitability for prediction of in vivo 3′UTR behaviour.

Finland är ett skogsrikt land, men de existerande skogarna utgör dock kvalitativt enformiga habitat och det råder brist på varierade habitat i form av till exempel blandskog, skogar i naturligt tillstånd och äldre skog. Enligt tidigare studier utgör äldre skogar viktiga habitat för många skogslevande arter och mångfalden i skogarna minskar som en följd av habitatförstörelse. Vidare påvisar forskning att skogarna i Finland är ojämnt skyddade och merparten av skogsskyddet förekommer i norra Finland på statlig mark. Forskning påvisar att dagens moderna skogsbruksmetoder bidrar till att våra skogar är artfattiga och till de största hoten mot mångfalden i våra skogar klassas skogsbruket samt klimatförändringen. Att skogslandskapet i Finland utarmas är ett stort problem i och med att mångfald ökar ett ekosystems beständighet mot yttre störningar. För att åtgärda förlusten av biologisk mångfald har genvägar eller verktyg, med vars hjälp större arealer och flera arter samtidigt kan skyddas tagits fram. Till de här verktygen hör konceptet paraplyarter och en paraplyart är kortfattat en art med vars hjälp flera andra arter kan skyddas och vars förekomst indikerar på att ett lokalt habitat i närheten av paraplyarten är av hög kvalitet. Paraplyarter används explicit för att skydda habitat av hög kvalitet till exempel på områden där biodiversiteten är hög. Tidigare studier har visat att stora dagrovfåglar vanligen är effektiva paraplyarter i och med att deras boplatser kan associeras med en hög biodiversitet. Syftet med den här undersökningen är att klargöra huruvida skogslandskap i närheten av duvhökens (Accipiter gentilis) boplatser uppvisar en högre förekomst av vissa arter jämfört med kontrollområden. Kontrollområdena med vilka boplatserna jämfördes var av två slag: (a) äkta skogskontroller och (b) slumpmässigt utvalda skogskontroller. De äkta skogskontrollerna utgjordes av områden som kvalitativt motsvarade skogen intill duvhöksbon, medan de slumpmässigt utvalda skogskontrollerna utgjordes av skog av vilken typ som helst. För vart och ett av de här områdena undersöktes förekomsten av flygekorre (Pteromys volans) och blåbär (Vaccinium myrtillus). Förekomst av flygekorre karterades på basen av spillningsfynd och blåbärsrisets riklighet uppskattades via analyser av fotografier tagna över fältskiktet. Det insamlade materialet för båda arterna analyserades statistiskt med hjälp av LME - modeller. Resultaten påvisade att flygekorre förekom rikligare vid duvhökens boplatser än i de två kontrollskogarna. Flygekorrens habitatpreferenser överlappar till stor del med duvhökens och mina resultat överensstämmer med tidigare forskning som har påvisat att flygekorren har en nytta av duvhökens närvaro i form av skydd mot predation från nattaktiva rovfåglar. Flygekorren prefererar således samma skogstyper som duvhöken och intressant nog verkar duvhökens närvaro i sig vara viktigare för flygekorren än själva skogstypen. Enligt resultaten från avhandlingen är det dock inte heller uteslutet att en annan art/egenskap tillsammans med duvhöken fungerade som en paraplyart för flygekorren. Blåbärsris däremot uppvisade en rikligare förekomst vid äkta skogskontroller och duvhöksskogarna var troligen överlag för lummiga för blåbärets trivsel, men blåbärsris förekom dock rikligt vid 50 m från boplatserna. Således kan slutsatsen dras att flygekorren kan skyddas i fall av att skogslandskap med duvhöksbon skyddas. Duvhöken kan även ses som ett verktyg för naturskydd eller som en indikator för skogslandskap med hög mångfald, i vilka flygekorre samt en del blåbärsris förekommer. Om vi i framtiden vill ha flygekorre och blåbär i våra skogar bör därmed äldre skogar bevaras i och med resultaten påvisar att nämnda arter inte verkar trivas i moderna ekonomiskogar. En tillämpning av resultaten kunde vara att bruka paraplyarter och förekomst av nyckelbiotoper för naturskydd simultant.

Post-transcriptional modifications (PTMs) in RNA are present in all known RNA species and conserved in all kingdoms of life. Transfer RNA (tRNA) has been shown to have numerous conserved modifications, which exemplifies the importance of modifications having impact on the structure of the tRNA and its function as carrier of the amino acids. Ribosomal RNAs (rRNA) are universally modified as well, and modifications are situated at functionally important spots of the ribosome. Given the fact that types and sites of modifications are conserved, it is likely that these modifications have been selected for and that they optimize the ribosomal structure and functions. Stress, such as temperature or infection by a pathogen, is known to change the presence or abundance of modifications in RNA molecules and thereby affect translation efficacy. In line with that, this master’s thesis project sought to gain insight into the dynamics of PTMs in tRNA and rRNA upon oxidative stress, with the goal of utilizing recently optimized UPLC/MS method for identifying modified ribonucleosides. As the specific aim of the thesis was to estimate the change in PTMs in tRNA and rRNA in response to oxidative stress with 0.5 mM and 2 mM hydrogen peroxide H2O2, 3 immediate goals were: (i) to isolate total tRNA from yeast grown in stress conditions, (ii) to isolate rRNA from yeast 80S ribosomes, and (iii) to identify present modifications using mass spectrometry. Yeast was cultured in presence of H2O2 as a stressor in mentioned concentrations, and both treatments considered showed a difference in survival when compared to the control. Rough cell concentration estimates (OD600) did not show the effect of the stressor on cell survival clearly, but when number of viable cells per mL was estimated, it was clear that growth of the stressed yeast cultures was hindered 2 hours after exposure to H2O2 but recovered during the 24 hours. Firstly, using UPLC/MS analysis, 29 modifications were identified in tRNA from control and H2O2 treated yeast. Most identified modifications showed no change in abundance in treatments, which is to be verified with additional replicates. However, distinct dynamics of stress-related change was found for several modifications, revealing additional modifications that may play a role in stress related modificome reprogramming to the previously known signature modifications of oxidative stress. It was expected that recovery of culture growth after 24 hours may be accompanied with modification level recovery. However, that was not demonstrated here as downregulation at 2 hours followed by upregulation at 24 hours was seen for 2-methylthio-N6-methyladenosine, N4-acetylcytidine and 5-methoxycarbonylmethyl-2-thiouridine, and the reverse was shown for N4-methylcytidine. Upregulation in both time points was also shown here for some modifications. Taken together, these results confirm a complex and dynamic control of tRNA modifications in cellular survival responses. Modifications found to be affected by oxidative stress are most frequently located on the wobble position 34 and anticodon loop position 37, so it is expected that changes in their modification levels could directly affect the tRNA function in translation, making them a specific target for future research. Secondly, modifications in rRNA from control yeast cultures were identified, such as expected methylations of all 4 canonical nucleosides. However, further analysis will be needed to confirm the other identified modifications, due to the potential mRNA and tRNA contamination. Optimizing the method for rRNA modifications identifications by acquiring more modified nucleosides specific for the rRNA to use as standards in the analysis, analyzing rRNA types separately and using tandem mass spectrometry would enable getting a deeper understanding of which modifications are present and where they are positioned. Finally, it would enable reliable identification of the signals of novel modifications present in rRNA, such as the tRNA modification 5-carbamoylmethyluridine signal found here. In conclusion, this thesis work lays the foundation to study the evolutionary conserved function of PTM changes during stress as modulators of translation, using the methodological approaches discussed in-depth within the thesis, primarily to confirm the intriguing results found here.

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.