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  • Vänttinen, Ida (2020)
    Multiple myeloma (MM) is a heterogeneous plasma cell cancer that results from the excessive proliferation of mutated B cells in the bone marrow and the accumulation of ineffective antibodies, monoclonal proteins, in the blood. Despite recent advances in research and novel therapeutics, MM remains incurable, mainly due to the mechanisms underlying disease progression and drug resistance. Therefore, novel biomarkers and therapeutics for the treatment of relapsed and refractory MM are urgently needed. MicroRNAs (miRNAs), short non-coding RNA molecules that play a key role in post-transcriptional gene regulation, have been found to be associated with different hallmarks of MM. Previous studies have indicated that abnormally functioning miRNA-mediated gene regulation followed by oncogene activation and tumor suppressor gene silencing results in drastic alterations in cell proliferation, apoptosis, growth, and metabolism. These changes in cellular functions have been indicated to be associated with the pathogenesis, progression, and formation of drug resistance in MM. Therefore, the role and potential of miRNAs to act as biomarkers to predict MM progression and drug sensitivity should be further investigated to ultimately improve the survival rates of patients. The aim of this master’s thesis was to investigate the relationships between drug sensitivity, disease progression and miRNA regulation in MM patients. Bioinformatically predicted miRNAs identified to be associated with sensitivity to panobinostat, a novel histone deacetylase inhibitor, and MM progression were validated in MM patient samples by using real-time quantitative reverse transcription PCR (RT-qPCR). In addition, the specific gene targets of miRNAs involved in the regulation of drug responses and MM progression were predicted by identifying statistically significant, negatively correlated interactions between the miRNA and RNA sequencing data of 45 MM patients in pairwise comparative correlation analysis. Finally, the predicted miRNA targets genes were validated in MM patient samples using RT-qPCR. Based on the bioinformatic analyses and RT-qPCR validation, mir-424 expression was significantly increased in relapsed MM patients as compared to respective patient samples taken at diagnosis, suggesting a potential role of mir-424 in MM progression. Similarly, mir-4433b expression was significantly elevated in panobinostat-resistant patients compared to sensitive patients, suggesting a potential effect of mir-4433b on the regulation of panobinostat drug response in MM patients. In addition, the RT-qPCR validation demonstrated that the disease progression and drug sensitivity associated mir-92b, mir-363 and mir-221, would potentially regulate the expression of FGF2, MFF, and TMEM248, respectively, providing novel insights into the functional roles of miRNAs in MM pathways.
  • Cowlishaw, Mark Cary (2020)
    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.
  • Torppa, Kaisa (2014)
    The purpose of this thesis was to examine the diversity and species composition of dung beetle (Scarabaeinae) communities in degraded rainforest landscapes in southeastern Madagascar. Several studies elsewhere in the world have revealed that forest-dwelling dung beetle communities and especially large species suffer from forest degradation and fragmentation by decreased species diversity. The most important factors affecting community structure of forest-dwelling dung beetles are habitat area, connectivity and vegetation quality i.e. microclimate. The hypothesis of this study was that the situation is the same in Madagascar. As dung beetles provide several important ecosystem services, like nutrient cycling and bioturbation, loss of dung beetle diversity imposes a secondary threat to the extraordinary nature of Madagascar by decreasing the regeneration ability of vegetation. Material for the study was collected in forest fragments of different size and quality between two areas of primary tropical rainforest – Ranomafana National Park and Vatovavy mountain – in November and December 2011 and January 2012. The sampling was conducted by transects of 30–60 fish- or carrion-baited pitfall traps which were set up in 55 localities in the study area. In each locality, several variables were measured to describe the vegetation and microclimatic conditions. The variables included temperature, humidity, estimate of vegetation quality by 6 observation-based classes, vegetation density, hights of three clearly visible vegetation layers, altitude and slope steepness. In addition, connectivities were measured for the localities using GIS and a satellite image –based vegetation classification. In order to demonstrate the differences between certain localities the study sites were divided into seven zones in terms of their distance from the Ranomafana National Park, average connectivity of the transects and elevation. Altogether 4,199 individuals belonging to 24 species were collected. Six of the species are currently under identification process in the Paris Museum of Natural History. According to the preliminary results they include two species new to science. Largest numbers of species were collected from good quality fragments between Ranomafana and Vatovavy. Also, a clear transition zone in species composition was detected a few kilometers west from Vatovavy, where altitude changes sharply. The study reveals that the species assemblages in the forest fragments and degraded forest areas are surprisingly species rich. This may, however, be partly because of extinction debt, and many of the still surviving species may soon die out due to restricted dispersal possibilities. Connectivity and vegetation quality were shown to have an effect on Canthonini species richness, with less species in less connected areas and lower vegetation quality. Vegetation quality was also shown to have an impact on the proportions of species with different body length: more small and medium-sized (< 8 mm) species were found in fragments where vegetation was more degraded. In addition to revealing how rainforest fragmentation and degradation affect local communities, the study gives interesting information about the distribution of certain species of Epilissus (Scarabaeinae: Canthonini). It has been known before that four species of Epilissus show elevational differentiation in their occurrence in Ranomafana. In this study, two more species of the same genus, E. prasinus and E. emmae obscurpennis, were shown to continue this pattern in lower elevations near Vatovavy mountain, about 50 kilometers east of Ranomafana.
  • Hackman, Jenny (2020)
    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.
  • Koljonen, Laura (2017)
    Tausta ja tavoitteet: D-vitamiinia sitova proteiini (DBP) kuljettaa D-vitamiinia ja sen aineenvaihduntatuotteita, esim. 25-hydroksi-D-vitamiinia (25(OH)D), verenkierrossa. Geneettinen muuntelu GC-geenissä, joka koodaa DBP:tä, on yhdistetty 25(OH)D-konsentraatioiden vaihteluun. Tavoitteena olikin tutkia GC:n geneettisten tekijöiden vaikutusta 25(OH)D-konsentraatioon suomalaisilla vastasyntyneillä. Lisäksi tutkittiin GC:ssä esiintyvän muuntelun aiheuttamia eroja lapsen kasvussa raskauden aikana. Menetelmät: GC:n SNP:ssä, eli yksittäisen nukleotidin vaihdoksissa, esiintyvää polymorfiaa ja raskauden aikaisen 25(OH)D-konsentraation ja napaveren 25(OH)D-konsentraation suhdetta arvioitiin 933 valkoihoisella vastasyntyneellä pojalla (468) ja tytöllä (465). 25(OH)D-konsentraatio määritettiin raskausviikoilta 6-13 ja napaverestä. Äidin D-vitamiinin saanti ravintolisistä määritettiin ruuankäytön frekvenssikyselylomakkeen avulla kahdelta viimeiseltä raskauskuukaudelta. GC:n kolme SNP:tä genotyypitettiin Taq-polymeraasin aktiivisuuteen perustuvan qPCR:n avulla (Bio-Rad, CFX384 C1000 Touch™ Real-Time PCR Detection System, USA): rs4588, rs7041 ja rs705124. Genotyyppien rs4588 ja rs7041 yhdistelmästä muodostettiin kuusi tunnettua diplotyyppiä ja kaikista kolmesta genotyypistä edelleen haplotyypit. Eroja geno-, diplo- ja haplotyyppien välillä 25(OH)D-konsentraatioissa ja kasvussa testattiin kovarianssianalyysin (ANCOVA) avulla käyttämällä relevantteja kovariaatteja. Tulokset: SNP rs4588 ja rs7041 geno- ja diplotyyppien välillä löydettiin tilastollisesti merkitsevä ero 25(OH)D-konsentraatioissa (keskiarvo (nmol/l) ± keskivirhe). Napaveren 25(OH)D-konsentraatio oli alhaisin geno- ja diplotyypillä GC 2/2 (77.8 ± 4.3, p=0.010, p=0.028, ANCOVA) ja korkein diplotyypillä GC 1F/1S (93.8 ± 2.0). Haplotyypillä ATG oli alhaisin napaveren 25(OH)D-konsentraatio (77.9 ± 3.8, p=0.016, ANCOVA). Raskauden ajan 25(OH)D-konsentraatiossa ei löytynyt eroja geno-, diplo- tai haplotyyppien välillä (ANCOVA). Genotyypin rs7041 sekä diplotyyppien ja päänympäryksen välilllä havaittiin ero (p=0.005, p=0.002, ANOVA), mutta geneettisen muuntelun ja syntymäpainon ja –pituuden välillä ei löytynyt eroja. Kun valittiin relevantit kovariaatit, niin genotyyppien rs7041 sekä diplotyyppien ja päänympäryksen välillä ei löytynyt enää tilastollisesti merkitseviä eroja. Yhteenveto: Tulokset osoittavat, että DBP:tä koodaavan geenin geneettisellä muuntelulla ja napaveren 25(OH)D-konsentraatiolla olisi vaikutusta jo vastasyntyneiden D-vitamiinitilaan ja aineenvaihduntaan. Saatujen tulosten perusteella GC:n geneettinen muuntelu ei kuitenkaan ollut yhteydessä lapsen kasvuun. Tulevaisuudessa GC:n SNP:n vaikutusta 25(OH)D-konsentraatioon voisi tarkastella syntymän jälkeisten vuosien aikana.
  • Milosavljevic, Silvija (2021)
    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.
  • 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.
  • Savolainen, Elina (2023)
    Climate change is one of the biggest challenges our planet and humanity are experiencing. The time window for finding ways to miti-gate carbon emissions is getting smaller and there is an urgent need to find solutions that aim not only to reduce greenhouse gas (GHG) emissions, but to address the complex problems of land use change, deforestation, loss of biodiversity, and inequality. REDD+ is a multi-objective initiative under UNFCCC designed to reduce GHG emissions through deforestation and forest degradation togeth-er with non-carbon co-benefits of livelihoods, poverty, biodiversity, and local peoples and indigenous peoples rights. By applying a Qualitative comparative analysis (QCA) this thesis compares 12 various REDD+ countries national political contexts and particularly REDD+ policies and measures over time in order to identify conditions that enable or hinder the countries from achieving results in reversing forest cover trajectories. All the countries analyzed here are tropical, developing, or emerging countries with a political commitment to REDD+ with characteristics that hinder carbon-effective, cost-efficient, and equitable (3E) implementation of REDD+. The thesis builds on three previous REDD+ qualitative comparative analyses of various REDD+ countries' progress in developing and implementing national REDD+ policies and measures. The analysis follows an underlining theoretical assumption that both insti-tutions and agencies affect the REDD+ policy outcome. The longitudinal data used in the analysis is based on expert assessments conducted in three various data collection rounds in 2012, 2014, and 2016. The results show a set of enabling conditions under which countries can achieve a positive outcome. The findings from the previous studies have highlighted the importance of already initiated policy change and effective forest legislation from the institutional context, and from the actor-centered policy arena, the presence of powerful coalitions and the availability of performance-based payments. Here, two enabling remote conditions are identified (1) pressure from the forest resources and (2) the presence of effective forest legislation. The pressure from the forest resources leads to a positive outcome together with (3) strong national ownership and politi-cal will combined with (4) performance-based payments or (5) REDD+ policies and measures. The effective forest legislation.leads away from business-as-usual practices towards a broader transformational change when combined with (6) powerful transformation-al coalitions and (7) inclusive policy processes. Policy relevance: Well over a decade has passed since REDD+ was launched in 2007 but the progress has been much slower than it was initially expected. The evidence on what works and what does not is essential to achieve the GHG reductions needed to keep the global warming below 2C. The findings from this study can guide towards more effective, efficient, and equitable REDD+ policy design formulation and implementation.
  • 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.
  • Pihl, Enni-Eveliina (2023)
    Microglia, the resident macrophage-like glial cells of the central nervous system (CNS), form the first line of defense against pathogens in the brain, and regulate both innate and adaptive immunity. Any abnormalities in their microenvironment lead to microglial activation, characterized by alterations in their gene expression, morphology, and functional behavior. Once activated, microglia respond to CNS injury and inflammation by, e.g., migrating to the site of damage, releasing pro-inflammatory cytokines, as well as phagocyting cell debris and pathogens. Prolonged activation of microglia expressing pro-inflammatory phenotypes can lead to exacerbated CNS damage. Hence, limiting CNS inflammation by stimulating microglial polarization towards their pro-resolving phenotypes would be of great clinical relevance. The research of our laboratory focuses on CNS injury and repair, as well as finding novel therapies for ischemic stroke. Specialized pro-resolving mediators (SPMs) derived from essential fatty acids have been proposed to offer a potential therapeutic approach for ischemic stroke via promoting resolution of post-stroke inflammation. Previous studies have revealed the ability of SPMs to induce a transformation of macrophages, the immune cells strongly resembling microglia, towards their anti-inflammatory phenotypes. The aim of this study was therefore to assess whether SPMs have similar effects on BV2 microglia, specifically on their lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). In addition to assessing the cytokine levels, our aim was to determine the optimal conditions for studying the effects of SPMs on microglial migration. In the present study, the levels of TNF-α and IL-6 were determined by specific ELISAs, and the transwell assay was used to measure microglial migration. Resolvins E1 (RvE1) and D1 (RvD1), as well as protectin D1 (PD1) and 15-epimer of lipoxin A4 (15-epi-LXA4) were all associated with decreased levels of TNF-α and IL-6, with RvE1 having the most potential as a resolving agent. In addition, we observed that serum starvation notably decreases the release of IL-6 and affects microglial migration. Overall, our results support the idea that SPMs could provide a novel therapeutic strategy for stroke therapy as they contribute to the resolution of CNS inflammation.
  • Hein, Emil (2022)
    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.