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Optimization of sample preparation for next-generation sequencing with Illumina Genome Analyzer II (2010)The growing interest for sequencing with higher throughput in the last decade has led to the development of new sequencing applications. This thesis concentrates on optimizing DNA library preparation for Illumina Genome Analyzer II sequencer. The library preparation steps that were optimized include fragmentation, PCR purification and quantification. DNA fragmentation was performed with focused sonication in different concentrations and durations. Two column based PCR purification method, gel matrix method and magnetic bead based method were compared. Quantitative PCR and gel electrophoresis in a chip were compared for DNA quantification. The magnetic bead purification was found to be the most efficient and flexible purification method. The fragmentation protocol was changed to produce longer fragments to be compatible with longer sequencing reads. Quantitative PCR correlates better with the cluster number and should thus be considered to be the default quantification method for sequencing. As a result of this study more data have been acquired from sequencing with lower costs and troubleshooting has become easier as qualification steps have been added to the protocol. New sequencing instruments and applications will create a demand for further optimizations in future.
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Optimized sample preparation protocol for the metatranscriptomic analysis of bloodstream infections (2021)Bacteraemia, the presence of bacteria in the bloodstream, may lead to severe and costly health issues. Sepsis, a serious complication of bacteraemia, is one of the top causes of mortality globally. Early and specific diagnostics as well as fast acting are essential in successful treatment. However, current diagnosis relies mainly on time-consuming blood culturing and clinical symptoms, which are unspecific for the causative agent. With the advanced technology and decreasing cost, state-of-art sequencing-based (Next generation sequencing) methods provide a new way to investigate the bacteria present. Metagenomics, which means sequencing and studying all DNA extracted from a microbial community sample, is widely used, but it only describes the genetic potential of a community and does not differentiate live from dead microbes. Metatranscriptomics, in which essentially all RNA from a sample is sequenced, provides information about expression and activity together with identification of viable bacteria, However, the high amounts of host cells and host RNA complicate the detection of bacterial transcripts from complex host-microbe samples. In this thesis, I investigated solutions for the efficient isolation and enrichment of bacterial RNA from whole blood to be used in sequencing and metatranscriptomics analysis. Firstly, I tested the capability of bacterial cell lysis of two commercial blood sampling tubes with Escherichia coli and Staphylococcus epidermidis suspensions. Both tubes, Tempus and RNAgard, were able to lyse gram-negative E. coli cells and good-quality RNA was extracted in measurable quantities with their respective RNA extraction methods. With Tempus tubes the RNA yield was clearly higher. With gram-positive S. epidermidis, RNA quantities from both extractions were below the measurement limits indicating insufficient lysis and need for further optimization. Secondly, I investigated the depletion of polyadenylated (poly-A) transcripts in order to reduce the host transcripts and thus to enrich the bacterial transcripts prior to costly sequencing step. I evaluated the performance of a previously designed in-house protocol, based on the capture of poly-A -transcripts with oligo-dT -beads, and tested different parameters to see whether the depletion efficiency could be enhanced. Most significantly, the amount of oligo-dT -bead suspension was reduced to half from the original protocol. In-house protocols were also compared to a commercial solution, which they clearly outperformed. Depletion performances were tested with a RT-qPCR and dot blot assay, which I designed along this thesis work. Finally, to make the poly-A depletion better suited for blood samples infested with globin transcripts (representing up to 80% of all poly-A transcripts extracted from whole blood), I tested and successfully pipelined the leading commercial method for depleting globin transcripts with the in-house poly-A depletion protocol. The optimized sample preparation protocol provides a platform for further bloodstream infection and sepsis studies. Next steps of the process, such as sequencing and testing with clinical samples, are already ongoing with promising preliminary results. In the future, the metatranscriptomics approach can be utilized in fast and specific identification of the pathogens and their antibiotic susceptibilities. In addition, infection mechanisms and host-pathogen interactions may be studied possibly providing novel insights for sepsis diagnostics and treatment.
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(2022)Acute leukemia is a life-threatening disease of blood and bone marrow, which is caused by malignant transformation of immature white blood cells. These malignant white blood cells invade space in bone marrow decreasing its ability to produce normal blood cells, eventually leading to death within weeks after the diagnosis without treatment. The acute leukemia can be broadly divided into its lymphoblastic and myeloid form, based on the affected cell lineage. Furthermore, acute leukemias can be classified based on different genomic features, such as gene fusions. Fusion genes are strong drivers in various cancers such as acute leukemias, and they are formed when two or more original genes join together forming a novel hybrid gene. If the novel hybrid gene is transcribed, it can lead to a translation of an abnormal fusion protein with altered function. The detection of the gene fusions is very important, since it affects to diagnosis and treatment of the patient. Various techniques can be used for fusion gene detection, of which the RNA sequencing is the method of choice, due to its ability to provide an unbiased identification of all known and novel gene fusions from the sample in a single experiment. In this thesis, the overarching aim was to develop an optimal sampling protocol for fusion gene detection using RNA sequencing for acute leukemia diagnostics. First, the whole blood samples in EDTA-tubes were collected from acute leukemia patients based on the findings from routine diagnostics. Next, the RNA was extracted at three different timepoints (0h, 8h, and 32h). The samples were stored at 4°C between the extractions. Finally, the RNA sequencing libraries were constructed, and the RNA sequencing was performed. After the sequencing, the data was analyzed using the FusionCatcher algorithm for fusion gene detection and the EdgeR-package for differential expression analysis. The FusionCatcher detected the same gene fusion in all the four fusion gene positive patients compared to routine diagnostics. However, the FusionCatcher failed to recognize the gene fusion in some of the samples with very low number of fusion breakpoint-spanning reads. These reads were visualized with IGV, suggesting that the detection failure resulted from the very low number of break-point-spanning reads. Furthermore, the sample storage did not affect on gene fusion detection. In addition, FusionCatcher detected PIK3AP::BLNK gene fusion from one of the fusion gene negative patients, suggesting a possibility that the patient truly was fusion gene positive. The differential expression analysis revealed changes in gene expression between the different timepoints. The results showed changes in various pathways related for example to cell death and protein biosynthesis, but also to pathways related to cancer. The results showed that prolonged sample storage alters the gene expression profile thus affecting the results of a gene expression study.
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(2024)Schizophrenia, a mental disorder affecting over 1% of the world’s population, has a 41-65% chance of being acquired in monozygotic twins, and shows a complex heritable pattern. Research has shown the involvement of various neuronal and glial cell types in the disorder’s progression. Recent studies are focusing on cortical interneurons, as clinical features of schizophrenia such as working memory deficits emerge due to the abnormal activity of these cells . The advent of induced pluripotent stem cell (iPSC) technology has made it easier to study schizophrenia disease mechanisms, with studies revealing differences in morphological and physiological properties of cortical interneurons in patients with schizophrenia. In this thesis , the aim was to optimize iPSC-interneuron differentiation protocol and live-cell imaging method suitable for disease modelling. Interneurons were differentiated from iPSCs with overexpression of inducible transcription factor, Achaete-scute homolog 1 (ASCL1). The iPSCs were derived from twin pairs discordant for schizophrenia and from healthy controls. Expression of interneuron-specific markers was verified using RT-qPCR and validated at the protein level by an immunocytochemistry (ICC) assay in the control cell lines first. Additionally, to estimate the formation of neurites and differences in neurite length and branching, the differentiated interneurons from the controls were subjected to live-cell imaging by IncuCyte S3 live-cell imaging system. Imaging parameters such as cell body cluster filter was optimized to visualize the neurites. To study interneuron involvement in schizophrenia, iPSCs from one twin pair discordant for schizophrenia were successfully differentiated. Interneurons strongly expressed Gamma-aminobutyric acid (GABA) neurotransmitter related neuronal markers: glutamate decarboxylase 67 (GAD67) and GABA at protein level. The neurons were identified as somatostatin (SST) subtype GABAergic neurons by their mRNA and protein expression. While it was possible to observe differences in gene expression, there were no clear differences in the morphology of the differentiated cells as well as the localization of markers in comparison to the healthy controls. Further studies should focus on having a protracted time for differentiation where more mature interneurons can be produced by establishing co-cultures with excitatory neurons. This will help replicate the in vivo cortical machinery which in turn will aid in better understanding of disease mechanisms.
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(2024)Alzheimer's disease (AD) is a degenerative brain disorder that exhibits deterioration as one gets older. Although much remains to be learned about the pathophysiology of AD, there is strong evidence links amyloid beta (Aβ) plaques, which are responsible for cognitive impairment, to GABAergic interneurons. Model systems are of prime importance for adequately studying the pathophysiology of this disorder; however, existing in vitro models have limitations in producing patient-specific cells. The development of induced pluripotent stem cell (iPSC) technology has provided a novel opportunity for the effective production of disease-relevant cell types while preserving the molecular traits of the patient. In this thesis, the differentiation protocol established by Nicholas et al. (2013) was used to promote the development of interneurons derived from iPSCs. To enhance the efficiency of differentiation, the protocol was modified with the use of small molecules combined in different ways. The end result of the differentiation was characterized using immunocytochemistry (ICC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The combination of molecules that produced greater efficiency in differentiation was selected, and the optimized protocol was carried out with iPSCs derived from an AD patient harbouring the APP Swedish mutation. The differentiation of cortical interneurons, demonstrated by the expression of pan-neuronal and specific GABAergic neuronal markers, signifies the successful generation of differentiated interneurons in the context of AD. AD iPSCs upregulated several markers related to AD pathology, such as APP and BACE1. However, the cell lines tolerated the small molecules differently, and thus, the protocol needs more optimization in the future. In summary, iPSC-based differentiation protocols are capable of producing disease-specific cell types that would be helpful in developing accurate AD models for revealing the mechanisms of Aβ pathology.
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Orgaanisten yhdisteiden hajoaminen puhdistetun jäteveden hygienisointiin käytetyssä UV-käsittelyssä (2017)Ympäristöön päätyvät orgaaniset yhdisteet ovat maailmanlaajuinen ongelma. Tällaisiin yhdisteisiin kuuluvat muun muassa lääkeaineet, kuluttajakemikaalit sekä muoviteollisuuden raaka-aineet. Nämä yhdisteet päätyvät vesistöihin pääasiallisesti jätevedenpuhdistamoiden epätäydellisten puhdistus prosessien seurauksena ja voivat aiheuttaa vesieliöille haittavaikutuksia jo pieninäkin pitoisuuksina. Lisäksi vesilaitokset käyttävät pintavesiä raakavesilähteinään, joten mahdolliset riskit koskevat myös ihmisiä. Näistä syistä on alettu kiinnittää yhä enemmän huomiota erilaisten haitta-aineiden esiintymiseen vesiympäristössä ja niiden puhdistusmenetelmiin. Yhtenä potentiaalisena menetelmänä pidetään UV-käsittelyä, joka on tehokas desinfiointimenetelmä ja käytössä paitsi juomavedenvalmistuksessa, myös jätevedenpuhdistamoilla, joissa sen avulla saadaan tehokkaasti poistettua koliformiset bakteerit. Kyseisen menetelmän on aiemmissa tutkimuksissa havaittu hajottavan myös orgaanisia yhdisteitä. Tämän pro gradu -työn tavoitteena oli tutkia valittujen orgaanisten yhdisteiden esiintymistä Lahden Ali-Juhakkalan jätevedenpuhdistamon puhdistetussa jätevedessä, niiden vuodenaikaisvaihtelua, sekä kyseisellä puhdistamolla käytössä olevan UV-käsittelylaitoksen tehokkuutta näiden yhdisteiden hajottamisessa. Tutkittavia yhdisteitä olivat lääkeaineista diklofenaakki, ibuprofeeni, naprokseeni ja karbamatsepiini, sekä muoviteollisuuden raaka-aine bisfenoli A ja synteettinen hajuste HHCB. Lisäksi suoritettiin laboratoriokokeita sekä puhdistetulla jätevedellä, että ultrapuhtaalla vedellä kotitalouskäyttöön tarkoitetulla UV-laitteistolla. Työ suoritettiin yhteistyössä MOTREM-projektin kanssa. Näytteitä kerättiin viitenä ajankohtana syksyn 2016 ja kesän 2017 välisenä aikana. Näytteet esikäsiteltiin kiinteäfaasiuuttomenetelmällä ja analysoitiin GC-MS:lla, jolle kehitettiin tässä työssä tutkittaville yhdisteille soveltuva analyysimenetelmä. Lisäksi pyrittiin löytämään diklofenaakin mahdollisia hajoamistuotteita ja nämä näytteet analysoitiin GC-TOF-MS:lla. Puhdistamolta mitatuissa näytteissä yhdisteiden pitoisuudet vaihtelivat välillä 27–1820 ng/l, korkein pitoisuus oli diklofenaakilla, ja korkeimmat pitoisuudet mitattiin helmikuussa. UV-käsittely laski diklofenaakin ja karbamatsepiinin pitoisuuksia kaikkina ajanhetkinä hieman, muiden yhdisteiden kohdalla vastaavaa ei havaittu. Laboratoriokokeista saatujen tulosten perusteella voitiin todeta, että diklofenaakki ja naprokseeni olivat herkkiä UV-säteilytykselle, puhtaassa vedessä diklofenaakista saatiin hajotettua jopa 97 %. UV-lamppukokeilla saatiin myös tietoa diklofenaakin hajoamisesta. Tästä työstä saatujen tulosten perusteella voidaan todeta, että puhdistettu jätevesi sisältää orgaanisia yhdisteitä pieninä pitoisuuksina ja UV-käsittely voisi olla potentiaalinen hajotusmenetelmä joillekin orgaanisille yhdisteille, kuten lääkeaineille. Veden uudelleenkäyttömahdollisuuksia on tulevaisuudessa pohdittava entistä tarkemmin ja tällaisten puhdistusmenetelmien kehittäminen on tärkeää.
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(2020)Previous studies have shown that involving citizens, especially young people, in environmental decision making, increases the ability to participate in environmental action (e.g. Riemer et al. 2014, Paloniemi and Koskinen 2005). There have been various school and after school projects to promote the engagement of young people in societal environmental action. Many youth engagement projects in the third sector have succeeded in achieving positive results in promoting environmental citizenship. This thesis evaluates the involvement of young people and young adults in environmental action at WWF Finland. The theoretical framework of this thesis is based on the model by Paloniemi and Koskinen (2005) of environmental policy action as a social learning process. According to the model, positive experiences of involvement can strengthen environmental action competence and encourage participants to take environmentally responsible action. In the spiral model, the context is taken into account in the results. The data for this thesis is from a survey of 30 young people at WWF, who took part in the project. The survey mapped the effects of the project on the participants' self-empowerment, social empowerment, and environmental citizenship. It also examined skills in environmental action, and the effects of the context on the results. These results were processed using a mix of qualitative content analysis and statistical analysis methods. In the content analysis, the open-question-answers were themed. The differences between numerical answers of those who suspended the project, and those who were involved until the end were compared, for example using a T-test. In addition, narrative inquiry was developed based on the answers to describe comprehensive profile of respondents. There were no statistically significant differences between those who discontinued the project and those who participated until the end. However, the content analysis of the study provided evidence that the activities succeeded to empower participants and strengthen their environmental citizenship and environmental action competence. According to the results, 26 out of 30 respondents received more information about environmental problems, and 24 received information about their own opportunities on environmentally responsible action. Almost everyone also learned other skills that support environmental citizenship. The project had a positive effect on self-empowerment and social empowerment for 21 respondents. More than half of the respondents also reported an increase in societal engagement, or encouragement to work in the field of environmental or sustainable development. The results of this thesis conclude that the context of WWF’s project was overall successful, as the desired results were achieved. Motivating and engaging factors that contributed to the goals of the operation were also identified. The results of the thesis are in line with previous research, which indicates that environmental participation in the third sector plays an important role in promoting environmental citizenship. However, more long-term research on the topic is needed, as well as research with different control groups.
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(2020)Participatory budgeting is one of the major democratic innovations of the recent decades. This participatory method from Brazil has been started to actively utilize in Finnish municipalities during 2010s. The basic idea of this methodology is that citizens can together decide how to use public assets. The goal of this thesis is to understand how participatory budgeting can have an impact on ecological sustainability in Finland. I’m focusing on the proposals of participatory budgeting which the citizens have voted for to be implemented. Research material has been collected from public online sources on all the Finnish participatory budgeting projects. Some of these projects have been excluded as they do not fulfill the characteristics of participatory budgeting. The material is analyzed using content analysis, building categories, themes and types. There are three key findings in this thesis. First, participatory budgeting has created ecologically sustainable solutions, but sustainability has been an unintended by-product. Proposals concerning environment focus primarily on people’s living environment. Second, the changes are minor, and individuals are the ones carrying the responsibility for the sustainability. Third, the means how proposals are carried out influences ecological sustainability. Here, municipal authorities have a significant role. Finnish participatory budgeting projects therefore have the chance to create ecologically sustainable proposals. However, the capability is limited by both small, project specific budgets and processes that do not acknowledge ecological sustainability. In the long run, the changes might be more significant as participatory budgeting still is relatively new method in Finnish society.
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(2019)Tiivistelmä – Referat – Abstract OCRL1 is a phosphatase that cleaves phosphatidylinositol 4,5-bisphosphate (PI4,5P) to phosphatidylinositol-4-phosphate (PI4P). ORP4 is a lipid binding/transport protein implicated in G-protein coupled signaling, cellular calcium homeostasis, proliferation and viability. OCRL1 and ORP4 are found in the endoplasmic reticulum and membrane contact sites throughout the endosomal system and the Golgi complex. OCRL1 is also present in the plasma membrane and vesicular structures. ORP4 has high affinity for binding sterols through the OSBP related domain (ORD). ORP4 also interacts with vimentin intermediate filaments via the ORD and influences the localization and organization of vimentin. The membrane lipid phosphatidylinositol (PI) can be phosphorylated either singly or in combination at three different positions on its inositol ring (D-3, D-4, and D-5)—yielding 8 possible phosphoinositides; the interconversion between the species is regulated by kinases and phosphatases that either add or remove phosphate groups from the various positions on the ring. Phosphoinositide metabolism is heavily involved in signal transduction pathways and cytoskeletal organization. Interestingly, it has also been found to be spatially regulated with distinct phosphoinositides being enriched in particular membrane compartments. BiFC assays can provide an important tool for visualizing protein-protein interactions. Not only is BiFC able to determine protein-protein proximity but it is also able to localize the interaction with regard to compartment membranes and structures. This study examined the interaction of ORP4 with OCRL1 by using BiFC analysis. We were able to determine that the protein pair seems to be in close proximity in the endoplasmic reticulum near the Golgi. Interaction only took place when the OCRL1 was tagged with VB at the C-terminal.
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(2005)Otsoni (O3) on ilmakehässä sekä elintärkeä että haitallinen ihmisille, eläimille ja kasvillisuudelle. Yläilmakehässä eli stratosfäärissä (15-50 km korkeudessa) otsoni estää vahingollisen UV-säteilyn pääsyä maanpinnalle. Alailmakehässä eli troposfäärissä (alle 15 km korkeudessa) otsoni on puolestaan kasvihuonekaasu sekä aiheuttaa ihmisille ja eläimille mm. hengitysteiden ärsytystä ja kasvillisuudelle vaurioita. Otsonin on todettu koeolosuhteissa aiheuttavan kasveille lehti- ja neulasvaurioita sekä vähentävän niiden fotosynteesiä ja kasvua. Myös luonnonkasveista on löydetty otsonivaurioita mm. Puolassa, Ukrainassa ja Sveitsissä. Erityisesti lämpimät ja aurinkoiset ajanjaksot lisäävät otsonivaurioita. Alailmakehän otsonipitoisuus on ihmistoiminnan vaikutuksesta noussut viimeisen sadan vuoden aikana kaksinkertaiseksi. Pohjoisella pallonpuoliskolla vuosittainen otsonipitoisuuden kasvu on viimeisen 30 vuoden aikana ollut noin 0.5-2.0 %. Otsonia ei sellaisenaan pääse ilmakehään, vaan se muodostuu ilmakehässä fotokemiallisissa reaktioissa hapesta, typen oksideista ja hiilivedyistä auringon säteilyenergian avulla. Typen oksideja ja hiilivetyjä pääsee ilmakehään lähinnä liikenteestä ja petrokemian teollisuudesta. Tässä Pro gradu -tutkielmassa tutkittiin kesällä 2002 sveitsiläisiltä LESS-aloilta (light exposed sampling site) kerättyjä pyökin (Fagus sylvatica), saarnen (Fraxinus excelsior), villaheisin (Viburnum lantana) ja kuusaman (Lonicera xylosteum) lehtinäytteitä. Alat kuuluvat kansainväliseen metsien kuntoa kartoittavaan yhteistyöhankkeeseen (IPC-forest). Aloilta oli käytettävissä otsonipitoisuus ja sademäärätiedot. Lehdistä mitattiin näkyvien vaurioiden määrä, ilmarakotiheys, huulisolupituus, lehden paksuus sekä lehden poikkileikkauksen soluja soluvälimäärät. Tutkimuksen lajeista ilman näkyviä otsonivaurioita oli säästynyt vain pyökki. Osassa saarnen ja villaheisin lehtiä oli erittäin paljon näkyviä vaurioita (51-100%). Otsonipitoisuus korreloi tilastollisesti merkittävästi (p < 0.05) vain saarnen ilmarakotiheys/huulisolupituus-suhteen kanssa, tämä oli negatiivinen korrelaatio. Suuntaa-antavia korrelaatioita otsonipitoisuudella oli useiden lehtiparametrien kanssa. Sademäärän korreloi tilastollisesti merkittävästi negatiivisesti saarnen pylväs-/hohkatylppysuhteen kanssa ja positiivisesti pyökin pylvästylpyn osuuden kanssa. Suuntaa-antavia korrelaatioita oli myös sademäärän ja lehtiparametrien välillä useita. Näkyvät vauriot korreloivat villaheisillä negatiivisesti ilmarakotiheyden kanssa. Otsonin haitallisuus ehkä vähenee sen levittyessä lehteen tasaisemmin. Tämä tutkimus vahvistaa aiempia tutkimustuloksia siitä, että Sveitsissä puusto kärsii luonnossa vallitsevista alailmakehän otsonipitoisuuksista. Koska merkit kasvillisuuden otsonivaurioista ovat selvät, täytyisi otsonipitoisuudet saada laskemaan. Kasvien vaurioitumiseen vaikuttavat monet ympäristötekijät ja eri lajien (myös yksilöiden) otsoniherkkyys vaihtelee. Tämä tulisi huomioida otsonipitoisuuden raja-arvoja tarkennettaessa. Otsonin vaikutusten tutkimus on painottunut ihmisille taloudellisesti arvokkaisiin lajeihin. Myös muita lajeja ja luontotyyppejä tulisi tutkia enemmän
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(2023)The sense of hearing is dependent on the sensory cells of the cochlea: inner and outer hair cells. The critical functional structure of these cells is the stereocilia bundle, the mechanotransduction (MET) organelle. The outer hair cells (OHCs) are highly vulnerable to environmental assaults, the effects of aging, and gene mutations. This vulnerability is thought to be mediated by challenges in maintaining intracellular calcium homeostasis. Endoplasmic reticulum (ER) stress is a form of cellular stress that was previously shown to cause hair cell impairment. A possibility is that this impairment is mediated by perturbations in calcium homeostasis. In this thesis, the aim was to find out how the OHC calcium homeostasis is affected by specific ER stress-inducing mutations and age in mouse models exhibiting progressive hearing loss. I studied OHC calcium homeostasis in Manf conditional knock-out (cKO) mice under C57BL/6 (B6) strain in which ER homeostasis-promoting protein MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) is depleted in cochlear hair cells. Previous studies have shown that these mice develop progressive hearing loss that includes OHC loss and prominent stereocilia pathology, stereocilia fusion. By itself, the B6 mouse strain is a model of age-related hearing loss due to a Cdh23 missense mutation that is known to cause progressive hearing loss and, according to earlier evidence, may be a source of ER stress. I studied B6 mice at 6–9 months of age and Manf cKO mice at 2 months of age to comparatively examine changes to OHC calcium homeostasis that may correlate with the changes in the stereocilia bundle morphology and with hearing loss. I studied hearing function by auditory brainstem recordings in vivo. I estimated the functionality of MET channels in OHCs by FM1-43 uptake. I used immunofluorescence microscopy to study subcellular expression of key calcium-buffering and calcium-extrusion proteins in OHCs. I used a novel super-resolution imaging technique, expansion microscopy (ExM), to study stereocilia bundle morphology. OHCs of Manf cKO mice showed changes in calcium homeostasis in all the studied aspects: (1) FM1-43 uptake through MET channels was reduced, (2) the expression of the calcium extrusion pump PMCA2 and its obligate partner, the cell adhesion protein neuroplastin, was decreased, (3) and the expression of calcium-buffering protein oncomodulin was increased. All this data indicated OHC calcium dyshomeostasis. These molecular changes were consistent with the hair bundle pathology—stereocilia fusion—revealed by phalloidin staining of the actin-rich core of the stereocilia followed by ExM imaging. OHCs of 6–9-month-old B6 mice exhibited reduced FM1-43 uptake, yet not to the extent seen in cKO mice, and there were no changes in PMCA2 and neuroplastin expression and no prominent stereocilia fusion. Together, I show in this study that OHC hair bundle dysmorphology is linked with changes in calcium homeostasis in the mouse model of ER stress-induced hearing loss. This is consistent with the fact that calcium dyshomeostasis is an integral part of cellular ER stress. An intriguing, yet unanswered question is whether these changes in stereocilia bundle physiology could actually be the trigger for the death of these sensory cells.
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(2022)Continuous development of urban areas poses challenges for sustainable use of resources and the management of complex waste streams. Recycling is seen as a solution for promoting sustainability, especially at the individual-level where waste sorting creates preconditions for successful material recovery operations. Behavior change strategies aim to encourage individuals to implement recycling practices in their daily lives. The effectiveness of behavior change strategies is achieved by broadly influencing capability, opportunity, and motivation to recycle, however, studies claim that many existing strategies are unable to do so. This study aims to gain an understanding in how extensively a municipal recycling service provider’s online communication on social media platforms (Instagram, Facebook, YouTube) enhances capability, opportunity, and motivation to recycle by communicating about means to overcome recycling barriers. Specifically, the study is interested in examining what types of recycling barriers are addressed in online communication and through which means it aims to deliver assistance for overcoming these barriers. Lastly, this study aims to explore the role of social media platform-based online communication as a channel to promote individual recycling behavior. This study focuses on social media materials published by a municipal service provider in the European Green Capital of 2021. The empirical material builds on a set of data collected from public and locatable online sources. The analysis includes 96 different types (pictural, textual, video) of recycling-themed online content. The data was analyzed by conducting qualitative content analysis. The results indicated that online communication addresses a broad range of recycling barriers. The most common means to overcome these was information provision through which the company aimed to increase knowledge and understanding of recycling practices. Although information provision alone is claimed to be insufficient to profoundly change behavior, results showed that it could serve as a means to generate a broad influence on areas behind behavior formation. The result of this study suggests that social media platforms as channels for online communication have the potential to create preconditions for overcoming recycling barriers especially through the means of information provision. The development of more profound recycling behavior, however, needs to include a broader range of collaborative information, motivation, and engagement elements that could engage and encourage people to implement more profound recycling behavior. This calls for future research that discovers means to stimulate behavior formation widely to support overcoming recycling barriers and the implementation of profound recycling behavior in everyday lives.
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(2023)The developing human hindbrain and its role in neuronal pathogenesis have been relatively difficult to study for ethical reasons. By using the dual-SMAD inhibition and WNT signalling induction, a new method to culture brain organoids to resemble the human hindbrain has recently been established. In this study the new method has been used to detect the developing hindbrain’s response to flaviviral infection. Model virus used in this study is the Zika virus (ZIKV) which is known to alter the development of central nervous system and cause microcephalia. Pathogenic activity of the virus is measured by detecting the morphology of the organoids during infection as well as screening the organoids activation against oxidative stress, in a form of KEAP1/Nrf2-ARE pathway activation. Three different clones of ZIKV, which differ from each other by one amino acid in their non-structural protein 1 (NS1) gene, were used in the infections as well as two different time points of development. Controversially to previous findings on ZIKV infections to brain organoids, our findings show that developing hindbrain-like organoids do not change in shape or size during ZIKV infection. There are no differences in the lack of morphological changes between one-month olds or two months old organoids or between the different ZIKV clone infected organoids. The activation of the KEAP1/Nrf2-ARE pathway was measured by screening the two final products of the pathway, Nqo1 and HO-1. By screening the mRNA levels of these two genes, it showed that different ZIKV clones affect the activation of the KEAP1/Nrf2-ARE pathway in different levels at different times of development. Also, the expression of the same gene can be altered by the age of the organoids. Additionally, the expression of the two genes were different from each other at given time points and in response to the different clones. These findings suggest that the different isoforms of NS1 of ZIKV may alter the developing hindbrain’s response to oxidative stress. Findings also show that the time of the infection can additionally play a critical role to the ZIKV infection. The altered response to oxidative stress may contribute to microcephaly: the oxidation homeostasis of the developing hindbrain is modified, and apoptotic cell death can take place.
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(2009)Life cycle assessment is a method to estimate potential environmental impacts of a product from cradle to grave. The life cycle of waste starts when product, e.g. cardboard box is discarded. The main goal of this study was to estimate by LCA, how discarded cardboard should be utilized and the collection organised to minimize the harmful environmental impacts. Environmental impacts studied were climate change, eutrophication of soil and particles. The impacts were evaluated by using four different scenarios. In scenarios, discarded cardboard was not sorted at all (Scenario 0) or sorted and collected from regional collection points (kerbside collection) and/or from residential buildings (Scenarios 1-3). The sorted cardboard was utilised to produce cardboard cores for industry. The cardboard that was not sorted was transported by mixed household waste collection to the waste incineration plant where the generated heat and electricity were utililized. Environmental benefits were gained if emissions to environment could be avoided by replacing normal processes by recycling and incineration. For cardboard recycling, the process replaced was CTM-pulp production from virgin forest and for incineration, the fuel used to the average heat and electricity production in Finland. In all studied environmental impact classes, all scenarios produced environmental benefits. In climate change and particles, scenario 0, where all cardboard was utilized in incineration plant, achieved most benefits and the scenario 3, where the maximal amount of cardboard was sorted and collected from residential buildings, achieved the least. However, the differences between the scenarios - especially in climate change class - were small. In eutrofication class, the results were inverse to climate change and particles classes. The most beneficial scenario was the one, where the maximal amount of cardboard was sorted and collected from residential buildings. Due to the dissimilar results of different environmental impact classes it is not simple to say how to utilize discarded cardboard. When applying the results, it should be noticed that the results depend on which energy and material products are produced, and foremost, on what kind of products they replace. The final conclusions concerning which alternatives are the best depend on which environmental impacts are emphasized.
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(2019)Rinteen ekspositio eli rinteen kaltevuus ja ilmansuunta, johon rinne viettää, vaikuttaa rinteen vastaanottaman auringonsäteilyn määrään yhdessä leveyspiirin kanssa. Eksposition erot voivat saada aikaan selviä ja säännönmukaisia eroja eliöyhteisöjen koostumukseen. Ilmiötä on tutkittu laajalti luonnollisissa elinympäristöissä, muttei juurikaan ihmisen luomissa elinympäristöissä. Tässä tutkielmassa selvitän rinteen eksposition vaikutusta maakiitäjäisyhteisöjen koostumukseen täyttömäillä, täyttömäille kehittyvien maakiitäjäisyhteisöjen ominaispiirteitä sekä paikallisten tekijöiden ja ympäröivien alueiden merkitystä täyttömäkien maakiitäjäisyhteisöjen muodostumiselle. Kaupungistuminen tuhoaa ja pirstoo elinympäristöjä, mikä korostaa rakentamatta jäävien viheralueiden merkitystä kaupunkialueilla. Täyttömäille ei voi rakentaa, ne ovat useiden hehtaarien laajuisia, ja niitä on kaupunkialueilla, minkä takia täyttömäillä on huomattavaa potentiaalia biodiversiteetin säilyttämisessä ja lisäämisessä kaupunkialueilla. Tarkoituksenani on antaa tuloksiin perustuva alustava arvio täyttömäkien merkityksestä kaupunkien biodiversiteetille. Käytin 10 Suomen pääkaupunkiseudulla sijaitsevan täyttömäen etelä- ja pohjoisrinteiltä Jarmo Saarikiven kuoppapyydyksillä 19.5.-28.9.2010 keräämää 5557 maakiitäjäisyksilöä käsittävää aineistoa. Määritin kaikki maakiitäjäisyksilöt lajitasolle. Selvitin kunkin runsaimman lajin ja lajiryhmän runsaudessa näkyvää vastetta rinteen ekspositioon ja jatkuviin muuttujiin (pH, muurahaisten runsaus, täyttömäen ikä ja korkeus) yleistettyjen lineaaristen sekamallien (GLMM) avulla. Lisäksi vertasin etelä- ja pohjoisrinteiden välisiä sekä eri täyttömäkien välisiä lajiyhteisöjen eroja moniulotteisen skaalauksen (NMDS) avulla sekä tarkastelemalla laji- ja yksilömääriä. Rinteiden suosimisen ja lajin elinympäristövaatimusten välillä havaittiin selvä yhteys. Kuivan ja avoimen elinympäristön lajit suosivat enimmäkseen etelärinnettä, kun taas kostean metsäelinympäristön lajit suosivat selvästi pohjoisrinnettä, ja generalistilajeilla ero oli vähäinen. Täyttömäillä dominoivat pääasiassa kuivan elinympäristön lajit: niin kuivan elinympäristön generalistilajit kuin myös kuivan ja avoimen elinympäristön lajit. Silti niin yhteisöjen koostumus kuin myös lajimäärä vaihtelivat huomattavasti eri täyttömäkien välillä. Koko maakiitäjäisyhteisön koostumuksessa ei ollut tilastollisesti merkitsevää säännönmukaista eroa etelä- ja pohjoisrinteiden välillä NMDS-tarkastelussa, mutta rinteiden välinen ero oli sitä suurempi, mitä suurempi täyttömäki oli, ja pelkästään suuria (17-38ha) täyttömäkiä tarkasteltaessa maakiitäjäisyhteisön koostumuksessa oli selvää rinteiden välistä säännönmukaista eroa. Tässä tutkimuksessa saatiin selvää näyttöä, että rinteen ekspositio vaikuttaa lajiyhteisön koostumukseen täyttömäillä siten, että etelärinteissä menestyvät parhaiten avoimen ja kuivan elinympäristön lajit ja pohjoisrinteissä taas kostean ja metsäisen elinympäristön lajit, kun taas generalistilajit menestyvät molemmissa rinteissä yhtä hyvin. Etelä- ja pohjoisrinteiden lajiyhteisöjen välille vaikuttaa kehittyvän selvä ja jossain määrin säännönmukainen ero vain riittävän suurilla (arviolta vähintään 6-17ha) täyttömäillä. Täyttömäkien lajiyhteisöt ovat keskenään uniikkeja, mutta kaikkien täyttömäkien lajiyhteisöjä karakterisoivat kuivan elinympäristön lajit sekä muissa avoimissa kaupunkielinympäristöissä tyypillisen lajin Poecilus cupreus täydellinen puuttuminen. Avoimen ja kuivan elinympäristön lajien joukossa ovat todennäköisimmin täyttömäistä hyötyvät uhanalaiset ja harvinaiset lajit, joten tulosten perusteella täyttömäkien etelärinteillä olisi eniten merkitystä lajien suojelun kannalta. Täyttömäet pystyvät elättämään sellaisenaan suunnilleen yhtä monipuolista ja samankaltaista maakiitäjäislajistoa kuin muutkin kaupunkien avoimet elinympäristöt, mutta täyttömäkien lajistoa voitaisiin ehkä monipuolistaa sopivalla kunnostuksella. Lajistoltaan monipuolisimmat yhteisöt vaikuttavat löytyvän kaupunkien reuna-alueiden täyttömäiltä, mikä voi kertoa ympäröivien alueiden merkityksestä niiden tarjoamasta lajipoolista dispersoivien lajien kolonisoidessa täyttömäen.
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(2016)Biotopes and biodiversity within the Metropolitan Area of Helsinki (cities of Helsinki, Espoo, Vantaa and Kauniainen) were prioritized with a spatial conservation prioritization software called Zonation. The aim of the thesis was to examine how different, largely urban or semi-urban, areas supported biodiversity and thus, indirectly, ecosystem services provisioning. Furthermore, the effects of the new City of Helsinki Strategic Plan 2050 on the prioritization were examined. The analyses were based on expert elicitations, in which different urban biotopes were evaluated in terms of how well they supported species richness and the occurrence of specialist species of 8 taxonomic groups. Following the biotope classification, an urban biotope map was compiled from various GIS sources. Based on the expert answers, these biotope maps were then converted to suitability maps for each of the eight taxonomic groups. The urban biotope map, as well as the suitability maps, were made according to two land-use versions: current land-use in the research area, and a scenario describing the actual City of Helsinki Strategic Plan 2050. Separate Zonation prioritizations were then made for both scenarios. Urban biotopes a-priori evaluated as important for biodiversity, such as lakes and their shores, ruderal areas, dry meadows, manor yards, shoreline meadows and herb-rich forests, became emphasized in Zonation prioritizations as well, whereas the currently heavily built areas received lowest conservation priority in the analysis. Thus, according to this study, densification of the current residential and built-up areas does not threaten the urban biodiversity or ecosystem services of the Metropolitan Area of Helsinki. According to the present analysis, the new Helsinki Strategic Plan would have some relatively small effects on biodiversity in the research area. While the plan proposes development in some top-value biodiversity areas, the estimated effects of the plan were on average only a 3.3% loss of biotopes across taxonomic groups. Nevertheless, the plans of Helsinki could influence biodiversity priority areas in neighboring cities, and I recommend that the broader Metropolitan Area should be treated as a whole from the perspective of urban biodiversity. Rather than preserving sets of defined species, multi-functionality and ecosystem services provided by urban biodiversity should be emphasized when planning for urban nature conservation. As biodiversity ultimately underlies and supports both of these objectives, it should be central in the urban planning as well. Spatial conservation prioritization can well be used for ecologically sustainable urban planning, as long as the quality of the input data is taken care of and different uncertainties and limitations of the analyses are recognized in interpretation.
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(2020)PAH-yhdisteet ovat aromaattisia renkaita sisältäviä haitallisia hiilivetyjä, joita vapautuu ympäristöön enemmän antropogeenisistä kuin luonnollisista lähteistä. Tässä Pro Gradu -tutkielmassa tutkittiin Lahdessa ja Espoossa sijaitsevien päiväkotien pihamaiden ilman ja pintamaan PAH-pitoisuuksia ja niiden vaikutuksia lasten ihon ja maaperän bakteeristoon. PAH-yhdisteitä kerättiin ilmasta passiivikeräimillä sekä pihojen eri puolilta otetuista maaperänäytteistä. Näytteet käsiteltiin laboratoriomenetelmin ja analysoitiin GC-MS-laitteistolla, ja tulokset suhteutettiin ennalta kerättyyn bakteeritietoon. Ilman PAH-pitoisuudet osoittautuivat niukoiksi, eivätkä maaperänkään PAH-pitoisuudet yleensä ylittäneet kynnysarvoja. Korrelaatioita ilman PAH-yhdisteiden ja ihon bakteeriston väliltä löydettiin runsaasti, mutta selitysasteet jäivät mataliksi. Löydetyt korrelaatiot maaperän PAH-yhdisteiden ja maaperän bakteeriston välillä olivat määrältään niukemmat mutta selitysasteiltaan suuremmat. PAH-pitoisuuksien ei todettu olevan riski ympäristölle tai terveydelle tutkimuskohteissa, mutta PAH-yhdisteet muuttivat bakteeristoja sekä iholla että maaperässä, mikä voi vaikuttaa ihmisen immuunipuolustukseen. Jatkotutkimustarpeiksi tunnistettiin mm. vuodenaikojen, maaperän ominaisuuksien sekä ympäröivän maankäytön vaikutukset PAH-pitoisuuksiin.
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(2018)2000-luvun alussa tapahtuneiden suurten tulvien jälkeen Euroopan Unionissa on tapahtunut paradigmanmuutos, jossa teknisiin ratkaisuihin keskittyvästä tulvasuojelusta on siirrytty kohti kokonaisvaltaisempaa tulvariskien hallintaa. Myös Suomessa on ollut suuria tulvia 2000-luvulla ja tulvariskien arvioinnin ja hallinnan direktiivin puitteissa on nimetty 21 merkittävää tulvariskialuetta. Samaan aikaan resilienssi on omaksuttu katastrofiriskien hallinnan yhdeksi tavoitteeksi. Resilienssi on systeemin kykyä vastata muutokseen tai häiriöön. Tämän pro gradu-tutkielman tarkoituksena on selvittää yhteisön resilienssin kehittymistä sekä keskeisiä resursseja ja toimijoita Kittilän tulvariskien hallinnassa. Tutkimuksen viitekehyksenä toimii yhteisön resilienssi, joka tarjoaa mahdollisuuden kiinnittää tarkempaa huomiota luonnonkatastrofien ja niiden hallinnan sosiaaliseen ulottuvuuteen. Tässä tutkimuksessa yhteisön resilienssi on määritelty yhteisön selvitymis-, sopeutumis- ja transformaatiokykynä häiriöiden kontekstissa. Tutkimuksen analyyttinen viitekehys jakaa yhteisön resilienssin kolmeen osa-alueeseen, jotka ovat resurssit ja kyvyt, toiminta sekä oppiminen. Osa-alueet ovat vuorovaikutuksessa toisiinsa ja niihin vaikuttavat myös yhteisön ulkopuoliset rakenteet, kuten katastrofiriskien hallinta sekä sosio-poliittinen, taloudellinen ja ympäristöllinen konteksti sekä niiden muutokset ja häiriöt. Vastauksia tutkimuksen kysymyksiin selvitettiin tapaustutkimuksen avulla. Tutkimuksen tapauksena on Kittilän vuoden 2005 tulva. Tutkimuksen aineistona ovat paikallisten ja alueellisten toimijoiden teemahaastattelut sekä tulvariskien hallintaa koskevat asiakirjat. Aineisto analysoitiin laadullisen sisällönanalyysin avulla käyttäen apuna Atlas.ti-analyysiohjelmaa. Yhteisö tulvariskien hallinnassa käsittää sekä institutionaaliset, järjestäytyneet että epäviralliset toimijat. Tulvariskien hallinta Suomessa on vahvasti institutionalisoitu ja vastuu viranomaisilla, mutta Kittilän tapauksessa asukkaiden ja heidän sosiaalisten verkostojensa toiminta oli tärkeässä osassa tulvasta selviytymisessä. Yhteisön resilienssin kannalta merkittävin resursseista oli yhteisön sosiaalinen pääoma, sillä sen avulla yhteisö pääsi käsiksi myös muihin resursseihin. Toimijoiden sosiaaliset verkostot olivat linkittyneet toisiinsa ja ulottuivat useammalle maantieteelliselle tasolle. Tulvan jälkeen toiminta oli kehittynyt järjestelmällisemmäksi ja ennakoivaksi. Vuoden 2005 tulvan jälkeen erityisesti varautuminen tulviin, instituutionaalisten toimijoiden organisaatiorakenteet ja yhteistyö, materiaaliset resurssit, tekniset ratkaisut sekä riskitietoisuus kehittyivät parempaan suuntaan. Vuoden 2005 tulvan jälkeen tapahtui pääasiassa yksikehäistä oppimista, jossa toimintaa muutettiin tulvakokemuksen perusteella. Kaksikehäistä oppimista ja kriittistä tarkastelua oli myös jonkun verran, mutta monikehäinen oppiminen ja yhteisön resilienssin transformatiivinen ulottuvuus puuttuivat kokonaan. Tulvariskien hallinta perustui vuoden 2005 tulvan jälkeen kunnan hallinnossa hyväksytyille tulvapenkereiden rakentamiselle, jotka olivat viivästyneet huomattavasti ja edelleen rakentamatta. Yhteisön resilienssiä tulville tulisi pystyä kehittämään, myös sellaisilla riskialttiilla alueilla, joilla ei ole viimeaikaisia tulvakokemuksia. Samalla yhteisöjen tulisi olla resilienttejä kaikenlaisille muutoksille, myös vaikeammin havaittaville ja hitaille sosio-ekologisille muutoksille, kuten ilmastonmuutokselle tai huoltosuhteen heikkenemiselle. Yksi ratkaisu voisi olla transformaatio, joka mahdollistaa ulkoisista häiriöistä selviytymisen lisäksi sosiaalisen systeemin siirtymisen kohti ekologisesti kestävämpää ja sosiaalisesti oikeudenmukaista tilaa. On myös muistettava, että yhteisön resilienssi on sidoksissa kontekstiin eli siihen vaikuttaviin ulkopuolisiin prosesseihin ja rakenteisiin. Tiedon yhteistuotannon ja tutkimushankkeiden yhteissuunnittelun avulla voitaisiin saada tietoa yhteisön resilienssistä, joka on sosiaalisesti kestävää, paikalliseen kontekstiin sopivaa ja jota voidaan suoraan hyödyntää yhteisön toiminnassa. Osallistavat tutkimusprosessit voivat myös vähentää konflikteja esimerkiksi tulvariskien hallinnan keinoista sekä lisätä riskitietoisuutta niillä alueilla, joilla ei ole aiempia katastrofi- tai häiriökokemuksia. Tutkimuksen aikana tuli esille myös jatkotutkimustarpeita. Yhteisön resilienssiä tulisi edelleen tutkia laajemmin sekä Kittilässä että muilla tulvariskialueilla esimerkiksi määrällisten tutkimusmenetelmien avulla tai suuremmalla aineistolla. Sosiaalisia resursseja on korostettu yhteisön resilienssille luonnonkatastrofeissa ja tässäkin tutkimuksessa ne tulivat monin tavoin esille. Tämän takia olisi mielenkiintoista tutkia myös sosiaalisten resurssien kehittymistä ja tarkempaa suhdetta luonnonkatastrofeihin. Esimerkiksi pitkittäistutkimukset riskialueilla voisivat tuoda lisää tietoa sosiaalisista resursseista ennen ja jälkeen luonnonkatastrofin
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(2019)Changing human behaviour has an important role in solving the planet’s environmental crisis. Neither technological innovations nor political incentives have any effect if people don’t start using them or just direct the saved resources into another environmentally harmful activity. In addition, cities account for most of the energy related carbon dioxide emissions already today and the populations in cities continue growing. Hence, we have to find ways of enhancing environmentally responsible human behaviour comprehensively. Place identity has been noticed to have a positive effect on environmental behavior. However, there is no research on the place identity of sustainable neighbourhoods. This master’s thesis’ aim is to find out what is the place identity and environmentally responsible behaviour like in a sustainable neighbourhood of Eko-Viikki. And to reflect the relationship of place identity and environmentally responsible behaviour in in the context of sustainable neighbourhoods. The framework for the place identity part of the thesis is a structural equation model where place identity is formed through commitment to place and five dimensions that impact commitment to place: attachment, continuity with personal past, perception of familiarity, cohesion and social acceptance. The model indicates that place identity affects people’s environmentally responsible behavior both independently and through attitudes. Environmentally responsible behaviour is examined through three human activities that have the biggest impact on the environment: housing, transportation and eating. The data of the thesis is nine semistructured theme interviews and one email interview. The data was analysed by qualitative content analysis using the dimensions of place identity and the three activities with the biggest impact on the environment. The results suggest that attachment, continuity with personal past, perception of familiarity and cohesion define the place identity of the interviewees the most. The interviewees are clearly attached to their neighbourhood and would like to live there as long as possible. Neighbourhood’s closeness to nature and neighbour relations are valued the most. Additionally, the interviewees have known the neighbourhood already before choosing to move there and many have dreamt of living there one day. The neighbourhood has its part in the past, present and future of the interviewees. Furthermore, four types of environmental behaviour were identified: The Realist, the Diligent, the Overestimator and the Underestimator. The Realist doesn’t perceive their behaviour as environmentally responsible and their described behaviour matches the perception. The Diligent perceives their behaviour as environmentally responsible and their described behaviour also matches the perception. The Overestimator perceives their behaviour as environmentally responsible but the described behaviour doesn’t match the perception. The Underestimator doesn’t perceive their behaviour as environmentally responsible enough but the described behaviour also doesn’t match the perception. Half of the interviewees think that the neighbourhood has had a positive effect on their environmental behavior at least to some extent but have no examples to show. The other half doesn’t think the neighbourhood has had any effects. Most of the interviewees that think the neighbourhood has had a positive effect on their behaviour are either Realists or Overestimators whereas most of the interviewees that don’t think the neighbourhood has had an effect are Diligents and Underestimators. In the future, more research is needed to examine the relationship of the place identity in sustainable neighbourhoods and environmental behaviour. Future research should focus on finding out if living in a sustainable neighbourhood has an effect on the residents’ environmental behaviour and if the effect is caused by their place identity. In addition, comparative research between sustainable neighbourhoods and so called ordinary neighbourhoods is needed to find out if there is a difference in how the place identity of a sustainable neighbourhood affects the behaviour compared to the place identity of a so called ordinary neighbourhood.
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