Browsing by department "Bio- ja ympäristötieteellinen tiedekunta"
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(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.
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(2018)Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility.
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(2019)Neurotrophic factors (NTFs) play an important role in regulating the survival, differentiation and maturation of developing neurons. Based on strong pre-clinical evidences, some of NTFs have been suggested to be efficient therapeutic agents for treatment of Parkinson’s disease (PD). PD is a neurodegenerative disorder characterized by loss of dopamine (DA) neurons from nigrostriatal pathway resulting in motor symptoms of the disease. A hallmark of the disease is the presence of Lewy bodies in the brain and they comprise majorly of aggregated alpha-synuclein (aSyn) protein. MANF, an unconventional NTF, was discovered over a decade ago and differs from traditional NTFs. Removal of MANF has been shown to trigger unfolded protein response in cells. Evidences indicate that increased endogenous level of aSyn may have a role in enhancing the process of aggregation of aSyn into Lewy body. Determining the initiation event of aSyn aggregation is an important step in Lewy body pathology and it is still under investigation. In the first part of this study, I aimed to elucidate if MANF knockout can trigger any change in endogenous level of aSyn. Transmission of Lewy bodies from cell to cell has been well studied by researchers and is suggested to spread across brain in a prion like fashion. CDNF has been neuroprotective and restorative for tyrosine hydroxylase (TH)-positive neurons in a toxin-based models of PD. However, presently exists no study which has evaluated the effects of CDNF on propagation of aSyn aggregates in vivo. In the second part of this study, I aimed at evaluating effects of long-term intrastriatal infusion of CDNF at two concentrations (1.5 μg/24h or 3 μg/24h) on propagation of endogenous phosphorylated aSyn inclusions in vivo. CRISPR/Cas9-mediated MANF knockout in SH-SY5Y cells did not yield any significant changes in the endogenous level of aSyn. Additionally, brain samples derived from MANF knockout mice yielded similar non-significant difference in level of aSyn compared to wild-type mice. MANF knockout primary DA neurons when inoculated either with only pre-formed fibrils (PFFs) or with a combination of PFFs and aSyn overexpression, showed no significant difference in the number of Lewy body like aggregates, suggesting no change in endogenous aSyn levels. Rats were injected with PFFs and then chronically infused with CDNF, 1 month and 2 months after PFFs at 2 different concentrations (1.5 μg/24h or 3 μg/24h). Immunohistochemical analysis of substantia nigra pars compacta (SNpc) derived from rats showed similar numbers of endogenous phosphorylated aSyn inclusions in animals treated chronically with either CDNF or PBS. In summary, only MANF knockout from cells or animals has no direct effect on endogenous level of aSyn. But external stressors may perhaps trigger upregulation of aSyn in MANF knockout cells. Furthermore, chronic infusion of CDNF either 1 month or 2 months after PFF injection doesn’t reduce the total number of phosphorylated aSyn inclusions in SNpc compared to control. Nevertheless, we need more data to corroborate this evidence.
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(2019)Eph (Erythropoietin producing hepatocellular) receptors and their membrane-bound ligands, ephrins, form the largest family of receptor tyrosine kinases in mammals. They regulate functions such as cell migration and axon guidance during development, and wound healing and tissue boundary maintenance in mature tissues for example. Due to the membrane-bound nature of the ephrin ligands, Eph-ephrin signalling can proceed in two directions: forward (Eph-expressing cell) and reverse (ephrin expressing cell). In addition to its critical physiological functions in development and tissue homeostasis, the Eph-ephrin system has also been implicated in multiple diseases, including several cancers, in which the aberrant expression of Eph receptors and ephrins are often present. The EphA2 receptor for example has been identified as an oncogene with a dual mode of action as either tumor suppressor or an oncogene through distinct phosphorylation statuses of the receptor. High-grade serous ovarian cancer is the most common subtype of ovarian cancer, and the deadliest gynaecological malignancy with a dismal five-year survival rate. High-grade serous ovarian cancer does not present symptoms at an early stage, yet it quickly progresses into forming peritoneal metastases by cell shedding from the primary tumour. Small patient cohort studies have given indications of the correlation between the Eph-ephrin system and survival in ovarian cancer. This aim of this study was to investigate the impact of the Eph-ephrin system in high-grade serous ovarian cancer using a large patient cohort mRNA expression dataset to obtain survival association data of proteins of interest used with cell-based studies and the analysis of clinical samples in the form of tumor microarrays and fresh primary samples to investigate the functions of the found proteins of interest. A 428-patient The Cancer Genome Atlas high-grade serous ovarian cancer microarray mRNA dataset was analysed using the Kaplan-Meier estimator for each Eph-ephrin family member. Cell based studies were performed with recombinant ephrin treatments and ephrin knockdowns. These data were analysed using Western blotting and immunofluorescence stainings. Clinical high-grade serous ovarian cancer samples obtained from Turku University Hospital were analysed using immunohistochemistry, immunofluorescence stainings, and mRNA sequencing. EfnA5 had a significant correlation of high expression and poor survival, which is atypical to ephrins. Low EfnA3 correlated with poor survival. High levels of known oncogenes EphA2 and EphA4 also correlated with poor survival. EfnA5 treatment resulted in increased oncogenic EphA2 signaling in comparison with canonical Eph-ephrin signalling mediated by EfnA1. Knockdown of EfnA5 increased canonical, tumour suppressive EphA2 signaling, while EfnA1 knockdown increased oncogenic EphA2 signaling. Immunohistochemical analysis of tumour microarrays with multiple ovarian cancer subtypes displayed an association between the highly malignant high-grade serous subtype and EfnA5 expression. In addition to this, EfnA5 expression was increased during high-grade serous ovarian cancer progression. The Eph-ephrin system is implicated in the survival associations of multiple cancers, but the exact functions facilitated by Eph-ephrin signalling in cancer have remained relatively unknown, with the exception of EphB4-EfnB2 driven angiogenesis. This study offers insights into oncogenic Eph-ephrin signalling in ovarian cancer, displaying that oncogenic EphA2 functions can be altered by ephrins in addition to the known kinase crosstalk pathway. The noncanonical nature of EfnA5 is highlighted by its oncogenic functions in comparison to typical Eph-ephrin signalling, and the significant increase of EfnA5 expression during high-grade serous ovarian cancer progression and association with this highly malignant subtype of ovarian cancer. Although the reverse signalling effects of EfnA5 were not studied, this study highlights the importance of ephrins in Eph-ephrin signalling in cancer, presenting that the focus should not be only on the Eph receptors when studying the oncogenic signalling facilitated by the Eph-ephrin system.
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Eheyttävää biologian opetusta projektioppimisen kautta : MOOC-verkkokurssin kehittäminen opettajille (2019)Interdisciplinary studies are currently an important topic in education. One reason behind this is the idea that students should learn 21st century skills, such as critical thinking, communication and problem-solving skills for their future careers. Interdisciplinary education has also been shown to increase students’ interest towards natural sciences. Furthermore, organizing cross-curricular learning units has become compulsory at a curricular level in Finland and other countries. Project-based learning is a widely supported teaching method in which learning is organized around projects. It is also a natural method to carry out interdisciplinary learning units. Project-based learning is usually defined by its characteristic features, and its successful implementation requires the teacher to have a good knowledge of them. However, these characteristics tend to be unknown for teachers. Research has also shown that teachers’ conceptions of what interdisciplinary education and project-based learning are, tend to vary. As such, it has been noted in the literature that there is a need to offer training and materials for the implementation of interdisciplinary and project-based learning for in-service teachers. Although MOOCs (Massive Open Online Courses) have a lot of potential as a means of professional development, they are rarely planned to serve primarily as in-service teacher training. Empirical research into their design is also scarce. The main aims in this study were to 1) provide more information about interdisciplinary education within biology education 2) develop a MOOC for the professional development of biology teachers on the topic of interdisciplinary education and project-based learning. In addition to that the study aimed to develop the StarT programme of LUMA Centre Finland. The study was carried out as design-based research. The main research question that directed the design process was: what should a MOOC that supports teachers to carry out interdisciplinary project-based learning within the theme “nature and environment” in biology be like? Nature and environment was chosen as the context of the study as from the perspective of biology education it offers an important and current topic for interdisciplinary project units. The main research question was approached through a theoretical and empirical problem analysis. Theoretical problem analysis focused on researching the question in earlier literature, and the empirical problem analysis was carried out as a case study in which qualitative data was studied through deductive content analysis. The materials studied in the empirical problem analysis were project-based learning units of comprehensive schools and high schools. Their project units were studied through the following research questions: 1. How did teachers and students carry out the characteristics of project-based learning in the context of the theme nature and environment in biology? 2. How is biology taught in an interdisciplinary way in the theme nature and environment? The first question was studied through the characteristics that were identified for project-based learning in the theoretical problem analysis, and the second by looking at which subjects collaborated with biology, how the collaboration was carried out and how the learning communities experienced it. The goal was to find a) challenging characteristics of project-based learning and interdisciplinary education that should be considered the design of the MOOC b) good examples from the studied learning communities to be shared on the MOOC. The study subjects consisted of 12 learning communities who had participated in the international StarT programme of LUMA Centre Finland. In accordance with earlier studies, also the subjects of this study struggled especially with driving questions, the unity of the project activities and in using the projects as a means to learn central contents. Scientific practices were visible well apart from students’ questions, but it could not be defined how strongly student-led the inquiry was. The connection between instructions of StarT and the characteristics of project-based learning that were well represented in the materials seemed evident: collaboration, sharing results, end products and using technology were all visible throughout the analyzed learning communities. However, the materials offered little information about how the collaboration between different subjects was carried out in practice. The study indicated also that students and teachers paid attention to different aspects of the learning taking place in project-based learning, and that teachers might include topics of certain subjects into the project units without realizing it. This should be further researched, however. The issues identified in the problem analyses were used to design an international MOOC that is suitable especially for biology teachers. The course ”Project-based learning and the theory behind it – create your own project!” was created on the Moodle-platform of the University of Helsinki. In addition to this, a phased model for creating professional development in a situation where the studied materials can be used both to direct the design process and as materials on the course was created. The framework that was created for the analyses in this study can also be further used and developed to study how characteristics of project-based learning are represented in other cases where teachers’ and students’ project units are being studied. The study provided also new information about what is possible and feasible to study from the materials of StarT. New information was provided also of the project-based learning that takes place at the schools of the StarT participants, as they have not been researched from the perspective of the characteristics of project-based learning earlier.
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(2020)Tutkielman tarkoituksena oli selvittää ja verrata keskenään kahden luontaisesti kalattoman järven eläinplanktonyhteisöjen rakennetta sekä kalaistutusten vaikutuksia siihen. Tarkoituksena oli myös selvittää äyriäiseläinplanktonin vasteita saalistukseen erilaisen predaatiopaineen vallitessa. Luontaisesti kalattomissa järvissä eliöyhteisö on muovautunut hyvin erilaiseksi visuaalisesti saalistavien kalojen puuttuessa. Näissä järvissä pääpredaattorina toimivat usein selkärangattomat pedot, joiden saalistustekniikka eroaa selvästi kalojen saalistuksesta, ja ne ovat usein kokorajoitteisia ravinnon käytön suhteen. Kalojen visuaalinen saalistus kohdistuu kooltaan suurimpiin ja näkyvimpiin äyriäiseläinplanktoneihin ja ne voivat näin tehokkaasti vaikuttaa eläinplanktonin kokorakenteeseen. Luontaisesti kalattomiin järviin tehdyillä kalaistutuksilla voi olla merkittäviä vaikutuksia koko järven eliöyhteisöön. Oletuksena oli, että äyriäiseläinplankton olisi kooltaan suurempaa kalattomassa järvessä verrattuna istutettuun järveen. Oletimme pienikokoisen Bosmina spp. -vesikirpun suojautuvan selkärangattomien petojen predaatiota vastaan erilaisten korostuneiden morfologisten piirteiden avulla. Myös äyriäiseläinplanktonin vertikaalisen sijainnin oletettiin eroavan järvien välillä. Kuutsjärvi ja Tippakuru sijaitsevat Värriön luonnonpuiston alueella Itä-Lapissa. Molemmat tutkimusjärvet ovat luontaisesti kalattomia latvajärviä, mutta Kuutsjärveen on istutettu taimenta 1980-luvulla ja se muodosti järveen pysyvän taimenkannan. Eläinplanktonnäytteet otettiin limnos -putkinäytteenottimella järven syvimmästä kohdasta koko vesipatsaan syvyydeltä metrin välein kesällä 2010, kolmena eri näytteenottokertana. Näytteet mikroskopoitiin ja niistä määritettiin lajit/suvut mahdollisimman tarkasti ja äyriäiseläinplanktonin koko mitattiin. Näytteistä määritettiin eläinplanktonin tiheydet, äyriäiseläinplanktonin biomassat, keskikoot ja kokojakaumat. Lisäksi mitattiin pienikokoisen Bosmina spp. -vesikirpun ulokkeiden (mucro, antennula) pituudet predaatiopaineen vaikutusten arvioimiseksi. Äyriäiseläinplanktonin vertikaalinen sijainti vesipatsaassa määritettiin. Selvimmin pelagiaalin eläinplanktonyhteisöt erosivat toisistaan kokonaistiheyksien osalta, minkä aiheutti rataseläinten voimakas lisääntyminen kalattomassa Tippakurussa kesän aikana. Eläinplanktonlajisto oli järvissä kuitenkin hyvin samanlainen ja lajirunsaus vain hieman suurempi kalattomassa Tippakurussa. Äyriäiseläinplanktonin biomassat vaihtelivat kuukausien välillä, mutta eivät eronneet merkittävästi järvien välillä. Molemmissa järvissä hallitsevina äyriäiseläinplanktoneina esiintyivät hankajalkaiset ja vesikirppuja esiintyi selkeästi vähemmän. Äyriäiseläinplanktonin keskikoot erosivat joiltain osin järvien välillä ja olivat pääosin suuremmat kalattomassa Tippakurussa. Kalattomille järville tyypillistä hyvin suurikokoista eläinplanktonia ei Tippakurussa kuitenkaan esiintynyt ja erot jäivät tästä syystä oletettua pienemmiksi. Pienikokoisen Bosmina spp. -vesikirpun mucrojen pituuksissa havaittiin kuitenkin selkeä ero. Mucrot olivat kooltaan selvästi suuremmat kalattomassa Tippakurussa koko kesän ajan ja tätä ollaankin pidetty yhtenä luotettavimpana Bosmina spp. -vesikirppujen vasteena selkärangattomien petojen saalistukseen. Äyriäiseläinplanktonin sijainnissa havaittiin eroja lähinnä hankajalkaisten ja vesikirppujen välillä, mutta järvien välillä erot olivat vähäisiä, joitain yksittäisiä tapauksia lukuun ottamatta. Erilaisesta predaatiopaineesta huolimatta järvien eläinplanktonlajisto erosi vain vähän järvien välillä ja erot olivat joiltain osin jopa päinvastaisia oletettuun nähden. Kuutsjärven eläinplanktonlajisto muistutti monelta osin muiden istutettujen järvien lajistoa, koostuen lähinnä pienikokoisesta äyriäiseläinplanktonista ja rataseläimistä. Järvessä esiintyvä taimen ei ole tehokas planktivori, joten se on todennäköisesti vaikuttanut Kuutsjärven eläinplanktonlajistoon lähinnä trofiatasojen välisten vuorovaikutusten kautta, hävittämällä selkärangattomat pedot pelagiaalista. Tippakurussa sen sijaan havaittiin kalattomille järville poikkeuksellisen runsaasti rataseläimiä ja äyriäiseläinplankton oli kooltaan verrattaen pientä. Myös äyriäiseläinplanktonin sijainti oli monilta osin poikkeava muihin tutkimuksiin nähden. Tippakurun pelagiaalissa, poikkeuksellisesti myös päiväsaikaan esiintyneet selkärangattomat pedot G.lacustris ja C. flavicans ovat tehokkaalla, myös suuriin eläinplanktereihin kohdistuneella predaatiollaan todennäköisesti muovanneet Tippakurun eläinplanktonyhteisöstä ja sen vasteista hyvin omaleimaisen sekä poikkeavan muihin kalattomiin järviin nähden. Voimakkaammasta selkärangattomien petojen predaatiosta Tippakurussa kuitenkin kertovat Bosmina spp. -vesikirpun suuremmat mucrot verrattuna Kuutsjärveen, jossa istutettu taimen on hävittänyt selkärangattomat pedot pelagiaalista.
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(2019)Parvoviruses are among the smallest known viruses. The parvovirus genome is a single stranded DNA, approximately 5 kb in size. The virion has a small (20 to 30 nm), rugged, non-enveloped icosahedral capsid. Parvoviruses can cause a number of diseases. Possibly the most recognized human parvovirus is parvovirus B19 (B19V), which can cause the so-called fifth disease, anemias and fetal death. Another relatively well characterised parvovirus is human bocavirus 1 (HBoV1), which causes respiratory tract infections in young children. Bufavirus (BuV) tusavirus (TuV) and cutavirus (CuV) are emerging parvoviruses, discovered during the years 2012-2016 using next generation sequencing methods. All three viruses were originally discovered in feces of patients suffering from diarrhea. BuV was originally found in Burkina Faso and has since been detected in fecal samples with polymerase chain reaction (PCR)-based methods from Europe, Asia and Africa. The seroprevalence of BuV differs between countries. TuV was found in a single stool sample from Tunisia, but no further reports of it have since emerged. CuV was found in 2016 and it has been linked to cutaneous T-cell lymphoma, but it is not known if the virus is the cause of the cancer or if the virus simply prefers quickly dividing cancer cells for its replication. BuV, TuV and CuV belong to the Protoparvovirus genus, but it is still unclear whether TuV is a human pathogen. More research is needed to study the epidemiology of these viruses and their role in illnesses. There were two main aims in this thesis: to set up an IgM µ-capture enzyme immunoassay (EIA) for human protoparvoviruses using BuV1 as an example and to screen three stool sample cohorts for BuV, TuV and CuV using an in-house multiplex quantitative PCR (qPCR). The IgM EIAs developed for B19V and HBoV1 was used as the base for developing human protoparvovirus IgM EIA, using Virus-like particles (VLP) as antigens. Setting up the EIA required a great amount of optimization and finally troubleshooting, since the assay did not work as expected. The troubleshooting revealed that the ambiguous results in the IgM µ-capture EIA were possibly due to degraded VLPs or that the sensitive µ-capture format requires extremely carefully purified VLPs. More optimizing is needed for this assay, however, the work done in this thesis offers a good base for further development of protoparvovirus IgM EIA. All three viruses were found in the stool samples during multiplex qPCR screening. Based on the qPCR and sequencing results one sample was positive for BuV DNA, one sample for TuV DNA and a total of 12 samples for CuV DNA. This is the first time TuV DNA has been found since its discovery. In addition to that, CuV DNA was identified in fecal samples for the first time since the discovery, previously CuV DNA had been found mostly in skin biopsies. As for TuV, based on the parvovirus phylogenetic analyses, its sequence is more closely related to rodent parvoviruses than CuV or BuV. More research is needed, possibly with animal and human samples, to establish the role of TuV as a human virus.
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(2020)Russia’s energy security is considered to rest on hydrocarbon exports, as the country’s economy and political regime depends on revenue generated in energy trade. Today, Russia faces a problem, as interests toward climate change mitigation and renewable energy have increased globally. Particularly the EU, Russia’s primary gas trade partner, seeks to reduce fossil fuel consumption due to its climate policy targets and concerns over energy security. Via the Yamal LNG project, Russia begun to diversify its markets toward East Asia in 2017. The project is expected to spur energy infrastructure development in the Arctic region, as it ships liquefied natural gas (LNG) along the Northern Sea Route. This study finds out how the stakeholders of the Yamal LNG project consider it to connect with the energy security of Russia, to which climate change mitigation poses a potential threat. Since securitization of energy is the result of a social process where political actors contest over the meanings of energy, the study also looks at whose interests the Yamal LNG project actually secures as “energy security”, and how that concept becomes projected as a general national interest, instead of having energy transition among the top objectives of energy policy. Neo-Gramscian analytical approach and frame analysis are used to deliver results from a data consisting of 11 research interviews and 40 archival sources. The stakeholders make sense of Yamal LNG’s relation to energy security and energy transition through four frames, which reflect distinct interest groups. The stakeholders appeal on others by utilizing the frames discursively, as they strategically contest over the meanings of energy. Ultimately, a hegemonic group consisting of the Russian state, JSC Yamal LNG shareholders, industrial organizations, and fossil energy lobbies determines the meanings of Yamal LNG’s production as general interests. Subordinate groups, including environmental NGOs and local indigenous residents, consent as they face combinations of discursive, organizational and material power. With the concession of others, the hegemonic group is able to project a “reality” that presents natural gas production as compatible with energy transition and climate change mitigation as an inferior interest to energy security. The Yamal LNG case shows that incumbent fossil energy regimes can effectively counter attempts to direct energy policy on low-carbon paths by pleading to security, which is a topic that research often neglects.
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(2019)Synthetic biology is an emerging interdisciplinary field of biology that aims to system-atically design artificial biological systems. As synthetic biologists seek increasingly complex control over cellular processes to achieve robust and predictable systems. A new frontier in synthetic biology is engineering synthetic microbial consortia. This ap-proach employs the concept of division of labor, instead of introducing large genetic cir-cuitry to homogenous cell populations. In this approach, different cell types are assigned to execute a portion of the overall circuit. Each cell type communicates with their co-worker subpopulations to complete the circuit. The main advantage of this strategy is the reduced metabolic burden on each cell type. Thus, leading to more reliable and stable overall performance. In this work, to simplify cellular communication between the mem-bers of the consortium, we used the simple architecture of quorum sensing machinery. We constructed a toolbox that contains promoter, receptor and quorum sensing signal synthase genes along with fluorescent reporters. Using this toolbox, we constructed dif-ferent cell types that can be used in synthetic consortia forming various communication topologies. We characterized the constructed cell types individually and in co-cultures.
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(2019)In this master’s theses, Finnish biology teachers’ needs for material for continuing education and educational material for upper secondary school in epigenetics were studied. Two sets of educational materials, continuing educational material for teachers and educational material for upper secondary school teaching was produced accordant with the results. Epigenetics is used to describe stable alterations in gene expression which do not consider mutations in DNA sequence. DNA methylation, histone modifications and chromatin remodelling are the main epigenetic mechanisms to affect gene expression. Epigenetic modification patterns can alter de novo, or they can be originated by some environmental factor. Epigenetic regulation was introduced as a new subject matter in National Core Curriculum for General Upper Secondary Schools 2015 in Finland. Epigenetics is a relatively new branch in biology, and as a result many teachers have not studied the subject matter at the university. Continuing educational material is needed to update their knowledge. Biology teachers’ needs for material in epigenetics were studied with a survey which was distributed to the Biology and Geography Teachers’ Union’s e-mail list subscribers. Data was analysed qualitatively and quantitatively. In order to produce continuing educational material for teachers, literature regarding teacher competence, adult education and Finnish biology teachers’ subject expertise was examined. The concept of constructivist learning, and conceptual change were applied in the production of educational material for upper secondary school teaching. Relevant scientific literature in epigenetics was gathered and used to produce both sets of materials. In the study survey, the teachers reported specific individual educational needs which were acknowledged in the production of both sets of materials, alongside literature listed earlier. The survey showed that one of 33 biology teachers had studied epigenetics at the university, 20 of 33 teachers independently and 13 of 33 hadn’t studied epigenetics at all. The extent of the teachers’ studies in epigenetics was most often elementary and the main motivation to study epigenetics independently was a desire to handle the basics. The most common resource teachers used to study epigenetics was non-scientific magazines. Among the teachers who had not studied epigenetics at all, lack of time was the most common reason mentioned. However, 14 teachers described epigenetics as an important subject matter. Three teachers reported that they lack the expertise in teaching epigenetics and three felt that textbooks don’t offer support in teaching epigenetics. Online material and expert lectures were the most common continuing education material forms requested. Regarding the content of the continuing educational material for teachers, the most common requests were that the material should include the basics of epigenetics and practical examples. The form of educational material for upper secondary school teaching was most commonly requested to be online text or educational video. Regarding the content of the educational material for upper secondary school teaching, the most common requests among teachers were a summary of the theory of epigenetics and practical examples. The continuing educational material for teachers produced in this thesis consists of an introduction part and four parts about different subjects in epigenetics. The titles of the parts are: 1) What is epigenetics? 2) Molecular mechanisms of epigenetic gene regulation 3) Epigenetic gene regulation and 4) Epigenetic inheritance. The material was designed in a way that texts 3) and 4) are possible to comprehend without studying text 2) about molecular mechanisms of epigenetic regulation. The educational material for upper secondary school teaching consists of seven parts. At the beginning there is an introduction to teachers which is followed by six separate parts for students. Each part has a “Rehearse before reading”-box which introduces students to the subject and encourages rehearsal of biological concepts and vocabulary which are necessary for comprehending each part. After each part there are questions which test students’ learning as well as encourage to apply freshly acquired knowledge about epigenetics to other biological contexts. Titles for different parts are: 1) What is epigenetics? 2) Epigenetics and nutrition 3) Epigenetics and exercise 4) Epigenetics and mental health 5) Epigenetics and tortoiseshell cats and 6) Epigenetic inheritance. The material has been designed in such a way that the parts can be taught and learned separately. References are provided at the end of both material sets. Both materials produced in this thesis meet the teachers’ requests revealed in the survey. The form of the continuing educational material for teachers is online material, which was one of the most common requests among the teachers who answered the survey. The contents of the material correspond to the teachers requests as well, since most requested contents were the basics of epigenetics and practical examples. The educational material for upper secondary school teaching was requested as online material with a summary of the theory of epigenetics and practical examples, and all these requests were met. Both materials were produced considering relevant theories on pedagogy and adult education. Results of this study cannot be applied nationally in Finland since the sample size was small. Therefore, national relevance of the materials cannot be predicted. Predictions about the impact of the materials on teachers’ and students’ understanding about epigenetics cannot be made either, since learning is an active process which requires effort from the learner. However, a strong case can be made for the produced material, because materials include relevant information and their pedagogic choices can be justified by the literature. This thesis uncovered many questions for future research. For example, the efficacy of the materials could be studied in a practical classroom situation. Other possible questions for research or study designs could be about biology teachers’ expertise and continuing education in Finland.
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(2019)Diesel, which belong to the fossil fuels, have been exposed recently because of global warming and because of decrease of known fossil fuel reserves. It has been estimated that reserves of fossil fuels will last next 40-50 years. Burning of fossil diesels will cause not only carbon dioxide emissions but also particle and metal emissions. There have been attempt to develop alternative energy sources. Growing alternative, which have came to the market, is renewable diesels, which can be produced from many sources such as reeds and biowaste. Biodiesels differ from fossil diesels by their chemical structure. Contrary to fossil diesel, completely renewable diesels do not contain aromatic compounds and they burn more purely. Diesel might end up in soil in accidents. Behaviour of fossil diesels in a ground is well known, however, the behaviour of renewable diesels is not known so well. Even though solubility of renewable diesels is similar with fossil diesels, renewable diesels may have different behavior due to different chemical structure compared with fossil diesels. Soil removal and taking the soil to be cleaned up is the most popular way to clean diesel contaminated soil resulted from diesel leaking. Alternative way to clean diesel contaminated soil is to use bioremediation where the microbes are used. In bioremediation microbes are added to the soil or activity of the microbes is boosted by improving living conditions of microbes by adding nutrients and oxygen. In this master thesis spreading of three different diesels qualities in soil and with water in laboratory were viewed. Results of the laboratory experiment were compared to the result of Katariina Lahti-Leikas’ lysimeter experiment in Jokimaa. Diesel qualities used in experiment were completely fossil EN590, partly renewable ProDiesel and completely renewable NEXBTL. Experiment were executed in soil columns in laboratory conditions where pure lysimeter sand were exposed to diesel. Biostimulation were made to half of the soil columns using urea that every diesel had natural decomposition and biostimulation. Soil columns were watered and hydrocarbons C10-C40 from collected water below the column were analysed. In addition, soil samples were collected and hydrocarbons C10-C40 were analysed. The pH of the soil and water samples were followed throught the experiment. Biostimulation decreased concentration of hydrocarbons in the water in every diesel quality. Biggest concentration were in water from NEXBTL columns which move in soil mostly in NAPL. In soil samples there was not the same kind of decrease in hydrocarbon concentration as in water samples. Diesel moved from the upper part of the soil columns to the lower part of soil columns during the experiment. The reason why biostimulation did not decreased the hydrocarbon concentration in soil is probably because the experiment period was too short. Other things to affect the result are probably wrong comparison of nutrients or lack of oxygen, which have slowed down decomposition of hydrocarbons. The results of this study are consistent with the results of Katariina Lahti-Leikas’ lysimeter experiment in Jokimaa.
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(2020)Haitalliset vieraslajit aiheuttavat muutoksia luonnon monimuotoisuuteen, ekosysteemien toimintaan sekä niiden tarjoamiin ekosysteemipalveluihin vaikuttamalla esimerkiksi elinympäristöihin, ravintoketjuihin ja kilpailuasetelmiin. Haitalliset vieraskasvit voivat syrjäyttää alkuperäisiä kasveja kilpaillessaan niiden kanssa tilasta ja resursseista. Lisäksi vieraskasvit voivat heikentää paikallisten lajien elinmahdollisuuksia muokkaamalla ekosysteemejä niille sopimattomiksi. Komealupiini (Lupinus polyphyllus) on yksi yleisimmistä vieraskasveista Suomessa. Tämä kansallisesti haitalliseksi vieraslajiksi luokiteltu kasvi on levinnyt jo vuosikymmenien ajan erityisesti tienpientareilla ja joutomailla. Kasvustojen ikääntyminen voi muuttaa vieraskasvin vaikutuksia elinympäristönsä alkuperäiseen kasvillisuuteen, mutta komealupiinin kohdalla ikääntymisen vaikutuksista ei ole tietoa. Tämän pro gradu -tutkielman tavoitteena oli selvittää eri-ikäisten komealupiinikasvustojen vaikutuksia alkuperäiseen kasvillisuuteen sekä yhteisötasolla että eri lajiryhmissä. Tutkin myös kasvuston ikääntymisen vaikutusta komealupiinin peittävyyteen. Kartoitin putkilokasvilajistoa Päijät-Hämeen alueella 19 tutkimuskohteessa, jotka olivat maaperältään hiekkaisia tai soraisia. Tutkimuskohteet jaettiin komealupiinikasvuston iän mukaan kolmeen luokkaan: 1) nuoret (alle 6 v vanhat), 2) keski-ikäiset (noin 10 v) ja 3) vanhat (yli 13 v). Eri-ikäisten kohteiden kasviyhteisöjä vertaamalla tutkittiin komealupiinikasvuston ikääntymistä ja sen vaikutuksia muuhun kasviyhteisöön. Komealupiinin vaikutusten yleistämiseksi esiintyneet putkilokasvilajit luokiteltiin kymmeneen ryhmään suotuisten elinympäristöjen sekä elinympäristöjen ravinteikkuuden ja kosteuden mukaan. Aineiston analysointiin käytettiin ei-metristä moniulotteista skaalausta (NMDS) sekä yleistettyjä lineaarisia sekamalleja (GLMM). Komealupiinilla ei ollut huomattavaa vaikutusta kasviyhteisöjen rakenteeseen tai lajimääriin. Komealupiini vähensi kuitenkin kuivien elinympäristöjen lajien sekä generalistien peittävyyksiä. Komealupiinin peittävyys ei vähentynyt kasvuston ikääntyessä. Komealupiini ei vaikuttanut kovin voimakkaasti elinympäristöönsä, mikä johtui todennäköisesti siitä, että komealupiinin peittävyydet olivat melko vähäisiä (keskimäärin 35 % ± SD 12). Tämän tutkimuksen perusteella komealupiinin vaikutukset tälläisessä kuivassa elinympäristössä eivät ole kovin voimakkaat, mikäli sen peittävyys on vähäinen. Luultavasti komealupiinin haitallisuus liittyykin juuri sen kykyyn muodostaa tiheitä kasvustoja. Komealupiinin torjunnassa olisikin tehokkainta keskittyä niihin alueisiin, joissa komealupiini muodostaa tiheitä kasvustoja. Toisaalta huomiota tulisi kiinnittää myös paikkoihin, joissa kasvaa etenkin uhanalaisia kuivien elinympäristöjen lajeja sekä lajeja, jotka pärjäävät heikosti lajienvälisessä kilpailussa.
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(2019)Not much attention is paid on the conservation of invertebrates despite their importance to the ecosystems in general and their benefits and ecosystem services to us, humans. This study is part of a project aiming to start the Sampled Red List Index (SRLI) for spiders. The IUCN Red List Index (RLI) is used for measuring the overall extinction risk of groups of species and the sampled approach is a way to evaluate the trajectory towards extinction of megadiverse groups without the need to assess every species of the whole group of interest. A random sample of 200 spider species were selected from the global checklist and assessed according to IUCN Red List Categories and Criteria. Given the availability of data, I was able to calculate the extent of occurrence and area of occupancy for 80 species, of which 70 species had an EOO above 20000 km² and 75 species an AOO above 2000 km², the thresholds below which species can be considered threatened. The trends in EOO and AOO were assumed to be stable for most species (49 species) given the inexistence of monitoring data for any taxon. Evidence of decline was found for only 10 species, usually inferred from habitat loss. Habitat data was collected for 118 species. The most common habitat type was forest (73 species), followed by grasslands (24 species) and artificial habitats (22 species). For 44 species the habitat trend was inferred to be stable, only declining, according to available knowledge, for 14 species and increasing for one species. For the remaining 141 species the habitat trend could not be inferred and was thus assumed to be unknown. The most commonly mentioned threat types were agriculture (11 species), fires (7 species) and logging (6 species). For 39 species there were no known threats and for the rest of the 132 species the threats were unknown. Conservation actions in place were observed for 104 species, most commonly site and area protection (100 species) and resource and habitat protection (88 species). Conservation actions such as education and awareness (8 species), resource and habitat protection (7 species) and site and area management (6 species) were to take into consideration. All the 200 species were estimated to be in need of further basic research especially on threats (143 species and distribution (140 species), but also on life history and ecology (135 species). Due to several knowledge shortfalls, including the Wallacean (distribution of species), Prestonian (population trends) and Hutchinsonian (response to environmental change), no threat category could be reached for the vast majority of the species. The results show that an IUCN category could be reached for only 59 species, of which 55 were assessed as Least Concern and a threatened category was reached for only 4 species (t as Critically Endangered and one as Vulnerable). The baseline SRLI at this first point in time was 0.95 (in a 0-1 scale, where 0 means all species are extinct and 1 for all species are Least Concern). We hypothesize however that among the 141 Data Deficient species there should be a higher proportion of threatened species than among the 59 evaluated. This would be due to two reasons. First, the scarcity of information on many species might in part be due to their rarity. Second, widespread species were often the only for which an assessment could be reached, creating a bias in the dataset towards a large base SRLI value. The strategy currently imposed by IUCN is therefore clearly inadequate for taxa with scarce information, which represent the vast majority of species. I propose the future use of a different, non-random, approach to the selection of species in the SRLI and its adoption for other taxa which represent in fact most extant and threatened species.
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(2019)Extracellular vesicles (EVs) are a diverse set of cell-derived membranous vesicles which have exosomes or small EVs (40 -200nm) ,large EVs (>200 nm) and larger apoptotic bodies/oncosomes (>1000 nm). They are present in most biological fluids, such as blood, sweat, and urine etc. EVs are expressed by almost all cells.Recent evidence suggests that EVs are a form of cellular communication, resulting in an intensive investigation of their role in different biological processes and also due to their biomarker potential. Ultracentrifugation (UC) is the standard method used for the isolation of EVs from different biological fluids. UC has some limitations related to laborious operation and scalability issues. Hence, there is a need for new methods for the isolation of EVs.Nanocellulose(NC) is a biopolymer obtained from wood biomass and has the ability to be functionalised with various chemical groups on its surface. The chemical groups alter the surface properties of NC for usability in different applications. Currently, It finds extensive use in different biomedical applications due to its unique physicochemical properties. The aim of the thesis is to evaluate the use of functionalised nanocellulose (FNC) as a novel alternative compared to the standard method of ultracentrifugation in the isolation of EVs from conditioned cell culture media. Cancer cell lines and mesenchymal stromal cells (MSCs) were grown in EV-depleted cell culture media after expansion in cell culture media. The EV-depleted media was collected from the cells after 48 hours and further processed by filtration to obtain the necessary conditioned cell culture media for EV isolation. Extracellular vesicles were isolated from the conditioned cell culture media by using UC and FNC. The isolated EVs from both methods were characterised and evaluated based on their different parameters using Bicinchoninic Acid (BCA) assay, Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and Western Blot. The EVs isolated by FNC were smaller in size with few vesicle-like structures around 20-40 nm compared to EVs isolated by UC 60-200nm. Most EVs isolated by UC had a higher particle-to-protein ratio which indicated more purity and reduced levels of non-vesicular contaminants in the EV isolations. The EVs isolated by FNC for many samples had a low particle-to-protein ratio indicating potentially higher levels of non-vesicular contaminants being present. Hsp70, a surface marker of EVs and soluble stress protein was observed in EVs obtained from a primary MSC and another cancer cell line among all the isolation methods. No contamination from Endoplasmic reticulum(ER) components was observed in any of the samples. The EVs isolated by FNC could not be analysed more thoroughly by some of the characterisation methods due to issues such as lack of standardised sample preparation protocols, possible interference from isolation components and sample variations. The isolation method will really benefit from further optimisation of sample preparation steps and the adoption of alternative characterisation methods for evaluation. A narrower research question with focus on FNC-EDA isolation of EVs from HOS-143B and AT-MSC will be an excellent starting point for more detailed investigations.The availability of different surface modifications, faster processing and more research on the isolation mechanism will make this novel method a great alternative to UC for isolation of EVs.
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(2018)Kaurapohjaiset elintarvikkeet ovat terveellisiä. Monet niiden terveyshyödyt johtuvat kauran liukoisen ravintokuidun suuresta β-glukaanipitoisuudesta. β-glukaanin terveysvaikutukset ovat riippuvaisia sen molekyylipainosta ja viskositeetista. Viskositeetilla on myös muuta merkitystä kaurapohjaisissa elintarvikkeissa kuten kaurajugurteissa ja kauramaidoissa. Aiemmassa Folafibre-tutkimusprojektissa oli tutkittu kaurakuidun folaattipitoisuuden (B9 vitamiini) kasvattamista fermentoimalla sitä eri mikrobeilla. Hyvin folaattia tuottaneet mikrobit myös alensivat kaurakuituvalmisteen viskositeettia, ja erittivät glykosyylihydrolaaseja ja/tai proteaasia. Oli kuitenkin jäänyt epäselväksi, olivatko entsyymit ainut syy viskositeetin alenemiseen, ja kuinka paljon kullakin niistä oli vaikutusta viskositeettiin. Lisäksi tämän alan kirjallisuudessa on pidetty epäselvänä, onko esim. tärkkelyksen ja β-glukaanin välillä jotain interaktioita jotka mm. nostavat niiden viskositeettia yli yksittäisten polymeerien viskositeetin summan. Näitä voitaisiin selvittää puhdistamalla viskositeettia alentaneista mikrobikannoista kyseiset entsyymit ja tutkimalla niiden vaikutusta kaurakuidun viskositeettiin. Yhdeksi mikrobiksi valittiin Exiguobacterium sp. RB3 kanta, koska em. syiden lisäksi tässä bakteerisuvussa esiintyy psykrofiilejä ja mikrobeja jotka voivat kasvaa korkeassa pH:ssa, jolloin sen erittämät entsyymit saattaisivat olla aktiivisia matalissa lämpötiloissa ja korkeassa pH:ssa, ja olla siten teollisesti kiinnostavia. Toiseksi mikrobiksi valittiin Bacillus sp. ABM5119, koska sitä oli käytetty monissa Folafibre-projektin tutkimuksissa. Työn tavoitteena oli puhdistaa Bacillus sp. ABM5119:n endo-β-1,4-glukanaasi, ja Exiguobacterium sp. RB3:n α-amylaasi ainakin siinä määrin, että ne ovat muista endoaktiivisista glykosyylihydrolaaseista ja proteaaseista puhtaita, ja sitten mitata puhdistettuja entsyymejä ja proteaasia käyttäen niiden vaikutus yhdessä ja erikseen keitetyn kaurakuituvalmisteen viskositeettiin. Lisäksi tavoitteena oli karakterisoida Exiguobacterium sp. RB3 amylaasin olennaisimmat biokemialliset ominaisuudet, sekvensoida sen geenin amylaasia koodaava alue, ja selittää sen ominaisuuksia myös sekvenssistä johdettavissa olevan tiedon perusteella. Keitetyn kaurakuitupreparaatin viskositeettia alentavat eniten α-amylaasi ja endo-β-1,4-glukanaasi. Proteaasi ei vaikuta kaurakuitupreparaatin viskositeettiin, kun kuitupreparaatti on keitetty. Endo-β-1,4-glukanaasi alentaa viskositeettia yhtä hyvin kuin β-1,3-1,4-glukanaasi. Synergiaa viskositeetin alentamisessa α-amylaasilla ja endo-β-1,4-glukanaasilla ei havaittu, mutta havaittiin että β-glukaani saattaa estää tärkkelyksen retrogradaatiota. Hyvin pienetkin entsyymiaktiivisuudet vaikuttavat viskositeettiin. Jos halutaan β-glukaanin viskositeetin ja siten terveysvaikutusten säilyvän, täytyy esim. kauramaidon valmistuksessa käytettävien entsyymivalmisteiden olla β-glukanaaseista hyvin puhtaita. Mikrobifermentaatioissa tapahtuvat viskositeetin alenemat johtuvat lähinnä mikrobien erittämistä glykosyylihydrolaaseista, ei niinkään esim. niiden metabolian sivutuotteista kuten happiradikaaleista. Exiguobacterium sp. RB3 α-amylaasi on rakenteeltaan Bacillus licheniformis α-amylaasin kaltainen glykosyylihydrolaasiryhmän 13 entsyymi. Se sitoo rakenteeseensa kolme kalsiumiatomia, ja kalsiumpitoisuus vaikuttaa sen aktiivisuuteen. Se on aktiivisimmillan pH alueella 5,0 – 7,5. Se sietää detergenttejä, toisin kuin eräs aiemmin karakterisoitu Exiguobacterium-α-amylaasi. RB3 α-amylaasin turnover number oli korkea, 29000 1/s. Exiguobacterium-suvun psykrofiilisessä haarassa esiintyy kahta eri α-amylaasia, joista yksi on tässä karakterisoitu, ja toinen on selvästi erilainen rakenteeltaan ja biokemiallisilta ominaisuuksiltaan.
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(2019)Despite the advances in the management of breast cancer, discovery of novel and targeted drugs remains a challenge. It has been suggested that drug failure rates in clinical trials might be diminished by improving the predictive potential of preclinical cancer models. Three-dimensional (3D) scaffold-based cell culture has emerged as an attractive platform for mimicking tissue-like microenvironment, since it is well-known that cells respond to the cues in the extracellular matrix (ECM). The aim of this thesis was to develop fibrin-based hydrogels and evaluate their performance in 3D cell culture of breast cancer cells. The fibrin gel formulation was first optimized by testing the effect of different buffers on gel properties. Structural properties were examined with scanning electron microscopy and mechanical properties measured with oscillatory rheometry. Three different fibrin concentrations of the optimized formulation were then used as scaffolds for DU4475 breast cancer cells. After seven days of culture, the morphology, phenotype and proliferation of the resulting cell structures were assessed by using techniques such as light microscopy, immunofluorescent confocal microscopy and Western blot analysis. The desired properties for 3D cell culture were obtained by preparing fibrin gels at high pH in the absence of calcium. The main finding of the thesis was that fibrin concentration strongly affected the phenotype of DU4475 cells, with cells cultured in the softest gel retaining their original characteristics to the greatest extent. In the future, the developed scaffold could possibly be used in drug discovery and personalized medicine by culturing tumor explants from patients. However, the methods used in the study must be further optimized and the results validated with other breast cancer cell lines and with primary tissues.
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(2020)In this study, the goal was to determine which nutrient, phosphorus or nitrogen, limits the phytoplankton growth at the Vanajavesi freshwater site. The aims were to detect spatial and temporal changes and find out if the wastewater treatment plant (hereafter, WWTP) located by the study site affects the nutrient concentrations and the limiting nutrient. The reliability of determining limiting nutrient by bioassays and measuring the phytoplankton response to different treatments as fluorescence was also evaluated. The study was conducted because knowledge of nutrient limitation is essential when allocating resources to reduce nutrient loading and planning other remediation practices in eutrophicated waterbodies. According to the EU Water Framework Directive, all waterbodies in the EU must be in a good ecological status by the year 2027. This goal is yet to be achieved in Vanajavesi; the ecological status of the river Vanajanreitti is poor and that of lake Vanajanselkä is moderate. The samples for bioassays were taken from five different locations. Three sampling sites were in the river and two by the lake. Based on the direction which the water flows, one of the sampling sites was before the outlet from the WWTP and the rest after it. The bioassays were carried out with the water and natural phytoplankton community taken from the study site. The experiment was conducted five times: in November, March, May, July and August. The temperature and light conditions in the incubation room were set to mimic those in Vanajavesi at each given time. Part of the preparations was to filter out the zooplankton using 50 μm plankton net. There were four different treatments: control without nutrient additions, nitrogen addition, phosphorus addition and nitrogen and phosphorus additions. Fluorescence from the 2 litre incubation bottles was measured every 1-3 days during each experiment. Chlorophyll a was determined in laboratory before and after the experiments. Nutrient concentrations were also determined before each experiment. Small seasonal and temporal changes were observed in the nutrient concentrations and the limiting nutrient. These changes were most likely due to changing seasons, effluent from the WWTP and denitrification at lake Vanajanselkä. Phosphorus limited phytoplankton growth year around at all places. At the end of the summer also nitrogen was limiting. In July co-limitation was detected in all sampling sites. In situations of co-limitation there was either no secondary limiting nutrient, or it was phosphorus. Only once, in August at the sampling point before the outlet from the WWTP, was the secondary limiting nutrient nitrogen. On average the nutrient concentrations were higher in the river than in the lake. Chlorophyll a concentrations and some nutrient concentrations were higher after the WWTP. However, no significant negative impact due to WWTP could be detected, especially at lake Vanajanselkä and the WWTP did not result in a change from phosphorus limitation to nitrogen limitation. Bioassays and the phytoplankton yield measured with a fluorometer was a reliable way of determining the limiting nutrient. Chlorophyll a concentrations verified the fluorescence results. The probe used in this study measured only the fluorescence of chlorophyll a. Even more accurate result of the phytoplankton biomass would have been obtained with a probe that measures also the fluorescence of phycocyanin, the photosynthetic pigment in cyanobacteria, because cyanobacteria has less chlorophyll a than other phytoplankton groups. As Vanajavesi is phosphorus limited or co-limited by phosphorus and nitrogen year around, reductions in phosphorus loading will likely improve the water quality. The main source of phosphorus to Vanajavesi is the nutrient loading from agricultural practises on the drainage basin. Efficient management of this diffuse loading will cause the phytoplankton biomass, especially the biomass of harmful cyanobacteria, to decrease. Nitrogen-fixing cyanobacteria is not dependent on the nitrogen concentrations in the water column, but the concentration of phosphorus. Significantly reducing the phosphorus loading is a prerequisite for the Vanajanreitti and Vanajavesi to be in a good ecological status by the year 2027.
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(2020)Hypolimnetic withdrawal and purification is a new method developed to restore eutrophic lakes, which enables the removal and capture of nutrients such as phosphorus from hypolimnetic water. The method is currently being studied in a pilot-scale project at Lake Kymijärvi, Lahti, Finland. The project employs a closed-circuit system, in which the hypolimnetic water is pumped into a filtering system onshore, where the dissolved phosphorus is first precipitated. There are two possible methods for precipitation: aeration of water, in which dissolved phosphorus is precipitated by iron hydroxides as the water aerates, and addition of calcium hydroxide (Ca(OH)2) (hereafter “chemical treatment”), in which dissolved phosphorus is precipitated as hydroxyapatite. The water then flows through a nutrient filter, which traps the precipitate. The nutrient filter consists of two parallel filters, which enables the simultaneous comparison of two different filter materials. Finally, the water is returned to the lake via a wetland. A closed hypolimnetic withdrawal and purification system like this has not been previously studied as a restoration method. The aim of this master's thesis is to study the operation of the hypolimnetic withdrawal and purification system of Lake Kymijärvi by comparing the phosphorus retention capacity with 1) two different filter materials and 2) two different precipitation methods. The compared filter materials were fine gravel (manufactured by Rudus) and calcitic waste rock (manufactured by Nordkalk). Additionally, the thesis investigates the ratio of phosphorus fractions (Ca-P and Fe-P) of the phosphorus trapped in the filters, the distribution of trapped phosphorus between different filter layers and the effect of the filter materials on the concentrations of calcium, iron, manganese and sulfur of the water flowing through the filters. The operation of the hypolimnetic withdrawal and purification system was tested during four hypolimnetic water test pumping periods in summer 2019. The material for this thesis consisted of water and filter material samples collected from the system during the test pumping periods. The water samples were used to determine the capacity of the filter materials to capture phosphorus and other elements. The filter material samples were used to study the ratio of different phosphorus fractions and the distribution of trapped phosphorus between different filter layers. Both filter materials captured phosphorus efficiently via both precipitation methods. At their highest efficiency, both materials captured over 90 % of dissolved phosphorus and over 75 % of total phosphorus. No statistically significant difference was found in the phosphorus retention capacity between the filter materials, nor the precipitation methods. Of other elements, both filter materials captured iron very efficiently during both precipitation methods, and manganese during the chemical treatment. The phosphorus trapped in the filters was not evenly distributed between different filter layers and most of the phosphorus was trapped in the bottom-most layer in both filters. The phosphorus fractions between the two precipitation methods differed less than expected. After water aeration, all the phosphorus trapped in the filters was bound to iron. Even after the chemical treatment, less than half of the trapped phosphorus was bound to calcium, with the remainder bound to iron. According to the results, it is possible to remove phosphorus efficiently from hypolimnetic water with the system, using whichever of the studied precipitation methods or the filter materials.
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(2020)According to the latest estimations, cancer is the second leading cause of death worldwide. Despite the significant advances in the range of drugs and treatment modalities to treat cancer, the number of deaths is estimated to continue rising, posing serious challenges for the patients, their families, and the healthcare systems. Conventional treatments tend to be associated with severe adverse side effects and treatment resistance. Consequently, safer and more efficient therapy options are urgently needed, especially for the treatment of metastatic tumors refractory to conventional treatments. A new and revolutionizing field in oncology is immunotherapy, in which oncolytic viruses are included. Oncolytic viruses have an inherent or acquired selectivity to replicate exclusively in tumor cells, ultimately destroying them. Simultaneously, they also activate the dormant host’s immune system to fight against the tumor. Adenoviruses, particularly, have shown to be safe, inducing only mild adverse side effects in clinical trials, making them a great candidate for further clinical development. Adenoviruses can be genetically modified to increase their infectivity or improve the anti-cancer immune responses induced by the virus, e.g., through the expression of immunostimulatory molecules. The focus of this thesis was to develop and characterize several genetically modified oncolytic adenoviruses expressing either OX40L alone or OX40L and CD40L, two co-stimulatory molecules capable of engaging both the innate and adaptive arms of the immune system to fight the tumor. The insertion of the transgenes into the E3B-14.7k region of the Ad5/3-∆24 adenovector plasmid was performed using Gibson Assembly® cloning approach. After successful cloning, the recombinant viral genomes were transfected into A549 cells for viral amplification, followed by CsCl purification to produce a high titer viral preparation. The expression of the transgenes was studied in vitro by ELISA and functional assays, showing promising expression levels of functional OX40L and CD40L. However, when the infectivity and virus killing potency were analyzed, in vitro by immunocytochemistry and MTS assay; and in vivo using an immunodeficient mouse model, the data showed that the cloned viruses performed sub-optimally when compared to the control unarmed virus (Ad5/3-∆24). These findings suggest that the insertion of the two transgenes in place of the E3-14.7k gene was detrimental to the fitness of the virus.
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(2020)Tiivistelmä – Referat – Abstract Ewing sarcoma is a rare bone and soft tissues cancer that occurs mainly among children and young adults. It is an aggressive cancer. Treatment of Ewing Sarcoma Family of Tumours (ESFT) primarily includes surgery, radiation and chemotherapy. The treatment protocol depends on the presence of tumour metastases at the time of diagnosis. In the treatment of local tumours, the 5-year patient survival rate has increased from 50% to 70%. However, patients that have tumour metastases at the time of the diagnosis or have a recurrent disease, the five-year survival rate is only 25%. As the current treatment options have reached their limits, it is important to develop more advanced therapies. DNA methylation is an epigenetic event that affects gene expression. By comparing the methylation level of the DNA in gene promoter regions in ESFT cancer cells to the methylation level of DNA in gene promoter regions in normal cells it could be possible to discover genes and signalling pathways that are important in the development of ESFT and that could be potential drug target molecules. The aim of this study is to find out the genome-wide gene promoter DNA methylation status in Ewing sarcoma cell line samples and Ewing sarcoma patient tumour samples compared to a normal reference sample. Another aim is to find gene promoter regions that are differentially methylated in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample. Materials and Methods The Ewing Sarcoma cell line samples (12) were obtained from the Laboratory of Oncologi Research, Instituti Ortopedici Rizzoli Laboratory, Bologna, Italy. The Ewing sarcoma patient tumour samples were pre-isolated DNA samples already in Finland. The normal reference sample was a commercial mesenchymal cell line sample. From the Ewing Sarcoma cell line samples and the normal reference sample, DNA isolation was done by using phenol-chloroform method. DNA methylation profiling of the samples was performed by combining MeDIP (methylated DNA immunoprecipitation) protocol with 2-set promoter microarray hybridization protocol provided by Agilent Tecnologies company. DNA methylation data that was received from the microarrays was normalized and pre-processed with the Feature Extraction software provided also by the Agilent Technologies company. Visualization of the DNA methylation data was performed by using Chipster analysis software provided by CSC. To measure the level of DNA methylation at the gene promoter regions, a log2ratio value was calculated for every gene promoter region in all the sample types. To find gene promoter regions that were differently methylated, a log2 fold change value was calculated from the log2ratio values between the Ewing Sarcoma cell line cancer samples and the normal reference sample and between the Ewing sarcoma patient tumor samples and the normal reference sample for each gene promoter region. The log2 fold change value was also calculated between the Ewing Sarcoma cell line cancer samples and the Ewing Sarcoma patient tumor samples. After this a t-test was performed to determine the statistical significance of the log2 fold change values. Detection of genome-wide DNA methylation levels at the gene promoter regions in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample was performed by averaging log2 fold change values. The same calculation method was used to detect the differences in the genome-wide DNA methylation levels at the gene promoter regions between the Ewing sarcoma cell lines and the Ewing Sarcoma patient tumour samples. Results Differences in the DNA methylation levels at the gene promoter regions were detected between the Ewing Sarcoma cell line samples, patient tumour samples, and the normal reference sample. Genome-wide measurement of the DNA methylation levels at the gene promoter areas showed that the Ewing sarcoma cell lines had more DNA methylation at the gene promoter regions than the patient tumour samples and the normal reference sample. The patient tumour samples showed less DNA methylation at the gene promoter regions compared to the Ewing sarcoma cell lines and the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the reference sample were found 16. In the patient tumour samples, also 16 differently methylated gene promoter regions were found compared to the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the patient tumour samples were 56.
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