Browsing by Title
Now showing items 1013-1032 of 1101
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(2023)Biallelic germline mutations in ERCC6L2 cause bone marrow failure (BMF) and predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The patients often develop varying cytopenias, and underlying hypoplasia in the bone marrow is usually evident. The aim of this thesis was to characterize the transcriptome of patient -derived fibroblasts with biallelic germline ERCC6L2 mutation. Moreover, the aim was to study changes on the gene expression at the RNA level in fibroblasts in different media conditions, ROS levels in ERCC6L2 -mutated fibroblasts, and whether addition of glutamine impacts the ROS levels. Fibroblasts from 16 skin biopsies were cultured; eight samples were from healthy controls and eight samples from patients with known mutations in ERCC6L2. Fibroblasts were cultured in different media conditions, followed by RNA extraction and RNA sequencing. We observed downregulation in base excision repair, nucleotide excision repair, mismatch repair, DNA replication, homologous recombination, and cell cycle in ERCC6L2 -mutated cells. MAPK signaling pathway, p53 signaling pathway, apoptosis, AMPK signaling pathway, and TGF-beta signaling pathway were in turn upregulated in ERCC6L2 -mutated cells. The medium did not affect the gene expression significantly across samples. We suspect that the effect of medium was not detected at the RNA level, but it might affect post-translational modifications. We also detected increased ROS levels in ERCC6L2 samples compared to control and observed decreased ROS levels in ERCC6L2 and control samples with excess glutamine. This study shows that biallelic mutations in ERCC6L2 do not only affect the bone marrow but can also affect tissues outside of the hematopoietic system. The transcriptomic analysis identified important biological processes, which could be studied with more detail in the future to further explore the pathology of the ERCC6L2 disease.
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(2021)Transfer RNA (tRNA) is one of the most extensively modified RNA molecules. The role of tRNA modifications become apparent during physiological condition such as oxidative stress, where it serves as an adaptive response to the changing environment. These modifications are upregulated mainly at the wobble position of the tRNA to enhance the translational efficiency of the stress response genes through enhanced decoding rate and tRNA–mRNA interaction. Hence, tRNA modification has a crucial role in regulating translational fidelity, and such modifications can be utilized to fine-tune the translation for improved production of heterologous protein. Therefore, this study aimed to analyze the tRNA modification changes in two laboratory-significant E. coli strains (BL21 (DE3) and K12) during oxidative stress and utilize these modifications to enhance the production of heterologous protein using a defined cell-free protein synthesis system. Ultra-performance liquid chromatography-mass spectrometry was used to detect and quantify the tRNA modification changes in the hydrogen peroxide-treated E. coli cells. The results showed unique tRNA modification patterns and intensities between the two bacterial strains in response to oxidative stress. Modifications such as ac4c and m2,2G were upregulated in E. coli BL21 (DE3) following hydrogen peroxide treatment, whereas k2C and chm5U were increased in E. coli K12. Further analysis of the dataset revealed that most of the upregulated ribonucleoside modifications were predominant at the anticodon loop of the tRNAs, indicating the potentiality of these tRNA pools to impact on translation. Likewise, I optimized an E. coli-based cell-free protein synthesis system to investigate the effect of modified tRNA pools on translation. Hence, this study serves as a stepping stone to understand the tRNA modification landscape of E. coli and provides a platform to depict the function of post-transcriptional tRNA modifications in translation with the CFPS system.
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(2015)Neurotrophic factors are essential for the development of the central nervous system. By signalling through Trk receptors, they have multiple effects on for example the survival of neurons, growth of axons and dendrites and stability of synapses. BDNF, which signals through TrkB receptor, is a neurotrophic factor with an important role in the formation and stabilization of glutamatergic synapses. It is also known to be released in an activity dependent manner. The mechanisms by which BDNF and TrkB signalling regulate synaptic transmission depend on the type and developmental stage of the synapse, and they are not well known. The electrical activity of immature networks consists of intrinsic activity with intermittent bursts of synchronous activity, which is believed to fine tune the synaptic connectivity through Hebbian plasticity mechanisms, which are stabilized by homeostatic mechanisms. Homeostatic regulation can be especially important during the development of the neural network while the glutamatergic transmisson is very labile. Little is known of the signalling routes that participate in the homeostatic plasticity during the development of the neural network. The aim of the thesis was to investigate how the long-term inhibition of TrkB signalling affects the glutamatergic transmission and the homeostatic regulation in area CA3 of neonatal hippocampus by using gene manipulated TrkBF616A mouse strain. The TrkB receptors in the TrkBF616A strain are modified so that they can be blocked with a kinase inhibitor (1NMPP1). In part of the work C57BL/6 -mice were used as control. In addition to the acute measurements the hippocampal slices were incubated in control conditions and with inhibitory drugs (TTX and 1NMPP1) for 15 to 20 hours, after which miniature excitatory postsynaptic currents (mEPSCs) were recorded with whole-cell patch clamp from area CA3 pyramidal cells. The research shows that continuous TrkB signalling is essential for the maintenance of AMPA receptor mediated synaptic transmission in CA3 area of neonatal hippocampus in TrkBF616A mice. Long-term inhibition of TrkB signalling decreases the amplitude of mEPSCs. TrkB-signalling seems to be needed also for the homeostatic response caused by network activity deprivation. The results also indicate that the inhibition of TrkB signalling increases the frequency of mEPSCs, possibly by a homeostatic mechanism. However, the results also show that the TrkBF616A mice strain might differ from wild type mice and the kinase inhibitor 1NMPP1 might have non-specific effects that are not currently known, so more extensive research on the matter is still needed to confirm the results.
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(2022)Aims and methods: Global plastic production is increasing annually and microplastics (MPs, particles of <5 mm in size) have been reported in the environment worldwide. In aquatic systems plastic pollution is present especially in coastal habitats, and MPs can concentrate within littoral zone vegetation. Numerous marine animals are known to be able to ingest MPs, and plastics can also have adverse effects on the health and behaviour of the exposed animals. This Master’s thesis examined trophic transfer of MPs in a Baltic Sea littoral food chain. Laboratory experiments with 10 µm fluorescence microspheres were conducted to study trophic transfer between food chains of different lengths. The longest food chain had three trophic levels: zooplankton, chameleon shrimp (Praunus flexuosus) and rockpool prawn (Palaemon elegans). Also, the gut passage time of rockpool prawn was experimentally studied. The digestive tracts of the studied animals were analysed for MPs under an epifluorescence microscope. Results: The results show that trophic transfer may be an important pathway of microplastic exposure for animals at higher trophic levels. The number of ingested microspheres in both chameleon shrimp and rockpool prawn was higher when the animals were exposed through pre-exposed prey in comparison to direct exposure from the water. In addition, the prawns ingested more MPs in the experiment with three trophic levels than in the two-level experiment. The results support earlier findings, that the feeding mode affects the microplastic exposure of animals. There were no clear results from the gut passage time experiment.
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(2018)Riisi (Oryza sativa) on yksi maailman viljellyimmistä ravintokasveista. Siltä tunnetaan maailmanlaajuisesti 20 merkittävää tuhohyönteistä, joista monet ovat yöllä lentäviä perhosia ja jotka runsaina esiintyessään voivat aiheuttaa huomattavia satotappioita riisinviljelijöille. Tässä tutkielmassa selvitettiin, mitkä riisin tuholaisperhoslajit ovat runsaimpia Kaakkois-Madagaskarin runsastuottoisilla märillä ja pengerretyillä riisipelloilla ja mitkä ympäristömuuttujat vaikuttavat näiden runsaimpien lajien esiintymiseen. Aiemmissa maaseutuelinympäristöjen tutkimuksissa on havaittu, että maanviljelyn tehostuminen ja sen aiheuttama luontaisten elinympäristöjen häviäminen saattaa johtaa tuholaisten runsastumiseen. Kaakkois-Madagaskarin sademetsävyöhykkeellä metsien väheneminen lisääntyneen maanviljelyn takia on viime vuosikymmeninä ollut huomattavaa voimakkaan väestönkasvun seurauksena. Tutkimus suoritettiin kenttäkokeina Kaakkois-Madagaskarilla Ranomafanan kansallispuiston läheisyydessä vuoden 2015 marras- joulukuussa. Perhosia kerättiin vetisiltä riisipelloilta kolmella valopyydyksellä kolmen yön ajan kahdeksassa, maisemarakenteeltaan erilaisessa kylässä. Pyyntijaksojen aikana mitattiin ja kerättiin tietoa pyyntiöiden sääolosuhteista. Tutkimusalueelta aiemmin tehdyn maanpeiteluokituksen avulla tarkasteltiin maisemamuuttujien vaihtelua pyyntipaikkojen läheisyydessä. Pyydyksillä kerätyt hyönteiset säilöttiin ja tuotiin Suomeen lajintunnistusta varten. Suomessa peltojen runsaslukuisimpien perhosten lajit ja sukupuolet määritettiin ulkoisten tuntomerkkien, lisääntymiselinten ja DNA-viivakoodauksen avulla. Tulosten perusteella Parapoynx-suvun (Crambidae) lajit, tunnettu riisituholainen Parapoynx stagnalis ja P. fluctuosalis, jonka tuholaisstatus vaihtelee kirjallisuuslähteistä riippuen, olivat selvästi tutkimusalueen riisipeltojen runsaimpia perhosia. Kaikkein runsain oli P. fluctuosalis -lajin tumma värimuoto. Aiempien tutkimusten mukaisesti tarkasteltavien yöperhosten runsauteen vaikuttivat ympäristömuuttujista eniten säätekijät ja maisematekijöiden vaikutus oli lähinnä suuntaa antavaa. Säätekijöistä lämmin ja kuiva sää lisäsivät perhosten määriä, kun taas pyyntiyöllä loistanut täysikuu vähensi pyyntisaalista huomattavasti. Maisematekijöistä ainoastaan riisipeltojen suuri määrä pyydyspaikan läheisyydessä lisäsi merkitsevästi vain P. fluctuosalis -lajin raidallisen värimuodon runsauksia. Lajimääritysten yhteydessä tarkasteltu tutkimuslajien sukupuolijakauma oli poikkeuksellinen: naarasperhoset olivat merkitsevästi runsaampia kuin koiraat. Tulos oli vastoin aiempia tutkimuksia, joiden mukaan valopyynti houkuttelee yleensä enemmän koiras- kuin naaraperhosia. Naaraiden runsautta suosi todennäköisesti lyhyt yökohtainen pyyntiaika, joka oli vain neljä tuntia auringonlaskusta eteenpäin. Parapoynx fluctuosalis -lajin eri värimuotojen morfologinen ja geneettinen tarkastelu varmistivat todennäköisesti ensimmäistä kertaa, että värimuodot edustavatkin kahta eri lajia, eivätkä saman lajin eri värimuotoja. Tutkielman tulokset kyseenalaistavat nykykäsityksen Parapoynx-lajien taksonomiasta. Tutkielman tulokset osoittavat ensimmäistä kertaa, että Ranomafanan kansallispuiston alueella, Kaakkois-Madagaskarilla esiintyy tunnettuja riisin tuholaisperhosia. Tulokset eivät kerro tuholaisten aiheuttamista mahdollisista satovaikutuksista, mutta vaikutukset ovat oletettavia siellä missä lajeja esiintyy. Tuholaisten tunnistamisen ja niiden alueellisen kartoittamisen sanotaan kuitenkin olevan ensimmäinen askel, jotta niiden mahdollisesti aiheuttamia haittoja pystytään myös torjumaan ja ennustamaan. Riisin tuholaisperhosten kartoittaminen tutkimusalueella on myös tärkeää, jotta paikalliset riisinviljelijät ymmärtäisivät luontaisten elinympäristöjen tärkeyden niiden torjunnassa. Tutkimukset ovat nimittäin useaan otteeseen osoittaneet, että viljapeltojen ympäristöjen eliöt ovat tärkeässä roolissa tuholaisten biologisen torjunnan suhteen. Aihetta jatkotutkimalla olisi ehkä mahdollista löytää yhteys alueen metsien häviämisen ja tuhohyönteisten määrien suhteen, mikä auttaisi paikallisia maanviljelijöitä ymmärtämään luonnontilaisten elinympäristöjen tärkeyttä myös heidän elantonsa osalta. Tutkimuksen tuottama massiivinen, Luonnontieteellisen keskusmuseon Luomuksen kokoelmiin tallennettava hyönteisaineisto antaa joka tapauksessa mahdollisuuksia moniin lisäselvityksiin. Aineisto sisältää valtavan määrän hyönteisiä, joiden biologiasta, taksonomiasta ja esiintyvyydestä ei tiedetä välttämättä mitään.
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(2020)Within the field of cancer immunotherapy, immune checkpoint inhibitors have been a revolution since they provoke re-activation of T-cell immune responses towards cancer. Despite their success, they only work in 13% of the patients because of a poorly immunogenic tumor, mostly due to weak T-cell infiltration. Oncolytic viruses have shown the ability to work in synergy with checkpoint inhibitors because of their tumour-specific tropism, innate immunogenicity and ability to secrete immunostimulatory agents into the tumor microenvironment. Regardless of the great potential, we lack suitable pre-clinical models to test this effect. In this study we developed renal cell carcinoma-derived organoids as in vitro platforms due to their high pre-clinical predictability compared to that of murine and in vitro 2-dimensional cell culture models. To test the ability of oncolytic viruses to stimulate the immune system, we generated three cytokine-expressing (CXCL9, CXCL10 and IL-15) oncolytic adenoviruses using a novel cloning method that we developed. We have shown that these viruses successfully produce high amount of the cytokine and attract peripheral blood mononuclear cells freshly isolated from Buffy coats. Genetically modified oncolytic adenoviruses were also shown to infect and kill human renal cell carcinoma organoids. Together, our results demonstrate the potential of organoids as test platforms for oncolytic virus -based therapy and the importance of adequate cytokine expression in T-cell recruitment. The tumor organoid platform we developed will be useful for advancing patient-specific treatment strategies and serve as a base for innovative immunotherapy models.
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(2022)Domesticated horses have been used for various tasks over their thousands of years of shared history with humans. To be able to perform these tasks every horse needs to learn the needed skills, and this requires systematic training. Training of adult horses has been studied for a long time and comparisons between the efficacy of different training methods have been done. There have also been some studies comparing how much and when young foals need to be handled for them to grow into easily trainable adults. From adult horses it is known that emotional state affects cognitive processes and with that also their learning efficiency and speed. The early stages of training young horses have not been studied very well. There is no clear picture about how young horses feel during training and how that affects their learning. In my thesis I studied young horses’ emotional states while learning new tasks and how that affects their learning. I followed the early training of 19 young horses (11 one-year-olds and 8 two- and three-year-olds) by videotaping five training sessions and collecting saliva samples before and after three of those sessions to analyse cortisol and oxytocin. From the videos I analysed how fast horses responded to trainer’s asks and how unfocused they were. From the hormone samples I measure the change in cortisol and oxytocin levels during training. Salivary cortisol has been widely used to measure acute stress. Oxytocin on the other hand is a newer indicator for positive emotions. To the best of my knowledge salivary oxytocin has never been used in horses. My data showed that the horses learned the required tasks: they became quicker at their responses and focused better during the course of training. Because my data was quite small and individual variation in the hormone levels was high, the results might have been affected by these factors. Linear mixed effect models showed that higher oxytocin levels before training session predicted quicker responses during training and lower levels after training predicted lower focusedness. Bigger increase in cortisol levels during training compared to the before level explained quicker responses and better focusedness, but higher levels before training resulted to lower focusedness and slower responses. This is in line with previous studies of adult horses, that showed that horses in a better emotional state and with less stress learn faster and are more interested in working with humans. This shows that it is important to not only focus on physical wellbeing but also mental wellbeing from early on in horses’ life.
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(2015)Suoturpeen energiankäyttö kattaa n. 7 prosenttia Suomen energiantuotannosta. Tätä käytetään erityisesti talojen lämmitykseen kaupungeissa. Turpeenpoltto tuottaa kuitenkin runsaasti hiilidioksidia, kattaen n. 20 % koko energiasektorin hiilipäästöistä. Tämä taas kiihdyttää ilmastonmuutosta, joka tekee turpeenpoltosta kansainvälisen asian. Suomi on osana YK:n ilmastosopimusta sitoutunut vähentämään kasvihuonekaasupäästöjään. Vain noin 1 % Suomen suoalasta on turvetuotannossa, mutta silti sen vaikutukset paikallisesti suoekosysteemiin ja vesistöihin voivat olla huomattavat. Suomella on lisäksi kansainvälinen vastuu soiden säilyttämiseksi, sillä alun perin kolmannes pinta-alastamme on ollut suota. Soiden moniulotteisuuden vuoksi maa- ja metsätalousministeriö valmisteli vuonna 2011 soiden ja turvemaiden kestävän käytön strategian. Tässä pro gradu -tutkielmassa tarkastellaan turpeen energiakäytön ympärille muodostunutta konfliktia, jota kutsutaan aineistossani turvekiistaksi. Siihen ovat vaikuttaneet soiden käytön ristiriitainen luonne ja strategian julkaisu, joka on herättänyt keskustelua intressiryhmien, erityisesti turvetuotantosektorin ja luonnonsuojelijoiden, välille. Molempien osapuolten intressit ovat törmänneet toisiinsa monella tapaa, ja seurauksena keskustelu on jämähtänyt paikalleen. Tilanteen ratkaiseminen parantaa kestävää soiden käyttöä ja auttaa kestävän energiapolitiikan suunnittelussa. Jotta turvekeskustelussa päästään kehittävässä mielessä eteenpäin, on tärkeää selvittää Suomen turvekeskustelun kenttä. Siihen pohjaavat tutkimuskysymykseni: 1. Mistä turvekiista koostuu ja 2. millaisia ratkaisuja kiistalle on löydettävissä? Aineisto koostuu 2008 2014 aikana kerätystä kirjallisessa muodossa olevasta medianäytteestä aina sanomalehdistä adresseihin ja kommenttiketjuihin. Menetelmänä käytän laadullista sisällönanalyysiä, jonka avulla turvekiistan osapuolten intressikenttä ja kiistelyn takana olevat tulkintakehykset voidaan hahmottaa. Tulkintakehykset ovat yhtenäisiä tapoja ymmärtää ilmiö mielekkäänä kokonaisuutena, minkä kautta turvetuotannon kokonaisvaikutukset nähdään joko uhkana tai mahdollisuutena, mikä jakaa kiistan osapuolet turvetuotannon puoltajiin ja vastustajiin. Analyysin mukaan turvekiistan intressikentässä on teollisuutta edustava puoli, vesistön virkistyskäyttäjät sekä luonnonsuojeluorientoitunut puoli. Kiistan keskeiset vastakkainasettelut on jaettu ekologiseen ja teknis-taloudelliseen kestävyyteen sekä hallinnon heikkouksiin. Niissä kiistellään muun muassa turvetuotannon vesistövaikutuksista, suon luontoarvoista, turpeen uusiutuvuudesta, sen energiakäytön huoltovarmuudesta, ympäristöluvasta ja sen valituksista sekä poliittisen ohjauksen nykytilasta. Vesistön virkistyskäyttäjien hahmottuminen osana kiistaa oli tärkeä havainto, koska keskustelua tulkitaan helposti pelkästään luonnonsuojelijoiden ja teollisuuden väliseksi vastakkainasetteluksi. Kiistan osapuolia hahmottavat tulkintakehykset nimesin kotimaisen tuotannon ja luonnon kestävän käytön kehyksiksi, jotka omilla tulkinnoillaan osallistuvat edellä mainittuihin vastakkainasetteluihin. Turvekiistassa esiintynyttä retoriikkaa on havaittavissa muissakin ympäristökonflikteissa. Mahdolliset ratkaisut kiistan selvittämiseen ovat jaettavissa tiedolliseen, arvo- ja intressikiistaan. Ratkaisuja ovat esimerkiksi paikallisen tason huomiointi ympäristöluvassa, turvetuotannolle ominaisiin vesistövaikutuksiin keskittyvän tutkimuksen lisääminen ja osapuolia yhdistävän kehyksen tietoinen rakentaminen, joka toimisi ns. sillanrakentajana. Kiistassa on lopulta kyse siitä, kumpi osapuoli pystyy yhdistämään oman tulkintansa paremmin sosiokulttuuriseen kestävyyteen.
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(2024)Tutkimuksellinen opetus ja tutkimuksellisuus oppilaiden suorittamissa tehtävissä ovat tärkeä osa biologian opetusta. Tutkimuksellisuus voi sisältää kokonaisen tutkimuksen, tai vain osia siitä. Tutkimuksellisten työtapojen hallinta on osa opetussuunnitelman mukaista oppimisen arviointia peruskoulussa ja lukiossa. Lukion opetussuunnitelman ihmisen biologian kurssin sisällöissä mainitaan myös pienimuotoisen tutkimuksen toteuttaminen osana oppimisen tavoitteita sekä arviointikehystä. Tutkimuksellinen lähestymistapa tukee ja korostaa oppilaan itseohjautuvuutta ja aktiivista, oma-aloitteista oppimisprosessia ja sen kautta opetusta voidaan myös eriyttää erilaisten oppijoiden tarpeita tukevalla tavalla. Tutkimuksellisten tehtävien ja työtapojen on todettu lisäävän motivaatiota ja parantavan oppimistuloksia. Ihmisen biologian oppimateriaaleissa tutkimuksellisia tehtäviä on todettu olevan lukumääräisesti vähemmän, kuin muissa biologian aihepiireissä. Tässä tutkimuksessa tarkastelluissa oppimateriaaleissa tutkimuksellisia tehtäviä on vähän suhteessa tehtävien kokonaismäärään, osassa materiaaleista ei välttämättä ollenkaan. Tutkimuksellisuuden taso ja eri tasojen edustus vaihtelee jonkin verran oppikirjojen välillä. Tutkimuksellisten tehtävien osuus eri aihekokonaisuuksissa vaihtelee niin aihekokonaisuuksien, kuin myös oppikirjojen välillä, mutta samat aihepiirit jäävät toistuvasti ilman tutkimuksellisia tehtäviä, kun taas muutama aihepiiri erottuu joka oppikirjassa tutkimuksellisten tehtävien runsaammalla lukumäärällä. Osa tehtävistä sisältää tutkimuksellisia piirteitä olematta kuitenkaan varsinaisesti tutkimuksellisia tehtäviä.
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(2012)Core-fucosylation of N-glycoproteins is associated with different cancers and other pathologies. Identification of glycoproteins and determination of their glycan structure manually by mass spectrometry (MS) is time-consuming and laborious. In this Pro gradu thesis, the use of the mass spectrum-analyzing software Glycopeptide ID for identification of core-fucosylation from a known standard, immunoglobulin G, was studied. Also, a plasma sample with unknown glycoproteins was analyzed. For the MS analysis, the proteins were digested with trypsin, and the resulting glycopeptides were enriched using lectin affinity chromatography. From IgG and plasma, also samples treated with α-Lfucosidase were prepared in order to cleave the core fucose. The presence of glycopeptides was determined by high-performanve liquid chromatographymass spectrometry (HPLC-MS) analysis, and they were fragmented using collision-induced dissociation (CID) in a tandem-MS (MS/MS) analysis. The MS/MS spectra were analyzed with the Glycopeptide ID software. The software was found to identify core-fucosylation reliably from high-quality spectra, but identification of proteins were often incomplete from spectra with poor quality. From the plasma sample with unknown proteins, a probable corefucosylation was found from IgG2, fetuin A, serotransferrin, hemopexin and ceruloplasmin. As a conclusion, the software Glycopeptide ID can be considered as an appropriate tool for identification of core-fucosylation in N-glycopeptides.
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(2015)Huntington's disease (HD) is a progressive neurodegenerative disorder that causes involuntary muscle movements, deteriorates muscle coordination and cognitive decline. Typical onset age of the disease is in mid age, although a juvenile form of HD is also known. The disease is inherited in an autosomal dominant manner via a mutation in the huntingtin gene (HTT). The characteristic mutation in HTT is an expansion of the glutamine stretch at the 5 end of the gene. Excessive amounts of glutamine residues alters the conformation and chemical features of the huntingtin protein (HTT) leading to accumulation of cellular aggregates. Although to date there are several known alterations in the cell that contribute to the disease, the pathogenesis of HD is not fully understood. Ubiquitin proteasome system (UPS) dismantles proteolytically unneeded or damaged proteins, which are targeted to proteolysis when ubiquitin tags are added to them. Deubiquitinating enzymes (DUB) recycle ubiquitin molecules by releasing them from proteasome substrates. Recycling of ubiquitin is critical to a cell as it maintains the free pool of the targeting molecule. Ubiquitin-specific protease 14 (USP14) is one of the DUB family enzymes and its distinctive function is to remove ubiquitin molecules from the tip of the ubiquitin chain and thus antagonize protein degradation. Although the specific function of the protein is unclear, it seems that USP14 operates as a fine regulator of protein turnover rate and in ER stress both in catalytic and non catalytic manner. The role of USP14 is especially emphasized in the nervous system, as it regulates synaptic transmission and neuronal development. Although it is suggested that dysfunction of UPS is involved in the pathogenesis of HD, the role of USP14 in the disease remains to be unknown. IU1 is a novel inhibitor of the catalytic domain of USP14. Studies with IU1 indicate that inhibition of USP14 enhances the clearance of aggregate prone proteins. The approach of this thesis was aimed to elucidate the routes of HD pathogenesis from diverse approaches. The general aim of the thesis was to investigate the role of USP14 in the wild-type PC6.3 cell model, and in the pathogenesis of HD by expressing HTT proteins with different lengths of glutamine stretches in PC6.3 cells. The specific aim of the study was to examine by western blot and microscopy analysis the pathogenic routes of HD that involve ER stress, oxidative stress, autophagy and mutant HTT aggregate dynamics. The function of USP14 was studied with overexpression of USP14, or by inhibiting its catalytic activity by IU1. The findings of this thesis show that overexpression of USP14 enhances the clearance of mutant HTT aggregates, and this effect is obtained in catalytic activity dependent manner. I show that upregulated USP14 is connected to improved clearance of mutant HTT and inhibition of autophagy, suggesting that the degradation is mediated via UPS. The catalytic activity of USP14 might also be important in ER stress regulation, as the results indicate that IU1 activates phosphorylation of both JNK and eIF2α. I was also able to establish a connection between USP14 and GADD34, as I show that GADD34 upregulates USP14. Finally, I show that catalytic inhibition of USP14 decreases the expression of antioxidant SOD2. The data in this thesis is lacking statistical significance, and it can be considered solely as a guideline. However, together these results indicate that the deubiquitinating activity of USP14 increases survival in PC6.3 cells in both a healthy and a HD model.
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(2022)Skeletal muscle is the most abundant tissue in the body, accounting for up to 40-50% of total bodyweight. Regeneration of this tissue is dependent on skeletal muscle stem cells, which are termed satellite cells (SCs) based on their anatomical position between the basal lamina and plasma membrane of muscle fibers. SCs exist under homeostatic conditions in a reversible G0 phase of the cell cycle. Quiescent SCs are recognized by the expression of the paired box 7 (Pax7) transcription factor, in the absence of other myogenic transcription factors such as myoblast determination protein 1 (MyoD) or myogenin (MyoG). Quiescent SCs are metabolically less active with a low oxygen consumption rate. They contain less ATP and have few mitochondria with a low membrane potential in comparison to activated SCs. Activated SCs enter the cell cycle and start to proliferate, undergoing metabolic rewiring to primarily utilize glycolysis for energy production. During early activation, there is an increase in mitochondrial content and ATP production, while the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) increase later during active proliferation. Although similar population dynamics, SCs are a heterogenous population of stem cells, with differences in the expression of notch receptors, stem cell markers, ATP and mitochondrial content, which in turn affect the myogenic potential of the cells. Mitochondria are semi-autonomous, double membrane organelles with various regulation within the cell, such as calcium homeostasis, apoptosis, production of metabolic intermediates, reactive oxygen species (ROS) metabolism, and ATP generation through oxidative phosphorylation (oxphos). Differentiation of various other stem cell types is accompanied by an increase in both mitochondrial content and oxidative phosphorylation, with ultrastructural changes that favour this shift in metabolism. The aim of this thesis was to quantify the ultrastructural changes that occur within SC mitochondria during the early proliferative phase, and to implement a method of Correlative Light and Electron Microscopy (CLEM) for identifying and studying subpopulations of SCs. After isolation and during early activation, SCs contain few mitochondria with a diffuse ultrastructure. Classification of the observed mitochondrial phenotypes revealed heterogeneity both within and between timepoints. During later phases of proliferation, there was an increase in the proportion of mature mitochondria, with an increase in cristae density and a decrease in cristae width. Utilizing genetically modified R26-Snaptag-Omp25 x PAX7CreErt2 mice in which recombination with tamoxifen initiates the expression of mitochondrial outer membrane protein 25 (omp25) bound with a SNAP-tag, allowed for specific and temporal labelling of SC mitochondria by fluorescent SNAP substrates. Performing CLEM on fluorescently labelled SC mitochondria enabled their identification during transmission electron microscopy (TEM). In addition to this, temporal labelling of pre-existing (old) and newly imported (young) omp25 revealed a few cells that contained more old mitochondria, with the cristae density being higher in these. While this indicates a correlation between mitochondrial content and ultrastructure within subpopulations of SCs, further studies are needed to validate these early observations.
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(2024)High-grade Serous Cancer (HGSC) stands out as the most prevalent and lethal subtype of ovarian cancer, characterized by genomic instability and aggressive behaviour. Genetic alterations are pivotal in its development and progression, which leads to distinct molecular profiles and clinical behaviours. Recent attention has shifted towards understanding the influence of Tumour Microenvironment (TME) in cancer prognosis. This study aims to uncover the relationship between TME, molecular profiles, and clinical outcomes in HGSC. To achieve this, Random Forest (RF) analysis on single-cell and spatially resolved data from high-plex immunofluorescent images was employed. By leveraging RF's feature selection capabilities, we identified pertinent TME features associated with the clinico-molecular characteristics of HGSC. Our analysis revealed distinctive TME characteristics in HGSC patients with BRCA loss and homologous recombination repair proficiency. Notably, we found that Major Histocompatibility Complex (MHC) class II expression, originating from cancer cells, was a critical feature that shaped the immune environment and cancer behaviour. Moreover, we discovered that MHC molecules, specifically MHC-II, were also crucial in distinguishing short and long-term survival groups. Increased MHC-II expression was associated with improved survival, independently of molecular profiles. This association highlighted the importance of endogenous immunity in fighting cancer. In conclusion, MHC-II is a crucial prognostic marker in our study, offering a valuable and assessable metric for patient prognosis. In addition, the identification of phenotypically distinct survival groups based on TME characteristics underscores the potential of our approach in enhancing patient stratification and guiding personalized treatment strategies, ultimately improving the management of HGSC.
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(2022)Fungi play an important role, especially in boreal coniferous forests and peatlands. For example, they are responsible for the circulation of nutrients, and are an important part of forest vegetation, such as tree function and nutrient uptake. Drainage of peatlands for silvicultural purposes has increased over the years and forest management has been found to change the structure of fungal communities. In addition to clear-cutting (CC) as one of the forest management methods, the method of continuous cover forestry (CCF) has been proposed as one of the possible forest management methods, but its effects on the soil fungal community have not been extensively studied. The aims of this master's thesis were to study how the active soil fungal community and its structure vary between the different forest management methods (CC, CCF and uncut control forest) of the peatland forest in Janakkala and between the seasons (spring, summer and autumn). The active community was studied by taking RNA samples from the area in May, July, and September 2021. In addition, the goal was to examine how potential environmental factors such as soil temperature and groundwater variations affect the active fungal community. Overall, diversity was higher in the autumn in all samples. The most stable area was the control forest, where active community members varied between seasons, but where biodiversity was similar between samples in both spring and autumn. The results of the CCF site followed in many ways the uncut forest, but in the autumn, there were large differences in the biodiversity and community structure of the samples in the forest of the CCF site. The biodiversity of the CC area was high. This may be explained by the deforestation of young trees already formed at the time of sampling, which contributes to the return of for instance, mycorrhizal fungi to the area. It should also be noted that the full number of parallel samples from the CC site in the spring and the autumn were not successful. In addition, the summer samples failed completely and no results could be obtained from them. The failure of the samples may be explained by the long hot and dry period in the area in the summer of 2021.
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(2021)Study-related burnout is a growing problem among university students. Study-related burnout is defined by exhaustion related to studying, a cynical attitude towards studying and feelings of inadequacy. Burnout can be caused by an overtaxing workload and by a lack of personal, social, or material resources. Stress is a natural part of life, but chronic stress can lead to illness and burnout. Students experience stress in their studies and interventions aiming to reduce stress and prevent burnout are therefore necessary. Psychological flexibility increases wellbeing and prevents burnout and is also related to enhanced learning. Acceptance and Commitment Therapy (ACT) is focused on improving psychological flexibility. Good study skills can also prevent burnout. The aim of this study was to examine university students’ experiences of an online ACT-based intervention that aims to increase psychological flexibility and enhance wellbeing and learning. The aim was also to compare the experiences of two groups of students based on their change in study-related burnout during the course. The participants wrote reflective diaries that were qualitatively analyzed using inductive category development and categories that describe students’ experiences of the intervention were formed. The result of this study shows that students had benefited from the course and described effects on both wellbeing and learning. Students in both study groups experienced effects on wellbeing and learning. The importance of peer-support during studying was discussed, which highlights the need for support from peers who experience similar difficulties and concerns. There were also mentions of negative experiences which the students gave as explanations to why a part of the course had not been effective in enhancing their wellbeing and learning. These categories were more often discussed by students whose risk for burnout had increased during the course. Several categories under the themes of positive experiences of effects on wellbeing and learning were more often discussed by students whose risk for burnout had decreased during the course. The result of this study shows that the ACT-based intervention can be regarded as effective in improving wellbeing and studying, and experiences of the intervention were in general positive. Integration of similar interventions in study programs would be beneficial.
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(2024)It has been found by multiple research projects that subjective and objective measurements of greenery often do not match when measured for the same area. Some even say that this is due to these measurements considering different attributes of the greenery. However, one important factor to consider in this equation is the impact of personal characteristics of the people providing subjective greenery measurements. Factors such as age, gender, worldview and education level have been found to have an impact on how and how much people perceive greenery. As there seem to be very few studies regarding this topic which focus on people with a higher education, the participants of this study are all university students from four of the largest cities or areas in Finland: the Helsinki Capital Region, Tampere, Oulu and Turku. One of the objectives of this study are to see whether participants perceive the same amount of urban greenery as the objective amount, calculated using the “normalized difference vegetation index”. Another objective is to research whether subjective perception is dependent on whether the participant is a resident of the city, for which they are estimating the urban greenery percentage. This study also looks into whether there is a correlation between age and urban greenery perception or difference between genders regarding the perception of urban greenery. The results show that the participants tend to overestimate the amount of greenery, and that residents always perceive their home city to be greener than at least some non-residents. No significant connections were found in the tests concerning age and gender. This highlights the importance of considering personal characteristics and perceptions of people when developing new green spaces.
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(2024)As part of the European Green Deal, the European Union has set a goal to increase its domestic raw material extraction by 2030. The EU Critical Raw Materials Act lays out concrete goals for securing and strengthening EU’s domestic supply of critical raw materials. The Critical Raw Materials Act will potentially have significant impacts on land use in Europe’s mineral rich regions. These include northern Fennoscandia which is also home to the Indigenous Sámi inhabiting Sápmi. Extractive projects have multifaceted impacts on local communities and the environment, and mining-related conflicts are on the rise globally. The EU’s goal to increase mining domestically calls for the critical assessment of mining from the perspective of justice. The theoretical framework of this thesis comprises of environmental justice and extractivism. The geographical focus of this thesis is on mining in the northern parts of Fennoscandia – Norway, Sweden and Finland. My research question is: how do different stakeholders from Norway, Sweden and Finland view increasing mining in northern Fennoscandia from an environmental justice perspective? My method is critical discourse analysis consisting of three levels of analysing discourse: textual, interpretative, and critical. I combine perspectives of environmental justice to the study of discourse and apply an environmental justice framework consisting of procedural, recognition and epistemic justice. My data consists of stakeholder feedback submitted to the European Commission’s open online consultation on the Critical Raw Materials Act proposal. The aim is to understand how different stakeholders consider environmental justice aspects as part of increasing extractive activities in northern Fennoscandia. The analysis reveals that most stakeholders agree with the need to increase mining of critical raw materials, and the northernmost Fennoscandia is coined as an important location for mining critical raw materials. Regions emphasize the involvement of local communities and the positive contributions of mining locally. Sámi representatives underline Indigenous rights and strengthening Sámi participation in decision-making. Business actors emphasize the need to accelerate mining permitting processes and to reassess environmental legislation in the context of mining projects. Many stakeholders view technological solutions as a means to conduct mining sustainably, and business actors in particular call for a change of attitudes towards mining. A critical analysis reveals that in terms of environmental justice the discourse is rather limiting; local and traditional knowledge and livelihoods are not thoroughly addressed, and the environment is viewed mainly as a resource.
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(2024)Lignin is a key macromolecule in many land plants and plays a role in structural support, water conduction, and defence (Vanholme et al., 2010). In most vascular plants, lignin makes up a significant portion of the total plant biomass, about 20-30% (Robinson, 1990). This is important to consider for a variety of reasons, including the large energetic and monetary cost that the forestry industry incurs when removing lignin from tree biomass during processing. Despite its cost to paper and pulp mills, lignin has large potential in the pharmaceutical, construction, and packaging fields, among others (Albuquerque et al., 2021). With proper bioengineering, lignin could even replace fossil fuels as a feedstock in bioplastics production. However, current research and knowledge of lignification, the process wherein lignin deposition occurs in the plant, is lacking when it comes to our ability to produce commercially viable plants with manipulated lignin properties on a large scale. These gaps in knowledge are why it is important to study species with unusual lignin formation, such as the small North American shrub Dirca palustris, commonly known as eastern leatherwood. Such efforts will enable comparative analyses that will improve our understanding of lignification. Previous work on leatherwood has found that the space between adjacent cell walls, known as the middle lamella, is deficient in lignin even after cell wall lignification has concluded, despite the middle lamellae in most other vascular plant species being highly enriched in lignin (Mottiar et al., 2020). This thesis expands upon that finding by determining through histochemical staining, autofluorescence in confocal microscopy, and transmission electron microscopy, that leatherwood is almost completely devoid of lignin in the middle lamella. Histochemical staining and laser excitation were also used to investigate lignin deficiencies in other leatherwood tissues: root, leaf petiole, and peduncle, the small stem that supports flowers and developing fruit. To provide some context for the lignin patterns in leatherwood, both leatherwood and a distant relative, Daphne mezereum, commonly known as daphne, were studied in this work. The results indicate that lignin deficiencies in the middle lamella of fibres may be unique to the Dirca genus as they do not occur in daphne and, therefore, are not necessarily present throughout the entire Thymelaeaceae family. Research was also done with another important macromolecule, pectin, to assess if leatherwood might also have a unique distribution of pectin. Immunostaining revealed that pectin in leatherwood is found in the middle lamella, the same area where lignin is deficient. This new information highlights the need for more research, especially molecular studies, regarding the spatiotemporal relationship between pectin and lignin biosynthesis. Further work is needed to elucidate the underlying genetic factors for leatherwood’s unique lignin patterning, and potentially unveil pectin’s role in the initiation of lignification.
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(2024)Clonal hematopoiesis is characterized by the accumulation of blood cells originating from a single mutated hematopoietic stem cell clone. Clonal hematopoiesis has been hypothesized to contribute to systemic inflammation, and by doing so increase the risk for inflammatory diseases. Despite the growing body of evidence from clinical datasets and murine levels linking clonal hematopoiesis and inflammation, the causative role of clonal hematopoiesis in promoting inflammation remains to be fully elucidated. The primary goal of this master's thesis is to investigate the potential causative relationship between clonal hematopoiesis (CH) and rheumatoid arthritis, one of the most common autoimmune diseases previously associated with clonal hematopoiesis. The analysis is done primarily by employing Mendelian randomization (MR) and multivariate logistic regression analyses to perform two-sample Mendelian randomization in a bidirectional manner to assess the strength of the analysis along with exploring potential pleiotropy. We leverage data from two large population-level cohorts: the FinnGen study and the UK Biobank project. We also employed multivariate logistic regression to validate the instrumental variables of the UK Biobank within the FinnGen data. Despite prior epidemiological and functional literature linking clonal hematopoiesis and rheumatoid arthritis, we observed no association between these two in these large-scale Mendelian randomization analyses. This may be due to differences in cohort characteristics and detection methods for clonal hematopoiesis between the two cohorts. The thesis discusses the strengths and weaknesses of Mendelian randomization in evaluating causative links between human phenotypes. This work contributes to the growing area of research on the genetic determinants of inflammatory diseases and opens avenues for further research into the role of CH in disease pathogenesis
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(2024)The Finnish National Core Curriculum for Primary and Lower Secondary Education mandates equal treatment of all students and emphasizes the importance of increasing visibility for the appreciation of sex, gender, and sexual diversity. Despite the growing discourse on the mental health of Finnish adolescents, less attention has been paid to gender and sexual minorities, who face more bullying, loneliness, and school exhaustion than their peers, resulting in even poorer mental health. This has largely been explained by the prejudices and heteronormativity that remain widely prevalent, even within schools. Science subjects have been criticized for enforcing these heteronormative narratives through their practices. However, studies suggest that science subjects, especially biology, have the potential to challenge these narratives and foster a more inclusive and safer space for sex, gender, and sexual minorities. In this study, I aim to explore the experiences of lower secondary school students regarding the teaching of sex, gender, and sexual diversity in biology classes. Despite that the educational material used in science subjects has been fairly well studied, there is a gap in previous research regarding the actual experiences of students about these issues, and there are no studies done on this in Finland. Employing a mixed methods approach, this study integrated both semi-structured interviews, an online questionnaire, and observation of lessons. The results indicate that the perceived coverage of sex, gender, and sexual diversity is minimal or brief. Student engagement during lessons where these topics could be discussed was also shown to be minimal, and students raised several wishes for improving the teaching of these topics in biology. These findings highlight the unused potential that remains in the teaching of biology and present students’ wishes for change in the ways diversity issues could be covered in biology to create more inclusive science classrooms.
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