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(2020)Rakkohauru (Fucus vesiculosus) on ruskeisiin leviin kuuluva makrolevä, joka kasvaa Atlantin valtameressä laajalla alueella Grönlannista Portugaliin asti. Rakkohauru on valtamerissä vuorovesivyöhykkeen laji, joka sietää laajaa suolaisuuden ja lämpötilan vaihtelua. Itämeressä rakkohauru kasvaa kilpailun vähäisyyden vuoksi sublitoraalissa vyöhykkeessä jopa kymmenen metrin syvyyteen asti ja on tärkeä elinympäristöjä muodostava laji. Rakkohaurua pidetään useimmiten kovien pohjien lajina, joka kiinnittyy alustaansa tyvilevyn avulla. Rakkohaurusta tunnetaan kuitenkin myös toinen tyyppi, joka elää pehmeillä hiekkapohjilla tai suistoalueilla eikä ole kiinnittynyt tyvilevyllä pohjaan. Tässä työssä selvitettiin, ovatko kiinnittyneet ja vapaana elävät rakkohaurut geneettisesti erilaisia, mistä vapaana kasvavat yksilöt ovat peräisin ja voivatko ne lisääntyä itsenäisesti. Suomen ja Ruotsin rannikoilta kerättiin kymmenestä eri paikasta sekä vapaana kasvavia että kiinnittyneitä rakkohaurunäytteitä, joita verrattiin toisiinsa. Keräyspaikat sijaitsivat yhden, muutaman tai satojen kilometrien päässä toisistaan. Kerätyistä näytteistä eristettiin viisi DNA-mikrosatelliittimarkkeria, joiden perusteella tehtiin geneettiset analyysit. Tutkimustuloksista selvisi, että Suomen ja Ruotsin rakkohaurupopulaatioiden välillä ei ollut tilastollisesti merkittävää geneettistä eroa eivätkä populaatioiden väliset geneettiset erot riippuneet niiden välisestä etäisyydestä. Niinpä kysymykseen siitä, mistä vapaana kasvavat populaatiot ovat lähtöisin, ei saatu vastausta. Sen sijaan vapaana kasvavat ja kiinnittyneet rakkohaurun muodot erosivat toisistaan geneettisesti. Tämä viittaa siihen, että vapaana kasvavat populaatiot lisääntyvät ainakin osittain itsenäisesti. On mahdollista, että tämä johtaa ajan kuluessa vapaana kasvavien ja kiinnittyneiden rakkohaurun muotojen kehittymiseen omiksi lajeikseen, mutta tällä hetkellä se ei vaikuta kovin todennäköiseltä. Koska tutkitut populaatiot olivat pieniä ja käytettyjen mikrosatelliittien määrä vähäinen, lisätutkimukset ovat tarpeen tulosten luotettavuuden varmistamiseksi.
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(2020)Tekstiiliteollisuudessa käytetään merkittäviä määriä kemikaaleja, joista osa on ympäristölle ja terveydelle haitallisia. Tekstiiliteollisuuskehityksen kasvaessa ja kulutuskäytäntöjen kiihtyessä pois heitettävän tekstiilin määrä lisääntyy. Samalla haitalliset kemikaalit vapautuvat saastuttamaan ihmistä ja ympäristöä. Tämä käy ilmi aiemmasta tutkimuksesta, vaikka poistotekstiilien ympärillä tehty tutkimus on muutoin vähäistä. Tämä tutkimus tehdään yritysyhteistyössä Lounais-Suomen Jätehuolto Oy:n kanssa määrittämään poistotekstiilien lajittelutyön yhteydessä ilmeneviä terveysvaikutuksia ja sovellettavia testimenetelmiä yrityksen tarpeisiin. Tutkielman tavoitteena oli ensisijaisesti selvittää kemikaalien, pölyn ja homeen terveysvaikutukset ja ne testimenetelmät, joita tulisi terveysvaikutusten vähentämiseksi tehdä. Muiden tutkimuskysymysten osalta tarkoituksena oli selvittää poistotekstiilien sisältämät merkittävimmät haitalliset kemikaalit ja niiden enimmäisraja-arvot. Näitä tutkittiin tutkimuksen teoreettisessa viitekehyksessä. Tutkimuksessa käytettiin laadullista tutkimussuuntausta ja sen menetelmiä. Aineiston keruumenetelmänä oli asiantuntijahaastattelu, jossa tietoja kerättiin puolistrukturoidun kysymyksenasettelun kautta neljältä asiantuntijataholta: Turun ammattikorkeakoululta, Terveyden- ja hyvinvoinninlaitokselta, Työterveyslaitokselta ja Aalto-yliopistolta. Haastattelut toteutettiin puhelinhaastatteluina. Aineistoa analysoitiin temaattisen sisällönanalyysin avulla, jossa aineisto luokiteltiin tutkimuksen kannalta merkittävimpiin teemoihin. Saatuja tuloksia verrattiin olemassa olevaan tutkimuskirjallisuuteen. Hengitystievälitteinen altistuminen yksistään pölylle nähtiin merkittävimmäksi terveysriskiksi laitosolosuhteissa. Kemikaalien, pölyn ja homeen aiheuttamien terveysvaikutusten vähentämiseksi soveltuvimpina testimenetelminä nähtiin erilaisten keräävien menetelmien käyttö ja biomonitorointi. On selvää, että poistotekstiilit sisältävät ihmisen terveydelle ja ympäristölle haitallisia kemikaaleja, pölyä ja homeita. Testimenetelmiä hyödyntämällä voidaan terveysriskejä paikantaa ja niiden osuutta työperäisessä altistumisessa vähentää. Tutkimustulosten tieteellisen luotettavuuden lisäämiseksi on suositeltavaa, että laitoksella otetaan käyttöön kokeelliset testausmenetelmät.
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(2020)In reductive dehalogenation, halogenated compounds act as electron acceptors in respiration or cometabolic reactions. The halogen atom is removed and usually replaced with hydrogen. Polybrominated diphenyl ethers (PBDEs) are persistent and toxic compounds, which have been extensively used as flame retardants. Even after bans and use restrictions, they are released to the environment from products manufactured earlier. PBDEs have been found ubiquitously in the environment. In anaerobic conditions like in sediments, they can be degraded through reductive dehalogenation. While remediating contaminated sediments, knowledge is needed about factors affecting the degradation of PBDEs. In the present work, anaerobic debromination of two PBDEs, BDE-47 and BDE-209 in sediment was investigated in microcosms. Sediment samples were taken from the estuary of the Kymijoki River from the Baltic, and from two sites in Lake Kernaalanjärvi, one representing clean and the other contaminated sediment. In addition to sampling site, the effect of incubation temperature on the degradation rate and products was investigated with different treatments, and possible abiotic degradation was examined with autoclaved controls. The samples were analyzed with gas chromatograph with electron capture detector (GC-µECD). The amounts of Dehalococcoides and total bacteria as 16S rRNA gene copies were determined with qPCR. Hypotheses were that debromination is faster in contaminated sediments, in warmer temperature, and in the microcosms with more bacteria of the genus Dehalococcoides. The half-time of BDE-47 varied from 173 to 7701 weeks, and that of BDE-209 from 28 to 173 weeks. The debromination rate could not be explained with sampling sites or incubation temperature, but the degradation of BDE-47 was significantly slower in autoclaved microcosms. In the microcosms where BDE-47 was removed most, the main debromination product was BDE-8. In the microcosms with sediment from the clean site of Lake Kernaalanjärvi, the amount of BDE-47 did not decrease. Obviously, bacteria capable of degrading it are not abundant in sites with little or no previous contamination with organohalogens. The removal of BDE-209 in autoclaved microcosms is probably explained with abiotic degradation or adsorption so that the compound is not extractable. At the end of incubation, there were more total bacteria in the microcosms with BDE-47 than those with BDE-209. The proportion of Dehalococcoides was greater in the microcosms with BDE-209, those incubated in room temperature, and those not autoclaved. Debromination was faster in the microcosms with more total bacteria in the beginning or Dehalococcoides at the end. BDE-47 proved to be more persistent than in the literature reviewed. The hypotheses about the effect of the incubation temperature and the contamination of the sediment did not reach statistical significance, but the importance of Dehalococcoides for debromination was established according to the hypothesis.
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(2015)Ihmistoiminta muuttaa elinympäristöjä maailmanlaajuisesti. Vaikutukset näkyvät niin yksittäisten lajien populaatioissa, lajien välisissä vuorovaikutuksissa kuin kokonaisten ekosysteemien toiminnassakin. Monimutkaisuutensa ja vaikean havainnoitavuutensa takia lajien väliset vuorovaikutukset ja erityisesti niiden yhteys ekosysteemien toimintaan tunnetaan osin huonosti. Ympäristömuutoksista ilmaston lämpeneminen vaikuttaa erityisen voimakkaasti arktisella alueella. Muuttuva ja yksinkertainen arktinen eliöyhteisö tarjoaakin loistavan malliekosysteemin lajien välisten vuorovaikutusten havainnoimiselle ja niiden rakenteen mallintamiselle. Tässä tutkielmassa selvitän pölyttäjäyhteisön rakennetta ja toimintaa yhden kasvilajin, lapinvuokon Dryas octopetala × integrifolia, pölytyksessä. Lapinvuokko on runsain kukkakasvi monilla pohjoisilla alueilla, ja sitä voidaankin pitää avainlajina paikallisissa pölytysyhteisöissä. Liittääkseni pölyttäjäyhteisön rakenteen lapinvuokon siementuotannon onnistumiseen havainnoin lapinvuokon pölyttäjäyhteisön vaihtelua tilassa ja ajassa. Tähän käytin kukanmuotoisia liimapahvista valmistettuja pyydyksiä. Tein liimakukkapyyntejä 21 koealalla Zackenbergin laaksossa Grönlannissa. Tutkin koealoilla lisäksi lapinvuokon siementuotantoa. Tutkimuksessani havaitsin, että lapinvuokon kukinnan ajoittumisessa on suurta alueellista vaihtelua. Ensimmäiset kukat puhkeavat heti pälvien ilmestyessä ja viimeisten yksilöiden kukinta alkaa vasta juuri ennen ensilumia. Myös lapinvuokon siementuotannon onnistuminen vaihtelee paljon (12,7 - 52,7 % kukista) alueellisesti. Alueellinen vaihtelu siementuotannon onnistumisessa johtuu osittain ympäristöolosuhteista, mutta myös pölyttäjäyhteisöllä on selvä vaikutuksensa. Lapinvuokolla tapahtuu itse- ja tuulipölytystä (8,6 % onnistuneesta siementuotannosta), mutta pölyttäjien läsnä ollessa siementuotanto on moninkertainen (33,7 %). Tutkimusalueellani Zackenbergissa maailmanlaajuisesti tärkeimmät pölyttäjät, mesipistiäiset, puuttuvat lähes tyystin. Runsaimpien pölyttäjien paikan ovat vallanneet kärpäset erityisen runsaslukuisten sukaskärpästen johdolla. Havaintojeni perusteella sukaskärpästen runsaus selittääkin parhaiten lapinvuokon siementuotannon onnistumista. Kesän edetessä havaitsin pölyttäjäyhteisön kasvavan huomattavasti sekä yksilö- että lajimäärältään. Pölyttäjäyhteisön runsastuminen kasvukauden edetessä antaa olettaa, että lapinvuokon pölytys voi parantua ainakin lyhyellä aikavälillä. Ilmaston lämmetessä pölyttäjien esiintymisen on havaittu liukuvan aikaisemmaksi kasvien kukintaa nopeammin. Näin ollen aikaisin kukkivat kasvit kohtaavat yhä enemmän pölyttäjiä ja myöhäisimmätkin kasvit onnistuvat tuottamaan siemeniä ennen syksyä, kun niiden kukinta siirtyy absoluuttisesti aikaisemmaksi. Toisaalta sukaskärpästen määrän on havaittu vähenevän ilmaston lämmetessä. Tutkimukseni pohjalta sukaskärpäset ovat lapinvuokon tärkeimpiä pölyttäjiä, ja jos niiden väheneminen jatkuu, on lapinvuokon pölytys Zackenbergissa uhattuna. Kaiken kaikkiaan tutkimukseni viittaa siihen, että lapinvuokon pölytys voi lyhyellä aikavälillä parantua, mutta pitkällä aikavälillä muutokset pölyttäjäyhteisössä uhkaavat lapinvuokon pölytyksen onnistumista. Lapinvuokko on arktisen alueen tärkeimpiä kasveja niin pölyttäjien kuin monien muidenkin hyönteisten ravintona. Siten muutokset sen menestyksessä heijastunevat koko pölyttäjien ja kukkakasvien muodostamaan yhteisöön.
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(2021)Ilmastonmuutos koettelee ankarimmin planeettamme pohjoisia alueita. Näiden alueiden eliöyhteisöt ovat ennestään erityisen herkkiä ympäristön muutoksille. Vaikka arktisen ympäristön fysikaaliset muutokset ovat jo monin paikoin nähtävillä, ovat tiedot näiden muutosten vaikutuksista alueen eliöyhteisöihin ja ekologisiin vuorovaikutussuhteisiin edelleen hyvin puutteelliset. Pohjoisten keväiden aikaistuminen altistaa yhteisöt fenologiselle irtautumiselle, jossa läheisessä ekologisessa vuorovaikutuksessa olevat lajit ajautuvat esiintymisaikojensa suhteen erilleen. Pölyttäjähyönteisen sekä pölytettävän kukkakasvin tapauksessa tästä voi olla seurauksena pölytystehon väheneminen. Tämän ja ilmastomuutoksen muiden seurausten ennustamiseksi käytetään usein nk. ajan ja tilan rinnastus –menetelmää. Menetelmän oletuksena on, että eliöt reagoivat ympäristötekijöihin samalla tavalla, tapahtuivatpa ne sitten ajallisesti tai paikallisesti. Selvittääkseni, onko arktisella alueella havaittavissa fenologista irtautumista, tarkastelen lapinvuokon sekä lajin pölyttäjäyhteisöön kuuluvan 14 hyönteisheimon fenologiaa Koillis-Grönlannissa. Tarkastellakseni, miten ilmaston vaihtelu vuosien välillä kuvastuu hyönteisaktiviteetin ajoittumiseen ja lajiryhmien väliseen vaihteluun, hyödynnän kahdenkymmenen vuoden aikasarjaa (jatkossa: aikasarja-aineisto). Selvittääkseni, miten ilmaston vaihtelu tilassa heijastuu paikallisiin eroihin pölyttäjäaktiviteetin fenologiaan, seuraan hyönteisaktiviteetin ajoittumisen yhden kasvukauden aikana maaston eri osissa (jatkossa: tila-aineisto). Molemmista aineistoista selvitän, mikä ympäristötekijä määrittää voimakkaimmin pölyttäjäyhteisön fenologiaa, keskittyen lumenlähtöpäivään ja lämpösummaan. Lopuksi arvioin, kuinka hyvin ajallinen ja paikallinen ulottuvuus vastaavat toisiaan. Näin pyrin selvittämään sitä, kuinka käyttökelpoinen ajan ja tilan rinnastus –menetelmä on pohjoisten pölyttäjäyhteisöjen tutkimuksessa. Totesin lumenlähtöpäivän selittävän lapinvuokon pölyttäjäyhteisön fenologiaa kokonaisuutena parhaiten. Tämä pätee sekä tila- että aikasarja-aineistossa. Lumenlähtöpäivän ja lämpösumman vaihdellessa eri lajiryhmien ajoitus muuttuu eri tavoin. Muutokset tilassa ja ajassa olivat lapinvuokon tärkeimpien pölyttäjäryhmien kohdalla suhteellisen yhteneväiset, mutta eräiden muiden ryhmien (esim. kukkakärpästen) kohdalla ja samalla koko yhteisön tasolla ennusteet olivat heikompia. Tulokseni osoittavat, että ilmaston vaihdellessa arktisissa yhteisöissä on nähtävissä fenologista irtautumista. Yhteisön sisällä eri lajiryhmät reagoivat sekä lumenlähtöpäivään että lämpösummaan eri tavoin. Tämä altistaa yhteisöjä fenologiselle irtautumiselle ympäristön edelleen muuttuessa. Pölyttäjäfenologiaa tutkittaessa ajan ja tilan rinnastus -menetelmä näyttää toimivan hyvin lapinvuokon ja tämän kasvilajin tärkeimpien pölyttäjäheimojen kohdalla. Koko yhteisön tasolla rinnastuksen ennusteet ovat kuitenkin puutteelliset, sillä joidenkin ryhmien ennusteet eroavat toisistaan merkittävästi sekä ajassa että tilassa. Tulevaisuudessa tulisikin selvittää tarkemmin sitä, kuinka nyt havaittu pölyttäjäyhteisön fenologinen epäyhtenäisyys heijastuu lajien välisiin vuorovaikutussuhteisiin eri trofiatasojen välillä.
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(2021)Gliomas are the most common malignant brain tumours. The most aggressive and lethal type of glioma is glioblastoma. It has a dismal prognosis, and, despite aggressive treatment, the average patient survival is 1-2 years. Although glioblastoma has a heavy impact on individuals and their families, as well as on healthcare systems, our current lack of mechanistic knowledge hinders the development of improved treatments and diagnostics. Recent studies showed that glutaminolysis, a metabolic pathway utilizing glutamine to produce α-ketoglutarate, is promoted in tumour cells, suggesting a significant role of α-ketoglutarate concentration in tumour progression. Therefore, I hypothesise that reduction of α-ketoglutarate concentration in glioblastoma might suppress glioblastoma aggressiveness. To address this hypothesis, I focus on another metabolic pathway controlling α-ketoglutarate concentration, namely the GABA metabolism. Here, I show that the expression of ABAT and GAD1, which encode rate-limiting enzymes of the GABA metabolism, is associated with the lower-grade of glioma and a better prognosis for patients. Interestingly the expression of ABAT and GAD1 negatively correlates with the expression of CD109, a glioma stemness marker. Furthermore, suppression of glioblastoma stemness by CD109 silencing induces ABAT and GAD1 expression. Taken together these results suggest that the upregulation of the GABA metabolism reduces glioblastoma stemness and proliferation. In future, I am planning to examine the effect of ABAT and GAD1 overexpression and knockdown on glioblastoma stemness and proliferation, as well as the underlying molecular mechanisms to understand how the GABA metabolism suppresses the glioblastoma progression.
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(2019)In recent years, the two-spotted field cricket Gryllus bimaculatus has emerged as a central model for studies on insect development, regeneration and physiology. At the moment, G.bimaculatus has the most extensive molecular toolkit within the Exopterygota, making it the foremost model for evolutionary developmental biology and comparative physiology within the field of entomology. However, the postembryonic development of G. bimaculatus has received considerably less attention than embryonic development. In this thesis, I have studied the postembryonic development of G. bimaculatus to better understand the evolution and physiology of the understudied Exopterygota. My thesis encompasses five parts: postembryonic morphology, wing development, appendage regeneration, allometry, and growth. The postembryonic stages, the nymphal stages, have never been properly characterised in G. bimaculatus. By following postembryonic development daily at 30 C, 8 nymphal stages (instars) were identified. Size, coloration, sclerotisation of the thorax, and morphology of the wings, the hind tibia and the ovipositor were useful characters in distinguishing the stages. The Dpp/BMP signalling pathway patterns the wing venation in the endopterygotan insects Drosophila melanogaster and Athaliae rosae, but nothing is virtually known about wing development in exopterygotan insects. The wings and the wing venation pattern in different nymphal stages of G. bimaculatus were studied using the hydrogen peroxide clearing protocol along with both brightfield and fluorescence microscopy, while the role of the Dpp/BMP signalling pathway was studied using immunohistochemistry (IHC), in situ hybridisation (ISH), and RNA interference (RNAi). The longitudinal veins are patterned in the 3rd and 4th nymphal stages, while the secondary veins in the 8th stage. The IHC and ISH experiments displayed only non-specific staining, while the RNAi experiments did not produce any change in the phenotype, possibly because of molecular redundancy. The nymphal legs of G. bimaculatus are known to be highly regenerative, and the Dpp/BMP signalling pathway has been shown to provide positional information in leg regeneration. However, nothing is known about the regeneration of the other appendages in G. bimaculatus. Antennae and cerci were amputated in different nymphal stages, and the degree of final regeneration depended on the nymphal stage. RNAi experiments did not produce any change in the phenotype, possibly because of molecular redundancy. The interrelationship between static, ontogenetic and evolutionary allometry in insects is poorly understood. The allometry of hind femur length with respect to body length has been shown to be negative in Orthoptera (i.e. evolutionary allometry), but nothing is known about corresponding ontogenetic and static allometry. By measuring hind femur length and body length in G. bimaculatus in different nymphal stages, the ontogenetic allometry was determined to be slightly positive or isometric, while the static allometries of different stages tended to be negative but highly variable. This may indicate that allometric relationships constrain development in the microevolutionary perspective, but are nevertheless evolvable in a macroevolutionary perspective of millions of years. The growth conditions and rearing of crickets and other insects have been widely reported, but the shape of the growth curve itself has been less investigated. The exponential, the von Bertalanffy (VBGF), the West, Brown and Enquist (WBE), and the dynamic energy budget (DBE) models have been proposed as continuous models for insect growth. These models were t to growth data from G. bimaculatus and the DBE and was shown to be optimal with parameter values α=0 and pAm = 0.69. The insects have been thought to follow Dyar's law, i.e. that the growth ratio or moulting increment (MI) is constant throughout development, although numerous other competing moulting models have been devised for the crustaceans. By fitting different moulting models to head width data from G. bimaculatus, the log-linear model (Mauchline's model) turned out to explain the MI the best. Lastly, the oxygen-dependent induction of moulting (ODIM) model has been proposed to explain moulting patterns in insects, but the model has never been applied to exopterygotan taxa. By fitting the ODIM model to growth data from G. bimaculatus, the model could predict moulting mass but not instar durations, probably because of high postembryonic plasticity in G. bimaculatus.
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(2018)Mitochondrial dysfunction is involved in many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). Most of the mitochondrial disorders lack effective treatments so far. One of the important defects of mitochondrial dysfunction is the blockade of mitochondrial respiratory chain. An alternative oxidase (AOX), originated from Ciona intestinalis, has been found to counteract this defect by providing a bypath of the mitochondrial respiratory chain in plants, drosophila, mice and human cultured cells. Therefore, we suggest that AOX might be a promising tool for curing neurodegenerative diseases from the aspect of alleviating mitochondrial defects. An engineered mouse model has been created to express the Ciona intestinalis AOX ubiquitously in the whole animal. Our study aims at investigating the expression and function of AOX in the transgenic mouse brain and exploring therapeutic potential of AOX in human diseases. AFG3l2 gene encodes a subunit of m-AAA metalloprotease, which is involved in mitochondrial protein quality control process. Mutations in the AFG3L2 is known to be related to spinocerebellar ataxias 28 (SCA28), a neurodegenerative disease resulting from cerebellar damage. Loss of AFG3L2 gene causes OPA1 protein cleavage and mitochondrial fragmentation which are easily detected. Thus, we propose an in vitro neurodegenerative disease model generated by knocking down of AFG3L2 in target cells. We used western blotting to detect the expression level of AOX in the brain and primary neuro-glia cells of the transgenic mice, finding that the AOX protein reduces in the brain along with age. Antimycin A treatment on neuro-glia cells demonstrated the function of AOX in transgenic pups. However, the respiratory activity caused by AOX also decreases with the mice age. In addition, we constructed a lentiviral vector with DNA recombinant technology to express AOX in U2OS cells. To examine the function of AOX under disease condition, we established an in vitro neurodegenerative disease model by knocking down the AFG3L2 gene of U2OS cells with siRNA, and infected the cells with AOX lentivirus. By comparing the immunostaining images of wild-type and AOX infected cells, we saw a significant difference in the degree of mitochondrial fragmentation between these cells. Nevertheless, the cleavage pattern of OPA1 protein, which is mainly responsible for mitochondrial fragmentation, remains unchanged whether AOX is present in the cell or not. In conclusion, our study first investigated the expression and function of AOX in the brain of this transgenic mouse model. And we established a lentivirus vector to apply AOX gene to human cells, testing the therapeutic function of AOX in a neurodegenerative disease model in vitro. Moreover, the mitochondrial fragmentation of the disease model is alleviated by AOX despite of OPA1 cleavage, indicating that the mitochondrial phenotype caused by loss of AFG3L2 is not only due to OPA1 cleavage, but also related to respiratory defects. This highlights the possibility to rescue the mitochondrial morphological changes from new aspects.
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(2020)Female breast cancer incidence rate has been growing world-wide and it is the most common cancer in women. In the battle against the breast cancer mortality, early detection is the strongest tool. This is the reason why national mammography screening programs are widely established for selected age groups, usually for women between 50 to 70 years old. Although it is well established that these programs save lives, mammography screening is not feasible to apply to younger and/or older groups due to increasing radiation load as well as economically. There is a room for a non-invasive, easy and cost-effective breast imaging modality, which could be used for all age groups e.g. in connection with regular health checks. In this theses breast cancer as a disease and its present clinical diagnostic tools are presented. As a possible new pre-diagnostic tool, infrared imaging combined with modern analytical and machine learning tools, is introduced. Also, preliminary results of an on-going study are presented, which encourage to continue the development.
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(2023)Forests play a key role in mitigating climate change and maintaining biodiversity. The ability of forests to sequester carbon dioxide and store it in wood biomasses and soils reduces the amount of carbon dioxide in the atmosphere. However, forests are cut down for various reasons, such as economic ones, releasing carbon dioxide from the trees into the atmosphere. One way to combat climate change and biodiversity loss is to provide forest owners a voluntary incentive scheme that provides a new objective for forest owners to use their forests. The use of forests in the future will be decided by the new forest owners, i.e., the younger generation of forest owners. For this reason, it’s important to study their preferences for a voluntary incentive scheme. The study examined the characteristics of absentee and non-absentee forest owners under the age of 40 and compared the preferences for a voluntary incentive scheme directed to these two groups. The survey response rate was low (7,3%). Choice experiment method and a binary logistic regression model was used to find out the forest owners’ preferences in relation to accept the programme. In the binary logistic regression model, absentee residence was the only factor that positively and reliably increases the acceptance of the programme. Absentee and non-absentee forest owners appreciate similar aspects of forest property, for example relaxation and financial security. The biggest difference between these two groups came with consideration of forestry work and/or wood for household as nonabsentee forest owners valued that aspect higher. The study points out that knowledge of a METSO programme is relatively low, as 35% of absentee and 23% of non-absentee forest owners who responded have heard of the programme. Some young forest owners may not have a clear idea on how to manage forests, because forest ownership is new to them and therefore it is often difficult for novice forest owners to know which forest management practices to apply in one's own forests. According to the study, economic income from the forest property is important, but it isn’t an important source of economic security for a large number of young forest owners, especially when the share of absentee forest owners’ characteristics and preferences are on the rise among forest owners.
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(2020)Endocytosis is the process responsible for internalising membrane components and as such plays a key role in the biology of this structure. Mammalian cells have evolved various endocytic strategies, but Clathrin-Mediated Endocytosis (CME) is the most common type. Since the discovery of CME, around 50 years ago, the field has built a remarkable wealth of knowledge on the core CME components. In stark contrast, our understanding on the relationship between CME and the actin cytoskeleton, which is present throughout the process, is still in its infancy. In this thesis, I show the production and characterisation of recombinant, SpyCatcher tagged transferrin (TF), a canonical CME ligand. TF was expressed in E. coli and using an optimised protocol, successfully solubilised and refolded from inclusion bodies. The protein was then labelled with a fluorophore and purified to a high level of purity. Tests in mammalian cells showed that home-made TF has the same endocytic behaviour as TF purified from human plasma. Moreover, I could show that the SpyCatcher moiety attached to our home-made TF is capable to mediate its covalent linkage to its counterpart SpyTag. The successful production, refolding and functional characterization of recombinant TF in this study is an important first step to examine the participation of the actin cytoskeleton during CME.
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(2012)In multicellular organisms, complex signalling mechanisms have evolved to guide the behaviour of individual cells. Growth factors are secreted proteins that can stimulate the proliferation and/or differentiation of cells. Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 2 (VEGFR-2) and for VEGFR-3, which are predominantly expressed on blood vascular endothelial cells and on lymphatic endothelial cells, respectively. Thus VEGF-D can contribute to growth of both blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis). Although there have been many reports showing the angiogenic and lymphangiogenic effects of VEGF-D, its physiological role is still largely unknown. Most of these reports are severely hampered by incomplete characterization of the specific form of VEGF-D that was used. During or after secretion, VEGF-D undergoes complicated proteolytic processing. Alternative Nterminal cleavage results in two different fully processed forms, VEGF-D major and VEGF-D minor. Processing significantly increases the activity of VEGF-D towards its receptors. Surprisingly, it is still unknown whether the differential N-terminal cleavage of VEGF-D has any effect on receptor binding activity or on receptor activation. The goal of this study was to produce and purify high quality biologically active VEGF-D which is needed for studying the physiological role of this growth factor. Several different forms of recombinant human VEGF-D were produced using the Drosophila Schneider 2 insect cell system. A bioassay utilizing the Ba/F3 cells expressing chimeric VEGFR/EpoR receptors was used to determine the receptor binding activities of recombinant VEGF-Ds. Two constructs producing biologically active VEGF-Ds were chosen for chromatographic purification (untagged major and his-tagged major forms). During purification, the activity of both VEGF-D forms towards their receptors decreased significantly. In case of the untagged form, this was presumably due to some residual proteolytic activity during purifications. The results might indicate that only the major form is responsible for the activation of VEGFR-3. The fact that no activity of the minor forms was detected when screening the cell supernatants with Ba/F3-VEGFR-3-EpoR-bioassay, supports this explanation. If this explanation can be verified, the role of the alternative N-terminal cleavage becomes obvious: By proteolysis the activity of VEGF-D can be redirected from the lymphatics towards the blood vessels.
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(2023)The discovery and development of new antifungal drugs has lagged behind the clinical needs for effective treatments of fungal infections. Invasive fungal infections can be challenging to treat and can become life-threatening, particularly for immunosuppressed individuals. Despite the need for new and improved antifungal drugs, the pipeline for antifungal drug development has been relatively slow, with only a few new agents being approved in recent years. Many existing antifungal drugs have toxic side effects, limiting their use and highlighting the need for more targeted and effective therapeutics. The glycosylphosphatidylinositol biosynthesis pathway has been proposed as a potential new target for antifungal drugs. The glycosylphosphatidylinositol (GPI) anchor is a complex glycoconjugate that is attached to many proteins found on the surface of eukaryotic cells. GPI anchored proteins play important roles in various cellular processes, including signaling, cell adhesion, and cell recognition. The biosynthesis of GPI anchors involves multiple enzymatic steps, including the transfer of the GPI anchor to a target protein. Gpi3 is one of the key enzymes involved in the first step of GPI biosynthesis and is the catalytic subunit of the GPI GlcNAc-PI synthesis complex. The naturally occurring molecule Jawsamycin has been shown to selectively inhibit fungal Gpi3 while not interfering with its human ortholog. However, the development and research of Jawsamycin and other potential inhibitors of the GPI synthesis pathway are hampered by the lack of structural data on the proteins involved in the pathway. This thesis aimed to express and purify functionally active Gpi3 as a recombinant fusion protein using the SUMO tag expression system, with the end goal of utilizing the protein for structural studies through crystallography to better understand the function of Jawsamycin. In this thesis, Gpi3 was successfully expressed and purified as a fusion protein. However, enzymatic activity of Gpi3 was not observed, additionally, the purification and stability of the Gpi3 fusion proteins were shown to be problematic and no crystal structure of the protein of interest was acquired. These results indicate that a different approach is needed to gain structural insights into the function and interaction between Gpi3 and Jawsamycin. A likely path forward is the purification of the whole GPI GlcNAc synthesis complex which could give more insights into the organization and function of both Gpi3 and Jawsamycin.
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(2018)One open question related to microplastics found in seas is the sources and routes of microplastics into seas. In Helsinki about one third of the snow collected from urban areas is dumped directly into the Baltic sea. When snow is collected from an urban area also the possible waste in the snow becomes collected. This is the first study that examines the numerical quantity and the characteristics of waste in urban snow. In order to study waste contained in snow, samples were collected from three different urban areas in Helsinki in February 2016. Snow was collected from Hakaniemen tori, Kullervonkatu in Käpylä and Sörnäisten rantatie. Solid matter in snow samples was divided into particles sized over 4 mm and 0.3–4 mm. Waste particles were visually identified utilizing the morphology of the particles. Material of selected waste particles was analysed using a FTIR spectrometer. Snow samples collected from Käpylä contained more waste by weight and number than samples collected from other areas. Reasons for differences between sampling areas did not become clear in this study. The most common waste in urban snow were particles disengaged from asphalt pavement and road markings, along with paper, plastic and rubber waste. All snow samples contained microplastics. Urban snow contained more microplastics than purified wastewater leaving from Viikinmäki wastewater treatment plant and seawater in the Gulf of Finland. Stormwaters have been identified as a route for microplastics from urban areas into inland waters and seas, and according to this study also dumping of urban snow into seas acts as a route for microplastics. In urban snow road marking and plastic particles were the most common microplastics. In snow samples collected from road areas road marking particles were clearly the most common microplastic by number. In snow collected from Hakaniemen tori about half of the microplastics were plastic particles and almost a half road marking particles. Most of the microplastics in snow were different kinds of fragments. Like in earlier microplastics studies also in urban snow the most common materials of small plastic particles were polyethylene and polypropylene. In this study sources of the waste in snow are estimated to be the abrasion of asphalt pavement, road markings, tyres, plastic products and possibly also wood and metal products and different kinds of textiles. In addition to that numerous waste items had ended up in urban areas through littering or inadequate waste disposal. Littering is the most significant environmental impact of dumping urban snow into the sea in Helsinki. Marine Strategy Framework Directive states that properties and quantities of marine litter are not to cause harm to the coastal and marine environment. According to the guidelines of the City of Helsinki, snow that is brought to snow collection sites needs to be clean and not to contain waste. Nevertheless, according to this study through dumping snow into the sea in Hernesaaren ranta several thousand kilograms of waste and approximately over fifty million microplastic particles end up in the Baltic Sea. A monitoring program should be established to investigate and monitor the amount and the characteristics of waste in urban snow in Helsinki.
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(2020)Jätevesiliete sisältää arvokkaita kasviravinteita ja orgaanista ainesta, jotka olisi välttämätöntä saada kiertämään kiertotalouden periaatteiden mukaisesti. Erityisesti kasvien kasvun kannalta olennainen ja määrältään rajallinen fosfori olisi tärkeä ohjata uudelleen käyttöön, esimerkiksi soveltuvaan maatalouteen, sillä Suomessa maatalouteen käytetään vuosittain 95,5 % kaikesta fosforista. Puhdistamoliete on kuitenkin yksi yhteiskunnan haastavimmista sivuvirroista, sillä se sisältää myös haitallisia aineita, kuten raskasmetalleja, mikromuoveja, pysyviä orgaanisia yhdisteitä ja lääkejäämiä. Tässä opinnäytetyössä aihetta tarkastellaan kirjallisuuskatsauksen ja dokumenttianalyysin keinoin sekä vahvistetaan laadullisella aineistolla. Viime vuosina monet suuret suomalaiset viljanostajat ovat kieltäneet puhdistamolietepohjaisten lannoitetuotteiden käytön sopimuspelloillaan. Tutkielmassa kartoitettiin asiantuntijahaastatteluiden avulla, miten tähän lopputulokseen päädyttiin sekä millainen suhtautuminen yrityksissä on uusiin kehitteillä oleviin puhdistusteknologioihin. Tutkimusta varten haastateltiin kolmen yrityksen edustajia tammikuussa 2020. Vastaukset analysoitiin sisällön analyysin keinoin. Puhdistamoliete ja sen käyttö on aineiston perusteella osa ympäristökonfliktien historiallista jatkumoa. Nykytutkimuksen valossa suomalainen puhdistamoliete on käsiteltynä turvallista käyttää. Useista tutkimuksista huolimatta kaikkien haitallisten aineiden pitkäaikaisia vaikutuksia erityisesti maaperään ei ole pystytty täysin kartoittamaan, sillä osa lietteestä löydettävistä haitallisista aineista on yhteiskunnassamme suhteellisen uusia yhdisteitä. Lietepohjaisten tuotteiden loppukäyttö on yhteiskunnallinen, taloudellinen ja ympäristökysymys, josta eri toimijoilla on eri intressit ja vaihtelevat näkemykset. Tutkimuksen perusteella viljanostajat suhtautuvat lietepohjaisiin tuotteisiin kielteisesti niiden kyseenalaisen maineen vuoksi. Yritykset korostivat, että heidän omat asiakkaansa eivät hyväksy lietepohjaisten tuotteiden käyttöä. Lisäksi haastatteluissa nousi ilmi huoli lietteen tutkimuksen tietoaukoista, koetut ihmisperäisten kierrätysravinteiden imagohaitat, sekä yritysten vastuullisuuden painopisteen sijoittuminen muualle kuin kierrätysravinteisiin. Pro gradu tutkielmassani esitän, että lietteen käsittelyn uudet teknologiat eivät tule yksin ratkaisemaan lietepohjaisten tuotteiden käyttöä maataloudessa, sillä viljanostajien kiinnostus kierrätysravinteiden käyttöön on vähäistä ja teknologiset erot käsittelymetodien välillä jäävät helposti epäselviksi.
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(2014)Sewage treatment plants produce circa 150 000 dry tons of sewage sludge in Finland each year. Sewage sludge contains a great amount of organic matter and nutrients but also a wide variety of harmful substances such as persistent organic pollutants and pharmaceuticals. Sewage sludge is waste material that must be properly disposed of. While more efforts are made to close nutrient cycles, the use of sewage sludge in agriculture has become a more attractive option also in Finland. In this study it was assessed whether or not organic contaminants present in sewage sludge may prove to be a problem for sludge applications in Finnish agriculture. The study was performed using mathematical models and data collected from literature. 34 different organic contaminants were selected and their accumulation into agricultural soil was simulated assuming that sludge was applied yearly. In addition, leaching was simulated for seven of the compounds. The simulated concentrations in soil and drain water were compared to predicted no effect concentrations (PNEC) given for each compound. Compound-specific data on each substances' behavior in soil environment and concentrations in sewage sludge was collected from literature. Because the data given in different sources varied, simulations were run for each compound using combinations of different values. For each compound 3–23 simulations were run. Simulations were performed with two models developed for simulating pesticide behavior in agricultural soils, PECsoil-calculator and MACRO 5.2. Finnish data on weather and soil properties were used in the simulations. For many compounds the simulated concentrations in soil and water were in the range of concentrations measured in field trials. According to the simulations, the most problematic compounds present in sludge are triclosan, 17α-ethinylestradiol, ibuprofen and carbamazepine. Triclosan and 17α-ethinylestradiol were the only compounds that exceeded their PNECsoil-values. Persistent organic pollutants such as PFAS- and PBDE-compounds accumulated into the soil very efficiently but did not exceed their PNECsoil-values. Leaching to sub-surface drains and below them was a significant end-point for carbamazepine and ibuprofen. These compounds did not exceed their PNECwater-values. However, the highest yearly average concentration in drain water simulated for ibuprofen exceeded the environmental quality standard proposed for it. The peak concentration in drain water simulated for triclosan was circa 70 times higher than its PNECwater-value. The compound-specific values used in the simulations contained a lot of variation. This increases the uncertainty of the results. However, the simulations demonstrate that based on current knowledge the agricultural use of sewage sludge may in some extreme cases cause harm for the environment.
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(2007)Tutkielmassa on tarkasteltu asiointimatkoja tekevien pyöräilijöiden kokemuksia ja reitin valintaa Helsingissä. Tutkimus on tehty haastattelemalla kuutta pyöräilijää ja analysoimalla haastatteluja fenomenologisella metodilla. Metodin tuloksena syntyi teksimuotoinen merkitysverkosto, johon on yhdistetty haastateltavien kuvaamat pyöräilykokemukset. Antropologi Tim Ingoldin "asumisen näkökulmaa" soveltaen kokemuksien on oletettu syntyvän jatkuvassa kehollisessa vuorovaikutuksessa ympäristön kanssa, ja ympäristölle annettujen merkityksien muotoutuvan toiminnallisessa suhteessa ympäristöön. Analyysin tuloksia on tulkittu myös ympäristöestetiikkaa, kehollisuutta, orientaatiota, liikenneturvallisuutta ja kaupunkisosiologiaa käsittelevän kirjallisuuden perusteella. Pyöräilijöiden kokemuksista voi löytää yhteisiä rakenteita. Asennoitumisen lisäksi pyöräilijä voi vaikuttaa omaan kokemukseensa reitin valinnalla. Pyöräilijän reitin valintaan vaikuttaa haastattelujen perusteella seitsemän tekijää: reitin tuttuus pyöräilijälle, kaupungin liikenteen normit ja säännöt, reitin tehokkuus, muun liikenteen laatu ja määrä, esteettiset arvot, reitin selkeys sekä ajoradan pinta. Esteettisiä kokemuksia pyöräilijät hakevat luonnon läsnäolosta, rauhasta, sujuvasta kulusta tai urbaanista ympäristöstä. Liikenteessä tärkeitä tekijöitä ovat sen ennustettavuus ja yhdenmukaisuus pyöräilijän oman liikkeen kanssa, liikenteen nopeus sekä raskaan liikenteen läsnäolo. Näiden paikkaan sidottujen kokemusten lisäksi pyöräilijän kokemuksessa olevia elementtejä ovat turvallisuuden tunne ja pelko, jota voi hallita erilaisin keinoin, muun liikenteen toimien ennakointi muun muassa liikkeitä lukemalla ja kuuntelemalla sekä kaupunkitilan käyttöön liittyvät konfliktit. Kaupungissa pyöräily on erilaisten taitojen joukko, johon kuuluu reittien tuntemus. Pyöräilijä löytää reittinsä aiemman kokemuksen, kokeilun ja ympäristön vihjeiden avulla. Kartta toimii apuna reitin suunnittelussa ja eksymistapauksissa. Kehollisuus tarjoaa pyöräilijän kokemukseen laaja-alaisen tulkinnan välineen, jonka avulla voi ymmärtää pyöräilijän kokemuksia, käyttäytymistä liikenteessä ja reitin valintaa. Kehollisuus tarjoaa myös tavan ymmärtää merkitysten syntyä ihmisen ja ympäristön välisessä toiminnallisessa vuorovaikutuksessa.
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(2020)In many Fennoscandian lakes, diatoms account for majority of phytoplankton biomass during the spring and autumn maximum. Autumnal blooms of filament-forming diatoms are known to cause fishing net fouling in several boreal lakes. Observations of such net sliming diatom blooms in the northern part of Lake Vesijärvi have increased during 2010’s. This thesis was done in collaboration with Lake Vesijärvi Foundation. The goal was to determine the starting time of the autumnal diatom bloom of 2018 and reveal the species that causes net sliming. Additionally, differences in water quality and phytoplankton composition between Enonselkä and Kajaanselkä basins were examined. Fishing net sliming was studied with netting experiments. Their results were complemented with water quality sampling and observing water column mixing conditions. Local fishermen reported net sliming to begin at week 40 (starting 1.10.2018) in Kajaanselkä basin during autumnal turnover. Results of netting experiments and water quality sampling revealed simultaneous increase in diatom abundance in the area, although no visible sliming occurred during sampling. Based on fishers’ reports and sampling results, fishing net sliming was caused by filament-forming diatom Aulacoseira islandica, that increased in abundance as water temperature reached ca. 11 °C. In Enonselkä, no similar net sliming was observed, and phytoplankton was dominated by cyanobacteria Planktothrix agardhii. Both basins of the lake are affected by eutrophication, but Enonselkä basin more so than Kajaanselkä. Although no clear explanation for differing phytoplankton composition between the two basins was discovered, it’s possible that Aulacoseira islandica receives competitive edge over cyanobacteria in mildly eutrophic waters of Kajaanselkä basin. The fact that Planktothrix agardhii seems to thrive in higher nutrient concentrations might explain its domination over diatoms in phytoplankton community of Enonselkä basin. Future research on net sliming would ideally be conducted as a combination of sliming accounting done by local fishermen and spatially and temporally more comprehensive netting experiments. Furthermore, studying the effects of wind and currents to diatom distribution in lakes could provide useful information for sampling setup.
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(2020)Human induced pluripotent stem cells (hiPSC) can be propagated in a long-term culture and further differentiated into many cell types, including cardiomyocytes (CM) and endothelial cells (EC). Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are promising tools in cardiac research, since they retain the original genotype of the individual donor and thus enable the use of patient- and disease specific cells. Crucial for the optimal use of hiPSC-CMs in experiments are methods for assessing cardiomyocyte phenotype. Contraction is a prominent feature for CMs, and it is essential that contraction can be quantified accurately. Reliable quantification is relevant when hiPSC-CMs are used for studying disease phenotypes, cardiac safety pharmacology, genotype-phenotype correlations, cardiac disease mechanisms and cardiac function over time. In this thesis project, contractile behavior of hiPSC-CMs was analyzed using video microscopy and online tool MUSCLEMOTION. Contraction parameters were obtained from hiPSC-CMs derived from patients with hypoplastic left heart syndrome (HLHS) and healthy controls on multiple timepoints during differentiation. In addition, contraction was analyzed in iPSC-CMs cocultured with induced pluripotent stem cell derived endothelial cells (iPSC-ECs), since it has been suggested that ECs can promote morphological and functional maturation of CMs in culture. Contraction duration (CD), time to peak (TTP), relaxation time (RT) and contraction amplitude (CA) was compared between different timepoints as well as between CMs cocultured with ECs and CMs cultured alone. Compared to control cell lines, HLHS patient hiPSC-CMs exhibited longer CD, TTP and RT as well as higher CA values. This difference was present in most of the timepoints, suggesting slower contractile kinetics in HLHS patient iPSC-CMs compared to control iPSC-CMs. Significant changes were also observed in contraction parameters when comparing hiPSC-CMs in coculture and monoculture. Contraction parameters of coculture iPSC-CMs changed in a relatively consistent manner over time, increasing or decreasing throughout the monitoring period whereas in hiPSC-CM monoculture there was more variation between timepoints. This project and results support the use of modern methods in detailed functional characterization of hiPSC-derived cells. In addition, it highlights the potential of coculture in disease modeling and the fact that hiPSC-CMs express variation in phenotypes. However, experiments should be repeated, and additional methods should be used in order to further validate the results and conclusions.
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(2021)Head and neck squamous cell carcinoma (HNSCC) is a diverse group of cancers defined by their localization in the head and neck region. These cancers are recognized for the heterogeneity between tumors from separate patients (inter-patient heterogeneity) as well as between cell types within individual tumors (intra-tumoral heterogeneity). Heterogeneity poses a major clinical challenge by making accurate diagnosis and selection of treatment options difficult. This study aims to improve precision of prognosis, quantify heterogeneity in HNSCC, and address its functional implications using two approaches: (1) profiling a set of HNSCC patient tumors using multiplexed immunohistochemistry and single-cell computational methods to identify a set of phenotypic descriptors correlating with differences in survival; and (2) using patient-derived cancer cell lines to investigate which cellular features correlate with relevant functional properties such as plasticity, invasiveness, clonogenicity and tumorsphere-forming abilities of cells. Epithelial-mesenchymal transition (EMT) as well as excistence of stem cell like states have been implicated in cancer aggressiveness and poor outcomes. We thus focused on identification of putative EMT, partial EMT (pEMT) and stem cell-like states. Based on a combination of morphometric analyses and stem cell- and EMT marker profiling, our computational method assigned patients into groups with different survival probabilities, and these patients’ tumors were found to differ in their expression of the stem cell transcription factor Sox2, the EMT transcription factor Slug, and in their morphometric parameters. Functional studies of patient-derived cell lines found that significant differences exist in protein expression, morphological features and cell behaviours between cell lines in vitro, and that inhibiting EMT promotes clonogenicity and can increase Sox2 expression. Thus, this study highlights important heterogeneous patient phenotypes and cellular behaviours in HNSCC, and implicates the need for a multimodal approach to diagnosis and therapy of this cancer.
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