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(2017)Trichoderma reesei, an anamorph of Hypocrea jecorina, is a filamentous fungus widely used for producing industrial enzymes. T. reesei is used for both endogenous and heterogenous protein production. The optimization of the production conditions and the effects of extracellular agents to T. reesei s production and secretion capacity are crucial for economically sustainable biotechnical production. The available carbon sources, most commonly different types of sugars, have a significant effect on the production and secretion of enzymes by T. reesei. Genetic modification of the pathways through which the fungi recognizes extracellular signals could bring advancements to industrial enzyme production. Because of T. reesei s potential and use as a production strain, the species is an interesting platform for genetic modifications that would enhance the production capacities. With the current methods the genome editing of T. reesei is however slow, and introducing multiple mutations to a single strain can take years. The aim of this study is to optimize the fairly new CRISPR/Cas9 genome editing system for use in T. reesei. In the CRISPR/Cas9 method, a catalytically active Cas9 enzyme is bound to a specific locus of the genome, guided by a guide RNA and the Watson-Crick base pairing principle. Once in the RNA-guided locus, Cas9 introduces a double stranded break in the DNA, which can be repaired by the cells endogenous non-homologous end joining pathways. This repair is error prone and produces mutations to site of the double stranded break. A donor DNA is often introduced together with the Cas9 and guide RNA. This donor DNA includes sequence homology to the site of interest and allows for the use of the cells homologous repair pathways. In this case, the mutation can be better controlled, and for example the risk of chromosomal mutations is reduced. Currently the CRISPR/Cas9 system is widely used in mammalian cell studies and up to 100% mutation frequencies have been reported in yeast cells. In this study the method is optimized for use in T. reesei. To our best knowledge, the research community has not found an organism in which CRISPR/Cas9 would not function. The question mainly lies on what type of set up and component introduction is suitable for each cell type and research purpose. In this thesis, three putative and one already published genes believed to be involved in hexose sugar sensing will be deleted from a T. reesei production strain with the help of CRISPR/Cas9. The effect of these deletions will be assessed through studying the secretion and activity of endogenous cellulases with enzymatic assays. One sugar transporter that may play a part in glucose sensing was identified in this study. The deletion of this transporter caused a decrease in cellulase production and/or secretion. The three other transporters or sensors did not have a significant effect on cellulase production in spent grain extract and lactose or glucose media. It s possible that these genes are involved in the uptake and use of other carbon sources. The continuous expression of the CRISPR/Cas9 system in T. reesei proved difficult. In the continuous expression method at least one of the CRISPR/Cas9 components, the Cas9 protein or the guide RNA, is produced in the cells in vivo. Neither was achieved in this study. Instead, a fully synthetic method in which the Cas9 is transformed into the cells as a protein along with an in vitro produced guide RNA was set up and produced up to 1000 × higher mutation frequencies when compared to the traditional transformation method used for T. reesei. This study also demonstrates a simultaneous deletion of two genes in T. reesei. To the best of our knowledge, multiple simultaneous gene modifications have never been achieved in T. reesei.
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(2024)Background: The infant gut microbiome undergoes major temporal changes in the first year of life, crucial for supporting normal development and long-term health. The immense diversity of fiber structures in breast milk and later in solid foods pose unique selection pressures on the gut microbiome maturation by providing novel substrates for the microbiota. However, the longitudinal impact of complementary food-derived fibers on the taxonomic and functional maturation of the gut microbiome during the gradual transition from breast milk to solid foods is not well understood. Objectives: My objective was to examine how breast milk, its fiber and complementary food fibers in the broader context of overall infant diet may affect the gut microbiome bacterial species composition and support age-appropriate gut bacterial maturation trajectories during first year of life. Methods: Longitudinal and cross-sectional development of 68 infant gut microbiomes and 33 metabolomes were examined with linear mixed models to determine the impact of infant nutrition on gut microbiome taxa and functional development. Nutrition assessments were based on detailed quantitative weighted 3-day food records (months 3,6,9,12) and the intakes of total dietary fiber with its food sources and fiber fractions relied on current internationally approved CODEX-compliant values. Questionnaires were utilized to monitor when various complementary foods were introduced, enabling more comprehensive nutritional analyses. Bacterial species identification was based on MetaPhlAn2 quantification of bacterial species from metagenomic data and metabolomic profiles were generated using four liquid chromatography-mass spectrometry (LC-MS) methods. Results: My examinations place the previously described sequential trajectories in infant gut microbiome maturation into detailed fiber-dependent nutritional context relying on metagenomic species identification. I discovered 176 complementary food derived fiber-bacterial species associations. The majority of the associations (147, 84%) were positive whereas breastfeeding and related variables tended to be inversely associated with the same species, showing strongest inverse correlations to later trajectory species indicative of slower maturation. Both bacterial species and metabolomic profiles displayed pronounced longitudinal shifts in response to solid food fibers. Each introduction of novel dietary source of fiber associated to diversification of the microbiome revealing fiber-species specific temporal patterns. Conclusions: The longitudinal analyses highlight that sufficient fiber intake from appropriate sources during the weaning period likely function to build capacity for the species permanence in the more diverse and stable mature gut microbiome composition and function reached in later childhood.
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(2019)The Baltic ringed (Pusa hispida botnica) and grey seal (Halichoerus grypus) populations have experienced dramatic changes in their abundances since the early 20th century, when their populations were much larger than today but since then have declined due to over exploitation and reproductive challenges linked to environmental pollutants. Both populations have however, begun to recover, and their numbers have increased since the 1970s. This increase has led to more seal-inflicted damages to coastal fisheries resulting in the demand to control their populations. In Finland, fishermen have reported significant economic losses, and many consider seals as the main threat to their livelihood. However, our knowledge on the diet composition and foraging behaviour of Baltic ringed and grey seals in Finnish sea area is lacking. In order to achieve sustainable seal management, more information on their diet is thus needed. Therefore, to shed light on the diet composition of Baltic seals in Finland, I examined the stomach contents from 156 ringed and 73 grey seals collected in 2017 across the Finnish sea area. Furthermore, I analysed dietary differences between demographic factors (i.e. age and sex), and seals from different geographic regions. A total of 15 prey taxa, of which 13 fish species or groups were identified. Ringed seal diet was dominated by benthic isopod Saduria entomon that was recovered from over half of the stomachs. In addition to Saduria entomon, herring (Clupea harengus) were the most important fish species consumed. Other important prey were gobies (Gobiidae), smelt (Osmerus eperlanus) and common whitefish (Coregonus lavaretus). In terms of biomass, common whitefish became the most important prey whereas in numbers gobies dominated the diet. For grey seals, herring were the most common and numerous prey consumed that made up most of their diet. Other common species were sprat (Sprattus sprattus) and smelt. Other prey did not contribute substantially to grey seal diet. Additionally, the results of this study showed differences in diet composition between seals of different age and sex.
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(2020)The Miocene epoch (c. 23-5 million years ago) was a noteworthy geological time period in which significant changes took place both in the climate regimes as well as in vegetation characteristics, bringing about novel adaptations in many herbivorous lineages. These adaptations constituted morphological, dietary, and ecological factors in a relatively short period of evolutionary time. Among these herbivores were the proboscideans, the living and extinct elephants, which were among the most dominant and largest herbivores at the time. Despite that proboscideans were diverse and large group of hundreds of species, yet the understanding of dietary and ecological patterns of majority of Miocene sympatric species is still limited. The aim of this study was to analyse the molar surfaces of Miocene proboscideans (e.g. Deinotherium and Gomphotherium) from Eurasia to provide a reconstruction of the feeding preferences of the study species based on observed dental wear. The dental wear indicates the abrasiveness of the diet, thus allowing broad categorization to either browser (<10% grass in the diet), mixed-feeder (10-90% grass in the diet) or grazer (>90% grass in the diet). Secondly, this study aimed for providing estimation of the environmental characteristic and vegetation patterns of the study localities by comparing to the previous studies and to hypsodonty value (proxy of general openness and aridity of the environment). Proboscidean dietary signals from the key localities of Maragheh (Iran) and Pannonian basin (Austria) were compared with the paleobotanical studies. Thus, the general estimation of spatial and temporal variation of the environment characteristics in the study localities were based on these parameters. The materials of fossilized molars were analysed by mesowear angle method, in which the measured angles show the diet abrasiveness due the nutritional targets’ differences. The results allowed the reconstruction of the feeding preferences which suggested that majority of Miocene proboscideans were browsers and browse-dominated mixed feeders or pure mixed-feeders. Instead, Choerolophodon pentelici was found grass-dominant mixed-feeder. The wide spectrum of feeding preferences allowed diet flexibility according available vegetation and also these sympatric species to co-exist by niche partitioning. Thus, demonstrating clearly the connection between diets and environments thought the diet. As a conclusion, in the diet of the paleocommunities of proboscideans had, on average, more grass-dominant components in open and dry environment likely due the presence of grass-dominant vegetation. Instead, in the wet conditions the closed-canopy forest environments enhanced browsing. Further, the results indicated shift in feeding preferences of proboscideans prior to Miocene climate and environment changes. These results are in line with the findings of the previous studies of modern elephants’ diet- environment relationships. The further studies would provide insight to the relative amounts of the grass in the diet of Miocene proboscideans.
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(2022)Cyanobacteria are an important part of the phytoplankton community and aquatic ecosystems. Cyanobacteria can form large mass occurrences, i.e. blooms, which can be toxic or cause other harm. Research and monitoring of cyanobacteria has been based on microscopy analysis. However, molecular-based methods, such as 16S rRNA sequencing are replacing microscopy analyses in the near future. The Finnish Environment Institute has stated that molecular methods are part of environmental monitoring before 2030. In this Master’s thesis the aim was to determine whether conventional microscopy analyses and 16S rRNA sequencing differ when comparing nano- and micro-sized cyanobacteria. The material was collected from a laboratory experiment of the Finnish Environment Institute’s (SYKE) MiDAS project, which was conducted in the summer of 2020. The results of the microscopy and 16S rRNA analyses differed from each other. The relative abundances of the cyanobacteria genera differed between sample types. Microscopy analyses estimated that the alpha diversity was higher compared to the results of the sequencing analyses. The main reason for the difference between the types of analyses was due to the differences in cyanobacteria belonging to the order of Synechococcales. Some of the Synechococcales species were observed only by the sequencing analyses, e.g. Snowella and some of the Synechococcales species were only observed by the microscopy analyses, e.g. Romeria and Woronichinia. It was observed that both methods are prone to identification errors. The differences between the 16S rRNA sequencing and the microscopy analyses are vastly different. It may affect on the review of long-term data of the phytoplankton community. Therefore, it is important to examine the differences between the types of analyses. Studying the dissimilarities between the types of analyses should be focused on the research of the small cell-sized colonial cyanobacteria, i.e. the species of Chroococcales and Synechococcales.
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(2022)Kv7.1 is a potassium ion channel comprised of the KCNQ1 protein, which can coassemble with distinct β-subunits modulating the channel functions in different tissues. In 2017, Raivio’s group (from the University of Helsinki) found two missense mutations in the KCNQ1 gene, p.(Arg116Leu) and p.(Pro369Leu), responsible for causing pituitary hormone deficiency and maternally inherited gingival fibromatosis. The facial features and bone structure pointed to a cranial neural crest (CNC)-derived phenotype caused by an alteration in the potassium channel balance, given that these cells form the bone and cartilage of the cranial zone. To understand the implication of the CNC in the KCNQ1 syndrome, I attempted to replicate the CNC differentiation protocol of Suga and Furue (2019) with the aim of obtaining cranial neural crest cells (CNCCs). This would enable future generation of a KCNQ1-related disease model. The differentiation process was carried out thrice, and two BMP4 concentrations (10 and 100 ng/ml) were assayed. The differentiated cells exhibited a CNC-like morphology as well as upregulation of the marker genes (TFAP2A, SOX10, DLX1, MSX1, and DLX2) associated to this cell lineage. However, the gene expression was low according to the qRT-PCR Ct values, which were in most cases higher than 30. Additionally, no differences were found between the two BMP4 treatments. Furthermore, the cells did not express KCNQ1, and thus the impact of the two KCNQ1 mutations was not investigated under this protocol. In conclusion, the protocol had a low efficiency in the generation of CNCCs that was not improved by increasing the BMP4 concentration. Further optimization of the protocol, such as the BMP4 concentration or the cell density of the culture, will be needed to improve its efficiency and obtain an adequate disease model.
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(2022)Digitaalisia teknologioita hyödynnetään lisääntyvästi kansalaisosallistumisen vahvistamisessa sekä vahvan ja osallistuvan demokratian toteuttamisessa. Tämän niin sanotun e-osallistumisen tai digitaalisen osallistumisen päämääränä on aktivoida kansalaisia ja madaltaa osallistumiskynnystä sekä rohkaista heitä keskusteluun julkisen hallinnon kanssa. Teknologiset ratkaisut lupaavat tiiviimmän ja reaaliaikaisenkin keskusteluyhteyden hallinnon ja kansalaisten välillä. E-osallistuminen koetaan myös ratkaisuna maaseuduille tyypillisten niukkenevien resurssien ja pitkien etäisyyksien haasteisiin. Kiinnostus e-osallistumiseen on tutkimusten mukaan kuitenkin vähäistä. Osallistumista tukevia teknologisia ratkaisuja olisi tarjolla, mutta niiden hyödyntämiseen ei olla innostuttu kunnissa. Tässä tutkimuksessa kartoitetaan, kuinka kuntaorganisaation viranhaltijat kokevat kuntalaisten osallistumisen sekä millaisia haasteita ja mahdollisuuksia he liittävät e-osallistumisen teknologisiin ratkaisuihin. Tutkimusaineisto koostuu 11 teemahaastattelusta, jotka kerättiin Mikkelin kaupungin eri palvelualueita edustavilta viranhaltijoilta. Haastattelupuhe on sosiaalisesti rakentunutta ja aineistoa tarkasteltiin teoriasidonnaisesti kehysanalyyttisella otteella. Analyysissa selvitettiin keskeiset osallistumiseen liittyvät kehystämistavat viranhaltijoiden näkökulmasta. Haastateltavat puhuivat osallistumisesta velvollisuuksien kehyksessä ja käytäntöjen kehyksessä. Vaikka osallistumista pidettiin tärkeänä, ristivetoa esiintyi velvollisuuksien (kuntalaki ja kuntalaisten osallistumismahdollisuudet) ja käytäntöjen (nykyiset toimintakulttuurit ja niukat resurssit) välillä. Edustuksellisen päätöksenteon ja kuntalaisten osallistumisen suhde osoittautui kitkaiseksi. Myös kuntalaisten näkökulmaa ja mielekkäitä osallistumisen tapoja olisi tulosten mukaan kehitettävä. Lisäksi kuntaorganisaatioiden nykyiset toimintakulttuurit kaipaavat osallistumisen huomioon ottavaa uudistamista ja riittävän resursoinnin varmistamisen.
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(2020)As biodiversity is being lost worldwide at an accelerating rate due to anthropogenic activities, the frequency and severity of many infectious diseases has been observed to increase. Together these patterns have brought forth an urgent need to understand the possible linkages between biodiversity and disease risk. Two contradicting hypotheses have been proposed to explain the diversity-disease relationship. The dilution effect hypothesis suggests that increasing host community species diversity ‘dilutes’ disease risk, whereas the amplification effect hypothesis predicts disease risk to increase with increasing diversity. Even though most of the studies support the dilution effect, there remains an intensive debate regarding the generality of this effect. As most of the research efforts to understand the relationship between diversity and disease have focused on animals and crop plants or have been carried out experimentally, one of the research gaps is how relevant the dilution effect is in wild plant communities. In nature, plants and their diseases are affected simultaneously by multiple abiotic and biotic environmental factors that might confound or supersede the effects of diversity. It is also poorly understood, whether we might expect dilution effects to occur not only on diversity gradients driven by anthropogenic diversity loss, but also on natural diversity gradients. To study the possible association between host community species diversity and disease risk in the wild and to test whether this association could be detected after accounting for the effects of abiotic factors, I surveyed grassland vascular plant communities for their species diversity and foliar disease symptoms along a natural diversity gradient driven by elevation. I also recorded data on the mean soil surface temperature in the surveyed plant communities and used structural equation modelling to differentiate and compare the effects of biotic and abiotic variables on disease risk. The data were collected on Mount Calanda in the Swiss Alps during summer 2019. In this thesis I show that host community species diversity and disease risk are negatively associated with each other along a natural diversity gradient driven by elevation. Furthermore, this negative effect can be detected even after accounting for the effects of elevation and mean soil surface temperature on disease. Together the results support the occurrence and the ecological relevance of the dilution effect in wild plant communities along natural diversity gradients and suggest that diversity might protect wild plant communities from increased disease risk. Future studies should aim to identify the exact mechanisms of the association to help us better understand when and where we might expect dilution effects to occur in the wild. This knowledge can be used to predict how epidemics, that affect the well-being of ecosystems, humans and wildlife, are born in the changing world.
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(2023)Macrozooplankton is an understudied size class of plankton in the Arctic, but even though species composition is similar around the Svalbard archipelago, the relative composition can act as a proxy for climate change. This study investigated if the fjords around Svalbard are similar in species composition and if any differences could be explained. 11 fjords were sampled with a focus on common and indicator species in terms of abundance, relative composition and length distribution. I found low presence of T.libellula and high abundances of T.abyssorum and T.inermis. T.abyssorum was the only species whos abundance was significantly correlated to water mass (Atlantic water), but T.libellula, T.abyssorum, T.inermis, T.longicaudata and A.digitale all showed significant differences in length distribution. This study provides further understanding of species composition in the different high Arctic fjords around Svalbard.
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(2023)Morphological features are considered as markers of microglial functionality, and they show regional heterogeneity in the brain. Recently the sleep-wake cycle was shown to affect microglial morphology in mice and correlate with cortical sleep slow wave activity (SWA). Microglial sizes and ramification increased during the dark period and decreased during the light period in cerebral areas associated with SWA, suggesting that neuronal activation could be affecting microglial morphology through SWA. I studied microglia in the hindbrain areas with and without functional connection to SWA to further investigate the association between SWA and alterations in morphology, and to investigate if there are differences in microglial morphology and their diurnal alterations in brain regions other than those commonly investigated. I examined three hindbrain areas (cerebellar cortex (CC), deep cerebellar nucleus (DCN) and medial vestibular nucleus (MVN)) and somatosensory cortex (SC) of mice (n=15) at two timepoints: 6 hours after the light onset (high SWA) and offset (low SWA). My aims were to answer if there are morphological differences in microglia between 1) the four brain areas at both timepoints and 2) between the two timepoints in each brain area. My hypotheses were that CC and DCN which have functional connections to cortical SWA, would show similar diurnal morphology alterations as demonstrated in the cerebral areas, and MVN that has no known cortical SWA connection, would lack significant alterations. As microglia are heterogenous throughout brain, I expected microglia to differ between different brain areas, especially the hindbrain and the SC. I found that microglial morphologies significantly differed between the hindbrain and the cortex, while the hindbrain areas were more similar in morphology. Moreover, the brain areas demonstrated diurnal morphology alterations of microglia with varying extent: CC and DCN microglial morphology did not correlate with SWA as clearly as SC did, and interestingly, morphological features of MVN microglia showed a pattern opposite to other areas, microglia being larger during the light period than the dark period. These results highlight the importance of the diurnal time to microglial morphology and the heterogeneity of microglia between different brain regions.
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(2020)The diversity of different neuronal types lays the foundation for different functions in the brain. The development of different subpopulations and special features of neurons in the central nervous system are still partly unknown. Finding answers to these developmental issues could help in the process of characterisation of cell types and mapping of neuronal networks between the brainstem nuclei in the brain. Previous studies have shown that a ventrolateral neuroepithelial domain in the anterior hindbrain, rV2, produces excitatory (glutamatergic) and inhibitory (GABAergic) neurons, which are related to monoaminergic nuclei in the brainstem (Lahti et al., 2016). In this master’s thesis project, the development of a subpopulation of neurons expressing Gsc2 transcription factor in the interpeduncular nucleus was studied. This project was based on single-cell RNA sequencing results conducted in E13.5 mice. Predicted by single-cell RNA sequencing results, Gsc2 expressing cells are GABAergic interneurons and originate from the rV2 domain of the rhombomere 1 region in the hindbrain. Co-expression pattern with another transcription factor Sall3 with Gsc2 during development was also addressed in the study. Furthermore, the role of Notch signalling in the binary cell fate decision between GABAergic and the glutamatergic fate of rV2 neurons was investigated. Validation of single-cell RNA sequencing results was performed using in situ hybridisation and immunohistochemistry methods with mice embryos at the age of E12.5 and E15.5. This study verified previously shown origin of Gsc2 expressing cells to the rhombomere 1 region and in addition, showed that Gsc2 expressing cells are GABAergic. Co-expression pattern of Gsc2 with Sall3 neither in the rV2 domain nor in the interpeduncular nucleus was seen in our results. In the rV2 domain, the depletion of Notch signalling decreased the expression of differentiating GABAergic neurons. This indicates that Notch has a role in GABAergic neurotransmitter identity during the development of brainstem neurons in mice. Based on our results, Gsc2 could be used as a lineage marker for GABAergic interneurons originating from the rhombomere 1 region and as a marker for a subpopulation of the interpeduncular nucleus. Furthermore, results from the role of Notch signalling could help in discovering the mechanisms related to the determination of neurotransmitter identity in rV2 neurons. Further investigations, in different developmental time points and with additional markers, are needed to verify these results.
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(2019)Background- Colorectal cancer (CRC) is the third most common epithelial carcinoma. There is an increased risk of colorectal cancer in people with longstanding inflammation in the large intestine, including individuals with ulcerative colitis (UC). Epigenetic changes in CRC such as aberrant DNA methylation alterations are common changes in human cancer. The aim of this study is to identify the DNA methylation alterations of selected inflammation related genes in UC-CRC vs. Lynch syndrome (LS). Method- DNA was extracted from archival tissue specimens from normal and tumor samples from UC-CRC (n= 31), and LS-CRC (n=29). Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) assays were used to detect CIMP status and CpG promoter methylation status of seven inflammation related genes. Microsatellite instability analysis was carried out using two mononucleotide repeat markers BAT25 and BAT26. Results- Increased hypermethylation frequencies in carcinoma vs. normal colonic mucosa were detected for all the inflammatory marker genes in specimens of UC-CRC patients. Statistically significant differences for methylation frequencies were observed in the NTSR1 gene (p value =0.008) and SOCS2 gene (p value =0.04) in specimens of UC-CRC patients. NTSR1 gene showed significantly increased methylation of normal colonic mucosae from UC-CRC vs. LS patients (p value=0.01). Conclusion- UC-CRC and LS tumor specimens revealed varying frequencies of hypermethylation in all the inflammatory genes. Methylation of the NTSR1 in the normal colonic mucosa suggests a possible field defect in UC-CRC, and could thus be used as an early biomarker to detect increased UC-CRC risk in non-neoplastic epithelium.
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(2011)Pro gradu tutkielmani aiheena oli tutkia koirien (Canis familiaris) saalistushalukkuutta ja persoonallisuutta sekä näiden mahdollista yhteyttä dopamiinireseptori D4 –geeniin (myöh. DRD4-geeni). DRD4-geeni ilmenee lähinnä hermosoluissa ja se vaikuttaa erityisesti aivojen limbisen järjestelmän toimintaan. Limbinen järjestelmä säätelee tunnetiloja ja tunteisiin liittyviä autonomisia toimintoja sekä motivaatiota eli samoja asioita, joihin dopamiinin tiedetään vaikuttavan. Dopamiini vaikuttaa elimistöön sitoutumalla esimerkiksi aivojen limbisen alueen dopamiinireseptoreihin. Dopamiinin on havaittu liittyvän assosiatiiviseen oppimiseen ja DRD4-geenin puolestaan mm. aktiivisuuteen ja impulsiivisuuteen sekä uteliaisuuteen mistä syystä se valittiinkin kandidaattigeeniksemme tutkimukseen. Koira puolestaan sopii erityisen hyvin mallilajiksi kandidaattigeenitutkimukseen, koska koirarodut ovat geneettisiä isolaatteja ja eroavat toisistaan niin rakenteensa kuin käyttäytymisensäkin puolesta. Kytkentäepätasapaino eli alleeliassosiaatio on koirissa huomattavasti voimakkaampaa kuin ihmisissä ja siten koiralta on merkittävästi helpompaa löytää mm. tautigeenejä. Koirilla kytkentäepätasapaino yltää noin 2 Mb laajuudelle, kun vastaava luku ihmisellä on vain noin 0,28 Mb. Tutkimuksen 405 suursnautserin ja saksanpaimenkoiran saalistushalukkuus testattiin koiran luonteenkuvauksesta eli MH-testistä tutulla viehetestillä. Viehetestissä koiran edestä yllättäen "pakenee" saalista muistuttava mutkittelevasti kulkeva kankainen viehe. Koirien suoritukset viehetestissä videoitiin ja suoritukset luokiteltiin neljään luokkaan äärimmäisen saalistushalukkaasta saalistushaluttomaan. Muita persoonallisuuspiirteitä tutkittiin koirien omistajien täyttämien luonnelomakkeiden avulla. Kaikista viehetestillä testatuista koirista otettiin verinäytteet, joista eristettiin DNA. Saalistustestissä äärimmäisen saalistushalukkaat ja täysin saalistushaluttomat saksanpaimenkoirat (N=44) valittiin DRD4-geenin alleeliassosiaatiotutkimukseen. Tutkittavaa geenialuetta monistettiin käyttämällä alleelispesifejä alukkeita PCR-reaktiossa. Tutkimuksen koirista löydettiin DRD4-geenin alleeleja 2 ja 3a. Luonnelomakeaineiston taustalta paljastui faktorianalyysin avulla neljä faktoria: sosiaalisuus ihmisiä kohtaan, leikkisyys ja aktiivisuus, aggressiivisuus koiria kohtaan sekä rohkeus. Näistä ensimmäinen assosioitui merkittävästi alleelin 3a kanssa. Koirat jotka kantoivat tätä alleelia olivat vähemmän aggressiivisia ihmisiä kohtaan kuin koirat, joilla 3a-alleelia ei ollut. Saalistushalukkuuteen kumpikaan alleeleista eikä mikään genotyypeistä assosioitunut. Saalistushalukkuutta kokonaisaineistossa selitti parhaiten leikkisyys ja aktiivisuus. Rodut erosivat toisistaan ihmisiin ja koiriin suuntautuvalta aggressiivisuudeltaan siten, että saksanpaimenkoirat olivat suursnautsereita aggressiivisempia.
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(2005)1-Bentsyylipiperatsiini (BZP) on viime vuosina laittomille markkinoille ilmaantunut ns. design-huume, jonka on todettu muistuttavan vaikutuksiltaan amfetamiinia. Sen myynti tapahtuu tyypillisesti tanssiklubeilla ja Internetin välityksellä. Esimerkiksi Yhdysvalloissa BZP on luokiteltu laittomaksi, mutta suurimmassa osassa maailmaa aine on huumausainelainsäädännön ulkopuolella. Suomesta ainetta takavarikoitiin neljästi vuosina 2002-04. Tässä tutkimuksessa selvitetään ehdollistetun paikkahakuisuuskokeen avulla, onko BZP:lla palkitsevia ominaisuuksia. Kokeessa koe-eläimet opetetaan yhdistämään tutkittavan aineen vaikutukset tiettyyn ympäristöön, ja mikäli eläin alkaa tämän jälkeen suosia ympäristöä, eli aine aiheuttaa paikkahakuisuutta, tulkitaan aineella olevan palkitsevia ominaisuuksia. Palkitsevien ominaisuuksien katsotaan heijastavan aineen riippuvuuspotentiaalia, sillä useimpien ihmisten väärinkäyttämien aineiden on todettu aiheuttavan ehdollistettua paikkahakuisuutta. BZP:n todettiin aiheuttavan rotille ehdollistettua paikkahakuisuutta annosriippuvaisesti, minkä jälkeen selvitettiin reseptorisalpaajien avulla dopamiinireseptorien merkitystä paikkahakuisuuden synnylle. Dopamiini-1-, eli D1-reseptorisalpaus esti paikkahakuisuuden, kun taas D2-salpauksella ei ollut siihen vaikutusta. Kokeiden perusteella BZP:lla on palkitsevia vaikutuksia, jotka välittyvät D1-, mutteivät D2-reseptorien kautta. Tulokset viittaavat BZP:lla olevan väärinkäyttö- ja riippuvuuspotentiaalia, minkä vuoksi olisi perusteltua, että se kuuluisi lainsäädännöllisesti samaan kategoriaan mm. amfetamiinin kanssa.
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Drone-kuvien käyttö haurujen (Fucus spp.) kartoitukseen ja monitorointiin itäisellä Suomenlahdella (2023)Tämä opinnäytetyö käsittelee uuden ympäristöseurantamenetelmän kehitystyötä. Hauruja (Fucus spp.) on seurattu Suomen rannikolla jo pitkään, mutta nykyisiin kartoitus- ja seurantamenetelmiin liittyy haasteita ja epätarkkuutta. Hauruvaltaiset elinympäristöt ovat uudessa uhanalaisuusarvioinnissa määritelty erittäin uhanalaisiksi. Tarvetta uusille seurantamenetelmille siis on. Drone-teknologia on kehittynyt ja halventunut viime vuosina, mikä on avannut uusia mahdollisuuksia hyödyntää kaukokartoitusta. Edulliset valokuvaamiseen tarkoitetut dronet mahdollistavat sekä ajallisesti että paikallisesti erittäin tarkkojen ilmakuvien tuottamisen. Näitä on maailmalla käytetty menestyksellisesti myös rannikon kartoituksessa ja seurannassa. Suomessa drone-kuvia ei kirjallisuuden perusteella ole käytetty makrolevien kartoitukseen tai seurantaan. Tämän tutkimuksen tarkoitus on selvittää, sopivatko tällaiset drone-kuvat hauruyhteisöjen kartoitukseen ja monitorointiin itäisellä Suomenlahdella sekä tuottaa tietoa menetelmän jatkokehitystä ajatellen. Kuvasin 22 merenranta-alueen reunaympäristön alkukesällä 2020. Kuvatuista alueista valitsin kahdeksan jatkotutkimuksiin, jotka kuvattiin uudelleen syksyllä 2020. Tällöin niiden alueelta kerättiin kuvatulkinnan tueksi 263 tukipistettä, joilta mitattiin veden syvyys ja arvioitiin haurujen esiintyvyys Drop-kuvausmenetelmällä. Rajasin ilmakuvilta näkyvät haurualueet ja vertasin niitä tukipisteaineistoon. Havaitsin, että 80 % havainnoista tieto luokittui oikein. Cohenin kappa -testin perusteella kohteiden välillä sekä eri syvyyksillä olevilla kasvustoilla oli suuria eroja havaintojen luotettavuudessa. Syvemmällä kuin 2,5 metrissä olevien haurukasvustojen erottaminen onnistui huomattavasti huonommin. Myös matalimman kasvuston (0 – 0,5 m) erottaminen oli hankalaa. Tulokset ovat lupaavat, mutta niiden perusteella menetelmää ei kuitenkaan vielä voi todeta toimivaksi, vaan lisätutkimuksia tarvitaan. Tämän tutkimuksen perusteella kehitystyötä kannattaa jatkaa käyttämällä tarkkaa paikkatietoa ilmakuvien ja tukipisteaineiston tuottamiseen sekä uusia kuvantamismenetelmiä, jotka kykenevät tuottamaan tavanomaista RGB-kameraa paremman erottelukyvyn. Drone- ja meritöiden turvallisuuden parantamiseksi tehtiin riskienhallintaprosessi, jossa tunnistin, analysoin sekä raportoin töihin liittyvät merkittävimmät riskit.
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(2020)Multiple myeloma (MM) is a heterogeneous plasma cell cancer that results from the excessive proliferation of mutated B cells in the bone marrow and the accumulation of ineffective antibodies, monoclonal proteins, in the blood. Despite recent advances in research and novel therapeutics, MM remains incurable, mainly due to the mechanisms underlying disease progression and drug resistance. Therefore, novel biomarkers and therapeutics for the treatment of relapsed and refractory MM are urgently needed. MicroRNAs (miRNAs), short non-coding RNA molecules that play a key role in post-transcriptional gene regulation, have been found to be associated with different hallmarks of MM. Previous studies have indicated that abnormally functioning miRNA-mediated gene regulation followed by oncogene activation and tumor suppressor gene silencing results in drastic alterations in cell proliferation, apoptosis, growth, and metabolism. These changes in cellular functions have been indicated to be associated with the pathogenesis, progression, and formation of drug resistance in MM. Therefore, the role and potential of miRNAs to act as biomarkers to predict MM progression and drug sensitivity should be further investigated to ultimately improve the survival rates of patients. The aim of this master’s thesis was to investigate the relationships between drug sensitivity, disease progression and miRNA regulation in MM patients. Bioinformatically predicted miRNAs identified to be associated with sensitivity to panobinostat, a novel histone deacetylase inhibitor, and MM progression were validated in MM patient samples by using real-time quantitative reverse transcription PCR (RT-qPCR). In addition, the specific gene targets of miRNAs involved in the regulation of drug responses and MM progression were predicted by identifying statistically significant, negatively correlated interactions between the miRNA and RNA sequencing data of 45 MM patients in pairwise comparative correlation analysis. Finally, the predicted miRNA targets genes were validated in MM patient samples using RT-qPCR. Based on the bioinformatic analyses and RT-qPCR validation, mir-424 expression was significantly increased in relapsed MM patients as compared to respective patient samples taken at diagnosis, suggesting a potential role of mir-424 in MM progression. Similarly, mir-4433b expression was significantly elevated in panobinostat-resistant patients compared to sensitive patients, suggesting a potential effect of mir-4433b on the regulation of panobinostat drug response in MM patients. In addition, the RT-qPCR validation demonstrated that the disease progression and drug sensitivity associated mir-92b, mir-363 and mir-221, would potentially regulate the expression of FGF2, MFF, and TMEM248, respectively, providing novel insights into the functional roles of miRNAs in MM pathways.
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(2020)Upregulation of specific helpful proteins represents a possible method for preventing or treating human diseases. Endogenous upregulation (knockup) is the increase of a gene's expression only in cells in which it is already expressed, thus avoiding physiologically abnormal spatiotemporal patterning. A gene's three prime untranslated region (3′UTR) affects protein expression through stability regulation of RNA already transcribed, which suggests 3′UTR modification as a viable route for endogenous upregulation. Mammalian model organisms can be generated in order to test the effects of different 3′UTR modifications, but at great cost of time, effort, and money. If able to predict in advance with an in vitro assay whether an in vivo modification would cause a desirable or undesirable change, these costs could be substantially reduced. In this thesis project, an in vitro assay was used to compare the protein expression influence of twenty neurodegeneration-relevant mouse genes' 3′UTRs to that of a flip-excision cassette (flex-cassette) previously used for in vivo conditional knockup. The assay used was the Promega Dual-Luciferase Reporter Assay, in which plasmids expressing Renilla and Firefly luciferase as reporter and internal control are co-transfected into in vitro cells, then each luciferase's expression measured with its respective substrate and a luminometer. Transfections were carried out in three-well replicates and on multiple days. The aims of the project were the evaluation of the assay's ability to predict in vivo results, the suggestion of 3′UTRs which could be upregulated in vivo by the conditional knockup flex-cassette, and the identification of any trends in 3′UTR-based protein expression influence according to gene function. A number of gene 3′UTRs were identified which were either candidates for flex-cassette upregulation or candidates for use in the flex-cassette to upregulate other genes. However, the flex-cassette's in vitro results were only partially consistent with its previous in vivo results. Specifically, the lox sites in the flex-cassette was observed to lower expression level to a degree not observed in vivo. Additionally, in the course of the project a number of possible workflow improvements were identified, for which suggestions have been made in the text. As such, this in vitro approach requires further study in order to determine suitability for prediction of in vivo 3′UTR behaviour.
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(2014)The purpose of this thesis was to examine the diversity and species composition of dung beetle (Scarabaeinae) communities in degraded rainforest landscapes in southeastern Madagascar. Several studies elsewhere in the world have revealed that forest-dwelling dung beetle communities and especially large species suffer from forest degradation and fragmentation by decreased species diversity. The most important factors affecting community structure of forest-dwelling dung beetles are habitat area, connectivity and vegetation quality i.e. microclimate. The hypothesis of this study was that the situation is the same in Madagascar. As dung beetles provide several important ecosystem services, like nutrient cycling and bioturbation, loss of dung beetle diversity imposes a secondary threat to the extraordinary nature of Madagascar by decreasing the regeneration ability of vegetation. Material for the study was collected in forest fragments of different size and quality between two areas of primary tropical rainforest – Ranomafana National Park and Vatovavy mountain – in November and December 2011 and January 2012. The sampling was conducted by transects of 30–60 fish- or carrion-baited pitfall traps which were set up in 55 localities in the study area. In each locality, several variables were measured to describe the vegetation and microclimatic conditions. The variables included temperature, humidity, estimate of vegetation quality by 6 observation-based classes, vegetation density, hights of three clearly visible vegetation layers, altitude and slope steepness. In addition, connectivities were measured for the localities using GIS and a satellite image –based vegetation classification. In order to demonstrate the differences between certain localities the study sites were divided into seven zones in terms of their distance from the Ranomafana National Park, average connectivity of the transects and elevation. Altogether 4,199 individuals belonging to 24 species were collected. Six of the species are currently under identification process in the Paris Museum of Natural History. According to the preliminary results they include two species new to science. Largest numbers of species were collected from good quality fragments between Ranomafana and Vatovavy. Also, a clear transition zone in species composition was detected a few kilometers west from Vatovavy, where altitude changes sharply. The study reveals that the species assemblages in the forest fragments and degraded forest areas are surprisingly species rich. This may, however, be partly because of extinction debt, and many of the still surviving species may soon die out due to restricted dispersal possibilities. Connectivity and vegetation quality were shown to have an effect on Canthonini species richness, with less species in less connected areas and lower vegetation quality. Vegetation quality was also shown to have an impact on the proportions of species with different body length: more small and medium-sized (< 8 mm) species were found in fragments where vegetation was more degraded. In addition to revealing how rainforest fragmentation and degradation affect local communities, the study gives interesting information about the distribution of certain species of Epilissus (Scarabaeinae: Canthonini). It has been known before that four species of Epilissus show elevational differentiation in their occurrence in Ranomafana. In this study, two more species of the same genus, E. prasinus and E. emmae obscurpennis, were shown to continue this pattern in lower elevations near Vatovavy mountain, about 50 kilometers east of Ranomafana.
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(2020)Finland är ett skogsrikt land, men de existerande skogarna utgör dock kvalitativt enformiga habitat och det råder brist på varierade habitat i form av till exempel blandskog, skogar i naturligt tillstånd och äldre skog. Enligt tidigare studier utgör äldre skogar viktiga habitat för många skogslevande arter och mångfalden i skogarna minskar som en följd av habitatförstörelse. Vidare påvisar forskning att skogarna i Finland är ojämnt skyddade och merparten av skogsskyddet förekommer i norra Finland på statlig mark. Forskning påvisar att dagens moderna skogsbruksmetoder bidrar till att våra skogar är artfattiga och till de största hoten mot mångfalden i våra skogar klassas skogsbruket samt klimatförändringen. Att skogslandskapet i Finland utarmas är ett stort problem i och med att mångfald ökar ett ekosystems beständighet mot yttre störningar. För att åtgärda förlusten av biologisk mångfald har genvägar eller verktyg, med vars hjälp större arealer och flera arter samtidigt kan skyddas tagits fram. Till de här verktygen hör konceptet paraplyarter och en paraplyart är kortfattat en art med vars hjälp flera andra arter kan skyddas och vars förekomst indikerar på att ett lokalt habitat i närheten av paraplyarten är av hög kvalitet. Paraplyarter används explicit för att skydda habitat av hög kvalitet till exempel på områden där biodiversiteten är hög. Tidigare studier har visat att stora dagrovfåglar vanligen är effektiva paraplyarter i och med att deras boplatser kan associeras med en hög biodiversitet. Syftet med den här undersökningen är att klargöra huruvida skogslandskap i närheten av duvhökens (Accipiter gentilis) boplatser uppvisar en högre förekomst av vissa arter jämfört med kontrollområden. Kontrollområdena med vilka boplatserna jämfördes var av två slag: (a) äkta skogskontroller och (b) slumpmässigt utvalda skogskontroller. De äkta skogskontrollerna utgjordes av områden som kvalitativt motsvarade skogen intill duvhöksbon, medan de slumpmässigt utvalda skogskontrollerna utgjordes av skog av vilken typ som helst. För vart och ett av de här områdena undersöktes förekomsten av flygekorre (Pteromys volans) och blåbär (Vaccinium myrtillus). Förekomst av flygekorre karterades på basen av spillningsfynd och blåbärsrisets riklighet uppskattades via analyser av fotografier tagna över fältskiktet. Det insamlade materialet för båda arterna analyserades statistiskt med hjälp av LME - modeller. Resultaten påvisade att flygekorre förekom rikligare vid duvhökens boplatser än i de två kontrollskogarna. Flygekorrens habitatpreferenser överlappar till stor del med duvhökens och mina resultat överensstämmer med tidigare forskning som har påvisat att flygekorren har en nytta av duvhökens närvaro i form av skydd mot predation från nattaktiva rovfåglar. Flygekorren prefererar således samma skogstyper som duvhöken och intressant nog verkar duvhökens närvaro i sig vara viktigare för flygekorren än själva skogstypen. Enligt resultaten från avhandlingen är det dock inte heller uteslutet att en annan art/egenskap tillsammans med duvhöken fungerade som en paraplyart för flygekorren. Blåbärsris däremot uppvisade en rikligare förekomst vid äkta skogskontroller och duvhöksskogarna var troligen överlag för lummiga för blåbärets trivsel, men blåbärsris förekom dock rikligt vid 50 m från boplatserna. Således kan slutsatsen dras att flygekorren kan skyddas i fall av att skogslandskap med duvhöksbon skyddas. Duvhöken kan även ses som ett verktyg för naturskydd eller som en indikator för skogslandskap med hög mångfald, i vilka flygekorre samt en del blåbärsris förekommer. Om vi i framtiden vill ha flygekorre och blåbär i våra skogar bör därmed äldre skogar bevaras i och med resultaten påvisar att nämnda arter inte verkar trivas i moderna ekonomiskogar. En tillämpning av resultaten kunde vara att bruka paraplyarter och förekomst av nyckelbiotoper för naturskydd simultant.
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(2017)Tausta ja tavoitteet: D-vitamiinia sitova proteiini (DBP) kuljettaa D-vitamiinia ja sen aineenvaihduntatuotteita, esim. 25-hydroksi-D-vitamiinia (25(OH)D), verenkierrossa. Geneettinen muuntelu GC-geenissä, joka koodaa DBP:tä, on yhdistetty 25(OH)D-konsentraatioiden vaihteluun. Tavoitteena olikin tutkia GC:n geneettisten tekijöiden vaikutusta 25(OH)D-konsentraatioon suomalaisilla vastasyntyneillä. Lisäksi tutkittiin GC:ssä esiintyvän muuntelun aiheuttamia eroja lapsen kasvussa raskauden aikana. Menetelmät: GC:n SNP:ssä, eli yksittäisen nukleotidin vaihdoksissa, esiintyvää polymorfiaa ja raskauden aikaisen 25(OH)D-konsentraation ja napaveren 25(OH)D-konsentraation suhdetta arvioitiin 933 valkoihoisella vastasyntyneellä pojalla (468) ja tytöllä (465). 25(OH)D-konsentraatio määritettiin raskausviikoilta 6-13 ja napaverestä. Äidin D-vitamiinin saanti ravintolisistä määritettiin ruuankäytön frekvenssikyselylomakkeen avulla kahdelta viimeiseltä raskauskuukaudelta. GC:n kolme SNP:tä genotyypitettiin Taq-polymeraasin aktiivisuuteen perustuvan qPCR:n avulla (Bio-Rad, CFX384 C1000 Touch™ Real-Time PCR Detection System, USA): rs4588, rs7041 ja rs705124. Genotyyppien rs4588 ja rs7041 yhdistelmästä muodostettiin kuusi tunnettua diplotyyppiä ja kaikista kolmesta genotyypistä edelleen haplotyypit. Eroja geno-, diplo- ja haplotyyppien välillä 25(OH)D-konsentraatioissa ja kasvussa testattiin kovarianssianalyysin (ANCOVA) avulla käyttämällä relevantteja kovariaatteja. Tulokset: SNP rs4588 ja rs7041 geno- ja diplotyyppien välillä löydettiin tilastollisesti merkitsevä ero 25(OH)D-konsentraatioissa (keskiarvo (nmol/l) ± keskivirhe). Napaveren 25(OH)D-konsentraatio oli alhaisin geno- ja diplotyypillä GC 2/2 (77.8 ± 4.3, p=0.010, p=0.028, ANCOVA) ja korkein diplotyypillä GC 1F/1S (93.8 ± 2.0). Haplotyypillä ATG oli alhaisin napaveren 25(OH)D-konsentraatio (77.9 ± 3.8, p=0.016, ANCOVA). Raskauden ajan 25(OH)D-konsentraatiossa ei löytynyt eroja geno-, diplo- tai haplotyyppien välillä (ANCOVA). Genotyypin rs7041 sekä diplotyyppien ja päänympäryksen välilllä havaittiin ero (p=0.005, p=0.002, ANOVA), mutta geneettisen muuntelun ja syntymäpainon ja –pituuden välillä ei löytynyt eroja. Kun valittiin relevantit kovariaatit, niin genotyyppien rs7041 sekä diplotyyppien ja päänympäryksen välillä ei löytynyt enää tilastollisesti merkitseviä eroja. Yhteenveto: Tulokset osoittavat, että DBP:tä koodaavan geenin geneettisellä muuntelulla ja napaveren 25(OH)D-konsentraatiolla olisi vaikutusta jo vastasyntyneiden D-vitamiinitilaan ja aineenvaihduntaan. Saatujen tulosten perusteella GC:n geneettinen muuntelu ei kuitenkaan ollut yhteydessä lapsen kasvuun. Tulevaisuudessa GC:n SNP:n vaikutusta 25(OH)D-konsentraatioon voisi tarkastella syntymän jälkeisten vuosien aikana.
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