Bio- ja ympäristötieteellinen tiedekunta


Recent Submissions

  • Rydman, Elina (Helsingin yliopisto, 2016)
    Common materials acquire new properties when manufactured in the nanoscale. The same properties that are responsible for the exciting new possibilities are also a cause for some concern. The high surface area to volume ratio is a feature of engineered nanomaterials (ENM) that causes the amount of surface area to dominate their possible effects. In the case of carbon nanomaterials and other fibers, also the shape and length of the particle play important roles. The two ENM studied in this thesis, nanosized titanium dioxide (TiO2) and carbon nanotubes (CNT), are among the most widely used ENM in the world which means that they hold a high potential of occupational and possible customer exposure. Inhalation is the most likely exposure route in occupational settings. For this reason, inhalation exposure of mice was the main route of administration. The study settings were chosen to mimic occupational exposure as far as possible. Different immunological parameters were examined in the lungs of the exposed mice, such as the influx of different leucocytes, expression of cytokine and chemokine messenger molecules and changes in the lung tissue. The results from TiO2 studies indicated that even a normally inert material when nanosized may become inflammogenic. In addition, even small changes in the structure, or even a coating, may modify radically the nature of a material. Exposure to most nanosized TiO2 caused only modest to no inflammation, whereas a silica (SiO2) coated TiO2 triggered an inflammation characterized by pulmonary neutrophilia and mRNA expression of neutrophil chemoattractant CXCL1 and proinflammatory TNF-α. In tissues and bronchoalveolar lavage (BAL), TiO2 was readily engulfed by macrophages. In a model of allergic asthma, it was found that exposure to both nanosized and larger TiO2 seemed to prevent asthmatic symptoms. This underlines the importance of bearing in mind the heterogeneity of the human population when assessing the toxicity of ENM. CNT have raised some serious concerns in the scientific community and the media due to their similarity in structure to asbestos. Here, a remarkable new type of eosinophilic inflammation was observed in long rigid CNT exposed mice after a week of inhalation to these particles. This inflammation was characterized by strong eosinophilia, goblet cell hyperplasia, Th2 type cytokines and increased airway hyper-responsiveness to methacholine. All of the above symptoms have previously been described as symptoms of classical asthma. Transcriptomic analyses revealed radical up-regulation of innate immunity and cytokine/chemokine pathways. There were also roles found for mast cells and alveolar macrophages in orchestrating the inflammation. Lastly in the only exposure conducted by aspiration, mice were exposed to two long CNT (rigid/R and tangled/T) and to crocidolite asbestos. From a few hours to 28 days after a single exposure, there was a striking inflammatory cascade starting with macrophages and neutrophils, progressing to eosinophilic inflammation and eventually terminating as granulomas, goblet cell hyperplasia, Charcot-Leyden-like crystals and the mRNA expression of IL-1β, TGF-β, TNF-α and IL-13. The most dramatic inflammation was seen in the R/CNT group, followed by asbestos with T/CNT being clearly more weakly inflammogenic. In summary, inhalation exposure especially to certain fibrous nanomaterials seems to cause strong pulmonary inflammation. This may put exposed individuals at risk of developing lung diseases. In addition to the material of the nanoparticle, two important factors in risk evaluation are the shape of the particle and the possible modification made (e.g. coating) to the particle. The model of allergic asthma demonstrated that an underlying inflammatory condition can greatly affect the inflammatory outcome seen after nanoparticle exposure. The results of this thesis help to understand the underlying mechanisms in nanoparticle induced pulmonary inflammation.
  • Oikkonen, Jaana (Helsingin yliopisto, 2016)
    Most people have the capacity for music perception and production, but the degree of music competency varies between individuals. In this thesis, I studied abilities to identify pitch, tone duration and sound patterns with Karma s test for auditory structuring (KMT), and Seashore s tests for time (ST) and pitch (SP). These abilities can be considered as basic components of musicality. Additionally, I studied self-reported musical activities, especially composing and arranging. Musical ability is a diverse phenotype that includes both acquired and innate abilities. Earlier studies have shown that genetic component affects musical traits; here, heritability was estimated as 21-68%. Genetic predisposition of musical abilities was studied in family material (N=915) using linkage and linkage disequilibrium analyses. The best association for musical aptitude (KMT, SP and ST) was obtained at 3q21.3 near GATA2 and the best linkage at 4p14 adjacent to PCDH7. For musically experienced individuals without creative activity in music, linkage was found at 18q21. Interestingly, regions near 4q22.1 and 16p12.3 were linked with multiple musical traits. The genes within the resulting regions showed enrichment of inner ear development, schizophrenia and the long-term depression pathway, which is a molecular pathway important in learning and memory. Using three selection signature methods, FST, haploPS and XP-EHH, over 100 candidate genomic regions were detected to be under positive selection in individuals with high music test scores in the music tests. Enrichment analysis pointed to the development of the inner ear, corresponding to the enrichment results from the musical aptitude linkage analysis, even though there were no common regions between these two studies. The fourth part of this thesis integrates our musical ability gene mapping results with other music-related studies, including also animal model studies. A convergent genomics approach ranked EGR1, cortisol, FOS and FOXP2 as the most prominent molecules that affect musical abilities. The best 40 genes showed enrichment of cognition. Previous studies have shown that musical abilities share a common background with cognitive abilities, such as intelligence, and this study is the first to suggest the associated molecules. In conclusion, with these studies, I suggest many new genes and pathways to be associated with musical traits. The genetic predisposition for music is affected by a large number of genes of which only a few have been identified. Musical abilities were linked to multiple different chromosomes of which the 4q22 region is especially interesting. We made a literary survey combining information of molecules related to musical traits that can be used to evaluate genomics results in the future.
  • Vaahtera, Lauri (Helsingin yliopisto, 2016)
    The air pollutant ozone (O3) enters plant leaves through stomata and activates apolastic reactive oxygen species (ROS) signaling. Depending on growth conditions and genotype, this results in large transcriptional reprogramming,closure of stomatal pores and activation of cell death programs. These responses are also regulated through plant stress hormones. This thesis sheds light on how stress hormone signaling is connected with apoplastic ROS signaling in the model plant Arabidopsis thaliana, and investigates regulatory mechanisms which generate specificity among sequence-specific transcription factors (TFs), the executers of apoplastic ROS -induced transcriptional reprogramming. The essential methods of the thesis include O3 exposures of Arabidopsis wild type and mutant plants followed by quantification of cell death and characterization of transcriptional responses supplemented with several protein-level analyses of selected WRKY family TFs. The O3-induced cell death was found to be inhibited by plant hormone salicylic acid, and genes RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and WRKY70 were found to be required for O3-induced cell death in jasmonic acid insensitive genetic background. Even though stress hormones were verified to play important roles in the regulation of cell death, the transcriptional response to apoplastic ROS in a hormone deficient/insensitive mutant was highly similar to wild type, suggesting that much of the signaling involved is independent of the studied hormones jasmonic acid, salicylic acid, and ethylene. The potential major executers of transcriptional response to apoplastic ROS, WRKY family TFs, were studied for their transcriptional regulation, DNA-binding preferences, protein-protein interactions, subcellular localization, and effects on transcriptome. The results showed that the DNA-binding preferences of WRKYs vary substantially between phylogenetic groups, implying that the specificity in signaling between different WRKYs can be partly achieved through DNA binding preferences. Transcriptomic analyses of mutants with altered expression levels of the strongly ROS-inducible WRKY75 implicate this TF as a positive regulator of well-known pathogen-responsive genes, such as PATHOGENESIS-RELATED GENE 1 (PR1) and PATHOGENESIS-RELATED GENE 2 (PR2), and as a negative regulator of several hormone signaling pathways and TFs.
  • Aarnos, Hanna (Helsingin yliopisto, 2016)
    As dissolved organic matter (DOM) constitutes a vast reservoir of carbon and nutrients in lakes, rivers and ocean, it plays an important role in the global carbon and nutrient cycles. Only a part of DOM is directly biologically utilizable by bacteria, but solar radiation induced photochemical reactions may mineralize DOM to inorganic forms such as dissolved inorganic carbon (DIC) and ammonium (NH4+), and also degrade non-labile DOM molecules to labile organic substrates available for bacteria. This dissertation examined the photochemical transformation of DOM in the surface water in different scales of aquatic environments; from a Finnish boreal lake and the Baltic Sea to the coastal areas of ten globally big rivers. This dissertation studied photochemical reactions such as photoproduction of DIC, NH4+, and labile substrates supporting bacterial growth, and determined the photoreactivity of DOM, i.e., apparent quantum yields for the photoreactions in each environment. To consider the relevance of photochemistry of DOM, an optical model was used to quantify the photoreaction rates taking into account for the determined photoreactivity of DOM and solar radiation incidental to each environment studied. In the Baltic Sea, the pelagic heterotrophic bacterioplankton was carbon-limited indicating low bioavailability of DOM in the surface water. Irradiations of the waters with natural or simulated solar light resulted in photobleaching of chromophoric dissolved organic matter (CDOM) and phototransformation of DOM to DIC and NH4+ as well as to labile DOM substrates. This dissertation showed that across the entire Baltic Sea, the annual photoproduction of NH4+ corresponded to 9 18% of the annual river loading of DON, but the photoproduction of DIC exceeded the annual river loading of photoreactive terrigenous DOC. Furthermore, the studies along the salinity transects indicated that terrigenous DOC was more photoreactive than marine DOC but the marine DON was more reactive than terrigenous DON. The photoproduced labile substrates supported bacterial production and biomass leading to a 3-level trophic transfer of non-labile DOM and a simultaneous stimulation of autotrophic algae and primary production. The annual amount of photostimulated bacterial biomass corresponded to 3 5% of total bacterial biomass across the entire Baltic Sea. In the Baltic Sea as well as in the mesohumic lake studied, the photolytic water layer was shallow and limited the phototransformation of DOM to the top 30 cm. In the global scale, the annual DIC photoproduction from terrigenous CDOM in front of the ten rivers studied (12.5 ± 2.1 Tg C y-1) corresponded to 18 ± 4% of annual flux of terrigenous DOC of the rivers. When extrapolated to a global estimate, 44.5 ± 10.6 Tg of terrigenous DOC was annually mineralized to DIC by solar radiation in coastal waters. Globally, the amount of photomineralization of terrigenous DOC was larger in coastal ocean than in lakes and reservoirs. However, the areal rates of DIC photoproduction were larger in the lakes and reservoirs than in the coastal waters indicating that phototransformation of terrigenous DOC was likely limited by relatively short residence times in inland waters. To conclude, the phototransformation in coastal waters formed the final sink for riverine terrigenous CDOM and DOC, but was restricted in general, to a few hundred kilometres from river mouths to the ocean with the exception of largest discharging rivers.
  • Puntila, Riikka (Helsingin yliopisto, 2016)
    Translocation of non-indigenous species is a global threat to the structure and functioning of coastal ecosystems. Coastal ecosystems and estuaries are particularly prone for invasions due to impacts from a variety of anthropogenic stressors and frequent propagule pressure, notably from shipping. In addition, species poor environments, especially when already impacted by multiple anthropogenic stressors, such as the Baltic Sea, are thought to be particularly susceptible to invasions. In the Baltic Sea, to date more than 130 non-indigenous species have been reported and about 80 have been able to become established. Few have become invasive, spreading rapidly and/or begun to impact the native ecosystem. In this thesis the aim was to study how invasive benthic non-indigenous species, specifically the Harris mud crab (Rhithropanopeus harrisii) and round goby (Neogobius melanostomus) have settled in their new environment, how they have affected the ecosystem and how this information of the species can in future be used in management practices (e.g., risk assessments) in the Baltic Sea. The results show that the interactions between the two non-indigenous species and the environment are highly complex and may involve both generic and strictly context-specific components. The native predators and parasites in the area have begun to exploit the new species, although they are currently unable to control the growing mud crab and round goby populations. Furthermore, the FISK (Fish Invasiveness Scoring Kit) risk assessment exercise for the southern coastal areas of Finland showed that many non-indigenous fish species, some of which have already established, do have potential for becoming pests in the area. However, the continuously changing environment (incl. due to climate change) modi- fies both the non-indigenous and invasive biota in the area as well as may alter the nature and magnitude of ecosystem changes caused by the non-indigenous species. The infor- mation gathered in the thesis can be used in further risk assessments and aiding future management decisions.
  • Kylväjä, Riikka (Helsingin yliopisto, 2016)
    For understanding the interaction between bacteria and the host, it is essential to identify and characterize bacterial adhesive proteins, adhesins, and other bacterial molecules that interact or interfere with the host. Adhesins mediate the initial colonization steps of bacteria. The colonization may lead to infection or commensalism. When the molecular mechanisms are known, new treatments to prevent the infections can more easily be designed. For assessment of the functions of bacterial proteins in vitro, the proteins are usually purified from the cells or displayed on the surface of a heterologous host. Various surface display techniques, such as phage or fimbria-assisted display, have been developed for the analysis of polypeptides complicated to overproduce by conventional expression methods. Secretion of recombinant proteins to the culture medium of E. coli is an appealing approach since the purifying step is simple. However, reports on high-level extracellular protein secretion in bacteria are scarce. In the first part of this study, a random chromosomal library of Staphylococcus aureus was created in the secretion-competent E. coli strain MKS12. S. aureus expresses several adhesins, some of which are well characterized. Here, we wanted to identify possible new adhesins or proteins with novel functions. All the extracellularly secreted staphylococcal polypeptides of this study were tested for binding to some well-known receptors of S. aureus such as fibronectin, fibrinogen, collagens, and plasminogen. We found three putative moonlighting proteins i.e. proteins with functions additional to their conventional functions: a universal stress protein and an ATPase subunit of phosphoribosylaminoimidazole carboxylase, which both bound to fibronectin and fibrinogen, and the penicillin binding protein 3 capable of binding and activating plasminogen. Adhesins have been studied at the molecular level in many human pathogenic bacteria, such as S. aureus, but the knowledge on the surface molecules mediating adhesion of commensals, such as Lactobacillus, has not drawn as much attention. The second part of this study dealt with the interaction of Lactobacillus crispatus ST1 with the epithelium of the chicken crop and the human vagina. We investigated the molecular mechanisms underlying the interaction of L. crispatus ST1 with the epithelium and identified a novel high-molecular-mass adhesin that was named as lactobacillus epithelium adhesin (LEA). LEA was shown to bind efficiently to the stratified squamous epithelium of the crop and to the similar type of epithelium in the human vagina. In this thesis work we have applied a novel secretion method for heterologous protein expression and characterized novel adhesive proteins of two Gram-positive bacterial species.
  • Laitinen, Anita (Helsingin yliopisto, 2016)
    Mesenchymal stem/stromal cells (MSCs) are multipotent cells that can be found in various tissues. These cells have the capacity to differentiate into bone, adipose, and cartilage. They also have the capacity to suppress immune reactions and the capacity to support angiogenesis. The utilization of these cells in cell based therapies has therefore been intensively studied. There are several clinical studies on going to demonstrate the therapeutic potential of these cells. The utilization of MSCs has been studied in for example graft-versus-host-disease (a severe complication after hematopoietic stem cell transplantation), stroke, myocardial infarction, and cartilage lesions. The frequency of MSCs is variable in different tissues. The number of these cells in tissues is so low that these cells need to be cultured outside of the body, in vitro, to obtain adequate numbers of MSCs for cell therapy purposes. It has been demonstrated that different in vitro culture conditions have effects on the properties of MSCs. Traditionally cells are cultured in growth medium containing fetal bovine serum (FBS). There is a great interest to find alternative supplements to replace FBS for clinical grade production of MSCs to avoid the patients to become predisposed to xenogenic infectious agents or antigens. Platelet-derived supplements might be one potential alternative for FBS. An efficient method to culture MSCs from cord blood was established in this thesis. Additionally a method to produce clinical-grade bone marrow MSCs in platelet-derived supplement containing culture medium was established. Different culture conditions were demonstrated to have an effect on proliferative and immunosuppressive capacity of MSCs as well as on their capacity to support angiogenesis. In this thesis it is also indicated that MSCs can suppress immunoreactions producing an immunosuppressive molecule, adenosine, via a cell surface enzyme, CD73. The knowledge of the impact of culture conditions on the properties of cells as well as understanding the functional mechanisms of the cells is a prerequisite to produce safe and efficacious cell therapy products.
  • Kumar, Anmol (Helsingin yliopisto, 2014)
    Neurotrophic factors such as brain¬-derived neurotrophic factor (BDNF) and glial cell line¬¬¬-derived neurotrophic factor (GDNF) are a family of proteins which play an important role inside and outside central nervous system. While precise regulation of BDNF and GDNF levels in time and space in an organism is crucial in determining the biological outcome, mechanisms involved in controlling their levels are not fully understood. Messenger RNAs (mRNAs) play a critical role in gene expression by conveying genetic information from DNA to protein synthesis. 3ʹ untranslated region (3ʹUTR) is a part of mRNA sequence which regulates gene expression by binding to microRNAs (miRs), RNA-binding proteins (RBPs) and other trans-acting factors. In this thesis, we investigated the 3ʹUTR mediated regulation of BDNF and GDNF. We demonstrate the presence of regulatory elements in the 3ʹUTR of BDNF and GDNF and, show that BDNF is regulated by four different miRs, namely miR-1, miR-10b, miR-155 and miR-191 and, RBP tristetraprolin (TTP) in different cell lines. Further, we show that GDNF is regulated by multiple miRs in cell lines and identify binding sites for miR-146a and miR-96 in the GDNF 3ʹUTR. Finally, we demonstrate that replacement of GDNF 3ʹUTR in mice with a 3ʹUTR with reduced responsiveness to negative regulators including miRs leads to elevated level of endogenous GDNF mRNA and protein in various organs with profound biological effects including in the brain dopamine system function in mice. We conclude that 3ʹUTR mediated regulation of BDNF and GDNF is biologically important and propose that 3ʹUTR replacement is an informative way to study gene function in vivo.
  • Magalhães, Ana Cathia Dias (Helsingin yliopisto, 2016)
    The time of arrival of interneurons and oligodendrocytes to the neocortex is critical for proper functional brain development. Aberrances in this sequence can be detrimental, and involved in different developmental diseases. Thus, understanding the mechanisms for temporal control of the genesis and migration of neural cells is crucial. The aim of this study was to focus on the ventral telencephalon, a major source of interneurons and oligodendrocytes, in more detail. A more sensitive method was developed for detecting and quantifying oligodendrocyte precursor cells, e.g. Olig2. The device decloaking chamber was compared to the microwave oven-based heat-induced epitope retrieval (HIER) method by studying the labeling of Olig2 marker in paraffin-embedded sections from embryonic mouse brain. The results demonstrated that the decloaking chamber-based HIER method is the most suitable technique for the detection of single Olig2-labeled cells in the ventral telencephalon. This qualitative result was reflected in the quantitative analyses: more Olig2-labeled cells were quantifiable with the decloaking chamber- than with the microwave oven-approach. Thus, the decloaking chamber-based HIER method constitutes a sensitive technique for the detection of oligodendrocyte precursor cells, and therefore for its quantification in the developing ventral telencephalon. The development of telencephalon depends on fundamental processes, which include proliferation and migration of neural cells. The Na-K-Cl cotransporter isoform 1 (NKCC1) is an important protein for the process of volume regulation, and has been implicated in cell division. Within the developing brain, the ventral telencephalon showed the highest expression of NKCC1. This expression corresponded to neural progenitor cells in the lateral ganglionic eminence (LGE). Using NKCC1 knockout mice, it was demonstrated that NKCC1 influenced cell cycle reentry. Consequently, mice lacking NKCC1 have impaired Sp8-expressing interneurons and Olig2-labeled cells. Thus, NKCC1 is crucial in vivo for cell cycle decision, thereby altering the production of oligodendrocyte and interneuron progenitor cells in the LGE. Once interneurons are born, they migrate to the neocortex. The implication of syndecan-3 was assessed in their tangential migration. The results showed that the Glial cell line-derived neurotrophic factor GDNF interacts with syndecan-3 to promote the tangential migration of calbindin-expressing interneurons within the telencephalon. Consistently, mice lacking syndecan-3 have an accumulation of migrating interneurons in the LGE. In summary, two important mechanisms were found for temporal and spatial control of cortical oligodendrocytes and interneurons.
  • Yan, Lijuan (Helsingin yliopisto, 2016)
    Soil pollution by petroleum hydrocarbons (PHCs) as a result of anthropogenic activities poses significant threats in the environment. In particular, used motor oil that contains high concentrations of aliphatics, polycyclic aromatic hydrocarbons (PAHs) and heavy metals (e.g. lead, zinc, chromium, barium and arsenic) contribute to chronic hazards including carcinogenicity. Microorganisms are able to degrade and utilize many recalcitrant compounds as carbon and energy sources in a natural attenuation process. However, in boreal regions this process is limited by the cool climate. The main goal of most bioremediation designs should be an optimization of environmental conditions for microbial growth and metabolic activities. Plant growth can stimulate the activities of soil microflora in the rhizosphere, thus enhancing the bioremediation of oil-polluted soil. Nitrogen deficiency is a frequent limiting factor of biomediation in oil-contaminated soils. Legumes that form symbiotic association with N-fixing bacteria are able to assist the biodegradation of PHCs. The planting of oil-tolerant perennial crops, especially legumes, in oil-contaminated soil holds promise for great economic benefits for bioenergy production while accelerating the oil degradation process. Fodder galega (Galega orientalis Lam.), a perennial forage legume, and smooth brome (Bromus inermis L.), a cool-season perennial sod-forming grass, are both persistent in boreal zones and have been shown to grow well together in crop mixtures without N-fertilizer supply. The oil tolerance and oil-rhizoremediation potential of G. orientalis and its microsymbiont Neorhizobium galegae have been demonstrated at microcosm and mesocosm scales. Plant growth promoting bacteria (PGPB) have potential to increase nodulation of galega, mitigate plant stress response and increase the bioavailability of soil contaminants, therefore enhancing the degradation of contaminants. These components can form a powerful combination to be used for bioremediation of oil-contaminated soil. However, the competitiveness and effectiveness of the crop- and PGPB-assisted bioremediation system need to be evaluated in field conditions. To date, there were no systematically described studies on bioremediation of oil-contaminated soil combined with crop biomass production in boreal regions. This multidisciplinary research project was conducted to fill this knowledge gap by evaluating the sustainability of the legume-cropping bioremediation system economically in terms of crop yield, and environmentally in terms of oil degradation rate and the dynamics of bacterial communities. To reach these aims, we established a multi-year bioremediation field experiment at the Viikki Experimental Farm, University of Helsinki, Finland (60°14'N, 25°01'E, 8 m AMSL) with crop treatments (brome grass, fodder galega, their mixture and bare fallow) as the main plots in four replicated blocks, and used motor oil treatments (7000 ppm +/-) and PGPB (+/-) treatments as the sub-plot factors. Soil samples were taken from the top 20 cm layer at six time points (July 2009, May 2010, November 2010, May 2011, May 2012 and October 2012). Soil chemical properties e.g. pH, electrical conductivity (EC), total C, total N and C:N ratio of three sample sets (July 2009, November 2010 and May 2012) were measured. Oil concentration was determined based on the difference of total solvent extractable material (TSEM) concentration between the oil-spiked plot and the average of control plots at each sampling time using the gravimetrical method. Crop physiological properties e.g. annual DM yield, total C, total N, C: N ratio, chlorophyll and BNF of the legume were measured. Soil-borne bacterial communities were investigated using i) LH-PCR community fingerprinting technique (all samples) and ii) Illumina s MiSeq sequencing (spring-summer samples). Oil contamination had a significant impact on soil chemical properties, e.g. pH, EC, total C and C:N ratio. The oil degradation was incomplete 40 months after the oil spike, with a dissipation of 73% - 92% of oil concentration (Paper I). As the field soil condition was good for oil degradation, the advantage of using crops to assist oil degradation was not evident. The oil degradation followed firstorder kinetics with the reduction rates decreasing as follows: bare fallow > galega-brome grass mixture > brome grass > galega. Oil, surprisingly, increased crop dry matter and nitrogen yield, particularly in the fourth year (Paper I). The legume-grass mixture produced significantly higher crop dry biomass than the pure stands. For instance, the unfertilized galega-brome grass mixture out-yielded the Nfertilized pure grass swards over years by an average of 32% (Paper I), suggesting that the inoculated galega could fully replace N-fertilizer for brome grass. PGPB enhanced the efficiency of biological nitrogen fixation of the legume, especially in legume-grass mixture plots (Paper I). The LH-PCR community fingerprinting technique produced similar results as the 16S rRNA gene amplicon sequencing. Both time and oil contamination were the main drivers of bacterial community dynamics (Papers II and III). The effect of oil was initially negative on overall bacterial diversity (Papers II and III), but variable on the diversity of bacterial sub-communities (Paper III). The bacterial communities responded quickly to oil contamination, but the effect of oil on community composition was recoverable over time (Papers II and III). Crop cultivation had a small impact on the composition of bacterial community (Paper III). The oil-favored taxa that discriminated bacterial communities between oil-contaminated and non-contaminated soils were mainly assigned to the two prevalent phyla Actinobacteria and Proteobacteria (Paper III). The operational taxonomic units (OTUs) with significantly different oil-specific abundance changes over time were all favored by oil; therefore, these oil-specific taxa were suggested as suitable bio-indicators to monitor the ecological impact of oil contamination (Paper III). Besides oil concentration, the changes in soil chemical properties, e.g. soil pH and EC, significantly affected bacterial community structure (Paper II and III). To summarize, oil contamination affected soil chemical and biological properties (e.g. crop growth and bacterial community), but the impact of oil decreased with time. The cultivation of oil-tolerant perennial crops, especially galega-brome grass mixture, in oil-contaminated soil can hopefully produce considerable biomass for bioenergy industry. Bacterial communities underwent a significant time- and season-dependent succession, regardless of oil contamination. Therefore, studies restricted to a single snapshot of time without any non-contaminated samples as reference cannot reveal oil contaminationrelated changes in the dynamic patterns of bacterial communities in the field soil. With the development and decreasing cost of high-throughput sequencing (NGS), NGS-based metagenomics analysis has become the mainstream method in microbial ecology research, providing in-depth view on bacterial populations at different taxonomic levels in the community. However, the LH-PCR technique is still suggested as a cost-effective method to monitor microbial community dynamics for assessing the ecological impact of oil contamination. Oil degradation was rather slow in the boreal climate. Long-term stimulation and monitoring of soil chemical properties, oil concentration, crop growth and microbial community are still needed for risk control. All these suggestions can be applied to soil contaminated by PHC contaminants other than used motor oil, despite hydrocarbon compositional differences.
  • Tynell, Janne (Helsingin yliopisto, 2016)
    Respiratory virus infections are a major cause of morbidity and mortality worldwide. Decades of research have yielded many breakthroughs in our understanding of virus-host interactions, but many aspects of viral pathogenesis still remain unresolved. Vaccines and antiviral treatments have been developed, but they are imperfect or completely lacking for many viral agents. Novel emerging viruses form an additional challenge, which can only be overcome by proper preparation and quick response against these unexpected infectious threats. Understanding virus-host interactions is critical for elucidating aspects of viral pathogenesis and devising better treatment strategies against viral pathogens. Our research has focused on the virus-host interactions of two major respiratory pathogens with a recent history of outbreaks by a novel viral agent, the influenza A virus and coronavirus. Influenza A virus has plagued humankind throughout human history and continues to cause annual epidemics and occasional pandemics associated with significant mortality. NS1 protein is one of the major virulence factors of influenza A virus. It has a multitude of different interactions with host cell components that either aid viral replication or hamper the antiviral response exhibited by the host cell. These interactions are located both in the host cell nucleus and the cytoplasm, and three signals regulating the intracellular localization of NS1 protein have been identified. While the critical impact of nuclear localization signal 1 is well known, the other two localization signals have remained poorly characterized. In this work we provide a detailed description of the NS1 nuclear export signal (NES), showing that the NES region is well conserved within different influenza A strains and that certain mutations within the region cause attenuation of virus replication. Through the use of different mutant viruses, we show that the attenuated phenotype is not due to impaired localization alone, but rather involves defects in the functions of NS1. We also show that NS1 is not exported through the classical CRM1-dependent pathway and we establish the nucleolar proteins that bind NS1 and thus guide its nucleolar localization. Human coronaviruses are a major cause of the common cold. For a long time coronaviruses were thought to cause only mild upper respiratory tract infections in humans. However, this view changed with the emergence of the highly lethal SARS coronavirus in 2002 and the identification of MERS coronavirus in 2012. While the SARS outbreak was efficiently contained, MERS coronavirus continues to circulate in camels and causes repeated introductions into the human population in the Middle East. Our MERS coronavirus research concentrated on characterizing MERS infection of human macrophages and dendritic cells, two important cell types of the innate immune system. We show that MERS coronavirus does not replicate productively in these leucocytes, but a significant innate immune response is generated. Altogether this work identifies important aspects of virus-host interaction of two important respiratory pathogens. We provide new information on the mechanisms and impact of influenza A virus NS1 intracellular localization and we characterize MERS coronavirus infection in primary human leucocytes as well as highlight important differences in the host cell responses between MERS and SARS coronaviruses.
  • Wong, Swee Chong (Helsingin yliopisto, 2016)
    In the past decade, advances in next generation sequencing (NGS) and genotyping technology have enabled merging of genetic analyses in ecological field studies of natural populations. The development of transcriptome sequencing such as RNA-seq provides a golden opportunity for researchers studying non-model organisms. In the design of genetic analyses in the context of ecological studies, genetic relationships among individuals are often overlooked, especially when sampling wild populations. In ecological studies, studies of e.g., life-history traits, environmental and ecological factors are often the main issues of interest, and genetic analyses remain a secondary consideration. Samples from ecological studies often represent different types, such as inbred samples, samples with complex family structure, and samples with unknown relationships. These distinctly different experimental setups present a challenge for genetic association studies, as the underlying relationships among samples could affect allele frequency distributions in populations, leading to spurious associations. The primary aim of this thesis is to devise protocols for quantitative genetic studies of samples from experimental designs intended for ecological studies, using a non-model organism, the Glanville fritillary butterfly, as the study species. Strategies and methods were implemented to discover potential genetic factors affecting trait variation in the Glanville fritillary. Interactions between the organism and the environment, such as effects of temperature on flight metabolic rate, on phenotypic plasticity and on larval development were studied. All samples were obtained from a large metapopulation of the Glanville fritillary in the Åland Islands in Finland. Flight metabolic rate, life history, female reproductive traits, and phenotypic plasticity were measured in family-based material, while the study of larval development was implemented using a population-based model. Problems tied to to relatedness of samples in each experimental setting were addressed with various strategies. The key findings of this thesis include discovery of a novel association between a SNP in the sex chromosomal gene triosephosphase isomerase and flight metabolic rate in females. This discovery was confirmed with one additional material: a dataset with 16 population samples. This gene is potentially another candidate gene in regulating the complex flight metabolic pathway. An interaction between temperature treatment preceding flight activity and SNP genotypes in the phosphoglucose isomerase gene was found to influence flight metabolic rate. Individuals with the genotype AC performed better when treated with low temperature, but individuals with genotype AA showed superior performance in high temperature treatments. Moreover, in another experiment, individuals with the AA genotype were more tolerable to heat shock than the AC individuals. Three SNPs in the cytochrome P450 337 gene were associated with the total number of eggs and caterpillars produced by females in their life-time. Further sequencing showed that regions prior to the coding region displayed similar association pattern with the three candidate SNPs, suggesting that causal variants might be located in the 5 regulatory region. Finally, SNPs from the vitellin-degrading protease precursor were associated with the incidence of the extra eighth larval instar (a development stage of insects), though the result was not significant after adjusting for multiple testing. This work contributes to developing procedures for samples originating from ecological studies and which might have sub-optimal experimental design for genetic analyses. Rigorous steps such as relatedness control, confidence interval calculation, and genetic power estimation were performed in this thesis to help interpret results from different chapters. The thesis shows that sound protocols can be developed to address problematic issues related to relationships among the samples.
  • Valo, Satu (Helsingin yliopisto, 2016)
    Lifestyle and diet have a major effect on the development of colorectal cancer (CRC). Dietary habits of Western populations in particular are recognized as a risk factor for CRC. However, the mechanisms that mediate the effects of Western-style diet (WD) on colorectal tumor development are largely unknown. CRC develops via multiple steps which involve genetic changes, such as mutations in growth-regulatory genes, and epigenetic alterations, such as CpG island hypermethylation. Lynch syndrome (LS) is one of the most common inherited cancer susceptibility syndromes. It is caused by inherited defects of the DNA mismatch repair genes (MMR), which together with other genetic and epigenetic changes are known to accelerate tumorigenesis. MMR defects are known to accelerate tumor progression in LS but the early events leading to polyp formation and the timing and order of the molecular hits remain unknown. A long-term feeding experiment with mouse models for LS and sporadic CRC was conducted to characterize tumor-promoting changes in normal colonic mucosa caused by WD and/or genetic predisposition. Changes in the proteome of histologically normal colonic mucosa were monitored at different time points of feeding experiment (5 weeks, 12, 18 and 21 months) with two high-throughput proteomic methods followed by analysis of affected pathways. Data from proteomic analysis indicated that the proteome was more consistently changed by diet and aging than by genotype. Overall, 21 out of 26 colonic tumors were detected in mice fed with WD. Proteomic analysis indicated disrupted lipid metabolism and increased oxidative stress in the normal-appearing tissue in association with WD. Moreover, proteome analyses revealed increased cell proliferation and decreased apoptotic processes in the normal colon mucosa of mice fed with WD, which may promote colorectal tumorigenesis. Finally, proteomic data coupled with measurement of bile acids in tissue specimens indicated that WD induces downregulation of intracellular bile acid transport, resulting in disrupted bile acid homeostasis which may provide a possible mechanism underlying the tumor-promoting effects of the diet. Studies on sporadic CRC have demonstrated that promoter hypermethylation leading to gene silencing can act as an alternative mechanism to mutations in early stages of tumor development but its importance in hereditary CRC remains unknown. We analyzed tissue specimens gathered during colonoscopy surveillances and colectomies performed on human LS mutation carriers to define changes in CpG island methylation that occur at different stages of the tumor progression sequence. Methylation changes at different stages of tumor progression were analyzed in relation to MMR gene expression, and normal tissue biopsies were studied for carcinogenic fields . In addition, we aimed to clarify the role of CpG island hypermethylator phenotype (CIMP) in the LS-associated tumorigenesis. Results indicate that the expression of the MMR protein corresponding to the gene mutated in the germline decreases along with dysplasia but occurs as a relatively late event in the tumor progression sequence, suggesting the presence of other somatic events that drive neoplastic transformation. Indeed, significant increase in the average degree of methylation of two candidate genes (SFRP1 and SLC5A8) was observed in normal colonic mucosa biopsies from patients with CRC (high-risk mucosa) when compared to those without (low-risk mucosa), indicating a possible carcinogenic field. Moreover, methylation was found to increase in LS adenomas and carcinomas along with dysplasia. These findings emphasize the importance and early appearance of epigenetic alterations in LS-associated tumorigenesis. In summary, the results offer new insights into the initiating molecular mechanisms through which Western-style diet and DNA methylation contribute to hereditary and sporadic colorectal carcinogenesis.
  • Ozan, Martina (Helsingin yliopisto, 2016)
    Individuals opt to breed cooperatively to increase their chances of successful propagation when resources are scarce or unpredictable. Yet, these same individuals are not genetically identical and come into conflict over limited resources they are to utilize for own reproduction. In spite of the predicted reproductive conflicts, many species, notably social insects, have evolved to become obligate cooperative breeders unable to propagate solitarily. So how do individuals share reproduction? In social insects, reproduction and work is divided among the society members i.e. the queen(s) and workers. The evolution of a non-reproductive caste - workers, can be explained by kin selection theory, which holds that individuals gain fitness indirectly by helping their kin queen, to reproduce. In many species of ants, however, colonies permanently contain multiple reproductive queens (polygyny). Queens compete for limited colony resources, which may lead to a conflict over personal reproduction and unequal reproductive shares. The reproduction by several queens also dilutes within-colony relatedness, which comes at a cost to worker indirect fitness. Hence, underneath the exemplary cooperation among colony individuals, both queens and workers, are predicted to act to enhance their own inclusive fitness. The aim of my dissertation was to disentangle mechanisms underlying reproductive partitioning in a polygynous black ant, Formica fusca, within the framework of kin selection theory. I examined queen traits that likely impact queen fitness including timing of oviposition, queen presence during brood rearing, her fecundity, and viability and size of queen offspring, along with the underlying chemical communication, as well as workers ability to utilize and exploit the available information to manipulate queen reproduction to own fitness advantage. My thesis has revealed that both, queens and workers, have the means and ability to influence the outcome of reproductive competition by actively utilizing chemical cues present on ant cuticle to pursue selfish actions. At the same time, the results suggest that passive feedback mechanism between the queen reproductive output and worker behaviour, nevertheless, ensures alignment of fitness interests between both parties. Overall, my dissertation highlights that complex within-group interactions govern the reproductive partitioning in social insects and contributes to better understanding of how reproductive conflicts are resolved to ensure peaceful coexistence.
  • El-Showk, Sedeer (Helsingin yliopisto, 2016)
    The evolution of vascular tissues was a critical innovation in the colonization of land by plants. We investigated how vascular tissues, in particular xylem, are patterned in the root of the model plant Arabidopsis. The vascular tissues of the Arabidopsis root tip are consistently patterned as a xylem axis flanked by procambial cells, with phloem poles developing perpendicular to the xylem axis. Cytokinin signalling inhibits the specification of protoxylem; the AHP6 gene inhibits cytokinin signalling at the protoxylem position during normal vascular development. We sought to understand the factors regulating AHP6 expression in the root tip. Cytokinin signalling is known to flank the xylem axis; we discovered a complementary domain of auxin signalling throughout the xylem axis. Based on this, we showed that auxin upregulates AHP6, creating a domain of low cytokinin signalling, and also acts to specify protoxylem. We used a combination of mutants and pharmacological treatments to investigate how mutually exclusive auxin and cytokinin signalling domains are maintained in the Arabidopsis stele. We discovered a feedback loop between the hormones, in which cytokinin activates auxin exporters, while auxin represses cytokinin signalling. The mutual inhibition between auxin and cytokinin regulates the extent of their domains during vascular patterning. We turned to computational simulations to investigate the sufficiency, stability, and dynamics of this network. Our simulations confirmed that the network is sufficient to maintain the hormone domains during vascular patterning, but also revealed a role for auxin importers, which we confirmed through experiments. While cytokinin is frequently thought to form gradients guiding developmental processes in the Arabidopsis shoot and root, we showed that an informative cytokinin gradient cannot form on the scale of these tissues via diffusion. While auxin is patterned through the activity of polarly localised transporters, there is no evidence for similar transport of cytokinin. Nevertheless, our findings highlight the need for a cytokinin patterning mechanism, such as directed cytokinin transport or patterning of the cytokinin perception machinery, since diffusion cannot form the observed cytokinin patterns. Finally, we discovered a potential link between the auxin-cytokinin feedback loop in the root tip and the initiation of lateral roots. Since our experimental data are equivocal on whether or not PIN1 is polarly localised in the procambium, we investigated both possibilities in our computational model. We discovered that polar localisation of PIN1 results in a regular flux of auxin towards the centre of the stele and back out via the xylem axis. This circuit privileges pericycle cells flanking the xylem axis to accumulate auxin if they experience a brief activation of an auxin importer; activation of the importer AUX1 in the xylem-pole pericycle cells is one of the earliest steps in lateral root initiation. Altogether, my thesis reveals a key role for mutually inhibitory auxin-cytokinin interactions in vascular development and links these findings to other developmental contexts. This work also demonstrates how the combination of experimental & computational approaches enables us to critically evaluate our models and develop more general insights.