Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Kallio, Katri (Helsingin yliopisto, 2016)
    All positive-strand RNA viruses replicate their RNA genomes in close association with cellular membranes. A great variety of cellular membranes are utilized by different viruses and those membranes are extensively modified to support viral replication and to protect the viral RNA from host cell defense mechanisms. Alphaviruses, including Semliki Forest virus (SFV), are positive-strand RNA viruses replicating their RNA on membranes derived from endosomal and lysosomal compartments. SFV induces small invaginations called spherules on plasma membrane and on endosomal membranes. Viral replication complex assembly, spherule formation and initiation of replication are carefully orchestrated events and are guided by specific sequence elements within the genomic RNA as well as by important enzymatic activities of nonstructural proteins (nsPs). The aim of this research was to study in detail how alphavirus replication complexes are assembled and to define the minimum requirements for spherule formation by using a plasmid-derived transreplication system mimicking SFV replication. The role of the genomic RNA in replication was deciphered by using RNA templates, which were either modified or differed in length. Use of RNA templates differing in length clearly showed that they define the spherule diameter suggesting that the template has a significant role in spherule formation. By modifying or deleting specific sequences from the template it was shown that highly conserved RNA elements are important for SFV replication and do not tolerate modifications without compromising replication. Study with the nsPs of SFV showed that the enzymatic activities essential for virus replication are also needed for spherule formation and that enzymes like helicase, protease and polymerase are absolutely essential for replication. Membrane association of the replication complex is also required to establish virus replication in the cells. The work with mutated nonstructural proteins and modified templates revealed a clear correlation between the minus-strand synthesis and spherule formation. This work describes the alphavirus replication processes in detail and provides new principles, which may be generally applicable to study the positive-sense RNA virus replication and the formation of virus-induced membranous replication spherules.
  • Söderholm, Sandra (Helsingin yliopisto, 2016)
    Phosphorylation is one of the most important post-translational modifications of proteins. Phosphorylation is a rapid and reversible way of modifying proteins; it is involved in the regulation of many cellular processes, serving as the main transducer of intracellular signaling cascades. It is possible to identify thousands of protein phosphorylation sites from a single sample with mass spectrometry (MS)-based phosphoproteomics. This is the main reason why MS-based phosphoproteomics is such an excellent method for revealing global changes in phosphoproteomes. Computational analysis of the MS data is vital for identifying the proteins and post-translational modifications. The data analysis steps in the phosphoproteomics workflow are also crucial for biological interpretation, e.g. identifying activated kinases and kinase substrates, as well as unravelling signaling pathways and networks. Cells of the innate immune system are central players in the host defence against pathogens such as viruses. Their pattern-recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs), which are conserved structures present in pathogens. The most important PAMPs in viral infections are the viral genomes and replication intermediates, and their detection evokes pro-inflammatory and antiviral responses in the infected cell. Host factors and signaling cascades that promote or inhibit virus infections can serve as potential drug targets. Since protein phosphorylation is vital for the progression of nearly all signaling cascades, there is an increasing interest in applying phosphoproteomics and combining it with bioinformatics in studying the regulation of cellular signaling under various conditions, including viral infections. The main aim of the studies included in this PhD thesis was to characterize the global changes in the cellular phosphoproteomes of virus infected and dsRNA stimulated innate immune cells, and to identify the host proteins and cell signaling pathways involved in the early stages of the host response to Sendai virus (SeV), influenza A virus (IAV), and viral dsRNA. A computational analysis tool, named PhosFox, was developed for processing and comparing MS-based phosphoproteomic data generated by multiple database search algorithms. PhosFox was used for cross-sample comparisons of phosphopeptide identifications. PhosFox also facilitated the identification of those proteins whose phosphorylation was different between samples, and the clarification of phosphorylation sites not described in the literature. The findings from the phosphoproteomic data were explored further by implementing functional studies involving small interfering RNAs (siRNAs) and kinase inhibitors. There were extensive alterations in the phosphorylation of proteins in human epithelial cells and macrophages that were transfected with the synthetic dsRNA-mimic polyinosinic:polycytidylic acid (pI:C) or infected with SeV or IAV. Many of these proteins were determined to be members of pathways with little or no previously known role in the antiviral response to these particular viruses and viral PAMPs. Two novel host factors, RelA-associated inhibitor (RAI) and sirtuin 1 (SIRT1), were identified as negative regulators of dsRNA-induced apoptosis and NF-κB regulated cytokine expression by combining 14-3-3 interactome and phosphoproteome characterizations. The p38 mitogen-activated protein kinase (MAPK) signaling pathway was found to be regulating cytokine expression and apoptosis in dsRNA-transfected human keratinocytes. MAPK signaling pathways were also regulated in SeV and IAV infected cells. The mTOR signaling pathway was shown to be critical for the interferon response and virus replication in SeV infected human lung epithelial cells. A substantial number of those cellular proteins whose phosphorylation status changed after SeV or IAV infection or viral dsRNA challenge were involved in Rho GTPase signaling. Major changes in protein phosphorylation in IAV infected primary human macrophages were linked to cyclin-dependent kinases (CDKs). CDK activity was shown to be required for efficient viral replication and host response in IAV infection. Administration of one specific CDK inhibitor, SNS-032, also protected mice from IAV-induced death. The studies included in this PhD thesis are some of the first to apply phosphoproteomics for characterizing host-virus interactions. They emphazise the benefit of applying phosphoproteomics and bioinformatics in innate immunity and viral research, i.e. it is possible not only to identify cell signaling pathways, but also the specific host factors that regulate the cellular responses to viral infections. The results of these studies underline the importance and the potential MS-based phosphoproteomics has in the discovery of novel host factors, which can serve as possible antiviral drug targets. In conclusion, the MS-based phosphoproteomics approach led to the discovery of novel host factors and cell signaling circuits in virus infected innate immune cells.
  • Venesjärvi, Riikka (Helsingin yliopisto, 2016)
    Maritime transport is an efficient way to ferry goods, oil, and chemicals but shipping poses a threat to marine ecosystems. Oil spills have a potential to extinguish or debilitate fish and wildlife populations and habitat types important to the marine ecosystem. The thesis assesses the environmental impacts of oil spills and the effectiveness of management practices to mitigate their adverse ecological effects. Improved strategies combine theoretical disciplines, such as population biology with practical oil spill response. The original five scientific papers contribute to answering the questions, 1) what are the methods for gathering knowledge on the impacts of oil on sensitive species, 2) how could ecological knowledge be given to the decision-makers, and 3) what are the improved approaches to the required ecological knowledge in oil spill risk management? Based on the synthesis of this thesis, suggestions can be made to improve oil spill risk management and the development of knowledge-induced policy planning. The results demonstrate that environmental risk assessment models can be used to structure problems, integrate knowledge and uncertainty, and persuade decision-makers by visualizing the results. Since the objective of risk assessment is to synthesize information for environmental management and policy design, which should rely on the extensive use of scientific evidence, communication between academia and decision-makers is of great importance. The use of Bayesian networks would improve the current oil spill risk management in the Baltic Sea, since all the variables affecting oil spill risk can be presented in one framework in a transparent manner. Many geospatial services work as tools of informative policy instruments, as they deliver ecological data and knowledge for oil spill risk management. Researchers could also participate more often in the contingency planning or practical management of oil spills as experts. Thus, all the relevant knowledge could be integrated into the decision-making process. The thesis offers new insights into oil spill risk management in the Baltic Sea and provides examples showing how evidence-based management actions should be chosen and carried out in order to minimize the risks. Policy recommendations are also provided. First, in oil spill risk management, the marine ecosystem should be prioritized based on its conservation value, recovery potential and protection effectiveness. Second, because preventive measures against oil accidents are considered cost-effective, maritime safety should be increased, with stricter and regional ship inspection practices. The effects of policy innovations should be assessed using probabilistic policy-support tools.
  • Eklund, Johanna (Helsingin yliopisto, 2016)
    Protected areas are a key tool for conserving biodiversity and an increase in their coverage has long been the aim of international conventions and initiatives. With progress to achieve target 11 of the Convention on Biological Diversity of protecting 17 % of terrestrial areas, the focus has now shifted towards assessing the protected area effectiveness in maintaining species or avoiding land conversions. In my doctoral thesis I develop a novel way of assessing PA effectiveness, based on the counterfactual thinking, and use this to link it to different management and ecological factors. I link different aspects of PA effectiveness conceptually to the quality of governance and show how spatial prioritizations can change with the inclusion of these socio-political factors. Using Madagascar as a case study, and in line with other studies elsewhere, I find that the protected area network is effective to some extent in mitigating the pressure of deforestation. I show the importance of considering the temporal dimension of protected area effectiveness measures and how protected area effectiveness changes over time due to increasing or decreasing pressures. These results link directly to considerations of vulnerability and irreplaceability in Systematic conservation planning and I show that accounting for governance factors in a global spatial prioritization analysis change the identification of areas. My thesis shows the relative nature of protected area effectiveness measures and how important it is to get the assessments right, especially because of the massive focus on protected area effectiveness as a panacea to stopping biodiversity declines. Improving protected area effectiveness needs to be linked to governance factors affecting not only the management but also the drivers of threat, something that previous studies have overlooked. With my thesis I make an attempt to bridge the themes of protected area effectiveness, considerations of quality of governance, and how it all links to conservation prioritizations. Our methodology has been developed with the aim to be computationally efficient and conceptually more robust than existing matching methods, with the potential to be scaled up for larger studies. However, how the two methods perform needs to be tested in the future. My dissertation has clear practical implications for the conservation of Madagascar s biodiversity and the results are of potential interest for both NGOs and the Madagascar National Park administration. The conceptual contribution of this thesis should be incorporated into mainstream thinking and the discourse of setting global priorities for biodiversity conservation, such as by the International Union for Conservation of Nature and Natural Resources (IUCN), the World Parks Congress (WPC) and ultimately the Convention on Biological Diversity (CBD).
  • Howlader, Mohammad Sajid Ali (Helsingin yliopisto, 2016)
    Amphibians are the most threatened class of vertebrates. About 48% of the known amphibian species are threatened by extinction, and many species still remain undescribed, especially from tropical and sub-tropical countries such as Bangladesh. In contrast to India and Sri Lanka, amphibian diversity in Bangladesh is poorly known, and little effort has been put towards documenting the species diversity and resolving evolutionary affinities among amphibian taxa in this country. Hence, the actual diversity of amphibians in Bangladesh remains unknown. The aim of this dissertation work was to improve our knowledge of amphibian diversity in Bangladesh by identifying and describing new amphibian species and investigating their evolutionary relationships with closely related taxa. I used morphological and molecular phylogenetic methods to identify and describe one new genus and five new species from different genera. In addition to using traditional morphological comparisons, I also utilized mitochondrial gene fragments to estimate phylogenetic affinities among the studied taxa, with Maximum-likelihood and Bayesian methods. The first two chapters of the thesis focus on the amphibian genera Fejervarya and Zakerana, the latter which was previously embedded within Fejervarya. These chapters also include descriptions of two new species Fejervarya asmati sp. nov. (now Zakerana asmati), as well as Fejervarya burigangaensis sp. nov. , respectively. In the third chapter, a new species (Zakerana dhaka sp. nov.) is described from the urban core of Dhaka, the capital of Bangladesh and one of the most densely populated mega cities in the world. In the fourth chapter, I describe Euphlyctis kalasgramensis sp. nov., which was earlier recognized as E. cyanophlyctis, and show that it is genetically highly divergent from the E. cyanophlyctis described from southern India. In the last and fifth chapter, I describe Microhyla nilphamariensis sp. nov. as a new species. It is a member of a highly genetically heterogeneous group of frogs that have been recognized as M. ornata for the past 173 years. In general, the results of the studies included in this dissertation advance our understanding of amphibian diversity in Bangladesh and adjacent regions, and show that discovery and description of new amphibian species from this region is still fairly easy. Consequently, it seems likely that more thorough sampling and further investigations in this region can uncover additional new amphibian species to science. Such studies, together with the discoveries described in this thesis, should also provide useful information for understanding and conserving the amphibian biodiversity in this poorly studied region.
  • Mathijssen, Paul (Helsingin yliopisto, 2016)
    Peatlands contain approximately a third of all soil carbon (C) globally and as they exchange carbon dioxide (CO2) and methane (CH4) copiously with the atmosphere, changes in peatland C budgets have a large impact on the global C balance and on the concentration of greenhouse gases in the atmosphere. How peatlands will react to future climate changes, however, is still relatively uncertain and as such there has been a growing interest in the reconstruction of past peatland C dynamics and linking these to past climate variations. In order to increase the understanding of peatland development and response patterns, I quantitatively reconstructed the Holocene (the last c. 11700 years) C dynamics of three different peatlands in Finland: a subarctic rich fen, a boreal poor peatland complex and a boreal managed pine bog. Several cores from each peatland were studied in order to reconstruct peatland succession, lateral expansion, peat and C accumulation rates, long term uptake of atmospheric CO2, CH4 fluxes and radiative forcing (RF). Peatland lateral expansion was most rapid during periods with relatively cool and moist climate conditions. The peatlands showed distinct successional pathways, which were sometimes triggered by fires. Successional stages were partly reflected in C accumulation patterns. In some cases, variations in C accumulation rates coincided with autogenic changes in peat type and vegetation, although accumulation rates were also related to the large-scale Holocene climate phases. The warm and dry conditions during the Holocene Thermal Maximum (between c. 9000 and 5000 years ago) reduced C accumulation rates in the subarctic fen and the boreal peatland complex. Reconstructed CH4 emissions suggest that CH4 emissions played a major role in the total C budget of the peatlands throughout the Holocene. The RF models based on long term CO2 uptake and CH4 emissions showed that the two boreal peatlands had a warming effect on the atmosphere for the first 4000-7000 years after the start of peat accumulation, after which they had an increasing cooling effect as a result of the long term effect of C uptake and storage. In contrast to the two southern sites, the subarctic fen had a warming effect through its entire history as a result of very low C accumulation rates. The results of my study show that peatland processes react differently to allogenic factors, such as climate and fire, depending on peatland type, microtopography and local hydrology. It highlights the necessity to study multiple peat cores per site before making exhaustive conclusions on historical development patterns and implications. The combination of lateral and vertical peat growth data with reconstructed CO2 and CH4 fluxes provided the necessary information for a comprehensive quantification of the climate - peatland feedback. In the studied sites this feedback seemed to be very sensitive to short term variations in CH4 emissions and lateral expansion.
  • Sakha, Prasanna (Helsingin yliopisto, 2016)
    Neurons have distinctive polar morphology with distinctive subcellular features comprising of cell soma, axons and dendrites. Primary objective of this study was to develop a novel microfluidic device for spatial isolation of axons from the somatodendritic compartment of cultured hippocampal neurons. A new method was developed for asymmetrical genetic manipulation improving specificity in studies of how individual proteins affect axonal morphology, presynaptic development and function. Subsequently, the microfluidic culture system was used to study the signaling events involved in synaptogenesis, focusing on the roles of kainate type of glutamate receptors (KARs). Functional studies have shown that KARs are present in axons and may regulate presynaptic function. However, the molecular composition and detailed subcellular localization of axonal KARs as well as their roles in presynaptic differentiation are largely unknown. The results show that axonal KARs promote early stages of synaptogenesis. Expression of low (GluK1-3) and high affinity (GluK4-5) KAR subunits promoted filopodiogenesis function at the isolated axons. In addition, axonal low affinity subunits enhanced clustering of synaptic vesicles and transmission efficacy at nascent glutamatergic synapses, an effect which was associated with widening of presynaptic active zone. High affinity KAR subunits had no effect on synaptic vesicle clustering, nor presynaptic transmission efficacy. However their heteromerization with low affinity subunits completely prevented the synapse promoting effects and instead lead to strong inhibition of presynaptic transmission efficacy. The presynaptic effects of GluK1-3 on synaptic vesicle clustering involved both PKA and PKC pathways. GluK1 expression was developmentally regulated in neonatal and juvenile hippocampus and heteromeric combination of GluK1c with high affinity subunits suppressed glutamatergic synaptic transmission. KARs are linked to various neurological and neuropsychiatric disorders. Our observations and previous findings strongly suggest that KARs are involved in morphological maturation of neurons and in refinement of neuronal circuitry in the brain. The present results provide novel insights into the involvement of different types of KAR subunits in synaptic development and morphological differentiation. Hence, they are potential therapeutic targets in various developmentally originating neurological disorders.
  • Mäkeläinen, Sanna (Helsingin yliopisto, 2016)
    Anthropogenic land use has caused detrimental impacts especially on forest ecosystems, and native forested landscapes have been lost and fragmented worldwide. Forest-dwelling animals are generally susceptible to habitat loss and fragmentation because of their strict habitat requirements and dependency on forests for food, nest sites and movements. Consequently, forest specialists, such as arboreal squirrels and gliding mammals, provide a worthy group of model species in order to assess the influence of landscape modification. The Siberian flying squirrel (Pteromys volans) is an arboreal rodent inhabiting spruce-dominated mature forests and due to destruction of its most suitable breeding habitat, the population has been decreasing in Finland. In my thesis, I investigate the effects of landscape modification on the occurrence, space use and survival of this species at multiple spatial scales. The occurrence probability of flying squirrel increased with a proportion of suitable movement forests on a 400-m-scale and was negatively associated with the isolation of occupied sites in a partly urban study area. However, no negative effects of urban habitat types were found, which indicates that the species is not disturbed by urbanization. It was found that regional environmental authorities were unaware of the presence of flying squirrel in most cases of forest harvesting in southern Finland. Despite occupied sites were delineated according to given guidelines, many of these sites became deserted after forest harvesting. This shows that the legal habitat protection of flying squirrel is ineffective and to improve this conservation practice, larger forested areas should be maintained around inhabited sites. The presence of urban habitats on movement routes increased movement distance and speed whereas urban habitats within home-range impeded only male movements. Structural forested connections had varying effects on nest-site switching and their importance remained unclear, which points out that defining and maintaining a species-specific connectivity is challenging in human-modified landscapes. Regional variation in flying squirrel survival was most likely caused by differences in predation pressure, predator community and landscape composition. Male mortality increased with a proportion of low-quality habitats in the surrounding landscape, whereas fine-scale habitat composition of the most used areas did not show any survival impacts. Natal dispersal distances of juveniles did not increase their mortality. Results of this thesis indicate that the effects of landscape modification on forest-dwelling animals are varying, sex-specific and depend on the scale. As landscape modification can also affect species indirectly, it is important to investigate the costs and risks of animal movements in human-modified environments.
  • Merikanto, Ilona (Helsingin yliopisto, 2016)
    Many existing and emerging microbial infectious diseases are caused by environmentally growing opportunist pathogens. These pathogens are, contrary to obligatory pathogens, able to survive and replicate in the outside-host environment as free-living microbes that use within-host growth as an alternative replication strategy. This disease class has eco-evolutionary implications in natural populations and causes a serious health and economical threat to humans, our food production and to wildlife. Because of the ability of environmentally growing opportunists to survive and replicate independently of hosts, these diseases are hard to eradicate with conventional methods. The conditions that favor or disfavor environmental opportunism are still poorly understood. Better understanding of the dynamics of these diseases is needed in order to develop proper control methods against them. In this thesis I have developed novel epidemiological models to describe the disease dynamics of environmentally growing pathogens. These models modify the traditional Susceptible-Infected host (SI-model) framework by combining it to the outside-host community of an environmentally growing pathogen. I have considered how the environmental growth of the pathogens and the antagonistic ecological interactions these pathogens face in the outside-host environment, such as competition, predation and parasitism, affect the disease dynamics, invasion of novel pathogens and biological control of environmentally growing infectious diseases. The analyses show that the disease dynamics of environmentally growing pathogens differ from obligatory pathogens. Importantly, ability to grow in the outside-host environment promotes disease outbreaks and can lead to the extinction of the host, which is untypical in the case of obligatory pathogens. Antagonistic interactions the pathogen faces in the outside-host environment can on the other hand limit disease outbreaks and prevent extinction of the hosts that would otherwise occur due to the disease. I conclude that the eradication can be accomplished 1) by increasing the outside-host competition, 2) through predation of pathogens, or 3) through viral infections in pathogens.
  • Saarikivi, Jarmo (Helsingin yliopisto, 2016)
    Ecologically simplified urban landscapes are often claimed to be useless nature with hardly any wildlife. Golf courses in particular are often claimed to be a poor use of land, ecologically speaking. They cover large areas, have a restricted entrance, are heavily managed and require a lot of resources in their up-keeping. These green deserts and elitist people practicing their sport have been subject to much debate and environmental concern. Populist, active and loud lobbying against the building of golf courses has created a popular opposition, although there is little scientific evidence to support their claims. Therefore, it is of interest to determine to what extent these claims are justified. In this thesis, the ecological value of golf courses was assessed to determine how golf courses contribute to the diversity of open green spaces in an urban setting, by studying the biodiversity of established and newly-created golf courses in the greater Helsinki region, southern Finland. In a series of field experiments, the biodiversity of various animal groups (insects, birds, frogs) with distinct life histories was measured at urban or semi-urban golf courses. Each animal group was used to study different aspects of diversity. Species diversity and assemblages were studied using carabid beetles (articles I, IV), genetic diversity using the common frog (Rana temporaria) (II) and a case study to measure the quality of the golf course environment as habitat was performed using three hole-nesting bird species (great tit, blue tit and pied flycatcher), evaluating their nesting and reproductive success at golf course forest edges (III). Furthermore, the effects of constructing a golf course on carabid beetles were evaluated before (I) and after (IV) course development. Golf courses appeared to be rich in carabid beetle species, very few of them rare or of conservation concern though. Beetle assemblages differed considerably between the golf courses studied and between the habitat types sampled on these courses, but not between course development stages (established vs. newly created vs. reference area). Genetic variation of common frog populations in golf courses showed little differentiation, suggesting that golf courses contribute positively to urban amphibian populations by providing suitable water bodies for reproduction and green corridors for dispersal, thus preventing isolation and loss of genetic variability within populations. Hole-nesting passerine birds showed a clear preference for golf course forest edges over the nearby forests. Birds also performed better in terms of nest occupancy and number of offspring at golf course forest edges, thus indicating a valuable habitat, which could be further improved with the addition of nest boxes. The studies show that golf courses host high biodiversity and have conservation potential in ecologically simplified urban landscapes. Urban fauna seems resilient and capable of adapting to these highly modified environments, thus providing possibilities to enhance biodiversity though the combined and well-planned efforts of various practitioners.
  • Meller, Kalle (Helsingin yliopisto, 2016)
    As the climate of our planet is warming, there is an urgent need to understand how temperature changes affect nature and how the species are able to cope with the changing environment. In my thesis I used Finnish long-term monitoring data to examine the effects of fluctuations in temperature and other environmental variables on the migration behavior and breeding performance of birds. I found that during warm springs birds started to breed earlier, which increased the length of the time period spent in the breeding area because the timing of autumn migration remained mostly constant (only the young from the latest broods migrated later). After warm springs productivity (the number of offspring per adult) was moderately but uniformly higher than after cold springs in the study community passerine birds. The annual productivities and population sizes varied synchronously among the species. Despite I found connections between the annual temperature, productivity and population size, the long-term population trends were not connected to changes in productivity. This implies that changes in survival during the non-breeding period would be driving the trends. Mild winter temperatures caused higher proportion of partially migratory waterbirds to overwinter in Finland than in cold winters. Climate warming related lack of severe ice winters arguably was behind the steep increase in the proportion of resident in waterbirds, while there were no trends in terrestrial species during the study period. The annual wintering area choice of terrestrial species was little affected by temperature, but in several species the seed crop of trees had a strong impact. Modelling the distribution of migration phenology is often essential for studying whether the temperature related adjustments in phenology are sufficient to cope with the effects of climate change. My comparison of methods showed that usually relative simple distributions describe the migration phenology well. I also provided a simple novel method to account for the day-to-day variation in migration data. My findings indicate that the life-history traits of the northern bird species are responsive to the variation in temperatures, but there are differences between taxa and functional groups in the responses, which underlines the importance of studying numerous taxa in order to draw general conclusions and predict the effects of continuing climate change.
  • Noreika, Norbertas (Helsingin yliopisto, 2016)
    Negative anthropogenic disturbances (e.g., drainage and urbanization) are causing biotic homogenization through the replacement of specialist species with generalists. The identification and conservation of biodiversity hotspots within degraded (e.g., highly urbanized) landscapes, and ecological restoration (i.e., positive anthropogenic disturbance) have the potential to be important tools to counteract these negative effects. Mires are suitable targets for the investigation of these homogenization-reducing activities since they host many mire specialist species of, e.g. invertebrates. The main aim of this PhD thesis was to investigate the effects of negative anthropogenic disturbances [urbanization (Chapter I) and drainage for forestry (Chapters II-IV)] on the invertebrate communities of boreal mires and how effective efforts are to reverse these negative effects through ecological restoration [i.e. positive anthropogenic disturbance (Chapters II-IV)]. In addition, the purpose was to determine which environmental variables are key in supporting mire specialist invertebrate species and communities. Therefore, this thesis started by reviewing current knowledge on the responses of mire invertebrate species and communities to anthropogenic disturbances. The effects of urbanization were studied on spiders and carabid beetles (Chapter I), while the effects of drainage for forestry and subsequent restoration were investigated on five solitary invertebrate groups (Chapter II) and social insects, i.e. ants (Chapter III). Finally, a powerful Before-After Control-Impact (BACI) design was used to reveal the effects of drainage and restoration on butterflies (Chapter IV). Generally, both high levels of urbanization (Chapter I) and mire drainage for forestry (Chapters II-IV) had negative effects on mire specialist species (lower abundances) and invertebrate communities (homogenized and very different in structure from pristine mire communities). However, these detrimental effects can be reduced or even reversed through appropriate urban mire conservation and ecological restoration. Local habitat conditions were shown to be particularly important for the survival of specialist invertebrate species in urban mires (Chapter I) and for the successful recovery of restored mire invertebrates (Chapters II-IV). Individual mire specialist species responded negatively to environmental variables associated with deteriorated (i.e. drained or highly urbanized) mire conditions [number of high (> 3m) trees for carabid beetles, crane flies, micromoths (Chapter II), ants (Chapter III) and butterflies (Chapter IV)] and positively to pristine mire-associated variables [Sphagnum cover for carabid beetles and spiders (Chapters I-II), crane flies (Chapter II) and suggestively for ants (Chapter III); larval food plant cover and number of lower (1.5 - 3m) trees for butterflies (Chapter IV)]. The more specialized the mire species were, the more negatively they were affected by deteriorated-mire-associated variables and the more positively they responded to pristine-mire-associated variables. I conclude that the restoration actions taken (removing tall trees but leaving smaller trees, and raising the water table level) are appropriate in creating suitable habitat conditions for mire invertebrates, as both individual specialist species and communities showed positive responses already 1-3 years since restoration (Chapters II-IV). Finally, the appropriate restoration actions in well-prioritized locations as well as urban mire conservation should reverse the trend of biotic homogenization.
  • Mäkelä, Sanna (Helsingin yliopisto, 2016)
    Influenza viruses are human respiratory pathogens that cause seasonal epidemics and pandemics. The host restricts the virus infection by inducing immune responses aiming at virus clearance. The immune response has two arms. The innate immunity is the first line defense mechanism that is activated immediately after the recognition of the pathogen. The adaptive immunity, which consists of humoral and cell-mediated immunity, takes more time to develop. The epithelial cells of the respiratory tract and innate immune cells, such as macrophages and dendritic cells, are equipped with a plethora of receptors and signaling molecules that are designed for pathogen recognition. These receptors include Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). The pathogen recognition by these receptors leads to the activation of complex cellular signaling cascades that culminate in the production of cytokines, small proteins that mediate the communication between cells. In influenza infection, one important class of cytokines is interferons (IFNs) which induce the production of antiviral proteins that are able to inhibit virus infection. On the other hand, influenza viruses are capable of evading innate immune surveillance and there are differences between influenza virus types or strains in their immune evasion mechanisms. In this thesis work influenza virus-induced IFN responses were studied in human macrophages and dendritic cells in vitro. Firstly, we showed that in macrophages influenza B virus infection induced a very early IFN-β and IFN-λ1 gene expression that coincided with the nuclear entry of the virus and the activation and nuclear import of IFN regulatory factor 3 (IRF3). This early activation did not take place in influenza A virus-infected cells. Furthermore, our study indicated that RIG-I receptor was essential for the early IFN gene expression. Secondly, we compared the cytokine responses induced by pandemic H1N1 influenza A virus to the ones induced by seasonal influenza A viruses in human macrophages and dendritic cells. We showed that the pandemic influenza A virus induced weak IFN responses but was highly sensitive to the antiviral actions of IFNs. During the infection, different types of microbial structures are present and can be recognized by different cellular receptors. Another aim of this thesis was to elucidate the mechanism of receptor cooperation in inducing synergistic cytokine production. We confirmed the previous findings that TLR3 or TLR4 together with TLR7/8 induces synergistic interleukin (IL)-12 and IFN gene expression in human dendritic cells. We studied, which regulatory factors bound to IL-12 and IFN-λ1 gene promoters during a synergistic stimulation and which cell signaling pathways took part in the cytokine production. We conclude that at the transcriptional level, several different IRF proteins and cell signaling pathways cooperate in the synergistic IL-12 and IFN-λ gene expression. In addition, we propose that IFNs produced after stimulation of the TLR3 pathway induce the expression of TLR7 receptor and other cell signaling components that create a positive feedback loop that further augments the cytokine and IFN production during synergistic stimulation. This thesis discusses the host-pathogen interactions in the human system and clarifies the cell signaling pathways leading to synergistic cytokine gene expression. Moreover, the early events in influenza B virus infection and IFN responses induced by pandemic H1N1 influenza A virus are described. More detailed knowledge of the human innate immune responses induced by host-pathogen interactions is needed for the development of effective vaccines and antiviral treatments against influenza virus.
  • Liu, Xinxin (Helsingin yliopisto, 2016)
    The worldwide use of triazines as pesticides has resulted in their widespread occurrence in groundwater. Within the northern boreal region in Southern Finland, 30% of groundwater sampling points contained pesticides, and acceptable drinking water limits were exceeded in 11%. Atrazine and its degradation products were among the most common pesticides observed. Chemical pesticide degradation in soil is slower than microbial degradation. Biodegradation often decreases with increasing depth. Nutritional factors and soil physicochemical properties affect microbial pesticide degradation. However, their effects on separate microorganisms, and the genetic basis of these responses, are not well documented. The aim of this thesis was to isolate and characterize atrazine-degrading microorganisms, and to find appropriate microbial, physicochemical, and nutritional demands for pesticide degradation. Microbial community composition was studied in farmland, forested farmland, and primary forest soils by pyrosequencing and in gardens, groundwater deposits, and vadose zone sediments by cultivation on mineral medium with atrazine or terbutryn as the nitrogen source. Atrazine dissipation efficiency was additionally compared under stagnant and circulating water conditions. The dominant phyla that increased in atrazine-treated farmland, gardens, deposits, and sediments were Proteobacteria and Actinobacteria. The overlap in genera was less than in phyla, while the isolated Pseudomonas strains only slightly overlapped between isolates from surface soils and subsurface deposits and sediments. Atrazine dissipation was better in circulating than in stagnant water, and aerobic microbes from genera known to have atrazine degradation genes, all from phyla Proteobacteria and Actinobacteria, were simultaneously enriched,. Based on the results, the application of microbial remediation of atrazine and terbutryn requires special attention to soil physicochemical properties and selection of proper microbial strains.
  • Araujo, César (Helsingin yliopisto, 2016)
    Prostatic acid phosphatase (PAP) was discovered during the mid-1930s, but the molecular mechanisms in which this protein is involved remain poorly understood. This enzyme was originally described as a highly-expressed protein in the human prostate that was secreted to the seminal fluid. It has always been associated to prostate cancer, since high levels of acid phosphatase activity were found in the sera of patients with metastatic disease. Therefore, for 40 years, research was focused on the improvement of biochemical assays in an effort to find specific substrates for clinical application. However, in the 1980s PSA (prostate specific antigen) superseded PAP as a biomarker for early detection of the disease and became the preferred marker for diagnosis. Later, in the mid-1990s with the advent of new molecular techniques such as cloning and high-scale protein purification it was possible to obtain high-quality crystals for 3D-structural determinations of PAP. In addition, new concepts emerged concerning the physiological role of the enzyme, with renewed speculation regarding the molecular mechanisms in which this protein could be involved. The fact that the serum levels of the enzyme are increased in prostate cancer patients with metastatic disease rendered this enzyme an attractive target for immunotherapies against advanced prostate cancer. However, this hypothesis has thus far neglected the existence of any potential isoforms that could be expressed in other organs and tissues. Therefore, as part of this thesis, the molecular mechanisms where PAP is involved will be investigated. For this purpose, two biological tools were employed: a PAP-knockout mouse model and stable virus-transfected LNCaP cell lines. A novel transmembrane type-I isoform of PAP (TMPAP) was first characterized as a product of alternative splicing of the same gene (ACPP) that encodes for the well-known secretory isoform (SPAP). TMPAP is distributed throughout mouse tissues, including prostate, lung, kidney, endometrium, salivary glands, and dorsal-root ganglia. The enzyme comprises an N-terminal domain containing the catalytic active site, a transmembrane helical domain, and a short C-terminal cytosolic domain that carries a tyrosine-based motif (Yxxφ) that targets the enzyme to the endosomal-lysosomal/exosomal pathway. This was confirmed by co-localization studies that revealed that PAP localizes to the plasma membrane as well as to the intracellular membranes of vesicles, lysosomes, and intraluminal vesicles of the multivesicular endosomes. Microarray experiments were performed on mouse tissues to study the differential gene expression profile between wild-type and PAP-knockout mice. The differential gene expression that was observed between the prostates of wild-type and PAP-knockout mice suggested that PAP is involved in secretory mechanisms, as many genes related to this process appeared dysregulated. Moreover, the results obtained from two-hybrid system experiments suggested that snapin (a SNARE-associated protein) was a likely candidate protein that could interact with TMPAP. This interaction was recently proved by co-localization and florescence resonance energy transfer (FRET) studies. The PAP-knockout mice developed prostate adenocarcinoma and showed dysregulation of genes related to vesicular traffic. Consequently, this investigation was focused on murine submandibulary glands (SMG) as a model of an exocrine organ. The expression of PAP in SMG was found to be even higher than in mouse prostate. In addition to microarrays and miRNA analyses, physiological and biochemical determinations help to demonstrate that there is an increased salivation volume in PAP-knockout mice upon stimulation with secretagogue drugs. This supports the hypothesis that PAP is involved in the regulation of secretory and exocytic processes. PAP was found to account for 50% of the total acid phosphatase activity in male mouse saliva and it is expressed by the granular convoluted tubular cells of the male SMG but not by the acinar cells. Unlike prostate gland, however, the mouse SMG does not develop signs of hyperplasia or adenocarcinoma in spite of an observed increased acinar cell proliferation. This discrepancy was explained by studying the degree of lymphocyte infiltration, the dysregulation of miRNAs, and the differentially expressed genes in microarray data. In SMGs of PAP-knockout mice, the innate immune system was shown to be responsive and able to remove proliferating acinar cells, which may explain the absence of adenocarcinoma. In addition, the upregulation of anti-inflammatory molecules may prevent the extension of tissue damage. Finally, we compared the effect of the overexpression of SPAP and TMPAP in LNCaP cells with empty-vector cells. As a result, the TMPAP-LNCaP cells exhibited slower growth than SPAP-LNCaP or empty-vector cells. Cells overexpressing either SPAP or TMPAP isoform showed increased 2D-projection area and increased HRP-uptake when compared with empty-vector cells. These two observations suggested an increased vesicular traffic in endo/exocytic pathways to maintain cell membrane homeostasis. Thus, vesicles loaded with TMPAP are most likely sorted to lysosomes by means of its Yxxφ motif. Consequently, there is an increased degradation of cargo molecules such as receptor tyrosine kinases expressed on the cell surface that could explain the observed slow growth of LNCaP cells that overexpress TMPAP. The molecular mechanisms identified in this study will definitely contribute to a better understanding of the physiological role of PAP in diseases and to a critical re-evaluation of existing immunotherapies. The knowledge of the molecular determinants responsible for the presence of TMPAP in the endo/exocytic pathway can also be exploited for the future development of radio-imaging and drug delivery protocols.