Browsing Bio- ja ympäristötieteellinen tiedekunta by Issue Date

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  • Koskinen, Patrik (Helsingin yliopisto, 2014)
    As more and more sequences are submitted to public databases, so will grow more computationally challenging sequence retrieval systems. When for example the UniProtKB/TrEMBL doubles in size annually, the tools used today might not be sufficient tomorrow. Faster and computationally lighter methods are needed for sequence retrieval. This study presents a computationally more efficient tool. The Suffix Array Neighbourhood Search (SANS) tool is a hundred fold faster than the most commonly used tool BLAST. The sequence databases do not only grow in size but also in the number of different functional annotations they contain. Recent studies have shown that a large number of these annotations are assigned incorrectly. When the error level of functional annotations in the databases grows to a statistically significant figure, better methods and the use of error detection statistics are highly recommended. In the present study we introduce novel methods for weighted statistical testing of functional annotations. Also novel methods for the calculation of information content value are presented. The information content value enables the discrimination of informative from uninformative annotations. A growing number of functional annotation tools are introduced annually. Since no gold standard evaluation sets exist, it is impossible to determine the reliability of the different methods. The Critical Assessment of Functional Annotations (CAFA) challenge is the first attempt to evaluate functional annotation tools by using blind testing on a large scale. The first CAFA challenge included the evaluation of 54 state-of-the-art methods in two different Gene Ontology categories. The results show that there is a plenty of room for improvement in the prediction accuracy of the existing tools.
  • Härkönen, Laura (Helsingin yliopisto, 2014)
    Visibility conditions of lakes in the Northern Hemisphere have been predicted to decline, due to climate change- induced variations in vegetation of the surrounding catchment areas, precipitation, soil erosion, as well as sediment resuspension. At the same time, climate models predict increasing wind and storm activities, resulting in increasing turbulent velocities in lakes. The aim here was to experimentally clarify how these changes in abiotic factors may affect planktivorous predation in lake ecosystems. We studied how turbulence affects the ability of pelagic invertebrates (Chaoborus flavicans) to avoid fish predation by altering their distribution, the feeding efficiency of C. flavicans and planktivorous perch (Perca fluviatilis) under varying turbidity conditions, and the response of the zooplankton community to various predators, i.e. pelagic invertebrates (Chaoborus), and fish (perch and roach (Rutilus rutilus) in highly colored water. Increasing turbulence negatively affected the ability of Chaoborus larvae to exploit their vertical refuge and also to determine their horizontal position, which in turn was assumed to affect their predator escape efficiency. Indeed, a positive interaction of turbulence and turbidity with planktivorous perch feeding was discovered. Our novel findings challenged the previous assumption that fish larger than a few centimeters in body length are unaffected by turbulence. This was attributed to increased encounter rates between predators and prey, as well as difficulties of chaoborids in escaping predators under high turbulence; the time lost in searching for the prey was compensated. Additionally, intermediate turbulence combined with humic water altered the selective feeding of planktivorous fish on zooplankton compared with calm conditions. Under turbulent conditions, planktivorous fish preferred copepods over cladocerans, whereas under calm conditions the contrasting situation prevailed. Turbulence-mediated changes in the selective feeding of planktivorous fish under low visibility conditions may result in drastic changes in the lower trophic levels in fish-dominated systems. The studies also revealed that intermediate turbulence benefits the feeding of C. flavicans, but only when introduced to a natural, versatile zooplankton community. In contrast to the effect of fish predation, the dark-water experiments in mesocosms showed that the combined effect of turbulence and Chaoborus predation was strongest on cladocerans. High turbulence, on the other hand, caused decreases in Chaoborus feeding. We suggest that turbulence together with varying visibility conditions can have crucial implications for planktivorous predation and should thus be treated as a significant factor in food web studies. Furthermore, intermediate turbulence together with contemporaneous increases in water color may possibly result in cascading effects on primary producers. Depending on the dominant planktivores present, these changes in abiotic factors can have significant consequences on the lower trophic levels.
  • Piccinini, Elisa (Helsingin yliopisto, 2014)
    Parkinson s disease (PD) is a neurodegenerative disorder affecting seven to ten million people worldwide. The average age of diagnosis is 60, but some forms can affect even young adults. In the US alone the direct and indirect expenses for PD exceed $25 billion each year. PD is best characterized by the death of dopaminergic neurons of the substantia nigra pars compacta, which causes symptoms ranging from rigidity to postural instability. As the disease progresses, other areas of the brain become affected, generating psychiatric and cognitive dysfunctions. Current therapies effectively reduce motor symptoms of PD, but do not stop its progression. Neurotrophic factors regulate neuronal growth, differentiation, and survival, and several of them have been shown to protect and regenerate dopaminergic neurons in animal models of PD. The glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) have reached clinical trials, but they did not replicate the promising results of the preclinical studies. Several reasons, including stability of recombinant proteins and their diffusion in the brain tissue, could explain the results of the clinical trials. Stability might have been a problem especially with GDNF, which has been delivered mostly as bacterially-produced recombinant protein. In this work we analysed GDNF produced in mammalian cells and compared it to bacterially-produced GDNF. E. coli produced-GDNF is less stable than mammalian GDNF. This difference is probably due to the purification/renaturation method used with the E. coli-produced factor. Processing and stability of GDNF are affected also by cell line and medium used for its production. In mammalian cells glycosylation of GDNF is fundamental for its processing into the mature molecule. The diffusion problem affects both GDNF and NRTN, which do not diffuse far enough from the infusion site because of their heparin-binding properties. Heparin and the closely related heparan sulphates are abundant in the extracellular matrix and on the cell surface, and hinder the diffusion of GDNF and NRTN. The diffusion issue might not be a significant problem in the animal experiments, but might limit the results achieved with humans, who have significantly bigger brain size compared to rats and monkeys. In this work we have developed NRTN mutant variants with lower affinity for heparin and characterized their activity in vitro and in a unilateral 6-OHDA rat model of PD. All NRTN variants were biologically active. Especially the variant N4 showed better diffusion and rescued a higher number of dopaminergic fibres than E. coli-produced GDNF. Toxin-treated rats administered with N4 also showed functional recovery in behavioural assays. However, as a caveat the mutations introduced could have drawbacks influencing NRTN recycling/degradation and signalling. In this respect lack of heparin-binding could affect NRTN accumulation on the cell surface and inside the cells, therefore causing a slower initiation of the signal. Taken together our results help understanding basic features of GDNF and NRTN, such as the roles of glycosylation and of heparin binding. They also point out several important features that have to be taken into account when producing and/or modifying growth factors for clinical use, and underlines that mammalian molecules with reduced heparin binding could be beneficial for treating PD patients.
  • Repo, Heidi (Helsingin yliopisto, 2014)
    Lysosomes are membrane enclosed acidic cell organelles found ubiquitously in higher eukaryotes. The lysosomal lumen contains more than 60 soluble lysosomal hydrolases, which degrade and recycle cellular macromolecules. Mutations in genes encoding lysosomal or lysosome related proteins result in over 50 different lysosomal storage disorders (LSDs) affecting 1 out of every 7700 newborn children. For instance, the first described LSD, Pompe disease, is caused by a mutation that impairs the function of lysosomal α-glucosidase (GAA) and that results in lysosomal accumulation of glycogen. In this study, several lysosomal proteins were studied via a variety of techniques to increase the knowledge of lysosomal function and correspondingly, the lysosome associated diseases. In order to better understand its function, the previously not well characterised phospholipase B-like protein 1 (PLBD1) was purified from bovine kidneys. It was crystallised and the structure solved by X-ray crystallography to a 1.9 Å resolution. The structure showed that PLBD1 is a member of the N-terminal nucleophile aminohydrolases superfamily. This would imply that PLBD1 is not an esterase as the name suggests, but an amidase. The finding that the hydrophobic tail of the potential phospholipid substrate does not fit into the acyl binding cavity also argues against phosphoesterase function. As a first step in the protein transport pathway to lysosomes mannose-6- phosphate-tag is added to lysosomal proteins. This is initiated by N- acetylglucosamine-1-phosphotransferase (GlcNAc phosphotransferase), which requires a recognition signal on the folded surface of the lysosomal proteins. In this study, the conservation of the signal in four lysosomal proteins was analysed. The phosphorylated N-glycosylation sites and the lysine residues on the GlcNAc phosphotransferase recognition site are well conserved at the sequence level in orthologous proteins, but not necessarily in the protein family. Based on surface analysis of PLBD1 and comparison to the paralogous PLBD2, the most likely recognition site for the GlcNAc phosphotransferase for the PLBD1 could be suggested. LSD associated mutations affect protein function through several mechanism. Several disease-associated missense mutations disturb the protein fold. A general analysis of four enzymes associated with LSD showed that the disease-associated missense mutations are not equally distributed among the 20 amino acids. Glycine, arginine and proline are clearly over-represented among the mutations comparedto their abundance in protein sequences. The hydrophobic amino acids tend to be under-represented among disease-associated mutations. The amino acids where mutation frequently involves a disease have unique properties that contribute to the protein structure in a way that cannot be compensated by other amino acids. Enzyme enhancement therapy with chemical chaperones is a novel treatment for LSDs and has shown potential also for Pompe disease. In this study, the stabilisation capacity of potential chemical chaperones for GAA were tested. Most of the compounds stabilised rhGAA against thermal unfolding and some stabilised even better than would be expected from their binding affinity. In addition, the compounds were modelled to the active site of a GAA structural model and based on this three factors to be considered in chemical chaperone design were defined. Firstly, the ligand size can vary, but the four OH-groups in the ligand are critical in orienting the molecule and making the binding specific. Last but most importantly, a positive charge and its location determine the strength of binding to GAA. This thesis with its structural studies of lysosomal proteins provides molecular understanding of lysosomal protein biology, which is critical for full understanding lysosome function and its involvement in diseases.
  • Korhonen, Jenni (Helsingin yliopisto, 2014)
    The variation in biodiversity has intrigued ecologists for centuries. Currently, studying biodiversity is increasingly important because of its seminal role for maintaining ecosystem functions. Thus, one of the central questions in modern ecology is how species richness and composition can affect ecosystem functioning. Besides spatial variation in diversity, scientists are increasingly interested in the temporal patterns in diversity and community structure. The aim of the PhD thesis was to study spatial and temporal turnover in aquatic communities. I investigated productivity-diversity relationships in three planktonic groups and at two spatial scales. Also, spatial patterns in community composition were compared among the three taxon groups and two spatial scales. Further, I studied the relationships between resource availability, species richness, biomass and resource ratio in phytoplankton communities. Temporal turnover in aquatic assemblages was studied in relation to several ecological, physical and geographical factors. Finally, within and between year variation in lotic diatom communities was investigated in Finnish streams showing wide variability in trophic status and size. The results show that the relationships between ecosystem productivity and plankton diversity are highly variable, ranging from linear negative to linear positive and unimodal. Both alpha and beta diversity showed scale-dependency, highlighting that community patterns may be weaker at smaller scales covering shorter environmental gradients. I also found several key drivers affecting temporal variation in aquatic communities, such as study duration, latitude and organism body size. For example, turnover was faster in low latitude environments than at high latitudes at short time scales, but slower at long time scales. Ecosystem size seems also to be of high importance for turnover rate in many kinds of aquatic ecosystems. This study revealed the suite of factors affecting aquatic species richness and composition both locally and regionally in several types of aquatic ecosystems. The results indicate how different types of communities and ecosystems change and are able to adapt to changing environmental conditions, such as increasing water temperatures or nutrient input due to global climate change. The factors affecting spatial and temporal components of diversity have an effect not only on the diversity and the identity of the biological organisms, but also on socio-economic well-being of humankind as we benefit from many resources and processes that are supplied by natural ecosystems, i.e. ecosystem services.
  • Mäntylahti, Sampo (Helsingin yliopisto, 2014)
    In the field of bioscience there is an ongoing explosive growth in discovery and information. Novel means in biotechnology as well as in medicines are introduced at an unseen rate. One of the aspects contributing to this development is the increased understanding of protein function and structure. Proteins have a role in almost every biological process. The function and structure of proteins are linked. Recent studies have discovered that the understanding of the protein structure has been biased. Namely, the studies have unearthed a previously dismissed protein structure state: intrinsically disordered proteins (IDPs). In this highly dynamic state a protein is without a globular fold, but does not meet the requirements of a random coil either. Rapid transition between folds renders most of the established research techniques to be poor methods to study the IDPs. Nuclear magnetic resonance (NMR) is a spectroscopy method, which enables the study of molecules at atomic resolution. The technique is based upon manipulation of the nuclear spins in specifically produced sample under strong magnetic field. In this method, spins of the system generate quantum coherence state(s), which is utilized to obtain information about the system. NMR is suitable for studying samples in solid and liquid mediums, but in case of biomolecules, water solution is preferable as it resembles in vivo environment. Highly mobile structure and chemical composition of IDPs cause many established NMR experiments to fail. Development of NMR pulse sequences is an obvious approach to solve the problem. This thesis presents a number of NMR pulse sequences, which are designed to improve acquisition of information from highly mobile sections of proteins. The key aspect is to utilize H atom instead of HN in coherence transfer. Additional improvements include limited residue specific identification and novel coherence transfer pathways. Articles I, II, and III present triple resonance experiments, which correlate protein backbone atoms. Combination of the spectra enables full sequential assignment. Article IV introduces an improved pulse sequence for measuring J couplings between nitrogen and amide proton. The experiments were subjected to experimental verification. Comparisons were drawn between established pulse sequences. In both globular proteins and IDPs the results show improvement over established pulse sequences. The proposed sequences yielded improved assignment coverage, resolution and sensitivity enhancement.
  • Yang, Ying (Helsingin yliopisto, 2014)
    Cyclin-dependent kinases (Cdks) are an evolutionary conserved group of serine/threonine protein kinases involved in critical cellular processes such as cell cycle and transcription. Cdk7 and CCRK (cell cycle related kinase; also known as Cdk20) form a separate branch together in a phylogenetic alignment of Cdks family. This study here has identified distinct cellular functions of these two kinases and does not support overlapping functions as suggested by orthologs in yeast. Cdk7 together with cyclin H and Mat1 forming the kinase subcomplex of TFIIH basal transcription factor complex is proposed to regulate RNA polymerase II (Pol II) mediated mRNA synthesis by phosphorylating the serine-5 (Ser5) residues of POL II large subunit C-terminal domain (CTD). Investigations in the genetic systems generated here allowing acute depletion of the Cdk7 subcomplex demonstrate that Cdk7 is the mammalian Ser5 phosphorylating kinase and is required for general transcription noted by analysis of newly transcribed RNAs. The analysis also reveals a requirement of Cdk7 for RNA polymerase I mediated rRNA synthesis. The reduced transcription following Cdk7 disruption is associated with changes on chromatin but not reflected in steady-state RNA levels due to increased RNA stability. These results also reveal a coupled regulation of transcription and RNA degradation. A tissue-specific function of the Cdk7 subcomplex is identified as a physiological roadblock to adipogenesis by phosphorylating the master transcription factor of adipogenic program-peroxisome proliferator-activated receptor gamma (PPARγ). The observation that the Cdk7 subcomplex is absent from adipose tissues indicates the so-called basal transcription machinery has very diverse composition in differentiated cells. CCRK is involved in regulating formation of primary cilium, a sensory organelle acting as a signaling hub in the cell. CCRK promotes cell cycle progression by inhibiting ciliogenesis. In glioblastoma cells, reducing the deregulated high level of CCRK or two related substrate kinases of CCRK restores cilia, leading to decreased glioblastoma cell proliferation. Here is identified the first kinase cascade used by tumor cells to disrupt cilia for a growth advantage and offered new therapeutic possibilities.
  • Lindström, Riitta (Helsingin yliopisto, 2014)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) proteins form a family of neurotrophic factors. Neurotrophic factors have been intensively studied as a putative therapeutic approach to treat neuronal injuries and neurodegenerative diseases. Mammalian MANF and CDNF have been shown to have protective and restorative effects on the nigrostriatal dopaminergic system. In addition, several studies have reported a role for MANF in the endoplasmic reticulum (ER) stress response. A recently established MANF knockout mouse model revealed that MANF functions in the pancreatic insulin-producing beta cells and might be involved in the pathogenesis of diabetes mellitus. Beyond their neurotrophic properties, MANF and CDNF appear to play a more general role in the maintenance of cellular homeostasis. In this study, Drosophila melanogaster was used as a model organism to explore the function and interaction of the MANF/CDNF protein family in vivo. The sole member of the MANF/CDNF family in Drosophila, DmManf, was discovered to be crucial for fly development. The human orthologues, HsMANF and HsCDNF, were found to be able to substitute the endogenous DmManf. Likewise, DmManf had the cytoprotective properties of mammalian MANF in cultured murine neurons. These results support that the findings from the Drosophila model can be adapted for research in mammalian systems. MANF/CDNF proteins consist of amino (N) - and carboxy (C) -terminal domains. In this work, several functional features identified in mammalian MANF structure were explored in the Drosophila model. Separate N- or C-terminal domain constructs, even when co-expressed together, failed to complement for the loss of endogenous DmManf. The ER retention of DmManf, mediated by the C-terminal signal sequence, and the positive charge of the N-terminal surface amino acid residues were found to be important for appropriate DmManf function. Furthermore, entering the secretory pathway via ER was essential for the stability of DmManf protein. A CXXC motif characteristic for oxidoreductases is located in the C-terminal domain of MANF. In this study, effects of a point mutation (C129S) in CXXC motif of DmManf were analysed in vivo. Intact CXXC motif was discovered to be vital for DmManf function. Furthermore, the expression of DmManf-C129S in wild type background was harmful for fly viability suggesting that this specific mutation represents either a dominant negative or a gain-of-function allele of DmManf. Utilising the unique potential of Drosophila model for in vivo screening, interactions of DmManf were studied in this work. Consistent with a previous in vitro study, a genetic interaction was found between DmManf and the fly homologue of the major ER chaperone GRP78. Moreover, DmManf interacted with other genes that encode components of ER function and the unfolded protein response. Finally, novel interactions with DmManf and genes involved in the ubiquinone synthesis pathway and mitochondria were discovered. Taken together, this study demonstrates the functional conservation of mammalian and fly proteins and provides meaningful information on structural and functional features of the MANF/CDNF protein family in vivo. The genetic interaction studies confirmed and expanded the previous knowledge on the ER-associated functions of MANF. Furthermore, novel interactions with mitochondria-related genes and DmManf were discovered.
  • Molotkov, Dmitry (Helsingin yliopisto, 2014)
    Among other glial cell types such as microglia, oligodendrocytes and radial glia, astrocytes are known to be involved in brain function; metabolically supporting neurons, regulating blood flow dynamics, participating in the development of pathological states, sensing and modulating synaptic activity. At the same time the complex astrocytic morphology, with a number of highly ramified peripheral processes located near the synaptic terminals, suggests them as a possible source for morpho-functional plasticity in the brain. This thesis summarizes the work on the in vitro development and further in vivo implementation, using a gene delivery system, of a tool for suppressing activity-dependent astrocytic motility. Calciuminduced astrocyte process outgrowth and its dependence on Profilin-1, novel in vivo gene delivery approaches, a demonstration of astrocytic motility in vivo and the independence of visual processing from astrocytic motility rates are the main findings of the project. The results described in this work increase our understanding of the interactions occurring between astrocytes and neurons as well as the consequences for brain function.
  • Suila, Heli (Helsingin yliopisto, 2014)
    Stem cells have a unique ability to both self-renew and differentiate into diverse cell types and they harbor remarkable potential in therapeutic applications. Stem cells can be isolated from various sources of both embryonic and adult origin. During the past decade, research on stem cells has rapidly expanded, but many issues of stem cell biology and their clinical use remain unresolved. There is a need for methods to thoroughly characterize therapeutic cell populations, to better distinguish them from other cells, and to control variation within and between different cell preparations. The surface of stem cells, like all other human cell surfaces, is covered by a complex network of glycans. This is the outmost layer of cells, called the glycocalyx. The glycocalyx is characteristic to and different in every cell type and reflects even subtle changes in cell behaviour and for example cell differentiation. Cell surface glycans are the first cellular components encountered by approaching cells, pathogens, signalling molecules and other binders, making the terminal glycan units key players in cell interactions and signalling. Due to their prominent cell surface localization, glycan epitopes can be utilized for identifying and isolating specific cell types from heterogeneous populations. The aim of this study was to characterize relevant glycan structures on umbilical cord blood derived stem and progenitor cells, to study how they are regulated and to determine their influence on stem cell biology. As decribed in the original publications of this study, we were able to characterize two novel glycan determinants, O-GlcNAc and linear poly-LacNAc, on umbilical cord blood derived mesenchymal stromal cells (UCB-MSCs). We further discovered that galectins-1 and -3 secreted by these cells are bound on the cell surface and that the cell surface galectin-1 interacts with P-selectin. This interaction is likely to play a role in the immunomodulatory homing of UCB-MSCs to sites of injury or inflammation. In addition, we present the effects and potential use of metabolic glycoengineering of UCB-MSC. Taken together, these studies provide new insights into the glycobiology of UCB derived stem and progenitor cells. This information may help to distinguish better cell populations for distinct therapeutic applications and to design therapeutic cells with enhanced biological properties.
  • Kekkonen, Mari (Helsingin yliopisto, 2014)
    Systematics, phylogenetics and taxonomy are the scientific fields of species discovery, delimitation, description, classification and evolutionary history. The major task of these fields is to form meaningful groups, such as species and higher taxa, based on certain rules and characters. Species and higher taxa create the units of the Linnean hierarchic classification system, which is needed as the basis of all knowledge on biodiversity. Indeed, there is a great need for a complete classification, covering every species on earth, because all subsequent studies and applications are hindered as long as species remain undescribed. However, the estimated count of nine million species on earth, of which ca. 86% are currently unknown to science, together with the numerous threats to biodiversity pose a real challenge to taxonomy, and efficient tools and procedures are strongly needed. In order to be both effective and high-quality, the taxonomic workflow needs to be divided into different steps in the correct sequence. A sampling scheme, the choice of characters and analytical tools are dependent on the phase performed along the workflow. The main aim of Mari Kekkonen s doctoral thesis is to study and conduct different steps along this taxonomic flowchart by using various gelechioid moths as focal species. Specifically, the thesis includes four chapters: a phylogenetic examination of the superfamily Gelechioidea based on combined data of multi-locus DNA and morphology, and three studies focusing on delineation of putative species (i.e., operational taxonomic units, OTUs) based on single-locus DNA barcodes. The delineation studies cover various topics, from testing different OTU delineation methods (BIN, TCS, ABGD, GMYC) with reference species, developing criteria for discordant results and a protocol for associating short sequences from type specimens to delineated groups, to employing the methods, criteria and protocols in practice. The first chapter presents a phylogenetic hypothesis for the Gelechioidea with the best support for families to date. This was achieved mainly due to the use of both molecular and morphological data. We also provide a new family-level classification with redefined 16 families. The three OTU delineation studies revealed taxon-dependent performance within the four datasets (Finnish Gelechiinae, Australian Elachistinae, Australian Hypertrophinae, European Elachista dispunctella complex), but otherwise rather congruent results between the methods. The third chapter provides 120 putative species for poorly-known hypertrophine moths and criteria for evaluating discordant delineation results. Finally, in the last chapter, the E. dispunctella group with a high number of poorly-defined species is re-examined based on DNA barcodes from both fresh non-type and old type specimens. This doctoral thesis is a part of a long-term study of the Hypertrophinae, aiming to provide well-supported phylogeny and species boundaries for the group. The results here not only enhance the study on hypertrophines, but also offer tools to benefit the taxonomic research in general. As a result of the great challenge of taxonomy to describe all species, every new innovation to speed up the workflows without compromising the quality is of crucial importance.
  • Holmroos, Heidi (Helsingin yliopisto, 2014)
    The dynamics of phosphorus (P) and nitrogen (N) were examined in two (shallow and deep) eutrophic lakes. The effects of resuspension and macrophytes as well as oxygen availability were determined, as was the role of natural nitrogen (N) removal (denitrification). The effect of resuspension on the availability of N and P was examined in experimental columns in a shallow eutrophic lake. The release of soluble reactive P (SRP) from resuspended matter varied both seasonally and interannually, depending on the conditions in the lake. SRP release was most significant during periods of high primary production, probably due to pH-induced ligand-exchange reactions. The effect of resuspension on dissolved N was less clear, although resuspension also increased the concentrations of ammonium, nitrite and nitrate during some of the experiments. The studies also showed that strong resuspension decreases the total N to total P ratio in the water independently of the phase of the growing season. The nutrient dynamics were further studied among two different macrophyte stands (submerged and floating-leaved macrophytes) and in the open water. The concentration of combined nitrate and nitrite N (NOx N) decreased to the detection limit (PIENEMPI 2 µg l 1) at all of the stations during summer. Among the submerged macrophytes, the NOx N was depleted almost 2 months earlier than at the other stations, which restricted denitrification. The rate of denitrification was measured with the isotope-pairing technique in both lakes. In the shallow lake, denitrification was measured at the location of the resuspension experiments as well as at the macrophyte stations and adjacent open-water area. Denitrification was mainly dependent on the availability of NOx N and temperature, and also contributed to the changes observed in the total N to total P ratio in water during summer by removing substantial amounts of NOx N from the water. The effects of oxygen (O2) availability were studied in a deeper, stratifying lake, where the deeps were treated with continuous or pulsed aeration, or with no aeration. The SRP release in the deeps was linked with the O2 in the sediment and the SRP accumulated in the hypolimnion during the periods of anoxia. During the aeration pauses and pulses, the concentration of NOx N decreased and increased respectively and the concentration of ammonium varied contrastingly. Continuous aeration prevented the ammonium from accumulating and provided NOx N for denitrification to occur. Accordingly, denitrification showed higher rates during aeration. However, aeration also increased the temperature of the hypolimnion and thereby also the O2 consumption.
  • Joensuu, Merja (Helsingin yliopisto, 2014)
    The boundaries of the cell and the intracellular organelles are created by biological membranes consisting of lipids, proteins and sugars. These membranes are used for a number of purposes, of which, probably the most important is the compartmentalization of the cell s functions. Understanding the mechanisms to create/ maintain the characteristic shape of organelles, and identifying the players involved, allows us to connect the structure of an organelle to its functions or dynamics and, importantly, to the disorders associated with malfunctioning organelle. How these processes in endoplasmic reticulum (ER) are coupled, has remained unclear. In this thesis, the structure and dynamics of ER and the connection to some of the ER s functions were analysed. By using live cell imaging and 3D-electron microscopy, biochemical approaches, and novel quantitative image analysis, we demonstrated the great variation in interphase ER network organization and sheet structures among several cultured mammalian cell lines. We described interphase ER sheet dynamics and showed that sheets were static and persistent structures. Our work revealed that a specific subset of actin filaments have a role in ER sheet persistence and the ER network organization. Furthermore, we discovered a novel role for molecular motor myosin 1c in regulating the ER-associated actin filament arrays. Moreover, we learned that in mitosis ER undergoes a progressive spatial reorganization and a structural transformation towards more fenestrated sheets and tubular forms. We showed that ER remains continuous during mitosis and that the partition of nuclear envelope was subordinate to ER, which, in addition to structural transformations of ER into smaller subunits, favours the stochastic model of inheritance of ER. Importantly, we showed that the natural increase of ER fenestrations and tubulation in mitosis correlated with the reduced number of membrane-bound ribosomes, and that the structural transformation could be mimicked by dissociating the membrane-bound ribosomes from the interphase ER by a drug treatment, suggesting that ribosomes and the associated luminal translation machinery have a role stabilizing the sheet structures. Collectively, this work describes the significant plasticity of ER morphology and organization in several common cell culture cells in interphase and upon inheritance of ER. Importantly, this work also demonstrates the dynamic rearrangements of ER in mitosis and interphase cells and provides novel information about the role of ribosomes and actin on ER sheets and the role of myosin 1c on the ER-associated actin arrays, serving as an opening for further studies on variety of regulatory possibilities of the interplay between ER subdomains and the identified player involved.
  • Mattila, Anniina L. K. (Helsingin yliopisto, 2014)
    Loss and fragmentation of natural habitats and changing climate pose severe threats to biodiversity. The ability of populations and species to respond to these challenges by dispersing across landscapes is imperative for their long-term survival. Dispersal is also the main mechanism leading to gene flow, and dispersal is therefore essential for maintaining genetic diversity and adaptive potential of populations. In this thesis, I build upon the vast knowledge gained during more than two decades of research on the Glanville fritillary butterfly (Melitaea cinxia), aiming towards a better understanding of the mechanisms and processes that shape dispersal in this model species. Previous studies have demonstrated a strong positive correlation between flight metabolic rate (FMR) and dispersal distances in the field. Here, I use FMR as a measure of flight and dispersal capacity. I study dispersal from multiple perspectives and use a variety of methods to address questions ranging from the genetic basis and heritability of flight capacity to interactions between genes, physiology and environment in affecting flight and dispersal. Variation in dispersal capacity and how it influences population and metapopulation-level processes are examined. Finally, I use a natural experiment to study the genetic and fitness consequences of complete lack of gene flow into a small isolated island population of the Glanville fritillary. Key findings of the thesis include the demonstration of significant heritable genetic variation in FMR, indicating that FMR and therefore dispersal capacity has the potential to respond to selection due to e.g. habitat fragmentation and climate change. In a genome-wide gene expression study, 755 genes were significantly up- or down-regulated in response to an experimental flight treatment. Differences between sexes and two contrasting populations in flight-induced gene expression in major metabolic pathways were associated with differences in FMR, suggesting that similar molecular mechanisms influence both gender and population differences in flight performance. An experiment examining changes in butterfly body temperature during flight showed that FMR and tolerance of high temperatures may significantly influence flight performance in different thermal environments. At the metapopulation level, male and female butterflies differed in the effects of flight capacity on realized dispersal rate between local populations, with likely consequences for the assortment of dispersive genotypes across fragmented landscapes. The small and completely isolated island population of the Glanville fritillary exhibited significant loss of genetic diversity and substantially reduced fitness. Complete and instant fitness recovery in hybrids strongly suggests that reduced population viability is due to high genetic load. This small isolated population serves as an example of the innumerable remnant populations in human-fragmented landscapes, in which extinction risk may increase due to lack of gene flow. This work contributes to the mechanistic understanding of dispersal (and its importance) in fragmented and isolated populations and in changing environmental conditions in the Glanville fritillary butterfly. Many findings of this thesis are also likely to be applicable to other similar species, particularly those living in fragmented landscapes.
  • Sun, Xiaoyu (Helsingin yliopisto, 2014)
    Many viruses protect their genome in a protein capsid. Viral capsid formation involves the association of multiple copies of viral capsid protein subunits, representing single or multiple protein species, and different assembly strategies are utilized. Pseudomonas phage phi6 uses an assembly pathway in which an empty capsid (procapsid, PC) is first assembled, serving as a compartment for the subsequent encapsidation of the RNA genome. During encapsidation, the compact, empty PC undergoes conformational rearrangement to reach its final expanded form. The phi6 PC is composed of the main structural protein, P1, and three minor protein species: the RNA-dependent RNA polymerase P2, the packaging nucleoside triphosphatase (NTPase) P4, and the assembly cofactor P7. In vitro systems of phi6 assembly, genome encapsidation, and transcription have been established, allowing infectious particles to be constructed from purified protein and RNA components. In this thesis, stoichiometric measurements were established to estimate the relative copy numbers of PC proteins in phi6 virions and PCs. Different concentrations of the phi6 minor proteins were employed in in vitro assembly reactions to probe potential PC binding sites. The results indicate that potential binding sites for proteins P2 and P7 are only partially occupied in phi6 virions and recombinant PCs. High P7 occupancy in self-assembled PCs resulted in reduced P2 incorporation, suggesting some correlation between P2 and P7 during PC assembly. Although high P4 hexamer occupancy was critical for initial particle formation, a large excess of P4 in the self-assembly reaction slowed the rate of PC self-assembly, which may be ascribed to excessive production of P1-P4 nucleation complexes. In addition, electrostatic interactions were demonstrated to be the main driving force in phi6 PC assembly. Furthermore, it was shown that P4 hexamers spontaneously dissociate from the empty capsid shell. P4-deficient particles have slower sedimentation velocity and an expanded appearance compared to the PC that has full-occupancy of P4. These particles are also defective in RNA packaging and transcription. However, purified P4 hexamers can efficiently assemble on P4-deficient particles, guiding the particles to their naive compact conformation and rescuing packaging and transcription activities. The results obtained from this study provide new insight into the principles of viral capsid assembly and demonstrate the reversibility of the PC maturation pathway.
  • Raatikainen-Ahokas, Anne (Helsingin yliopisto, 2014)
    Embryonic cells undergo sequential specification processes to generate multiple cell types of mature organs. Some cells retain pluripotency. They serve as stem or progenitor cells, and provide both new stem cells (self-renewal) and offspring for differentiation. The fate of some cells is to die by programmed cell death. In this thesis, the cell fates in nephrogenesis and spermatogenesis were studied. During kidney organogenesis, an outgrowth of the Wolffian duct, the ureteric bud, induces condensation of the metanephric mesenchyme into a cap condensate, the progenitor cell population that forms the epithelium of all future nephrons. The cap condensate is surrounded by stromal cells. The developmental fates of these cells that also surround the ureter and nascent nephrons, i.e. the kidney stroma, are poorly understood. Bone morphogenetic protein 4 (BMP4) inhibited the outgrowth of the ureteric bud from the Wolffian duct in organ culture. It also had an inhibitory effect on subsequent ureteric branching. The branching defect primarily reflected the effect of BMP4 on the mesenchymal components of the kidney. BMP4 promotes the recruitment of mesenchymal cells around the ureter and their differentiation into smooth muscle. This periureteric cell population likely has a regulatory function in subsequent ureteric growth and differentiation. The exogenous BMP4 also disrupted the cap condensates in kidney explants and large amounts of mesenchymal cells underwent apoptosis. BMP4 maintained the isolated metanephric mesenchymes while suppressing the nephrogenic potential, suggesting that BMP4 acts as a survival/differentiation factor for the stromal progenitors. The stromal cells are apparently essential for the formation and maintenance of the cap condensate. In some organs, such as the testis, the maintenance of stem cells throughout the life span is essential to the normal function, e.g. the formation of sperm cells. Spermatogonia with stem cell activity (SSCs) are among the undifferentiated spermatogonia located at the basement membrane of the seminiferous tubule. Daughters of SSCs both replenish the stem cell pool and enter the differentiation pathway into spermatozoa. Glial cell line-derived neurotrophic factor (GDNF), essential for ureteric branching morphogenesis, is also crucial to the self-renewal of the SSCs. Haploinsufficiency of the Gdnf gene in Gdnf+/- mice caused segmental exhaustion of stem cells, resulting in germ cell loss in old mice. In mice overexpressing GDNF in the testis, spermatogenesis was arrested and large clusters of spermatogonia accumulated in prepubertal animals. Thus, high GDNF concentration promotes the propagation of undifferentiated spermatogonia, whereas low GDNF levels allow SCCs to differentiate in excess and make them prone to depletion. In conclusion, signalling molecules, such as BMP4 and GDNF, affect the cell fates both in nephrogenesis and spermatogenesis by maintaining the precursor cells and promoting their differentiation.
  • Shakeel, Shabih (Helsingin yliopisto, 2014)
    Pathogenic human picornaviruses are known to cause a wide variety of diseases ranging from mild colds to severe paralysis. In addition to their importance in causing disease, they also serve as models for understanding the basic mechanisms of host-pathogen interactions, virus entry, viral genome release, viral synthesis and viral assembly. In picornaviruses, the majority of the structural and host-cell interaction studies have been conducted on polioviruses and human rhinoviruses. Picornaviruses like coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1 have not been so well studied because of difficulties in culturing them. Recently, the number of cases reported for infection by these viruses has increased dramatically due to better detection methods, thus making structural studies of these viruses and their interactions with their host cells important in order to understand their mode of infection so that better therapeutics can be designed against them. I have studied coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1, which are all pathogenic picornaviruses, in order to understand the mechanism of pathogenesis, tropism, viral entry and assembly for these viruses in particular and for picornaviruses in general. Two studies dealt with determining the structure of coxsackievirus A 7, a Human Enterovirus A species for which there was no structural information available at the time when this study was conducted. The genome-filled and empty structure of coxsackievirus A 7 were determined using cryo electron microscopy to sub-nanometer resolution which helped in building pseudo-atomic models for them using homology modelling and flexible fitting. With the help of these models, the majority of the strain variations in the capsid proteins were identified on the surface of VP1. Such variations are the likely cause of differences in pathogenesis and tropism between strains. Furthermore, superimposition of these models showed that the capsid underwent a conformational change on RNA release. In the process, generalised methods for optimising and comparing results from flexible fitting were developed. The next structural study elucidated the interaction of coxsackievirus A 9, a Human Enterovirus B species, with a cellular receptor. Integrins were found to bind sub-stoichiometrically to the capsid using electron cryo-tomography (cryo-ET). Asymmetric reconstruction indicated that this was probably due to steric hindrance. The affinity of this interaction was calculated to be 1nM using surface plasmon resonance. Additionally, the conformational changes which occur on its RNA release were quantified. The fourth study explained the importance of viral RNA in picornavirus assembly. Pentameric intermediates of human parechovirus 1 were isolated and used to identify packaging signals in the viral RNA required for capsid assembly using aptamer library screening and next generation sequencing analysis. Poly-U was identified as the common motif for these packaging signals present on the stem or the loop of the RNA secondary structure. Overall, this thesis gives an insight into many important aspects of host-virus interactions especially the events occurring on viral RNA exit and during its encapsidation. The work in this thesis could be utilized to identify potential targets for antiviral synthesis and also to define general virus assembly principles.
  • Majaneva, Sanna (Helsingin yliopisto, 2014)
    Gelatinous zooplankton, such as ctenophores, have attracted attention during the last decade,mainly as a result of the enigma around their potentially increased abundances around the world. Despite the increased attention, they remain either understudied or disregarded in most food web investigations and monitoring programs, and are defined as one of the most difficult groups of pelagic animals to study. Consequently, their diversity and ecological role are often grossly oversimplified and misunderstood, leading to biased views of ecosystem functioning. In addition, ctenophores share traits such as voracious predation behavior, the ability to starve and shrink during periods of low food availability and to tolerate increased temperatures, as well as high reproductive capacity, making them likely to take advantage of changing environmental conditions. In the Arctic, earlier ctenophore data consist of sparse abundance estimates and dietary studies lacking a systematic or integrative approach. In the Baltic Sea, despite the wellestablished routine plankton monitoring program conducted by the surrounding nations, the distribution and the role of the Arctic ctenophore Mertensia ovum in this ecosystem has been unknown since its first reported appearance in 2007. In this thesis, the biodiversity of cydippid ctenophores and their role in Svalbard waters and in the Baltic Sea were studied. Extensive in situ sampling, laboratory experiments, morphological and molecular identification analysis, traditional and molecular gut content analysis, as well as several direct measures of the ctenophores and the pelagic communities they inhabit were combined to address system-specific questions and to better understand how important a role the ctenophores might have in marine ecosystems. A combination of morphological species identification and molecular methods revealed Euplokamis sp. and an unidentified mertensiid species to co-occur with the dominating Mertensia ovum in Arctic waters. Similarly, the first recording of the cydippid ctenophore, Euplokamis sp., near the entrance to the Baltic Sea was reported in conjunction with extensive sampling of Mnemiopsis leidyi and Mertensia ovum. Interestingly, Pleurobrachia pileus, earlier reported to commonly co-occur with M. ovum in the Arctic and be present throughout the Baltic Sea (and earlier reported as the only ctenophore species in the northern Baltic Sea), was not present in either study site. It was demonstrated that morphological species identification alone is insufficient. In addition, the lack of proper species descriptions and public sequences limited the identification to the genus, family or order level. Thus, more emphasis should be placed on combining morphological and molecular methods together with photographic vouchers for rigorous taxonomic identification and accurate species descriptions. The lack of historical survey data and accurate abundance estimates of M. ovum have biased interpretations of its role in the Arctic and Baltic ecosystems. According to the results presented in this thesis, the potential predation impact of M. ovum was high when assuming relatively homogenous distributions of M. ovum and its prey, but it was even higher when patchiness of both predators and prey was taken into account. The potential predation impact was further affected by extensive spatial and seasonal migration patterns. Therefore, to adequately model prey predator interactions, more emphasis should be placed on the fine-scale distribution patterns of predators and prey. Also, different populations of a single species can have very different trophic roles in the food web due to the great difference in body size, as exemplified in this thesis with two populations of M. ovum. In the Arctic, M. ovum is a voracious predator of copepods (Calanus spp.), while in the Baltic, the substantially smaller M. ovum individuals mainly prey upon pico- and microplankton. Thus, generalizing and extrapolating ecological traits such as diet and foraging behavior from one population to another can be misleading. Moreover, the future of the two studied populations of M. ovum is likely to differ substantially because their distribution area will be differently affected by climate change; the distribution area is predicted to diminish in the Baltic Sea and remain more or less constant in the Arctic. To conclude, this thesis demonstrates that our current knowledge on the diversity, role, and potential future changes of the ctenophores in pelagic communities is still very incomplete. Thus, to properly understand the ecological impact of ctenophores at present and in the near future, this thesis suggests that all available techniques need to be applied in species identification, and that clear recommendations for a proper assessment of routine ctenophore monitoring are urgently needed.
  • Helmy, Mohamed (Helsingin yliopisto, 2014)
    Birth asphyxia is a major cause of infant and childhood death, disability and neurodevelopmental delay worldwide. During birth, impairment of respiration is reflected in elevated levels of CO2 and diminished levels of O2 in the neonate. The fundamental presentation and diagnostic criterion of birth asphyxia is severe acidosis, most commonly measured in umbilical blood. Resuscitation is associated with normalization of blood pH values and arterial blood gases. Within hours of a moderate or severe asphyxic insult during birth, severe seizures are triggered. In the present study, asphyxic conditions during birth are modeled as an induced hypoxia and hypercapnia in postnatal day 6 rat pups. Respiratory conditions are altered so that pups breathe 20 % CO2 with either 9 % O2 or 4 % O2 (N2 balanced) for 60 or 45 minutes, respectively. Brain extracellular and intraneuronal pH became rapidly acidotic during asphyxic conditions. After experimental asphyxia, immediate restoration of normoxia and normocapnia was associated with a large seizure burden. Seizures in the postasphyxia period were tightly correlated with a recovery and alkaline overshoot in brain pH. Enhanced acid extrusion from the brain was attributed to increased Na/H exchange across the blood-brain barrier. Pharmacologic blockade of Na/H exchange in the blood-brain barrier with amiloride or its analog abolished brain alkalosis and seizures. These findings suggest that a brain-confined alkalosis is generated by Na/H exchangers in the blood-brain barrier when normocapnic conditions are immediately restored after experimental birth asphyxia. A putative therapeutic strategy was tested, where the CO2 level of inhaled air in the postasphyxic period was reduced in steps, so that normocapnic conditions are gradually restored. This graded restoration of normocapnia was achieved by exposing the pup to 10 % CO2 in air for 30 minutes, followed by 5 % CO2 in air for a further 30 minutes, and finally with room air. A dramatic attenuation of brain alkalosis and seizures was induced by graded restoration of normocapnia. Immediate restoration of normocapnia after asphyxia was associated with adverse outcome in juvenile and adult rats, manifest as compromised sensorimotor coordination, altered emotional reactivity to acute stress, diminished inhibition of fear-motivated behavior, impaired memory and learning, abnormal social interaction, and increased seizure susceptibility. Graded restoration of normocapnia after asphyxia was associated with significant and favorable improvement of outcome, such that behavioral deficits were rescued, and seizure threshold was not significantly different from control animals. The findings of the this study suggest a central role for Na/H exchange in the blood-brain barrier in mediating the postasphyxia brain alkalosis as measured in the present study as well as in human babies. Importantly, the findings also suggest a putative therapeutic strategy in which recovery from acidosis during neonatal resuscitation is controlled through a graded restoration of normocapnia.
  • Suomalainen, Marjo (Helsingin yliopisto, 2014)
    Omptins are a family of conserved, integral outer membrane proteases and widely distributed within Gram-negative bacterial species. The family offers a good example of the evolution and the adaptation of a protein to novel functions and to differing pathogenic bacterial life-styles. This work investigates three different omptins: Pla of Yersinia pestis, PgtE of Salmonella enterica and OmpT of Escherichia coli. The omptin proteases differ in substrate specificity and need lipopolysaccharide (LPS) for activity. My thesis work addressed two main questions in omptin function: what is the molecular basis of the dissimilar substrate selectivity in the structurally very similar omptins; and what are the structural features in LPS that affect omptin activity. I studied the LPS dependency of omptins by expressing the proteins in bacterial cells that differ in LPS structure and by reconstituting purified, detergent-solubilized omptin protein with characterized, purified LPS molecules. Y. pestis alters its LPS structure in response to change of temperature from 20°C to 37°C, which reflects the transfer from a flea to a mammalian host. I found that the activity of Pla in cells from 20°C was very low, whereas cells from 37°C expressed high activity. I reconstituted detergent-purified His6-Pla protein with various model LPS structures and with LPSs of Y. pestis grown at different temperatures. Adding Y. pestis LPS from 37°C to the nonfunctional Pla protein induced high proteolytic activity, whereas 20°C-LPS gave very low activity, indicating that the activity of Pla is controlled by LPS. Similarly, I found that the activity of PgtE was high with rough LPS and low with smooth LPS; the difference mimics the LPS of intracellular (rough) and extracellular (smooth) S. enterica. Thus, in both bacterial species the omptin activity is controlled by the LPS type that the bacteria express during infection in mammals. I further studied the fine structure of Y. pestis LPS that affects Pla activity. This was done by reconstituting Pla activity with various structurally characterized Y. pestis and E. coli LPSs. I found that lower levels of lipid A acylation and phosphate substitution by aminoarabinose, are important for Pla activity, these features are characteristic for Y. pestis LPS from 37°C. A common and conserved feature in omptin structure is the presence of LPS-binding motif in protein barrel. Disrupting of the lipid A-binding motifs in PgtE and Pla abolished their proteolytic activity, emphasizing the importance of the LPS binding site for omptin activity. Omptins have a highly spatically conserved active center and catalytic domains but express functional heterogeneity. The omptin transmembrane barrel contains five surface-exposed loops that show slightly higher sequence variation than the transmembrane protein regions. To study the effect of loop structures in omptin proteolytic specificity, I changed OmpT of E. coli to a Pla-like enzyme by a stepwise substitution of the loop areas. The proteins were characterized by their ability to activate the human protease precursor plasminogen(Plg) to the active serine protease plasmin and to inactivate the main plasmin inhibitor, α2-antiplasmin(α2AP); both functions are important for bacterial virulence. Pla cleaves very efficiently both substrates, whereas OmpT is only poorly active with them. I showed that OmpT could be converted into a Pla-like enzyme by cumulative substitutions at the loop areas, especially the loops L3-L5 were important. The successful conversion of OmpT towards Pla indicates that the loop structures are critical for omptin activity by allowing correct recognition of the polypeptide substrate. More detailed substitution analysis was taken to identify the catalytic residues in Pla. My thesis demonstrates that the omptin proteolytic activity depends on two things: their specific interaction with LPS and the structure of their surface-exposed loops. The thesis offers an example of omptins extensive evolvability and of how they adapt to the lifestyle of their host bacterium.