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  • Ihalmo, Pekka (Helsingin yliopisto, 2007)
    The glomerular epithelial cells and their intercellular junctions, termed slit diaphragms, are essential components of the filtration barrier in the kidney glomerulus. Nephrin is a transmembrane adhesion protein of the slit diaphragm and a signalling molecule regulating podocyte physiology. In congenital nephrotic syndrome of the Finnish type, mutation of nephrin leads to disruption of the permeability barrier and leakage of plasma proteins into the urine. This doctoral thesis hypothesises that novel nephrin-associated molecules are involved in the function of the filtration barrier in health and disease. Bioinformatics tools were utilized to identify novel nephrin-like molecules in genomic databases, and their distribution in the kidney and other tissues was investigated. Filtrin, a novel nephrin homologue, is expressed in the glomerular podocytes and, according to immunoelectron microscopy, localizes at the slit diaphragm. Interestingly, the nephrin and filtrin genes, NPHS1 and KIRREL2, locate in a head-to-head orientation on chromosome 19q13.12. Another nephrin-like molecule, Nphs1as was cloned in mouse, however, no expression was detected in the kidney but instead in the brain and lymphoid tissue. Notably, Nphs1as is transcribed from the nephrin locus in an antisense orientation. The glomerular mRNA and protein levels of filtrin were measured in kidney biopsies of patients with proteinuric diseases, and marked reduction of filtrin mRNA levels was detected in the proteinuric samples as compared to controls. In addition, altered distribution of filtrin in injured glomeruli was observed, with the most prominent decrease of the expression in focal segmental glomerulosclerosis. The role of the slit diaphragm-associated genes for the development of diabetic nephropathy was investigated by analysing single nucleotide polymorphisms. The genes encoding filtrin, densin-180, NEPH1, podocin, and alpha-actinin-4 were analysed, and polymorphisms at the alpha-actinin-4 gene were associated with diabetic nephropathy in a gender-dependent manner. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the downregulation of filtrin seems to be characteristic for human proteinuric diseases. In the context of the crucial role of nephrin for the glomerular filter, filtrin appears to be a potential candidate molecule for proteinuria. Although not expressed in the kidney, the nephrin antisense Nphs1as may regulate the expression of nephrin in extrarenal tissues. The genetic association analysis suggested that the alpha-actinin-4 gene, encoding an actin-filament cross-linking protein of the podocytes, may contribute to susceptibility for diabetic nephropathy.
  • Uvarov, Pavel (Helsingin yliopisto, 2010)
    K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.
  • Ning, Lin (Helsingin yliopisto, 2013)
    In the central nervous system, neurons are connected through local contacting structures called synapses. The initial contacts between axons and dendrites undergo changes in morphology and protein composition, and differentiate into fully functional synapses. Precise regulation on synapse formation is essential for normal brain functioning. Cell adhesion molecules are the key regulators during this process, by connecting the membranes of synapses and initiating signaling cascades that mediate synapse formation. A neuron-specific cell adhesion molecule ICAM-5 (telencephalin) controls immune responses by suppressing T-cell activation or by mediating neuron-microglia interaction. On the other hand, ICAM-5 is so far the only identified CAM that slows synaptic development. Deletion of ICAM-5 from mice leads to accelerated synapse formation, enhanced synaptic capacity, and improved memory and learning. Clinically, changes in ICAM-5 levels are associated with various diseases, such as acute encephalitis, epilepsy, and Alzheimer s disease. The major goal of my thesis is to address the molecular mechanisms by which ICAM-5 regulates synapse formation as well as maturation of dendritic spines, the post-synaptic components of excitatory synapses. In my study, ICAM-5 was observed as a substrate for MMP-2 and -9. Activation NMDA receptors in neurons elevated the level of MMP activity, and subsequently induced ICAM-5 ectodomain cleavage, which in turn promoted spine maturation. In addition, I identified the pre-synaptic β1-integrins as counter-receptors for ICAM-5. This trans-synaptic interaction inhibited the MMP-induced ICAM-5 cleavage, and thereby prevented spine maturation. At the intracellular side, α-actinin, an actin cross-linking protein, was found as a binding partner for ICAM-5. This binding linked ICAM-5 to the actin cytoskeleton and was important for the membrane distribution of ICAM-5 and neurite outgrowth. The GluN1 subunit of the NMDAR is also known to bind to α-actinin. We found here that GluN1 and ICAM-5 competed for the same binding region in α-actinin. Activation of NMDAR changed α-actinin binding property to ICAM-5, resulting in α-actinin accumulation and actin reorganization. In conclusion, my thesis defines a novel, ICAM-5-dependent mechanism, which regulates synapse formation, spine maturation and remodeling.
  • Parkash, Vimal (Helsingin yliopisto, 2009)
    Four GDNF ligands (GDNF, neurturin, artemin and persephin), and mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF) protect midbrain dopaminergic neurons that degenerate in Parkinson's disease. Each GDNF ligand binds a specific coreceptor GDNF family receptor α (GFRα), leading to the formation of a heterotetramer complex, which then interacts with receptor tyrosine kinase RET, the signalling receptor. The present thesis describes the structural and biochemical characterization of the GDNF2-GFRα12 complex and the MANF and CDNF proteins. Previous and current mutation data and comparison between GDNF-GFRα1 and artemin-GFRα3 binding interfaces show that N162GFRα1, I175GFRα1, V230GFRα1, Y120GDNF and L114GDNF are the specificity determinants among different ligand-coreceptor pairs. The structure suggests that sucrose octasulphate, a heparin mimic, interacts with a region R190-K202 within domain 2 of GFRα1. Mutating these residues on the GFRα1 surface, which are not in the GDNF binding region, affected RET phosphorylation, which provides a putative RET binding region in domain 2 and 3 of GFRα1. The structural comparison of the GDNF-GFRα1 and artemin-GFRα3 complexes shows a difference in bend angle between the ligand monomers. This variation in bend angle of the ligand may affect the kinetics of RET phosphorylation. To confirm that the difference is not due to crystallization artefacts, I crystallized the GDNF-GFRα1 complex without SOS in different cell dimensions. The structure of the second GDNF-GFRα1 complex is very similar to the previous one, suggesting that the difference between the artemin-GFRα3 and GDNF-GFRα1 complexes are intrinsic, not due to crystal packing. Finally, MANF and CDNF are bifunctional proteins with extracellular neurotrophic activity and ER resident cytoprotective role. The crystal structures of MANF and CDNF are presented here. Intriguingly, the structures of both the neurotrophic factors do not show structural similarity to any of previously known growth factor superfamilies; instead they are similar to saposins, the lipid-binding proteins. The N-terminal domain of MANF and CDNF contain conserved lysines and arginines on its surface, which may interact with negatively charged head groups of phospholipids, as saposins do. Thus MANF and CDNF may provide neurotrophic activities by interacting with a lipo-receptor. The structure of MANF shows a CXXC motif forming internal disulphide bridge in the natively unfolded C-terminus. This motif is common to reductases and disulphide isomerases. It is thus tempting to speculate that the CXXC motif of MANF and CDNF may be involved in oxidative protein folding, which may explain its cytoprotective role in the ER.
  • 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.
  • Hiltunen, Jukka (Helsingin yliopisto, 2001)
  • Sairanen, Mikko (Helsingin yliopisto, 2007)
    Neuronal plasticity is a well characterized phenomenon in the developing and adult brain. It refers to capasity of a single neuron to modify morphology, synaptic connections and activity. Neuronal connections and capacity for plastic events are compromised in several pathological disorders, such as major depression. In addition, neuronal atrophy has been reported in depressive patients. Neurotrophins are a group of secretory proteins functionally classified as neuronal survival factors. Neurotrophins, especially brain derived neurotrophic factor (BDNF), have also been associated with promoting neuronal plasticity in dysfunctional neuronal networks. Chronic antidepressant treatment increases plastic events including neurogenesis and arborization and branching of neurites in distinct brain areas, such as the hippocampus. One suggested mode of action is where the antidepressants elevate the synaptic levels of BDNF thus further activating several signaling cascades via trkB-receptor. In our studies we have tried to clarify the mechanisms of action for antidepressants and to resolve the role of BDNF in this process. We found that chronic antidepressant treatment increases amount of markers of neuronal plasticity in both hippocampus and in the medial prefrontal cortex, both of which are closely linked to the etiology of major depression. Secondary actions of antidepressants include rapid activation of the trkB receptor followed by a phosphorylation of transcription factor CREB. In addition, activation of CREB by phosphorylation appears responsible for the regulation of the expression of the BDNF gene. Using transgenic mice we found that BDNF-induced trkB-mediated signaling proved crucial for the behavioral effects of antidepressants in the forced swimming test and for the survival of newly-born neurons in the adult hippocampus. Antidepressants not only increased neurogenesis in the adult hippocampus but also elevated the turnover of hippocampal neurons. During these studies we also discovered that another trkB ligand, NT-4, is involved in morphine-mediated anti-nociception and tolerance. These results present a novel role for trkB-mediated signaling in plastic events present in the opioid system. This thesis evaluates neuronal plasticity and trkB as a target for future antidepressant treatments.
  • Pihlatie, Mari (Helsingin yliopisto, 2007)
    Microbial activity in soils is the main source of nitrous oxide (N2O) to the atmosphere. Nitrous oxide is a strong greenhouse gas in the troposphere and participates in ozone destructive reactions in the stratosphere. The constant increase in the atmospheric concentration, as well as uncertainties in the known sources and sinks of N2O underline the need to better understand the processes and pathways of N2O in terrestrial ecosystems. This study aimed at quantifying N2O emissions from soils in northern Europe and at investigating the processes and pathways of N2O from agricultural and forest ecosystems. Emissions were measured in forest ecosystems, agricultural soils and a landfill, using the soil gradient, chamber and eddy covariance methods. Processes responsible for N2O production, and the pathways of N2O from the soil to the atmosphere, were studied in the laboratory and in the field. These ecosystems were chosen for their potential importance to the national and global budget of N2O. Laboratory experiments with boreal agricultural soils revealed that N2O production increases drastically with soil moisture content, and that the contribution of the nitrification and denitrification processes to N2O emissions depends on soil type. Laboratory study with beech (Fagus sylvatica) seedlings demonstrated that trees can serve as conduits for N2O from the soil to the atmosphere. If this mechanism is important in forest ecosystems, the current emission estimates from forest soils may underestimate the total N2O emissions from forest ecosystems. Further field and laboratory studies are needed to evaluate the importance of this mechanism in forest ecosystems. The emissions of N2O from northern forest ecosystems and a municipal landfill were highly variable in time and space. The emissions of N2O from boreal upland forest soil were among the smallest reported in the world. Despite the low emission rates, the soil gradient method revealed a clear seasonal variation in N2O production. The organic topsoil was responsible for most of the N2O production and consumption in this forest soil. Emissions from the municipal landfill were one to two orders of magnitude higher than those from agricultural soils, which are the most important source of N2O to the atmosphere. Due to their small areal coverage, landfills only contribute minimally to national N2O emissions in Finland. The eddy covariance technique was demonstrated to be useful for measuring ecosystem-scale emissions of N2O in forest and landfill ecosystems. Overall, more measurements and integration between different measurement techniques are needed to capture the large variability in N2O emissions from natural and managed northern ecosystems.
  • Lindahl, Maria (Helsingin yliopisto, 2004)
  • Jernberg, Joonas (Helsingin yliopisto, 2013)
    Growth in economics and prosperity has been a global trend during recent decades and the use of chemicals has increased tremendously as a part of industrial production, agriculture and everyday life. The use of hazardous chemicals has been restricted by many intergovernmental treaties and legislation but new replacement chemicals are synthesized constantly and no decrease in the future production volumes of chemicals is soon expected. The term chemicalization is used to describe the increased use of chemicals and resultant environmental contamination. In the analysis of environmental samples, usually only some selected regulated compounds are measured. The problem with these target analyses is that other compounds remain undetected. Complementary techniques without any preselection of the analytes are thus required to identify new compounds. The aim of this study was to develop novel instrumental techniques for the determina-tion of organic compounds without any analyte preselection and to identify unknown anthropogenic contaminants in different water matrices. The methods developed were based on analytical separation, using gas and liquid chromatography combined with accurate mass measurement using time-of-flight mass spectrometry. The data produced were then processed with a deconvolution program to locate the chromatographic peaks and to extract their mass spectra. The measured accurate masses were then used to confirm the elemental compositions of the detected ions. The identification processes were validated, using spiked water samples, and finally the methods were applied to the identification of organic xenobiotics from wastewater effluent, stormwater, surface water and landfill leachate samples. The results showed that analysis using time-of-flight mass spectrometry enables screening of large analyte groups without previous information on sample composition. The most comprehensive knowledge is yielded by analysing the sample with both gas and liquid chromatography. In many cases, tentative compound identification can be obtained if the deconvoluted spectra and accurate mass data are complemented with information, e.g. from spectral libraries and peak isotope patterns. This tentative identifications must, however, always be confirmed with a pure standard compound. The main limitations of the methods were related to insufficient features of the deconvolution program used. Most of the data-processing stages had to be performed manually or visually, which slows down the data processing and hinders their applicability, especially with large sample sets. Dozens of compounds were tentatively identified from water samples and several of them were also confirmed with a standard compound. The highest numbers of compounds were identified from wastewater effluent, stormwater and landfill leachate samples. The results confirmed the fact that anthropogenic waste streams are an important route for organic xenobiotics into the environment. Since the future volumes of chemicals will increase, the control and efficient treatment of these fluxes becomes evermore essential.
  • Lindholm, Päivi (Helsingin yliopisto, 2009)
    Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.
  • Li, Jing (Helsingin yliopisto, 2007)
    Plants are capable of recognizing phytopathogens through the perception of pathogen-derived molecules or plant cell-wall degradation products due to the activities of pathogen-secreted enzymes. Such elicitor recognition events trigger an array of inducible defense responses involving signal transduction networks and massive transcriptional re-programming. The outcome of a pathogen infection relies on the balance between different signaling pathways, which are integrated by regulatory proteins. This thesis characterized two key regulatory components: a damage control enzyme, chlorophyllase 1 (AtCHL1), and a transcription factor, WRKY70. Their roles in defense signaling were then investigated. The Erwinia-derived elicitors rapidly activated the expression of AtCLH1 and WRKY70 through different signaling pathways. The expression of the AtCHL1 gene was up-regulated by jasmonic acid (JA) but down-regulated by salicylic acid (SA), whereas WRKY70 was activated by SA and repressed by JA. In order to elucidate the functions of AtCLH1 and WRKY70 in plant defense, stable transgenic lines were produced where these genes were overexpressed or silenced. Additionally, independent knockout lines were also characterized. Bacterial and fungal pathogens were then used to assess the contribution of these genes to the Arabidopsis disease resistance. The transcriptional modulation of AtCLH1 by either the constitutive over-expression or RNAi silencing caused alterations in the chlorophyll-to-chlorophyllide ratio, supporting the claim that chlorophyllase 1 has a role in the chlorophyll degradation pathway. Silencing of this gene led to light-dependent over-accumulation of the reactive oxygen species (ROS) in response to infection by Erwinia carotovora subsp. carotovora SCC1. This was followed by an enhanced induction of SA-dependent defense genes and an increased resistance to this pathogen. Interestingly, little effect on the pathogen-induced SA accumulation at the early infection was observed, suggesting that action of ROS might potentiate SA signaling. In contrast, the pathogen-induced JA production was significantly reduced in the RNAi silenced plants. Moreover, JA signaling and resistance to Alternaria brassicicola were impaired. These observations provide support for the argument that the ROS generated in chloroplasts might have a negative impact on JA signaling. The over-expression of WRKY70 resulted in an enhanced resistance to E. carotovora subsp. carotovora SCC1, Pseudomonas syringae pv. tomato DC3000 and Erysiphe cichoracearum UCSC1, whilst an antisense suppression or an insertional inactivation of WRKY70 led to a compromised resistance to E. carotovora subsp. carotovora SCC1 and to E. cichoracearum UCSC1 but not to P. syringae pv. tomato DC3000. Gene expression analysis revealed that WRKY70 activated many known defense-related genes associated with the SAR response but suppressed a subset of the JA-responsive genes. In particular, I was able to show that both the basal and the induced expression of AtCLH1 was enhanced by the antisense silencing or the insertional inactivation of WRKY70, whereas a reduction in AtCLH1 expression was observed in the WRKY70 over-expressors following an MeJA application or an A. brassicicola infection. Moreover, the SA-induced suppression of AtCLH1 was relieved in wrky70 mutants. These results indicate that WRKY70 down-regulates AtCLH1. An epistasis analysis suggested that WRKY70 functions downstream of the NPR1 in an SA-dependent signaling pathway. When challenged with A. brassicicola, WRKY70 over-expressing plants exhibited a compromised disease resistance while wrky70 mutants had the opposite effect. These results confirmed the WRKY70-mediated inhibitory effects on JA signaling. Furthermore, the WRKY70-controlled suppression of A. brassicicola resistance was mainly through an NPR1-dependent mechanism. Taking all the data together, I suggest that the pathogen-responsive transcription factor WRKY70 is a common component in both SA- and JA-dependent pathways and plays a crucial role in the SA-mediated suppression of JA signaling.
  • Ursache, Robertas (Helsingin yliopisto, 2014)
    Plant vascular tissues are supporting and conductive tissues composed of two major components, xylem and phloem. These tissues transport water, food, hormones and minerals within the plant. In my thesis work, I used the Arabidopsis root as a model system to study vascular tissue formation. The first part of my thesis work is focused on the formation of xylem, the water transporting tissue. In the Arabidopsis root, the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. It has been previously reported that high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. In this work, we provide evidence that auxin biosynthesis promotes HD-ZIP III expression and metaxylem formation. We observed that plants with mutations in auxin biosynthesis genes, such as trp2-12, wei8 tar2, or the quintuple yucca mutant, as well as plants treated with a pharmacological inhibitor of auxin biosynthesis, show reduced expression of the HD-ZIP III genes accompanied by specific defects in metaxylem formation. We were able to induce a partial rescue of the metaxylem defects by introducing an endogenous auxin supply. In addition, some of the patterning defects can be suppressed by synthetically elevating HD-ZIP III expression in the stele of the Arabidopsis root. The second part of my thesis work is focused on phloem tissue formation. Phloem is the tissue responsible for long-distance molecular transport and signaling. The conductive components of the phloem, the sieve elements, rely on specific junctions between the conducting cells in the form of highly perforated sieve areas. We identified mutations in the CHER1 (CHOLINE TRANSPORTER LIKE 1) locus of Arabidopsis which result in altered phloem conductivity, reduced sieve pore density, and defects in sieve pore formation. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We provide data showing that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and is associated with the late stage of phragmoplast formation during cytokinesis. Interestingly, CHER1 has a sustained, polar localization in forming sieve plates, which is consistent with its function in the elaboration of the sieve areas.
  • Hienola, Anni (Helsingin yliopisto, 2007)
    The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.
  • Fagerlund , Riku (Helsingin yliopisto, 2008)
    The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). Proteins to be targeted into the nucleus by the classical nuclear import system contain nuclear localization signals (NLSs), which are recognized by importin alpha, the NLS receptor. Importin alpha binds to importin beta, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin alpha isoforms have been identified. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes and modulate the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor kB (NF-kB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-kB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-kB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin alpha5 molecule via unconventional dimer-specific NLSs. Importin alpha molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin alpha5 molecule. This work demonstrates that all NF-kB proteins are transported into the nucleus by importin alpha molecules. In addition, NLS was identified in RelB protein. The interactions between NF-kB proteins and importin alpha molecules were found to be directly mediated by the NLSs of NF-kB proteins. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-kB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.
  • 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.
  • Lehtonen, Jukka T. (Helsingin yliopisto, 2013)
    Native faunas and floras are especially susceptible to negative effects of invasive alien species in islands. The world´s fourth largest island, Madagascar, has very unique biota with high level of endemism. The black rat, Rattus rattus, is claimed to cause more extinctions of insular vertebrates than any other introduced rodent in the world. On Madagascar, R. rattus is suggested to competete with native rodents belonging to the endemic subfamily Nesomyinae. Extensive deforestation and fragmentation also threatens Malagasy forest-dwelling species. The aims of this thesis were to 1) study the occurence of native and introduced species and the different kind of factors determining occurence in southeastern Madagascar and 2) help to target future studies by estimating which native rodents are the most potential competitors with introduced rodents on Madagascar. Habitat use data of black rat and endemic rodents were collected both in fragmented and in unfragmented forest in Ranomafana National Park in southeastern Madagascar. A total of 698 rodent individuals were captured in 6204 trap nights. Logistic and Poisson regression models were used to determine the factors that influence the presence and abundance of rodent species and to investigate how sensitive one variable can be to other variables in regression models. This led to the introduction of a new approach called explanatory framework based regression analysis (EFRA) which rests on the social science based elaboration technique. EFRA enables systematization of the link between ecological knowledge and statistical analysis. From the point of it multicollinearity is more source of information than a problem for data analysis. The abundance of R. rattus increased with increasing forest disturbance. The spread of R. rattus was suggested to be associated with deforestation but not directly with fragmentation. It is not surprising when remembering that R. rattus utilizes open areas too. The measured value of the size of the fragment can be viewed as an index capturing certain features of the forest fragment. For Malagasy native rodents, clear-cutting is disastrous and forest fragmentation may have a diminishing effect on populations of Nesomys audeberti. However, none of the native species was more abundant in un-logged than in selectively logged forest. There was no evidence that Eliurus webbi suffers directly from forest fragmentation but it may be more susceptible to interactions with R. rattus in fragments than in continuous forest. In the last paper, the comparison of morphological measurements leads to the conclusion that there is a high probability of competition between introduced R. rattus and the following native taxa: all species of Nesomys, larger semiarboreal Eliurus species (e.g. E. tanala, E. webbi), and especially Gymnuromys roberti.
  • Spilling, Kristian (Helsingin yliopisto, 2007)
    Increased anthropogenic loading of nitrogen (N) and phosphorus (P) has led to an eutrophication problem in the Baltic Sea, and the spring bloom is a key component in the biological uptake of increased nutrient concentrations. The spring bloom in the Baltic Sea is dominated by both diatoms and dinoflagellates. However, the sedimentation of these groups is different: diatoms tend to sink to the sea floor at the end of the bloom, while dinoflagellates to a large degree are been remineralized in the euphotic zone. Understanding phytoplankton competition and species specific ecological strategies is thus of importance for assessing indirect effects of phytoplankton community composition on eutrophication problems. The main objective of this thesis was to describe some basic physiological and ecological characteristics of the main cold-water diatoms and dinoflagellates in the Baltic Sea. This was achieved by specific studies of: (1) seasonal vertical positioning, (2) dinoflagellate life cycle, (3) mixotrophy, (4) primary production, respiration and growth and (5) diatom silicate uptake, using cultures of common cold-water diatoms: Chaetoceros wighamii, C. gracilis, Pauliella taeniata, Thalassiosira baltica, T. levanderi, Melosira arctica, Diatoma tenuis, Nitzschia frigida, and dinoflagellates: Peridiniella catenata, Woloszynskia halophila and Scrippsiella hangoei. The diatoms had higher primary production capacity and lower respiration rate compared with the dinoflagellates. This difference was reflected in the maximum growth rate, which for the examined diatoms range from 0.6 to 1.2 divisions d-1, compared with 0.2 to 0.3 divisions d-1 for the dinoflagellates. Among diatoms there were species specific differences in light utilization and uptake of silicate, and C. wighamii had the highest carbon assimilation capacity and maximum silicate uptake. The physiological properties of diatoms and dinoflagellates were used in a model of the onset of the spring bloom: for the diatoms the model could predict the initiation of the spring bloom; S. hangoei, on the other hand, could not compete successfully and did not obtain positive growth in the model. The other dinoflagellates did not have higher growth rates or carbon assimilation rates and would thus probably not perform better than S. hangoei in the model. The dinoflagellates do, however, have competitive advantages that were not included in the model: motility and mixotrophy. Previous investigations has revealed that the chain-forming P. catenata performs diurnal vertical migration (DVM), and the results presented here suggest that active positioning in the water column, in addition to DVM, is a key element in this species' life strategy. There was indication of mixotrophy in S. hangoei, as it produced and excreted the enzyme leucine aminopeptidase (LAP). Moreover, there was indirect evidence that W. halophila obtains carbon from other sources than photosynthesis when comparing increase in cell numbers with in situ carbon assimilation rates. The results indicate that mixotrophy is a part of the strategy of vernal dinoflagellates in the Baltic Sea. There were also indications that the seeding of the spring bloom is very important for the dinoflagellates to succeed. In mesocosm experiments dinoflagellates could not compete with diatoms when their initial numbers were low. In conclusion, this thesis has provided new information about the basic physiological and ecological properties of the main cold-water phytoplankton in the Baltic Sea. The main phytoplankton groups, diatoms and dinoflagellates, have different physiological properties, which clearly separate their life strategies. The information presented here could serve as further steps towards better prognostic models of the effects of eutrophication in the Baltic Sea.
  • Taipale, Mikko (Helsingin yliopisto, 2005)