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  • Llano, Olaya (Helsingin yliopisto, 2015)
    Dendritic spines are the main site of reception of glutamatergic -excitatory- neurotransmission in the central nervous system. According to the current view on neuronal function, dendritic spines play a pivotal role in the formation of synaptic networks for memory storage. Consequently, dendritic spines are crucial for cognitive processes, e.g. learning. Numerous disorders such as intellectual developmental disorders, schizophrenia and cognitive impairment are associated with functional and structural abnormalities of dendritic spines. The main objectives of this project were to identify molecular regulators of the structure and function of dendritic spines and to characterise novel mechanisms leading to dendritic spine development and synapse formation. Actin is the most abundant protein in dendritic spines. Rearrangements of the actin cytoskeleton are responsible for the morphological changes of dendritic spines, making actin a major player in the regulation of glutamatergic synaptogenesis. Increasing evidence shows that dendritic filopodia are crucial in the formation of dendritic spines. Often filopodia act as precursors of mature dendritic spines. While filopodial protrusions in other cell types have been widely studied, the molecular mechanisms regulating the emergence and maintenance of dendritic filopodia are poorly understood. In this thesis work, we show that the polymerizing factor mDia2 promotes initiation and polymerization of actin in the filopodial tip. We also describe a novel observation of filopodial root polymerization. Spine maturation is accompanied by expansion of the spine head. We propose here that the actin polymerizing factor Arp2/3 complex takes active part in the branched actin polymerization during spine head expansion. Spine heads are dynamic structures, with long protrusions often visible on their surface. Our results demonstrate that the actin depolymerizing factor cofilin-1 has a double function in the regulation of dendritic spine actin dynamics. On one hand cofilin-1 replenishes the actin monomer pool, and on the other hand it shapes the spines by severing the actin filaments and therefore controls actin filament length. The maturation of synaptic networks is strictly dependent on the synchronous development of both inhibitory and excitatory transmission. Within this context the formation of and stabilization of dendritic spines is an important step in the maturation of glutamatergic transmission. However, in terms of functional maturation, chloride regulatory proteins, such as the K-Cl cotransporter KCC2, are crucial regulators of GABAergic -inhibitory- transmission. Interestingly, previous studies have identified KCC2 as an important agent required for the maturation of dendritic spines and consequently glutamatergic transmission. The mechanism how KCC2 exerts its chloride-extrusion independent effect on dendritic spines and excitatory synapses remained obscure. In this thesis work we have identified the molecular interaction between the potassium-chloride cotransporter KCC2 and the guanine nucleotide exchange factor βPix. Importantly, KCC2 inhibits the action of βPix towards the GTPase Rac1, a major regulator of the actin cytoskeleton in dendritic spines. The inhibition of βPix by KCC2 leads to decreased cofilin-1 inactivation and subsequent reduction in the fraction of actin that is stable. This novel molecular pathway leads to the regulation of glutamatergic synaptogenesis and spine formation by KCC2 via βPix. Synaptic cell adhesion molecules orchestrate trans-synaptic recognition as well as specification of glutamatergic synapses. Fine-tuning of synaptic networks requires a delicate balance between positive and negative signalling mechanisms that regulate dendritic spine formation. The intercellular adhesion molecule ICAM-5 negatively regulates the maturation of dendritic spines. We have found that ICAM-5 binds to pre-synaptic β1 integrins in filopodia and immature dendritic spines, preventing spine maturation. We have characterized the molecular mechanisms leading to the diminished interaction of ICAM-5 and β1 integrins during spine maturation. Moreover, genetic manipulation of ICAM5 affected the morphology and function of dendritic spines. The results included in this thesis work contribute to the deeper understanding of the molecular mechanisms regulating the development of dendritic spines. We have studied molecules that control all steps of these processes, from filopodia formation to mature spine regulation; encompassing structural and functional synaptogenesis.
  • 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)
  • Putkonen, Noora (Helsingin yliopisto, 2012)
    Neuronal cell death caused by excitotoxicity accompanies neurodegenerative disorders, such as Alzheimer s disease (AD) and Huntington s disease (HD), epilepsy and ischaemia. Glutamate is the major excitotoxin in the CNS and causes activation of glutamate receptors. Ionotropic glutamate receptors can directly cause calcium influx that further enables activation of cell death pathways. Kainic acid (KA) is a specific agonist for ionotropic non-NMDA glutamate receptors, namely KA and AMPA receptors. KA induces epiletic activity in rodents and causes hippocampal sclerosis, similar to human temporal epilepsy. HD, a neurodegenerative disease characterized by accumulation of mutant huntingtin protein, and causing cell death in the striatum of affected individuals, has also been shown to involve excitotoxic cell death. Intracellular organelles have been implicated in stress sensing and contribute to cell death signaling. Mitochondria have been closely linked to apoptotic pathways and recent research has also implicated other organelles, such as the endoplasmic reticulum (ER), lysosomes and Golgi apparatus in cell death. In this thesis, the involvement of ER stress was shown to accompany hippocampal cell death caused by KA in vivo and in vitro as well as in a cell model of HD. KA induced activation of ER stress sensors that aim to restore homeostasis via activation of the unfolded protein response (UPR). In prolonged stressful conditions, the UPR activates apoptotic pathways. Treatment with an ER stress inhibitor, Salubrinal (Sal), significantly attenuated cell death in hippocampal neurons in vivo and in vitro. ER stress was also activated in a cell model of HD and treatment with Sal reduced cell death and mutant hungtingtin aggregation. These data indicated for the first time the involvement of the ER in cell death pathways caused by excitotoxicity, and that inhibition of ER stress could be a potential treatment against neuronal cell death in HD and other disorders involving excitotoxicity. In search of other cell death mediators we focused on Cdk5 that has been implicated deregulated in excitotoxicity. Involved in multiple signaling pathways, Cdk5, has been implicated, for instance, in regulation of synaptic proteins, ER stress and cell death. In this thesis, a KA receptor important for mediating cell death in the hippocampus, GluR6, was shown to be regulated by Cdk5. Inhibition of Cdk5 reduced GluR6 downregulation by KA as well as cell death caused by KA in vitro. These data indicated Cdk5 involvement in KA excitotoxicity and could also present a potential drug target in neurological disorders. Moreover, this was the first time that Cdk5 was shown to contribute to KA receptor regulation.
  • Johansson, Marie (Helsingin yliopisto, 2006)
    Oxysterol binding protein (OSBP) homologues have been found in eukaryotic organisms ranging from yeast to humans. These evolutionary conserved proteins have in common the presence of an OSBP-related domain (ORD) which contains the fully conserved EQVSHHPP sequence motif. The ORD forms a barrel structure that binds sterols in its interior. Other domains and sequence elements found in OSBP-homologues include pleckstrin homology domains, ankyrin repeats and two phenylalanines in an acidic tract (FFAT) motifs, which target the proteins to distinct subcellular compartments. OSBP homologues have been implicated in a wide range of intracellular processes, including vesicle trafficking, lipid metabolism and cell signaling, but little is known about the functional mechanisms of these proteins. The human family of OSBP homologues consists of twelve OSBP-related proteins (ORP). This thesis work is focused on one of the family members, ORP1, of which two variants were found to be expressed tissue-specifically in humans. The shorter variant, ORP1S contains an ORD only. The N-terminally extended variant, ORP1L, comprises a pleckstrin homology domain and three ankyrin repeats in addition to the ORD. The two ORP1 variants differ in intracellular localization. ORP1S is cytosolic, while the ankyrin repeat region of ORP1L targets the protein to late endosomes/lysosomes. This part of ORP1L also has profound effects on late endosomal morphology, inducing perinuclear clustering of late endosomes. A central aim of this study was to identify molecular interactions of ORP1L on late endosomes. The morphological changes of late endosomes induced by overexpressed ORP1L implies involvement of small Rab GTPases, regulators of organelle motility, tethering, docking and/or fusion, in generation of the phenotype. A direct interaction was demonstrated between ORP1L and active Rab7. ORP1L prolongs the active state of Rab7 by stabilizing its GTP-bound form. The clustering of late endosomes/lysosomes was also shown to be linked to the minus end-directed microtubule-based dynein-dynactin motor complex through the ankyrin repeat region of ORP1L. ORP1L, Rab7 and the Rab7-interacting lysosomal protein (RILP) were found to be part of the same effector complex recruiting the dynein-dynactin complex to late endosomes, thereby promoting minus end-directed movement. The proteins were found to be physically close to each other on late endosomes and RILP was found to stabilize the ORP1L-Rab7 interaction. It is possible that ORP1L and RILP bind to each other through their C-terminal and N-terminal regions, respectively, when they are bridged by Rab7. With the results of this study we have been able to place a member of the uncharacterized OSBP-family, ORP1L, in the endocytic pathway, where it regulates motility and possibly fusion of late endosomes through interaction with the small GTPase Rab7.
  • Hynynen, Riikka (Helsingin yliopisto, 2009)
    ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.
  • Ramu, Päivi (Helsingin yliopisto, 2007)
    Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.
  • Lundell, Robin (Helsingin yliopisto, 2011)
    Winter is a significant period for the seasonality of northern plants, but is often overlooked when studying the interactions of plants and their environment. This study focuses on the effects of overwintering conditions, including warm winter periods, snow, and snowmelt on boreal and sub-Arctic field layer plants. Wintertime photosynthesis and related physiological factors of evergreen dwarf shrubs, particularly of Vaccinium vitis-idaea, are emphasised. The work combines experiments both in the field and in growth chambers with measurements in natural field conditions. Evergreen dwarf shrubs are predominantly covered by snow in the winter. The protective snow cover provides favourable conditions for photosynthesis, especially during the spring before snowmelt. The results of this study indicate that photosynthesis occurs under the snow in V. vitis-idaea. The light response of photosynthesis determined in field conditions during the period of snow cover shows that positive net CO2 exchange is possible under the snow in the prevailing light and temperature. Photosynthetic capacity increases readily during warm periods in winter and the plants are thus able to replenish carbohydrate reserves lost through respiration. Exposure to low temperatures in combination with high light following early snowmelt can set back photosynthesis as sustained photoprotective measures are activated and photodamage begins to build up. Freezing may further decrease the photosynthetic capacity. The small-scale distribution of many field layer plants, including V. vitis-idaea and other dwarf shrubs, correlates with the snow distribution in a forest. The results of this study indicate that there are species-specific differences in the snow depth affinity of the field and ground layer species. Events and processes taking place in winter can have a profound effect on the overall performance of plants and on the interactions between plants and their environment. Understanding the processes involved in the overwintering of plants is increasingly important as the wintertime climate in the north is predicted to change in the future.
  • Welling, Annikki (Helsingin yliopisto, 2003)
  • Gorbikova, Elena (Helsingin yliopisto, 2009)
    Energy conversion by living organisms is central dogma of bioenergetics. The effectiveness of the energy extraction by aerobic organisms is much greater than by anaerobic ones. In aerobic organisms the final stage of energy conversion occurs in respiratory chain that is located in the inner membrane of mitochondria or cell membrane of some aerobic bacteria. The terminal complex of the respiratory chain is cytochrome c oxidase (CcO) - the subject of this study. The primary function of CcO is to reduce oxygen to water. For this, CcO accepts electrons from a small soluble enzyme cytochrome c from one side of the membrane and protons from another side. Moreover, CcO translocates protons across the membrane. Both oxygen reduction and proton translocation contributes to generation of transmembrane electrochemical gradient that is used for ATP synthesis and different types of work in the cell. Although the structure of CcO is defined with a relatively high atomic resolution (1.8 Å), its function can hardly be elucidated from the structure. The electron transfer route within CcO and its steps are very well defined. Meanwhile, the proton transfer roots were predicted from the site-specific mutagenesis and later proved by X-ray crystallography, however, the more strong proof of the players of the proton translocation machine is still required. In this work we developed new methods to study CcO function based on FTIR (Fourier Transform Infrared) spectroscopy. Mainly with use of these methods we answered several questions that were controversial for many years: [i] the donor of H+ for dioxygen bond splitting was identified and [ii] the protolytic transitions of Glu-278 one of the key amino acid in proton translocation mechanism was shown for the first time.
  • Ahlfors, Reetta (University of Helsinki, 2008)
    Tropospheric ozone (O3) is one of the most common air pollutants in industrialized countries, and an increasing problem in rapidly industrialising and developing countries in Asia, Africa and South America. Elevated concentrations of tropospheric O3 can lead to decrease in photosynthesis rate and therefore affect the normal metabolism, growth and seed production. Acute and high O3 episodes can lead to extensive damage leading to dead tissue in plants. Thus, O3 derived growth defects can lead to reduction in crop yield thereby leading to economical losses. Despite the extensive research on this area, many questions remain open on how these processes are controlled. In this study, the stress-induced signaling routes and the components involved were elucidated in more detail starting from visual damage to changes in gene expression, signaling routes and plant hormone interactions that are involved in O3-induced cell death. In order to elucidate O3-induced responses in Arabidopsis, mitogen-activated protein kinase (MAPK) signaling was studied using different hormonal signaling mutants. MAPKs were activated at the beginning of the O3 exposure. The activity of MAPKs, which were identified as AtMPK3 and AtMPK6, reached the maximum at 1 and 2 hours after the start of the exposure, respectively. The activity decreased back to clean air levels at 8 hours after the start of the exposure. Both AtMPK3 and AtMPK6 were translocated to nucleus at the beginning of the O3 exposure where they most likely affect gene expression. Differences were seen between different hormonal signaling mutants. Functional SA signaling was shown to be needed for the full protein levels and activation of AtMPK3. In addition, AtMPK3 and AtMPK6 activation was not dependent on ethylene signaling. Finally, jasmonic acid was also shown to have an impact on AtMPK3 protein levels and AtMPK3 activity. To further study O3-induced cell death, an earlier isolated O3 sensitive Arabidopsis mutant rcd1 was mapped, cloned and further characterized. RCD1 was shown to encode a gene with WWE and ADP-ribosylation domains known to be involved in protein-protein interactions and cell signaling. rcd1 was shown to be involved in many processes including hormonal signaling and regulation of stress-responsive genes. rcd1 is sensitive against O3 and apoplastic superoxide, but tolerant against paraquat that produces superoxide in chloroplast. rcd1 is also partially insensitive to glucose and has alterations in hormone responses. These alterations are seen as ABA insensitivity, reduced jasmonic acid sensitivity and reduced ethylene sensitivity. All these features suggest that RCD1 acts as an integrative node in hormonal signaling and it is involved in the hormonal regulation of several specific stress-responsive genes. Further studies with the rcd1 mutant showed that it exhibits the classical features of programmed cell death, PCD, in response to O3. These include nuclear shrinkage, chromatin condensation, nuclear DNA degradation, cytosol vesiculation and accumulation of phenolic compounds and eventually patches of HR-like lesions. rcd1 was found to produce extensive amount of salicylic acid and jasmonic acid in response to O3. Double mutant studies showed that SA independent and dependent processes were involved in the O3-induced PCD in rcd1 and that increased sensitivity against JA led to increased sensitivity against O3. Furthermore, rcd1 had alterations in MAPK signature that resembled changes that were previously seen in mutants defective in SA and JA signaling. Nitric oxide accumulation and its impact on O3-induced cell death were also studied. Transient accumulation of NO was seen at the beginning of the O3 exposure, and during late time points, NO accumulation coincided with the HR-like lesions. NO was shown to modify defense gene expression, such as, SA and ethylene biosynthetic genes. Furthermore, rcd1 was shown to produce more NO in control conditions. In conclusion, NO was shown to be involved in O3-induced signaling leading to attenuation of SA biosynthesis and other defense related genes.
  • Kainov, Denis (Helsingin yliopisto, 2005)
  • Couchoux, Christelle (Helsingin yliopisto, 2013)
    In my thesis I investigated the foraging behaviour of the wasp Hyposoter horticola, an egg-larval parasitoid of the Glanville fritillary butterfly Melitaea cinxia, in the Åland islands in Finland. The particularity of this system is that the wasp is resource limited and faces strong intraspecific competition. ---------- I first focused on behaviour at an individual scale. In a series of experiments I tested how H. horticola s host searching behaviour was affected by developmental timing of both the parasitoid and the host, and direct intraspecific competition among foraging females. I found that the wasps visit host egg clusters before the hosts are susceptible to parasitism, presumably to cope with the limited time availability of the hosts. As the unparasitized hosts matured their value increased, competition became more frequent, and the wasps foraged more actively. Competition can also affect the parasitoid at earlier stages in its life. As larvae inside the hosts, the immature H. horticola suffered from competition due to superparasitism. Combining behavioural experiments in the laboratory and genetic analyses of sibship, I found that adult H. horticola deposit a chemical marking after oviposition that deters conspecifics from parasitizing a previously exploited host cluster. This protects parasitized host clusters from further exploitation. I found that the effectiveness of the deterrent persisted under natural conditions, where individual host egg clusters were each primarily parasitized by a single female H. horticola. Even when several females parasitized a cluster, the great majority of the offspring were full-siblings and the parasitism rate did not increase above the average 1/3 observed throughout the population. Considering that H. horticola is resource limited and faces intraspecific competition when foraging for hosts, it is surprising that only they parasitize a fraction of the hosts in each host egg cluster. After testing several physiological and evolutionary hypotheses for what might lead to this sub-maximal rate of host exploitation, I concluded that optimal foraging with avoidance of superparasitism was the most plausible explanation, as long as the search time between host clusters was low. ------ Then, I worked at a larger scale than individual behaviour. In the Åland islands, the butterfly host lives as a classic metapopulation with a high extinction rate of local populations. Due to strong competition, almost all the M. cinxia egg clusters in the population are found and parasitized by H. horticola. This suggests that the wasps must be good dispersers, which could influence the spatial genetic structure of the parasitoid population. I used DNA microsatellite markers and analysed H. horticola individuals sampled from over the entire population. My results indicate that, contrary to theory that higher trophic level species are more affected by habitat fragmentation than the species upon which they depend, the H. horticola population was less strongly genetically structured than the metapopulation of its butterfly host. It seems that H. horticola s dispersal ability allows it to compensate for the fragmented distribution of its host and not suffer from the metapopulation dynamics of the host local populations. Overall, the results of my thesis show that interactions between H. horticola and its host M. cinxia are strongly affected by competition among the adult female wasps. Intraspecific competition has an important role from an evolutionary perspective. Hyposoter horticola s deterrent marking behaviour has evolved in response to competition and the risk of superparasitism faced by immature offspring. Avoidance of superparasitism to limit competition is also the fundamental mechanism that controls H. horticola s optimal foraging strategy. And intraspecific competition modifies individual female host searching behaviour, increasing their foraging activity. -------- Interactions within a multitrophic system are complex and predictions concerning host-parasitoid interactions are difficult to generalise. However, as in this system, competition is factor that should receive more attention in empirical and theoretical studies of host-parasitoid interactions.