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  • 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.
  • Marttila, Minttu (Helsingin yliopisto, 2014)
    We collected all mutations in TPM2 and TPM3 genes hitherto found to cause congenital myopathies, to perform genotype-phenotype correlations, and to increase our understanding of the pathogenetic mechanisms of congenital myopathies caused by mutations in the tropomyosin and nebulin genes. Nemaline myopathy (NM), a rare, genetic muscle disorder defined on the basis of muscle dysfunction and the presence of structural abnormalities in the muscle fibres (i.e. nemaline bodies), is caused by mutations in ten genes known to date: Nebulin (NEB), α-actin (ACTA1), α-tropomyosin (TPM3), β-tropomyosin (TPM2), troponin T (TNNT1), cofilin 2 (CFL2), KBTBD13, KLHL40, KLHL41 and leiomodin 3 (LMOD 3). Tropomyosin controls muscle contraction by inhibiting the actin myosin interaction in a calcium-sensitive manner. Mutations in tropomyosin genes may cause NM, cap myopathy, congenital fibre-type disproportion, distal arthrogryposes and Escobar syndrome. We correlated the clinical picture of these diseases to novel and previously published mutations to the TPM2 (30 mutations) and TPM3 (20 mutations) genes. Mutations in TPM2 and TPM3 caused an increased Ca2+ sensitivity, resulting in a hypercontractile molecular phenotype. We studied the pathogenetic mechanisms to which five disease-causing mutations in β-tropomyosin (p.Glu41Lys, p.Lys49del, p.Glu117Lys, p.Glu139del and p.Gln147Pro) lead. We showed that four of the mutations cause changes in the affinity for actin leading to muscle weakness in patients, while two mutations show defective Ca2+ activation of contractility. Nebulin (NEB) is a giant 600 900-kDa filamentous protein in thin filament. We produced four wild-type nebulin super-repeats and five corresponding mutation constructs (p.Glu2431Lys, p.Ser4665Ile, p.Thr5681Pro, p.Arg2478_Asp2512del and p.Val3681_Asn3686del) in the study. The mutations were identified in patients with NM or distal myopathy. We performed F-actin and tropomyosin-binding experiments for the nebulin fragments. Our results demonstrate actin nebulin interactions and, for the first time, tropomyosin nebulin interactions in vitro, and show that the interactions are altered by disease-causing mutations. This suggests that an abnormal interaction between aberrant thin filament proteins is a pathogenetic mechanism in NM and related disorders.
  • Al-Hello, Haider (Helsingin yliopisto, 2012)
    Enteroviruses (EVs) are small non-enveloped RNA viruses forming a large group of different serotypes. EVs belong to the family Picornaviridae. The primary replication site of an enterovirus is typically the epithelium of the respiratory tract and the gastrointestinal mucosa. Virus replication in the gastrointestinal mucosa may continue, often asymptomatically, for several weeks occasionally causing viremia. During the viremia the virus spreads through the lymphatic system and circulation. Organ-specific symptoms rise after viral replication in the secondary target tissues. Occasionally, cellular adaptation is required for a virus to initiate replication in the secondary target tissue(s). Adaptation is linked to mutation(s) which may lead to alteration in cellular tropism, e.g., recognition of new surface receptor molecules or other host cell constituents essential for virus entry and replication. However, the critical step may also occur later during in the interaction of the host cell and the replicating virus. In the present study, genetic changes responsible for altered phenotypic features were sought using two strains of Human enterovirus B (HEV-B) species. Firstly, a laboratory isolate of coxsackievirus B5 (CV-B5), strain DS, was passaged 15 times in mouse pancreas in vivo, which resulted in a diabetogenic mouse pancreas passaged virus strain (MPP). The concept of diabetogenic means the ability of the MPP strain to replicate, cause insulitis and dysregulation of the glucose metabolism in the mouse pancreas in vivo. The interaction between the MPP virus strain and insulin producing β-cells was further studied in cell culture using a mouse-derived insulinoma cell line, MIN-6 cells, as an experimental model. The replication of the MPP virus strain was clearly slower in the MIN-6 cells compared to the other tested cell lines. After three days of incubation, extensive replication of MPP was evident in MIN-6 cells and resulted in a MIN-6 cell-adapted virus strain (MCA). Secondly, the ability of the D207 virus strain, isolated from a type 1 diabetic patient, to replicate in a primary human β-cell culture was tested. D207 was initially serotyped as coxsackievirus A9 (CV-A9) in a virus-specific neutralization assay. The D207 virus strain was found to cause cytolysis in the primary human β-cells and, simultaneously, severe functional damage of the surviving β-cells. The genomes of the four virus strains DS, MPP, MCA and D207 were cloned and sequenced. The sequence comparison of three CV-B5 strains (DS, MPP, and MCA) revealed only limited changes, three capsid and two non-structural (NS) amino acid substitutions between MPP and DS, and two capsid and six NS amino acid substitutions between MCA and MPP. In order to determine which of the amino acid substitutions were responsible for the changed phenotype in vivo and in vitro, full-length infectious clones were constructed from the MPP virus and its parental DS virus. By using reverse mutagenesis and chimeric viruses (MPP/DS and DS/MPP), it was shown that a change from MPP to the MCA phenotype in MIN-6 cells was mediated by only a single amino acid at position 94 in VP1, while the in vivo adaptation of the DS virus strain to the inflammation-inducing MPP virus strain may require multiple genetic determinants in the virus capsid and probably also in the NS proteins. Sequence analyses of D207 revealed that the virus belonged to a genogroup D of E-11, but was also neutralized with monotypic antisera to CV-A9. The isolate D207 was found to be closely related to a specific E-11 strains known to cause uveitis. Uveitis-causing E-11 strains were also found to be well neutralized with both CV-A9- and E-11-specific antisera. In a further study, a wide range of E-11 isolates were included to test the observed dual neutralizibility among isolates belonging to the D genogroup. Five of the six studied strains belonging to genogroup D were also neutralized with antisera against coxsackievirus A9 Griggs. The peptide scanning technique was utilized to identify antigenic regions of the capsid proteins of the D207 strain responsible for the observed dual neutralization. Several regions in the capsid of D207 were found to cross-react with an antiserum raised against CV-A9. However, epitopes responsible for the cross-neutralization remained unidentified. In conclusion, these studies indicate that the specific location of mutation may affect the phenotype of an enterovirus more than the overall quantity of changes. In the experimental settings, radical changes in the viral phenotypic features occurred only after a few amino acid substitutions. The majority of the studied viruses in the genogroup D of E-11 maintained exceptional phenotypic property, the cross-neutralization with CV-A9 specific antiserum, despite their genetic divergence.
  • Ollila, Saara (Helsingfors universitet, 2008)
    Hereditary nonpolyposis colorectal cancer (HNPCC) is a hereditary cancer syndrome, which associates with high penetrance of early onset colorectal and endometrial tumours. Susceptibility for HNPCC is dominantly inherited with germline defects in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. A truncating mutation in one of these genes leads to deficient MMR, predisposing the mutation carriers to HNPCC, but a nontruncating mutation can either be neutral or lead to increased cancer risk and HNPCC. The correct determination of the pathogenicity of a found mutation is very important, as the verification of the causative mutation enables genetic counselling and surveillance of mutation carriers. This has been shown to lead to significantly lowered mortality. MSH2 is the second most commonly mutated HNPCC susceptibility gene and defects in it account for 39% of all identified HNPCC mutations. The aim of this work was to gather functional evidence on the pathogenicity of patient-derived nontruncating MSH2 variants. The proteins corresponding to the original genetic variants were expressed and purified. The expression level, MMR efficiency, interaction with MSH6, mismatch binding, and mismatch release capabilities of the protein variants were studied. The results of the functional assays were compared to the clinical characteristics of the mutation carriers. 12 of the studied 18 mutations were found to exhibit severe defects in the functional assays, supporting the hypothesis that these mutations were the underlying cause of the cancer phenotype in mutation carriers. 2 mutations reduced but did not abolish the function of the protein, leaving their pathogenicity status inconclusive. 4 mutations showed no or only a minor defect in the assays, suggesting nonpathogenicity. The functional defects were mediated through different mechanisms. The majority of the MMR-deficient mutations which were located in the amino-terminal domains of MSH2 demonstrated defects in the protein expression level. Most of the carboxy-terminal mutations, situated in the ATPase domain, had an impact on the ability of the protein to bind or release mismatched DNA. When comparing the biochemical data to the tumour phenotype, a significant correlation between the functional deficiency in vitro and lack of expression of the corresponding protein in the tumour was observed. The analyses demonstrated that the location of the mutation affects the biochemistry of MMR, but may also have an effect on the phenotype of MSH2 mutation carriers. This study significantly contributed to the knowledge of MSH2-associated HNPCC tumorigenesis, especially facilitating the diagnostics and counselling of the associated families.
  • Kiialainen, Anna (Helsingin yliopisto, 2007)
    PATHOGENIC MECHANISMS OF PLOSL Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease, is a recessively inherited disease of brain and bone. PLOSL manifests as early-onset progressive dementia and bone fractures. Mutations in the TYROBP (DAP12) and TREM2 genes have been identified as the primary cause of PLOSL. DAP12 and TREM2 encode important signalling molecules in cells of the innate immune system. The mechanism by which loss-of-function of the DAP12/TREM2 signalling complex leads to PLOSL is currently unknown. The aim of this thesis work was to gain insight into the pathogenic mechanisms behind PLOSL. To first identify the central nervous system (CNS) cell types that express both Dap12 and Trem2, the expression patterns of Dap12 and Trem2 in mouse CNS were analyzed. Dap12 and Trem2 expression was seen from embryonic stage to adulthood and microglial cells and oligodendrocytes were identified as the major Dap12/Trem2 producing cells of the CNS. To subsequently identify the pathways and biological processes associated with DAP12/TREM2 mediated signalling in human cells, genome wide transcript analysis of in vitro differentiated dendritic cells (DCs) of PLOSL patients representing functional knockouts of either DAP12 or TREM2 was performed. Both DAP12 and TREM2 deficient cells differentiated into DCs and responded to pathogenic stimuli. However, the DCs showed morphological differences compared to control cells due to defects in the actin filaments. Transcript profiles of the patient DCs showed differential expression of genes involved in immune response and for genes earlier associated with other disorders of the CNS as well as genes involved in the remodeling of bone, linking the findings with the tissue phenotype of PLOSL patients. To analyze the effect of Dap12 deficiency in the CNS, genome wide expression analysis of Dap12 deficient mouse brain and Dap12 deficient microglia as well as functional analysis of Dap12 deficient microglia was performed. Regulation of several pathways involved in synaptic function and transcripts coding for the myelin components was seen in Dap12 knockout mice. Decreased migration, morphological changes and shortened lifespan of the Dap12 knockout microglia was further observed. Taken together, this thesis work showed that both Dap12 and Trem2 are expressed by CNS microglia and that Dap12 deficiency results in functional defects of these cells. Lack of Dap12 in the CNS also leads to synaptic abnormalities even before pathological changes are seen in the tissue level.This work further showed that loss-of-function of DAP12 or TREM2 leads to changes in morphology and gene expression in human dendritic cells. These data underline the functional diversity of the molecules of the innate immune system and implies their significant contribution also in demyelinating CNS disorders, including those resulting in dementia.
  • Kariola, Tarja (Helsingin yliopisto, 2006)
    Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.
  • Kysenius, Kai (Helsingin yliopisto, 2015)
    Aging-related increase of neuronal stress may promote the development of sporadic late-onset Alzheimer s disease (LOAD) and other forms of dementia. LOAD risk is also increased by genetic factors such as ApoE4 and diseases such as type 2 diabetes (T2D). Early LOAD pathology is characterized by alterations in brain lipoprotein receptor expression and neuronal hypometabolism. Multifunctional lipoprotein receptors regulate neuronal plasticity, cholesterol and metabolic homeostasis. Lipoprotein receptors apolipoprotein receptor 2 (ApoER2) and very-low density lipoprotein receptor (VLDLR) bind ApoE, but also interact with proteins centrally involved in LOAD pathogenesis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and the nutraceutical berberine modulate lipoprotein receptor levels in vivo. PCSK9 inhibitors and berberine have recently surfaced as promising treatment options for hypercholesterolemia and T2D, respectively. Additionally, PCSK9 and berberine are implicated in pathways modulating neuronal viability, suggesting they may hold therapeutic potential against neurodegenerative diseases. However, the effects of PCSK9 and berberine on neuronal cell death and lipoprotein receptors are currently poorly understood. The objective of this study was to elucidate the role of PCSK9 and berberine as modulators of lipoprotein receptors and cell death in neurons. The effects of RNAi-mediated PCSK9 downregulation and berberine on neuronal viability were studied in mouse and rat primary neuron cultures. Mechanistic basis of effects were further studied in combination with lentiviral RNA interference, kinase inhibitors and various inducers of cellular stress and cell death. Cell viabilities were assessed by immunofluorescence, Western blotting, and cell toxicity and mitochondrial assays. The main conclusions of this study are: (1) reducing endogenous PCSK9 levels genetically by lentiviral-mediated RNAi protects neurons against apoptotic cell death in an ApoER2-dependent fashion; (2) a potential PCSK9 inhibitor and a widely used nutraceutical berberine causes mitochondria- and NMDA receptor-dependent neuronal cell death at micromolar concentrations; (3) at subtoxic nanomolar concentrations, berberine sensitizes neurons to rotenone and glutamate toxicity calling for caution in berberine dosing and chronic use; and (4) subtoxic stress, including berberine, increase neuronal VLDLR expression, associated with a biphasic effect on the stabilization of the transcription factors hypoxia-inducible factor 1α and β-catenin. To conclude, ApoER2, VLDLR and their modulators PCSK9 and berberine contribute to the regulation of neuronal cell death via multiple mechanisms, suggesting a potential role in neurodegenerative disease pathogenesis at the interface of metabolism and survival signaling.
  • Povelainen, Mira (Helsingin yliopisto, 2008)
    The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.
  • Marjamaa, Kaisa (Helsingin yliopisto, 2007)
    Lignin is a complex plant polymer synthesized through co-operation of multiple intracellular and extracellular enzymes. It is deposited to plant cell walls in cells where additional strength or stiffness are needed, such as in tracheary elements (TEs) in xylem, supporting sclerenchymal tissues and at the sites of wounding. Class III peroxidases (POXs) are secreted plant oxidoreductases with implications in many physiological processes such as the polymerization of lignin and suberin and auxin catabolism. POXs are able to oxidize various substrates in the presence of hydrogen peroxide, including lignin monomers, monolignols, thus enabling the monolignol polymerization to lignin by radical coupling. Trees produce large amounts of lignin in secondary xylem of stems, branches and roots. In this study, POXs of gymnosperm and angiosperm trees were studied in order to find POXs which are able to participate in lignin polymerization in developing secondary xylem i.e. are located at the site of lignin synthesis in tree stems and have the ability to oxidize monolignol substrates. Both in the gymnosperm species, Norway spruce and Scots pine, and in the angiosperm species silver birch the monolignol oxidizing POX activities originating from multiple POX isoforms were present in lignifying secondary xylem in stems during the period of annual growth. Most of the partially purified POXs from Norway spruce and silver birch xylem had highest oxidation rate with coniferyl alcohol, the main monomer in guaiacyl-lignin in conifers. The only exception was the most anionic POX fraction from silver birch, which clearly preferred sinapyl alcohol, the lignin monomer needed in the synthesis of syringyl-guaiacyl lignin in angiosperm trees. Three full-length pox cDNAs px1, px2 and px3 were cloned from the developing xylem of Norway spruce. It was shown that px1 and px2 are expressed in developing tracheids in spruce seedlings, whereas px3 transcripts were not detected suggesting low transcription level in young trees. The amino acid sequences of PX1, PX2 and PX3 were less than 60% identical to each other but showed up to 84% identity to other known POXs. They all begin with predicted N-terminal secretion signal (SS) peptides. PX2 and PX3 contained additional putative vacuolar localization determinants (VSDs) at C-terminus. Transient expression of EGFP-fusions of the SS- and VSD-peptides in tobacco protoplasts showed SS-peptides directed EGFP to secretion in tobacco cells, whereas only the PX2 C-terminal peptide seems to be a functional VSD. According to heterologous expression of px1 in Catharanthus roseus hairy roots, PX1 is a guaicol-oxidizing POX with isoelectric point (pI) approximately 10, similar to monolignol oxidizing POXs in protein extracts from Norway spruce lignifying xylem. Hence, PX1 has characteristics for participation to monolignol dehydrogenation in lignin synthesis, whereas the other two spruce POXs seem to have some other functions. Interesting topics in future include functional characterization of syringyl compound oxidizing POXs and components of POX activity regulation in trees.
  • Kluen, Edward (Helsingin yliopisto, 2012)
    In order to adapt their behaviour optimally and to be able to increase fitness, individuals are assumed to respond flexibly to environmental variation they encounter. Contrasting with this classical behavioural ecological point of view is the concept of animal personality. The latter focuses on understanding the mechanisms underlying and evolutionary processes maintaining variation in the expression of a behavioural trait over time and across situations or contexts. Originating in human psychology, personality studies have recently been integrated into the fields of ecology and evolution. Studies on consistent variation in behaviour within and between individuals (personality) have resulted in numerous insights and these are still expanding. In the first chapter of this thesis I research underlying factors and possible consequences of the response (delayed hatching) of blue tits (Cyanistes caeruleus) to encountered climatic variation. I find that hatching delay (i.e. number of days hatching was delayed) is associated with early laying dates and low mean temperatures during the egg-laying phase. In addition hatching delay is negatively associated with clutch hatchability and female body condition. Using a reciprocal cross-fostering protocol on a large number of broods, I find that hatching delay may also negatively affect developmental parameters in offspring, in particular body mass of nestlings at fledging. Results from this study demonstrate that environmental conditions during egg laying can have effects lasting throughout the breeding and nestling period. In chapters II to V I investigate variation in behaviour among individuals. The focus in these four chapters is on personality traits in blue tits. I first design an experimental setup, using a bird cage, in which several behavioural traits can be measured in a quick and non-invasive manner and which can be applied in both winter and breeding season. In addition several behavioural traits are measured during handling of both adult and nestling birds. All these behavioural measures are then used to test several aspects of behaviour in a personality context in the blue tit. The behavioural traits derived from the bird cage are repeatable over time and qualify as personality traits in this species. In addition I find an association between one of the measured personality traits in the cage and a single nucleotide polymorphism in the 3rd exon of the dopamine receptor (D4) gene (DRD4), similar to what has been found in recent research on great tits (Parus major). This suggests that there is a genetic basis underlying this personality trait and that this genomic region might be involved in animal personality. I apply a reaction norm framework to assess context specificity of the traits measured in the bird cage, using measures from (partly) the same birds measured in two distinct contexts (winter and breeding season). I show that one needs to carefully consider the context under which individuals are assayed and that a recorded behaviour may or may not be repeatable in another context. Furthermore I use data from a cross-foster protocol on nestling blue tits in combination with quantitative genetics. I assess the heritability of three behavioural traits and show that these traits form a behavioural syndrome at both the phenotypic and genetic level. In addition, from the applied animal model analysis I can conclude that environmental factors, encountered by nestlings during the rearing period, may have a considerable impact on a nestling s personality. Thus, taken into account findings from the first chapter in this thesis, the development of both physical and behavioural traits in an individual seems to find its origin already in the earliest phases of life. Finally I test whether three personality traits and two immunological traits in the blue tit covary and form a syndrome which includes behavioural and immunological traits. I find that there are intrinsic correlations between behavioural and immunological traits; however there is no strong evidence for the existence of a syndrome of these traits in the blue tit.
  • Ylä-Anttila, Päivi (Helsingin yliopisto, 2015)
    Eukaryotic cells contain membrane-bound organelles to carry out specialized cellular functions. These organelles are inherited in cell division as templates and are augmented by proliferation through production of protein and lipid components by the cell, and the trafficking of these components within the cell. Autophagy is an evolutionarily conserved degradation pathway for cells to maintain homeostasis, produce nutrients for energy production, degrade misfolded proteins or damaged whole organelles, and fight against intruding pathogens. The process of autophagy entails the isolation of cargo by a specialized organelle, called the phagophore, which closes to form a sealed double membrane bound autophagosome. This organelle then undergoes maturation by fusion with endosomes and lysosomes to obtain its degradation capacity. Hence, there are many dynamic membrane modifications that need to take place during the autophagic process. The origin of the autophagic limiting membrane, as well as the clearance of the degradative structures, are yet to be defined. This study utilized high resolution electron microscopic methods and three dimensional modeling to reveal nanometer scale interactions of phagophores and autophagosomes with other organelles. Immunolabeling techniques at both light and electron microscopy level were utilized to determine which organelles should be sampled at an ultrastructural level. Direct membrane communication was detected between the phagophore and endoplasmic reticulum (ER), (putative) ER exit sites, mitochondria, the Golgi complex, as well as late endosomes or lysosomes. ER was the most frequent proximal organelle to phagophores and autophagosomes and this suggests an involvement of ER in the nucleation process of phagophores. This study also reveales a role of the small GTP-binding protein RAB24 in the clearance of autophagic structures in cells. Biochemical and microscopic methods in combination showed that RAB24 is needed in the clearance of autophagic structures in nutrient rich conditions i.e. during basal autophagy. RAB24 was confirmed to localize in both of the autophagosome limiting membranes. GTP binding and prenylation of RAB24 were found to be necessary for the targeting of the protein to LC3 positive autophagic structures, whereas tyrosine phosphorylation was less important for this targeting. Electron microscopy revealed that autolysosome-like structures accumulate in cells when RAB24 is silenced, suggesting that it has a role in the clearance of autolysosomes.
  • Heikinheimo, Liisa (Helsingin yliopisto, 2002)
  • Laurinavicius, Simonas (Helsingin yliopisto, 2008)
    In this study we used electro-spray ionization mass-spectrometry to determine phospholipid class and molecular species compositions in bacteriophages PM2, PRD1, Bam35 and phi6 as well as their hosts. To obtain compositional data of the individual leaflets, phospholipid transbilayer distribution in the viral membranes was studied. We found that 1) the membranes of all studied bacteriophage are enriched in PG as compared to the host membranes, 2) molecular species compositions in the phage and host membranes are similar, and 3) phospholipids in the viral membranes are distributed asymmetrically with phosphatidylglycerol enriched in the outer leaflet and phosphatidylethanolamine in the inner one (except Bam35). Alternative models for selective incorporation of phospholipids to phages and for the origins of the asymmetric phospholipid transbilayer distribution are discussed. Notably, the present data are also useful when constructing high resolution structural models of bacteriophages, since diffraction methods cannot provide a detailed structure of the membrane due to high motility of the lipids and lack of symmetric organization of membrane proteins.
  • Söderholm, Sandra (Helsingin yliopisto, 2016)
    Phosphorylation is one of the most important post-translational modifications of proteins. Phosphorylation is a rapid and reversible way of modifying proteins; it is involved in the regulation of many cellular processes, serving as the main transducer of intracellular signaling cascades. It is possible to identify thousands of protein phosphorylation sites from a single sample with mass spectrometry (MS)-based phosphoproteomics. This is the main reason why MS-based phosphoproteomics is such an excellent method for revealing global changes in phosphoproteomes. Computational analysis of the MS data is vital for identifying the proteins and post-translational modifications. The data analysis steps in the phosphoproteomics workflow are also crucial for biological interpretation, e.g. identifying activated kinases and kinase substrates, as well as unravelling signaling pathways and networks. Cells of the innate immune system are central players in the host defence against pathogens such as viruses. Their pattern-recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs), which are conserved structures present in pathogens. The most important PAMPs in viral infections are the viral genomes and replication intermediates, and their detection evokes pro-inflammatory and antiviral responses in the infected cell. Host factors and signaling cascades that promote or inhibit virus infections can serve as potential drug targets. Since protein phosphorylation is vital for the progression of nearly all signaling cascades, there is an increasing interest in applying phosphoproteomics and combining it with bioinformatics in studying the regulation of cellular signaling under various conditions, including viral infections. The main aim of the studies included in this PhD thesis was to characterize the global changes in the cellular phosphoproteomes of virus infected and dsRNA stimulated innate immune cells, and to identify the host proteins and cell signaling pathways involved in the early stages of the host response to Sendai virus (SeV), influenza A virus (IAV), and viral dsRNA. A computational analysis tool, named PhosFox, was developed for processing and comparing MS-based phosphoproteomic data generated by multiple database search algorithms. PhosFox was used for cross-sample comparisons of phosphopeptide identifications. PhosFox also facilitated the identification of those proteins whose phosphorylation was different between samples, and the clarification of phosphorylation sites not described in the literature. The findings from the phosphoproteomic data were explored further by implementing functional studies involving small interfering RNAs (siRNAs) and kinase inhibitors. There were extensive alterations in the phosphorylation of proteins in human epithelial cells and macrophages that were transfected with the synthetic dsRNA-mimic polyinosinic:polycytidylic acid (pI:C) or infected with SeV or IAV. Many of these proteins were determined to be members of pathways with little or no previously known role in the antiviral response to these particular viruses and viral PAMPs. Two novel host factors, RelA-associated inhibitor (RAI) and sirtuin 1 (SIRT1), were identified as negative regulators of dsRNA-induced apoptosis and NF-κB regulated cytokine expression by combining 14-3-3 interactome and phosphoproteome characterizations. The p38 mitogen-activated protein kinase (MAPK) signaling pathway was found to be regulating cytokine expression and apoptosis in dsRNA-transfected human keratinocytes. MAPK signaling pathways were also regulated in SeV and IAV infected cells. The mTOR signaling pathway was shown to be critical for the interferon response and virus replication in SeV infected human lung epithelial cells. A substantial number of those cellular proteins whose phosphorylation status changed after SeV or IAV infection or viral dsRNA challenge were involved in Rho GTPase signaling. Major changes in protein phosphorylation in IAV infected primary human macrophages were linked to cyclin-dependent kinases (CDKs). CDK activity was shown to be required for efficient viral replication and host response in IAV infection. Administration of one specific CDK inhibitor, SNS-032, also protected mice from IAV-induced death. The studies included in this PhD thesis are some of the first to apply phosphoproteomics for characterizing host-virus interactions. They emphazise the benefit of applying phosphoproteomics and bioinformatics in innate immunity and viral research, i.e. it is possible not only to identify cell signaling pathways, but also the specific host factors that regulate the cellular responses to viral infections. The results of these studies underline the importance and the potential MS-based phosphoproteomics has in the discovery of novel host factors, which can serve as possible antiviral drug targets. In conclusion, the MS-based phosphoproteomics approach led to the discovery of novel host factors and cell signaling circuits in virus infected innate immune cells.
  • Jansen, Gunther (Helsingin yliopisto, 2009)
    Understanding the overwhelming diversity of life calls for complex organisational schemes. The field of systematics may thus be seen as the cornerstone of evolutionary biology. In the last few decades, systematics has been rejuvenated through the introduction of molecular methods such as DNA barcoding and multi-gene phylogenetic approaches. These methods may shed new light on established taxonomic ideas and problems. For example, the classification of ants has aroused much debate due to reinterpretation of morphological characters or contradictions between molecular data and morphology. Only in the last few years a consensus was reached regarding the phylogeny of ant subfamilies. However, the situation remains deplorable for lower taxonomic ranks such as subfamilies, tribes and genera. This thesis describes the systematics and evolution of the Holarctic ant genus Myrmica and the tribe to which it belongs, Myrmicini. Using barcoding, molecular-phylogenetic data and divergence time estimations, it addresses questions regarding the taxonomy, morphology and biogeography of this group. Furthermore, the interrelationships between socially parasitic Myrmica species and their hosts (other species in the genus) were inferred. The phylogeny suggests that social parasitism evolved several times in Myrmica. Finally, this thesis investigated whether coevolution shaped the phylogeny of socially parasitic Maculinea butterflies that live inside Myrmica colonies. No evidence was found for coevolution.
  • Vahtera, Varpu (Helsingin yliopisto, 2008)
    Throughout the history of Linnean taxonomy, species have been described with varying degrees of justification. Many descriptions have been based on only a few ambiguous morphological characters. Moreover, species have been considered natural, well-defined units whereas higher taxa have been treated as disparate, non-existent creations. In the present thesis a few such cases were studied in detail. Often the species-level descriptions were based on only a few specimens and the variation previously thought to be interspecific was found to be intraspecific. In some cases morphological characters were sufficient to resolve the evolutionary relationships between the taxa, but generally more resolution was gained by the addition of molecular evidence. However, both morphological and molecular data were found to be deceptive in some cases. The DNA sequences of morphologically similar specimens were found to differ distinctly in some cases, whereas in other closely related species the morphology of specimens with identical DNA sequences differed substantially. This study counsels caution when evolutionary relationships are being studied utilizing only one source of evidence or a very limited number of characters (e.g. barcoding). Moreover, it emphasizes the importance of high quality data as well as the utilization of proper methods when making scientific inferences. Properly conducted analyses produce robust results that can be utilized in numerous interesting ways. The present thesis considered two such extensions of systematics. A novel hypothesis on the origin of bioluminescence in Elateriformia beetles is presented, tying it to the development of the clicking mechanism in the ancestors of these animals. An entirely different type of extension of systematics is the proposed high value of the white sand forests in maintaining the diversity of beetles in the Peruvian Amazon. White sand forests are under growing pressure from human activities that lead to deforestation. They were found to harbor an extremely diverse beetle fauna and many taxa were specialists living only in this unique habitat. In comparison to the predominant clay soil forests, considerably more elateroid beetles belonging to all studied taxonomic levels (species, genus, tribus, and subfamily) were collected in white sand forests. This evolutionary diversity is hypothesized to be due to a combination of factors: (1) the forest structure, which favors the fungus-plant interactions important for the elateroid beetles, (2) the old age of the forest type favoring survival of many evolutionary lineages and (3) the widespread distribution and fragmentation of the forests in the Miocene, favoring speciation.
  • Högnabba, Filip (Helsingin yliopisto, 2007)
    Phylogenetic studies of cyanobacterial lichens Lichens are symbiotic assemblages between fungi (mycobiont) and green algae (phycobiont) or/and cyanobacteria (cyanobiont). Fossil records show that lichen-like symbioses occurred already 600 million years ago. Lichen symbiosis has since then become an important life strategy for the Fungi, particularly for species in the phylum Ascomycota as approximately 98% of the lichenized fungal species are ascomycetes. The taxonomy of lichen associations is based on the mycobiont. We reconstructed, using DNA sequence data, hypotheses of phylogenetic relationships of lichen-forming fungi that include species associated with cyanobacteria. These hypotheses of phylogeny should form the basis for the taxonomy. They also allowed studies of the origin and the evolution of specific symbioses. Genetic diversity and phylogenetic relationships of symbiotic cyanobionts were also studied in order to examine selectivity of cyanobionts and mycobionts as well as possible co-evolution between partners involved in lichen associations. The suggested circumscription of the family Stereocaulaceae to include Stereocaulon and Lepraria is supported. The recently described crustose Stereocaulon species seem to be correctly placed in the genus, although Stereocaulon traditionally included only fruticose species. The monospecific crustose genus Muhria is also shown to be best placed in Stereocaulon. Family Lobariaceae as currently delimited is monophyletic. Within Lobariaceae genus Sticta including Dendriscocaulon dendroides form a monophyletic group while the genera Lobaria and Pseudocyphellaria are non-monophyletic. A new classification of Lobariaceae is obviously needed. Further studies are however required before a final proposal for a new classification can be made. Our results show that the cyanobacterial symbiotic state has been gained repeatedly in the Ascomycota while losses of symbiotic cyanobacteria appear to be rare. The symbiosis with green algae is confirmed to have been gained repeatedly in Ascomycota but also repeatedly lost. Cyanobacterial symbioses therefore seem to be more stable than green algal associations. Cyanobacteria are perhaps more beneficial for the lichen fungi and therefore maintained. The results indicate a dynamic association of the lichen symbiosis. This evolutionary instability will perhaps be important for the lichen fungi as the utilization of options will perhaps enable lichens to colonize new substrates and survive environmental changes. Some cyanobacterial lichen genera seem to be highly selective towards the cyanobiont while others form symbioses with a broad spectrum of cyanobacteria. No evidence of co-evolution between fungi and cyanobacteria in cyanolichens could be demonstrated.