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  • 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.
  • Mäkeläinen, Sanna (Helsingin yliopisto, 2016)
    Anthropogenic land use has caused detrimental impacts especially on forest ecosystems, and native forested landscapes have been lost and fragmented worldwide. Forest-dwelling animals are generally susceptible to habitat loss and fragmentation because of their strict habitat requirements and dependency on forests for food, nest sites and movements. Consequently, forest specialists, such as arboreal squirrels and gliding mammals, provide a worthy group of model species in order to assess the influence of landscape modification. The Siberian flying squirrel (Pteromys volans) is an arboreal rodent inhabiting spruce-dominated mature forests and due to destruction of its most suitable breeding habitat, the population has been decreasing in Finland. In my thesis, I investigate the effects of landscape modification on the occurrence, space use and survival of this species at multiple spatial scales. The occurrence probability of flying squirrel increased with a proportion of suitable movement forests on a 400-m-scale and was negatively associated with the isolation of occupied sites in a partly urban study area. However, no negative effects of urban habitat types were found, which indicates that the species is not disturbed by urbanization. It was found that regional environmental authorities were unaware of the presence of flying squirrel in most cases of forest harvesting in southern Finland. Despite occupied sites were delineated according to given guidelines, many of these sites became deserted after forest harvesting. This shows that the legal habitat protection of flying squirrel is ineffective and to improve this conservation practice, larger forested areas should be maintained around inhabited sites. The presence of urban habitats on movement routes increased movement distance and speed whereas urban habitats within home-range impeded only male movements. Structural forested connections had varying effects on nest-site switching and their importance remained unclear, which points out that defining and maintaining a species-specific connectivity is challenging in human-modified landscapes. Regional variation in flying squirrel survival was most likely caused by differences in predation pressure, predator community and landscape composition. Male mortality increased with a proportion of low-quality habitats in the surrounding landscape, whereas fine-scale habitat composition of the most used areas did not show any survival impacts. Natal dispersal distances of juveniles did not increase their mortality. Results of this thesis indicate that the effects of landscape modification on forest-dwelling animals are varying, sex-specific and depend on the scale. As landscape modification can also affect species indirectly, it is important to investigate the costs and risks of animal movements in human-modified environments.
  • Noreika, Norbertas (Helsingin yliopisto, 2016)
    Negative anthropogenic disturbances (e.g., drainage and urbanization) are causing biotic homogenization through the replacement of specialist species with generalists. The identification and conservation of biodiversity hotspots within degraded (e.g., highly urbanized) landscapes, and ecological restoration (i.e., positive anthropogenic disturbance) have the potential to be important tools to counteract these negative effects. Mires are suitable targets for the investigation of these homogenization-reducing activities since they host many mire specialist species of, e.g. invertebrates. The main aim of this PhD thesis was to investigate the effects of negative anthropogenic disturbances [urbanization (Chapter I) and drainage for forestry (Chapters II-IV)] on the invertebrate communities of boreal mires and how effective efforts are to reverse these negative effects through ecological restoration [i.e. positive anthropogenic disturbance (Chapters II-IV)]. In addition, the purpose was to determine which environmental variables are key in supporting mire specialist invertebrate species and communities. Therefore, this thesis started by reviewing current knowledge on the responses of mire invertebrate species and communities to anthropogenic disturbances. The effects of urbanization were studied on spiders and carabid beetles (Chapter I), while the effects of drainage for forestry and subsequent restoration were investigated on five solitary invertebrate groups (Chapter II) and social insects, i.e. ants (Chapter III). Finally, a powerful Before-After Control-Impact (BACI) design was used to reveal the effects of drainage and restoration on butterflies (Chapter IV). Generally, both high levels of urbanization (Chapter I) and mire drainage for forestry (Chapters II-IV) had negative effects on mire specialist species (lower abundances) and invertebrate communities (homogenized and very different in structure from pristine mire communities). However, these detrimental effects can be reduced or even reversed through appropriate urban mire conservation and ecological restoration. Local habitat conditions were shown to be particularly important for the survival of specialist invertebrate species in urban mires (Chapter I) and for the successful recovery of restored mire invertebrates (Chapters II-IV). Individual mire specialist species responded negatively to environmental variables associated with deteriorated (i.e. drained or highly urbanized) mire conditions [number of high (> 3m) trees for carabid beetles, crane flies, micromoths (Chapter II), ants (Chapter III) and butterflies (Chapter IV)] and positively to pristine mire-associated variables [Sphagnum cover for carabid beetles and spiders (Chapters I-II), crane flies (Chapter II) and suggestively for ants (Chapter III); larval food plant cover and number of lower (1.5 - 3m) trees for butterflies (Chapter IV)]. The more specialized the mire species were, the more negatively they were affected by deteriorated-mire-associated variables and the more positively they responded to pristine-mire-associated variables. I conclude that the restoration actions taken (removing tall trees but leaving smaller trees, and raising the water table level) are appropriate in creating suitable habitat conditions for mire invertebrates, as both individual specialist species and communities showed positive responses already 1-3 years since restoration (Chapters II-IV). Finally, the appropriate restoration actions in well-prioritized locations as well as urban mire conservation should reverse the trend of biotic homogenization.
  • 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.
  • 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.