Lost in Translation : Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins

The present study focuses on the translational strategies of Cocksfoot mottle virus (CfMV, genus Sobemovirus), which infects monocotyledonous plants. CfMV RNA lacks the 5'cap and the 3'poly(A) tail that ensure efficient translation of cellular messenger RNAs (mRNAs). Instead, CfMV RNA is covalently linked to a viral protein VPg (viral protein, genome-linked). This indicates that the viral untranslated regions (UTRs) must functionally compensate for the lack of the cap and poly(A) tail. We examined the efficacy of translation initiation in CfMV by comparing it to well-studied viral translational enhancers. Although insertion of the CfMV 5'UTR (CfMVe) into plant expression vectors improved gene expression in barley more than the other translational enhancers examined, studies at the RNA level showed that CfMVe alone or in combination with the CfMV 3'UTR did not provide the RNAs translational advantage. Mutation analysis revealed that translation initiation from CfMVe involved scanning. Interestingly, CfMVe also promoted translation initiation from an intercistronic position of dicistronic mRNAs in vitro. Furthermore, internal initiation occurred with similar efficacy in translation lysates that had reduced concentrations of eukaryotic initiation factor (eIF) 4E, suggesting that initiation was independent of the eIF4E. In contrast, reduced translation in the eIF4G-depleted lysates indicated that translation from internally positioned CfMVe was eIF4G-dependent. After successful translation initiation, leaky scanning brings the ribosomes to the second open reading frame (ORF). The CfMV polyprotein is produced from this and the following overlapping ORF via programmed -1 ribosomal frameshift (-1 PRF). Two signals in the mRNA at the beginning of the overlap program approximately every fifth ribosome to slip one nucleotide backwards and continue translation in the new -1 frame. This leads to the production of C-terminally extended polyprotein, which encodes the viral RNA-dependent RNA polymerase (RdRp). The -1 PRF event in CfMV was very efficient, even though it was programmed by a simple stem-loop structure instead of a pseudoknot, which is usually required for high -1 PRF frequencies. Interestingly, regions surrounding the -1 PRF signals improved the -1 PRF frequencies. Viral protein P27 inhibited the -1 PRF event in vivo, putatively by binding to the -1 PRF site. This suggested that P27 could regulate the occurrence of -1 PRF. Initiation of viral replication requires that viral proteins are released from the polyprotein. This is catalyzed by viral serine protease, which is also encoded from the polyprotein. N-terminal amino acid sequencing of CfMV VPg revealed that the junction of the protease and VPg was cleaved between glutamate (E) and asparagine (N) residues. This suggested that the processing sites used in CfMV differ from the glutamate and serine (S) or threonine (T) sites utilized in other sobemoviruses. However, further analysis revealed that the E/S and E/T sites may be used to cleave out some of the CfMV proteins.

Stiffer Optical Tweezers through Real-Time Feedback Control

This thesis integrates real-time feedback control into an optical tweezers instrument. The goal is to reduce the variance in the trapped bead s position, -effectively increasing the trap stiffness of the optical tweezers. Trap steering is done with acousto-optic deflectors and control algorithms are implemented with a field-programmable gate array card. When position clamp feedback control is on, the effective trap stiffness increases 12.1-times compared to the stiffness without control. This allows improved spatial control over trapped particles without increasing the trapping laser power.

Topographical, structural and geophysical characterization of fracture zones: implications for groundwater flow and vulnerability

The main objective of this study is to evaluate selected geophysical, structural and topographic methods on regional, local, and tunnel and borehole scales, as indicators of the properties of fracture zones or fractures relevant to groundwater flow. Such information serves, for example, groundwater exploration and prediction of the risk of groundwater inflow in underground construction. This study aims to address how the features detected by these methods link to groundwater flow in qualitative and semi-quantitative terms and how well the methods reveal properties of fracturing affecting groundwater flow in the studied sites. The investigated areas are: (1) the Päijänne Tunnel for water-conveyance whose study serves as a verification of structures identified on regional and local scales; (2) the Oitti fuel spill site, to telescope across scales and compare geometries of structural assessment; and (3) Leppävirta, where fracturing and hydrogeological environment have been studied on the scale of a drilled well. The methods applied in this study include: the interpretation of lineaments from topographic data and their comparison with aeromagnetic data; the analysis of geological structures mapped in the Päijänne Tunnel; borehole video surveying; groundwater inflow measurements; groundwater level observations; and information on the tunnel s deterioration as demonstrated by block falls. The study combined geological and geotechnical information on relevant factors governing groundwater inflow into a tunnel and indicators of fracturing, as well as environmental datasets as overlays for spatial analysis using GIS. Geophysical borehole logging and fluid logging were used in Leppävirta to compare the responses of different methods to fracturing and other geological features on the scale of a drilled well. Results from some of the geophysical measurements of boreholes were affected by the large diameter (gamma radiation) or uneven surface (caliper) of these structures. However, different anomalies indicating more fractured upper part of the bedrock traversed by well HN4 in Leppävirta suggest that several methods can be used for detecting fracturing. Fracture trends appear to align similarly on different scales in the zone of the Päijänne Tunnel. For example, similarities of patterns were found between the regional magnetic trends, correlating with orientations of topographic lineaments interpreted as expressions of fracture zones. The same structural orientations as those of the larger structures on local or regional scales were observed in the tunnel, even though a match could not be made in every case. The size and orientation of the observation space (patch of terrain at the surface, tunnel section, or borehole), the characterization method, with its typical sensitivity, and the characteristics of the location, influence the identification of the fracture pattern. Through due consideration of the influence of the sampling geometry and by utilizing complementary fracture characterization methods in tandem, some of the complexities of the relationship between fracturing and groundwater flow can be addressed. The flow connections demonstrated by the response of the groundwater level in monitoring wells to pressure decrease in the tunnel and the transport of MTBE through fractures in bedrock in Oitti, highlight the importance of protecting the tunnel water from a risk of contamination. In general, the largest values of drawdown occurred in monitoring wells closest to the tunnel and/or close to the topographically interpreted fracture zones. It seems that, to some degree, the rate of inflow shows a positive correlation with the level of reinforcement, as both are connected with the fracturing in the bedrock. The following geological features increased the vulnerability of tunnel sections to pollution, especially when several factors affected the same locations: (1) fractured bedrock, particularly with associated groundwater inflow; (2) thin or permeable overburden above fractured rock; (3) a hydraulically conductive layer underneath the surface soil; and (4) a relatively thin bedrock roof above the tunnel. The observed anisotropy of the geological media should ideally be taken into account in the assessment of vulnerability of tunnel sections and eventually for directing protective measures.

Geospatial environmental data modelling applications using remote sensing, GIS and spatial statistics

This thesis presents novel modelling applications for environmental geospatial data using remote sensing, GIS and statistical modelling techniques. The studied themes can be classified into four main themes: (i) to develop advanced geospatial databases. Paper (I) demonstrates the creation of a geospatial database for the Glanville fritillary butterfly (Melitaea cinxia) in the Åland Islands, south-western Finland; (ii) to analyse species diversity and distribution using GIS techniques. Paper (II) presents a diversity and geographical distribution analysis for Scopulini moths at a world-wide scale; (iii) to study spatiotemporal forest cover change. Paper (III) presents a study of exotic and indigenous tree cover change detection in Taita Hills Kenya using airborne imagery and GIS analysis techniques; (iv) to explore predictive modelling techniques using geospatial data. In Paper (IV) human population occurrence and abundance in the Taita Hills highlands was predicted using the generalized additive modelling (GAM) technique. Paper (V) presents techniques to enhance fire prediction and burned area estimation at a regional scale in East Caprivi Namibia. Paper (VI) compares eight state-of-the-art predictive modelling methods to improve fire prediction, burned area estimation and fire risk mapping in East Caprivi Namibia. The results in Paper (I) showed that geospatial data can be managed effectively using advanced relational database management systems. Metapopulation data for Melitaea cinxia butterfly was successfully combined with GPS-delimited habitat patch information and climatic data. Using the geospatial database, spatial analyses were successfully conducted at habitat patch level or at more coarse analysis scales. Moreover, this study showed it appears evident that at a large-scale spatially correlated weather conditions are one of the primary causes of spatially correlated changes in Melitaea cinxia population sizes. In Paper (II) spatiotemporal characteristics of Socupulini moths description, diversity and distribution were analysed at a world-wide scale and for the first time GIS techniques were used for Scopulini moth geographical distribution analysis. This study revealed that Scopulini moths have a cosmopolitan distribution. The majority of the species have been described from the low latitudes, sub-Saharan Africa being the hot spot of species diversity. However, the taxonomical effort has been uneven among biogeographical regions. Paper III showed that forest cover change can be analysed in great detail using modern airborne imagery techniques and historical aerial photographs. However, when spatiotemporal forest cover change is studied care has to be taken in co-registration and image interpretation when historical black and white aerial photography is used. In Paper (IV) human population distribution and abundance could be modelled with fairly good results using geospatial predictors and non-Gaussian predictive modelling techniques. Moreover, land cover layer is not necessary needed as a predictor because first and second-order image texture measurements derived from satellite imagery had more power to explain the variation in dwelling unit occurrence and abundance. Paper V showed that generalized linear model (GLM) is a suitable technique for fire occurrence prediction and for burned area estimation. GLM based burned area estimations were found to be more superior than the existing MODIS burned area product (MCD45A1). However, spatial autocorrelation of fires has to be taken into account when using the GLM technique for fire occurrence prediction. Paper VI showed that novel statistical predictive modelling techniques can be used to improve fire prediction, burned area estimation and fire risk mapping at a regional scale. However, some noticeable variation between different predictive modelling techniques for fire occurrence prediction and burned area estimation existed.

Individuals on the Move : Body Condition Dependent Dispersal and Quasi-Local Competition in Metapopulations

Individual movement is very versatile and inevitable in ecology. In this thesis, I investigate two kinds of movement body condition dependent dispersal and small-range foraging movements resulting in quasi-local competition and their causes and consequences on the individual, population and metapopulation level. Body condition dependent dispersal is a widely evident but barely understood phenomenon. In nature, diverse relationships between body condition and dispersal are observed. I develop the first models that study the evolution of dispersal strategies that depend on individual body condition. In a patchy environment where patches differ in environmental conditions, individuals born in rich (e.g. nutritious) patches are on average stronger than their conspecifics that are born in poorer patches. Body condition (strength) determines competitive ability such that stronger individuals win competition with higher probability than weak individuals. Individuals compete for patches such that kin competition selects for dispersal. I determine the evolutionarily stable strategy (ESS) for different ecological scenarios. My models offer explanations for both dispersal of strong individuals and dispersal of weak individuals. Moreover, I find that within-family dispersal behaviour is not always reflected on the population level. This supports the fact that no consistent pattern is detected in data on body condition dependent dispersal. It also encourages the refining of empirical investigations. Quasi-local competition defines interactions between adjacent populations where one population negatively affects the growth of the other population. I model a metapopulation in a homogeneous environment where adults of different subpopulations compete for resources by spending part of their foraging time in the neighbouring patches, while their juveniles only feed on the resource in their natal patch. I show that spatial patterns (different population densities in the patches) are stable only if one age class depletes the resource very much but mainly the other age group depends on it.

Kohtaamisia Santiagon tiellä pyhiinvaellus matkana ja spirituaalisena kokemuksena

Pyhiinvaellustutkimuksen tavoitteena on kuvata, analysoida ja ymmärtää pyhiinvaeltajien kokemuksia ja erityisesti spirituaalisia tai sellaisiksi tulkittuja kokemuksia. Tutkimuskysymys voidaan muotoilla myös kysymällä, missä on vaeltajien mahdollisesti kokema pyhiinvaelluksen pyhä . Pyhiinvaellus suuntautuu Santiago de Compostelan kaupunkiin. Kuvattu pyhiinvaellusreitti alkaa Ranskan ja Espanjan rajalta ja kulkee Espanjan luoteiskulmassa noin 800 kilometrin päässä olevaan määränpäähän. Tutkimuksen aineistona on sähköpostikyselyn vastaukset, jotka on saatu kahdeltakymmeneltäkahdelta keväällä 2006 Santiagoon jalkaisin vaeltaneilta pyhiinvaeltajilta. Tutkimusaineistona ovat myös tutkijan omat havainnot ko. pyhiinvaellukselta. Tutkijan havainnoista on kirjoitettu kuvaus Santiagon tien pyhiinvaelluksesta ja matkan arkisista käytännöistä. Sähköpostikyselyn vastaukset, vaeltajien spirituaaliset kokemukset, on analysoitu laadullista sisällön analysointia käyttäen. Kuudesta vastauksesta on kirjoitettu myös pyhiinvaeltajan henkilökohtainen tarina. Narratiivisuus, kerronnallisuus, on tässä tutkimuksessa yksi tapa esittää, kuvata ja ymmärtää tutkimustuloksia. Tutkimus on liitetty fenomenologiseen viitekehykseen. Spirituaalisia kokemuksia on lähestytty mm. pyhiinvaellustradition, pyhän käsitteen ja uskonnollisten kokemusten näkökulmasta. Spiritualiteetin käsitettä ja spirituaalisen kokemuksen kuvaamisen mahdollisuuksia on myös pohdittu tutkimuksessa. Pyhiinvaelluksen spirituaaliset kokemukset on tutkimusraportissa jaoteltu seitsemään ryhmään: kävely, ihmiset, vapaus, luonto, rukous, kirkot ja Santiago. Lisäksi on esitetty matkaan lähdön motiiveja sekä pyhiinvaelluksen vaikutuksia elämässä. Käveleminen oli pyhiinvaelluksen ydinkokemus. Käveleminen mahdollisti meditaation tai rukoilemisen. Se aiheutti myös kärsimystä ja oli keskeinen ihmisiä yhdistävä teema. Pyhiinvaellus irrotti ihmiset erittäin hyvin arjen rutiineista ja jokapäiväisestä normaalista elämästä. Pyhiinvaelluksella vapauden tunne olikin merkittävä spirituaalinen kokemus ja ajan käsitys saattoi saa uuden ulottuvuuden. Luonto näyttäytyi merkittävänä spirituaalisen innoituksen lähteenä ja usein myös yksinolon paikkana. Kaikki pyhiinvaeltajat vierailivat kirkoissa ja katedraaleissa ja useimmat myös messuissa ja pyhiinvaeltajille järjestetyissä rukoushetkissä. Pyhiinvaelluksen uskonnollinen ulottuvuus oli kaikille läsnä myös Santiagon messussa. Kristillinen tulkintakehys kokemuksille oli koko ajan pyhiinvaeltajalle tarjolla. Kuitenkaan Santiagon vaellukseen ei välttämättä liittynyt mitään omaa spirituaalista kokemusta. Pyhiinvaelluksen merkittävät spirituaaliset kokemukset liittyivät yhteisöllisyyteen ja jaettuihin kokemuksiin. Tyypillinen moderni pyhiinvaeltaja etsi itseään pyhiinvaelluksella. Yllätyksekseen hän saattoi löytää jotain silloin kun hän antautui avoimeen vuorovaikutukseen toisten ihmisten kanssa. Lähes kaikki tutkimuksen osallistuneet pyhiinvaeltajat pitivät hyvin tärkeänä toisia pyhiinvaeltajia ja sitä, mitä he saivat toisilta ihmisiltä. Pyhiinvaellusta leimaa poikkeuksellisen avoin vuorovaikutus ja elämän jakaminen. Pyhiinvaellukseen liittyy monia individualistisia piirteitä. Spirituaalisten kokemusten näkökulmasta individualismi ei kuitenkaan saa tukea tutkimuksesta, koska yhteisöllisyys näyttää niin merkittävältä spirituaalisten kokemusten kannalta. Pyhiinvaelluksen yhteisöllisyys on avointa, vapaaehtoista ja usein myös hetkellistä. Moderni pyhiinvaeltaja näyttää uskovan enemmän omiin kokemuksiinsa ja myös toisten kokemuksiin kuin minkään kirkon opilliseen auktoriteettiin.

Endoplasmic reticulum stress and cell death mechanisms in the cell model of Huntington’s disease

This thesis clarifies important molecular pathways that are activated during the cell death observed in Huntington’s disease. Huntington’s disease is one of the most common inherited neurodegenerative diseases, which is primarily inherited in an autosomal dominant manner. HD is caused by an expansion of CAG repeats in the first exon of the IT15 gene. IT15 encodes the production of a Huntington’s disease protein huntingtin. Mutation of the IT15 gene results in a long stretch of polyQ residues close to the amino-terminal region of huntingtin. Huntington’s disease is a fatal autosomal neurodegenerative disorder. Despite the current knowledge of HD, the precise mechanism behind the selective neuronal death, and how the disease propagates, still remains an enigma. The studies mainly focused on the control of endoplasmic reticulum (ER) stress triggered by the mutant huntingtin proteins. The ER is a delicate organelle having essential roles in protein folding and calcium regulation. Even the slightest perturbations on ER homeostasis are effective enough to trigger ER stress and its adaptation pathways, called unfolded protein response (UPR). UPR is essential for cellular homeostasis and it adapts ER to the changing environment and decreases ER stress. If adaptation processes fail and stress is excessive and prolonged; irreversible cell death pathways are engaged. The results showed that inhibition of ER stress with chemical agents are able to decrease cell death and formation of toxic cell aggregates caused by mutant huntingtin proteins. The study concentrated also to the NF-κB (nuclear factor-kappaB) pathway, which is activated during ER stress. NF-κB pathway is capable to regulate the levels of important cellular antioxidants. Cellular antioxidants provide a first line of defence against excess reactive oxygen species. Excess accumulation of reactive oxygen species and subsequent activation of oxidative stress damages motley of vital cellular processes and induce cell degeneration. Data showed that mutant huntingtin proteins downregulate the expression levels of NF-κB and vital antioxidants, which was followed by increased oxidative stress and cell death. Treatment with antioxidants and inhibition of oxidative stress were able to counteract these adverse effects. In addition, thesis connects ER stress caused by mutant huntingtin to the cytoprotective autophagy. Autophagy sustains cellular balance by degrading potentially toxic cell proteins and components observed in Huntington’s disease. The results revealed that cytoprotective autophagy is active at the early points (24h) of ER stress after expression of mutant huntingtin proteins. GADD34 (growth arrest and DNA damage-inducible gene 34), which is previously connected to the regulation of translation during cell stress, was shown to control the stimulation of autophagy. However, GADD34 and autophagy were downregulated at later time points (48h) during mutant huntingtin proteins induced ER stress, and subsequently cell survival decreased. Overexpression GADD34 enhanced autophagy and decreased cell death, indicating that GADD34 plays a critical role in cell protection. The thesis reveales new interesting data about the neuronal cell death pathways seen in Huntington’s disease, and how cell degeneration is partly counteracted by various therapeutic agents. Expression of mutant huntingtin proteins is shown to alter signaling events that control ER stress, oxidative stress and autophagy. Despite that Huntington’s disease is mainly an untreatable disorder; these findings offer potential targets and neuroprotective strategies in designing novel therapies for Huntington’s disease.

Short-term effects of variable retention on epigaeic spiders and carabid beetles in Finland

Forestry has influenced forest dwelling organisms for centuries in Fennoscandia. For example, in Finland ca. 30% of the threatened species are threatened because of forestry. Nowadays forest management recommendations include practices aimed at maintaining biodiversity in harvesting, such as green-tree retention. However, the effects of these practices have been little studied. In variable retention, different numbers of trees are retained, varying from green-tree retention (at least a few live standing trees in clear-cuts) to thinning (only individual trees removed). I examined the responses of ground-dwelling spiders and carabid beetles to green-tree retention (with small and large tree groups), gap felling and thinning aimed at an uneven age structure of trees. The impacts of these harvesting methods were compared to those of clear-cutting and uncut controls. I aimed to test the hypothesis that retaining more trees positively affects populations of those species of spiders and carabids that were present before harvesting. The data come from two studies. First, spiders were collected with pitfall traps in south-central Finland in 1995 (pre-treatment) and 1998 (after-treatment) in order to examine the effects of clear-cutting, green-tree retention (with 0.01-0.02-ha sized tree groups), gap felling (with three 0.16-ha sized openings in a 1-ha stand), thinning aiming at an uneven age structure of trees and uncut control. Second, spiders and carabids were caught with pitfall traps in eastern Finland in 1998-2001 (pre-treatment and three post-treatment years) in eleven 0.09-0.55-ha sized retention-tree groups and clear-cuts adjacent to them. Original spider and carabid assemblages were better maintained after harvests that retained more trees. Thinning maintained forest spiders well. However, gap felling and large retention-tree groups maintained some forest spider and carabid species in the short-term, but negatively affected some species over time. However, use of small retention-tree groups was associated with negative effects on forest spider populations. Studies are needed on the long-term effects of variable retention on terrestrial invertebrates; especially those directed at defining appropriate retention patch size and on the importance of structural diversity provided by variable retention for invertebrate populations. However, the aims of variable retention should be specified first. For example, are retention-tree groups planned to constitute life-boats , stepping-stones or to create structural diversity? Does it suffice that some species are maintained, or do we want to preserve the most sensitive ones, and how are these best defined? Moreover, the ecological benefits and economic costs of modified logging methods should be compared to other approaches aimed at maintaining biodiversity.

Effects of environmental variation on ecological and evolutionary dynamics

Environmental variation is a fact of life for all the species on earth: for any population of any particular species, the local environmental conditions are liable to vary in both time and space. In today's world, anthropogenic activity is causing habitat loss and fragmentation for many species, which may profoundly alter the characteristics of environmental variation in remaining habitat. Previous research indicates that, as habitat is lost, the spatial configuration of remaining habitat will increasingly affect the dynamics by which populations are governed. Through the use of mathematical models, this thesis asks how environmental variation interacts with species properties to influence population dynamics, local adaptation, and dispersal evolution. More specifically, we couple continuous-time continuous-space stochastic population dynamic models to landscape models. We manipulate environmental variation via parameters such as mean patch size, patch density, and patch longevity. Among other findings, we show that a mixture of high and low quality habitat is commonly better for a population than uniformly mediocre habitat. This conclusion is justified by purely ecological arguments, yet the positive effects of landscape heterogeneity may be enhanced further by local adaptation, and by the evolution of short-ranged dispersal. The predicted evolutionary responses to environmental variation are complex, however, since they involve numerous conflicting factors. We discuss why the species that have high levels of local adaptation within their ranges may not be the same species that benefit from local adaptation during range expansion. We show how habitat loss can lead to either increased or decreased selection for dispersal depending on the type of habitat and the manner in which it is lost. To study the models, we develop a recent analytical method, Perturbation expansion, to enable the incorporation of environmental variation. Within this context, we use two methods to address evolutionary dynamics: Adaptive dynamics, which assumes mutations occur infrequently so that the ecological and evolutionary timescales can be separated, and via Genotype distributions, which assume mutations are more frequent. The two approaches generally lead to similar predictions yet, exceptionally, we show how the evolutionary response of dispersal behaviour to habitat turnover may qualitatively depend on the mutation rate.

The community of medium-sized carnivores: the interactions between species, habitats and rabies

The composition of the carnivore community influences the different forms of inter-specific interactions. Furthermore, inter-specific interactions of carnivores have important implications for intra-guild competition, epidemiology and strategies of species-specific population management. Zoonooses, such as rabies, are diseases that can be transmitted from wildlife to people. Knowing the ecological characteristics of the species helps us to choose the right preventive actions and to time them accurately. In this thesis, I have studied how raccoon dogs Nyctereutes procyonoides, European badgers Meles meles, red foxes Vulpes vulpes and domestic cats Felis silvestris catus act as members of carnivore community, and how these interactions relate to the transmission risk of rabies. In the study area, these species form a community of medium-sized and rather generalist predators. They live in the same areas, in spatially and temporally overlapping home ranges and use the same habitats and dens and even have similar diets. However, there is no direct evidence of competition. Shared dens point to good tolerance of other species. Numerous observations of animals moving in each other’s proximity give similar clues. However, overlapping home ranges and similar habitat preferences lead to frequent inter-specific contacts, which increase the risk of possible rabies transmission. Also, the new insight of habitat use gained by this study illustrates the similar favouring of deciduous forests and fields by these sympatric medium-sized carnivores, creating a basis for contact zones, i.e. risky habitats for rabies transmission and spread. This study is so far the only simultaneous radio tracking study of raccoon dogs, badgers, foxes and cats. These results give new insight of the interactions in the carnivore community, as well as of the behaviour of each individual species. Also, these results have significant implications for the planning of rabies control. In order to reach viable management decisions, not only one or two species should be taken into consideration, but the whole community. In particular, this changes the perspective to inter-specific contacts, animal densities, densities of individuals susceptible to diseases and the magnitude of preventive actions. Rabies should be considered as a multi-vector disease, at least in Finland and the Baltic states. It is of interest for disease management to be able to model an epizootic with local parameters to reflect the real situation and also to suite best the local management needs.

The role of productivity in the ecological and evolutionary dynamics of predator-prey interaction

Productivity is predicted to drive the ecological and evolutionary dynamics of predator-prey interaction through changes in resource allocation between different traits. However, resources are seldom constantly available and thus temporal variation in productivity could have considerable effect on the species' potential to evolve. To study this, three long-term microbial laboratory experiments were established where Serratia marcescens prey bacteria was exposed to predation of protist Tetrahymena thermophila in different prey resource environments. The consequences of prey resource availability for the ecological properties of the predator-prey system, such as trophic dynamics, stability, and virulence, were determined. The evolutionary changes in species traits and prey genetic diversity were measured. The prey defence evolved stronger in high productivity environment. Increased allocation to defence incurred cost in terms of reduced prey resource use ability, which probably constrained prey evolution by increasing the effect of resource competition. However, the magnitude of this trade-off diminished when measured in high resource concentrations. Predation selected for white, non-pigmented, highly defensive prey clones that produced predation resistant biofilm. The biofilm defence was also potentially accompanied with cytotoxicity for predators and could have been traded off with high motility. Evidence for the evolution of predators was also found in one experiment suggesting that co-evolutionary dynamics could affect the evolution and ecology of predator-prey interaction. Temporal variation in resource availability increased variation in predator densities leading to temporally fluctuating selection for prey defences and resource use ability. Temporal variation in resource availability was also able to constrain prey evolution when the allocation to defence incurred high cost. However, when the magnitude of prey trade-off was small and the resource turnover was periodically high, temporal variation facilitated the formation of predator resistant biofilm. The evolution of prey defence constrained the transfer of energy from basal to higher trophic levels, decreasing the strength of top-down regulation on prey community. Predation and temporal variation in productivity decreased the stability of populations and prey traits in general. However, predation-induced destabilization was less pronounced in the high productivity environment where the evolution of prey defence was stronger. In addition, evolution of prey defence weakened the environmental variation induced destabilization of predator population dynamics. Moreover, protozoan predation decreased the S. marcescens virulence in the insect host moth (Parasemia plantaginis) suggesting that species interactions outside the context of host-pathogen relationship could be important indirect drivers for the evolution of pathogenesis. This thesis demonstrates that rapid evolution can affect various ecological properties of predator-prey interaction. The effect of evolution on the ecological dynamics depended on the productivity of the environment, being most evident in the constant environments with high productivity.

Environmental effects of thermal and radioactive discharges from nuclear power plants in the boreal brackish-water conditions of the northern Baltic Sea

During recent decades, thermal and radioactive discharges from nuclear power plants into the aquatic environment have become the subject of lively debate as an ecological concern. The target of this thesis was to summarize the large quantity of results obtained in extensive monitoring programmes and studies carried out in recipient sea areas off the Finnish nuclear power plants at Loviisa and Olkiluoto during more than four decades. The Loviisa NPP is located on the coast of the Gulf of Finland and Olkiluoto NPP on that of the Bothnian Sea. The state of the Gulf of Finland is clearly more eutrophic; the nutrient concentrations in the surface water are about 1½ 2 times higher at Loviisa than at Olkiluoto, and the total phosphorus concentrations still increased in both areas (even doubled at Loviisa) between the early 1970s and 2000. Thus, it is a challenge to distinguish the local effects of thermal discharges from the general eutrophication process of the Gulf of Finland. The salinity is generally low in the brackish-water conditions of the northern Baltic Sea, being however about 1 higher at Olkiluoto than at Loviisa (the salinity of surface water varying at the latter from near to 0 in early spring to 4 6 in late autumn). Thus, many marine and fresh-water organisms live in the Loviisa area close to their limit of existence, which makes the biota sensitive to any additional stress. The characteristics of the discharge areas of the two sites differ from each other in many respects: the discharge area at Loviisa is a semi-enclosed bay in the inner archipelago, where the exchange of water is limited, while the discharge area at Olkiluoto is more open, and the exchange of water with the open Bothnian Sea is more effective. The effects of the cooling water discharged from the power plants on the temperatures in the sea were most obvious in winter. The formation of a permanent ice cover in the discharge areas has been delayed in early winter, and the break-up of the ice occurs earlier in spring. The prolonging of the growing season and the disturbance of the overwintering time, in conditions where the biota has adjusted to a distinct rest period in winter, have been the most significant biological effects of the thermal pollution. The soft-bottom macrofauna at Loviisa has deteriorated to the point of almost total extinction at many sampling stations during the past 40 years. A similar decline has been reported for the whole eastern Gulf of Finland. However, the local eutrophication process seems to have contributed into the decline of the zoobenthos in the discharge area at Loviisa. Thermal discharges have increased the production of organic matter, which again has led to more organic bottom deposits. These have in turn increased the tendency of the isolated deeps to a depletion of oxygen, and this has further caused strong remobilization of phosphorus from the bottom sediments. Phytoplankton primary production and primary production capacity doubled in the whole area between the late 1960s and the late 1990s, but started to decrease a little at the beginning of this century. The focus of the production shifted from spring to mid- and late summer. The general rise in the level of primary production was mainly due to the increase in nutrient concentrations over the whole Gulf of Finland, but the thermal discharge contributed to a stronger increase of production in the discharge area compared to that in the intake area. The eutrophication of littoral vegetation in the discharge area has been the most obvious, unambiguous and significant biological effect of the heated water. Myriophyllum spicatum, Potamogeton perfoliatus and Potamogeton pectinatus, and vigorous growths of numerous filamentous algae as their epiphytes have strongly increased in the vicinity of the cooling water outlet, where they have formed dense populations in the littoral zone in late summer. However, the strongest increase of phytobenthos has extended only to a distance of about 1 km from the outlet, i.e., the changes in vegetation have been largest in those areas that remain ice-free in winter. Similar trends were also discernible at Olkiluoto, but to a clearly smaller extent, which was due to the definitely weaker level of background eutrophy and nutrient concentrations in the Bothnian Sea, and the differing local hydrographical and biological factors prevailing in the Olkiluoto area. The level of primary production has also increased at Olkiluoto, but has remained at a clearly lower level than at Loviisa. In spite of the analogous changes observed in the macrozoobenthos, the benthic fauna has remained strong and diversified in the Olkiluoto area. Small amounts of local discharge nuclides were regularly detected in environmental samples taken from the discharge areas: tritium in seawater samples, and activation products, such as 60Co, 58Co, 54Mn, 110mAg, 51Cr, in suspended particulate matter, bottom sediments and in several indicator organisms (e.g., periphyton and Fucus vesiculosus) that effectively accumulate radioactive substances from the medium. The tritium discharges and the consequent detection frequency and concentrations of tritium in seawater were higher at Loviisa, but the concentrations of the activation products were higher at Olkiluoto, where traces of local discharge nuclides were also observed over a clearly wider area, due to the better exchange of water than at Loviisa, where local discharge nuclides were only detected outside Hästholmsfjärden Bay quite rarely and in smaller amounts. At the farthest, an insignificant trace amount (0.2 Bq kg-1 d.w.) of 60Co originating from Olkiluoto was detected in Fucus at a distance of 137 km from the power plant. Discharge nuclides from the local nuclear power plants were almost exclusively detected at the lower trophic levels of the ecosystems. Traces of local discharge nuclides were very seldom detected in fish, and even then only in very low quantities. As a consequence of the reduced discharges, the concentrations of local discharge nuclides in the environment have decreased noticeably in recent years at both Loviisa and Olkiluoto. Although the concentrations in environmental samples, and above all, the discharge data, are presented as seemingly large numbers, the radiation doses caused by them to the population and to the biota are very low, practically insignificant. The effects of the thermal discharges have been more significant, at least to the wildlife in the discharge areas of the cooling water, although the area of impact has been relatively small. The results show that the nutrient level and the exchange of water in the discharge area of a nuclear power plant are of crucial importance.

Effects of recreational use and fragmentation on the understorey vegetation and soil microbial communities of urban forests in southern Finland

The impacts of fragmentation and recreational use on the hemiboreal urban forest understorey vegetation and the microbial community of the humus layer (the phospholipid fatty acid (PLFA) pattern, microbial biomass and microbial activity, measured as basal respiration) were examined in the greater Helsinki area, southern Finland. Trampling tolerance of 1) herb-rich OMT, 2) mesic MT, and 3) sub-xeric VT forests (in decreasing order of fertility) was studied by comparing relative understorey vegetation cover (urban/untrampled reference ratio) of the three forest types. The trampling tolerance of forest vegetation increased with the productivity of the site (sub-xeric < mesic < herb-rich). Wear of understorey vegetation correlated positively with the number of residents (i.e., recreational pressure) around the forest patch. An increase of 15000 residents within a radius of 1 km around a forest patch was associated with ca. 30% decrease in the relative understorey vegetation cover. The cover of dwarf shrub Vaccinium myrtillus in particular decreased with increasing levels of wear. The cover of mosses in urban forests was less than half of that in untrampled reference areas. Cover of tree saplings, mainly Sorbus aucuparia, and some resilient herbs was higher than in the reference areas. In small urban forest fragments, broad-leaved trees, grasses and herbs were more abundant and mosses were scarcer than in larger urban forest areas. Thus, due to trampling and edge effects, resilient herb and grass species are replacing sensitive dwarf shrubs, mosses and lichens in urban forests. Differences in the soil microbial community structure were found between paths and untrampled areas and the effects of paths extended more than one meter from the paths. Paths supported approximately 25-30% higher microbial biomass with a transition zone of at least 1 m from the path edge. However, microbial activity per unit of biomass was lower on paths than in untrampled areas. Furthermore, microbial biomass and activity were 30-45% lower at the first 20 m into the forest fragments, due to low moisture content of humus near the edge. The decreased microbial activity detected at forest edges and paths implies decreased litter decomposition rates, and thus, a change in nutrient cycling. Changes in the decomposition and nutrient supply may in turn affect the diversity and function of plant communities in urban forests. Keywords: boreal forest vegetation, edge effects, phospholipid fatty acids, trampling, urban woodlands, wear

Dispersal and related life history traits in the Glanville fritillary butterfly

Most studies of life history evolution are based on the assumption that species exist at equilibrium and spatially distinct separated populations. In reality, this is rarely the case, as populations are often spatially structured with ephemeral local populations. Therefore, the characteristics of metapopulations should be considered while studying factors affecting life history evolution. Theoretical studies have examined spatial processes shaping the evolution of life history traits to some extent, but there is little empirical data and evidence to investigate model predictions. In my thesis I have tried to bridge the gap between theoretical and empirical studies by using the well-known Glanville fritillary (Melitaea cinxia) metapopulation as a model system. The long-term persistence of classic metapopulations requires sufficient dispersal to establish new local populations to compensate for local extinctions. Previous studies on the Glanville fritillary have shown that females establishing new populations are not a random sample from the metapopulation, but they are in fact more dispersive than females in old populations. Many other life-history traits, such as body size, fecundity and lifespan, may be related to dispersal rate. Therefore, I examined a range of correlated traits for their evolutionary and ecological consequences. I was particularly interested in how the traits vary under natural environmental conditions, hence all studies were conducted in a large (32 x 26 m) outdoor population cage built upon a natural habitat patch. Individuals for the experiments were sampled from newly-established and old populations within a large metapopulation. Results show that females originating from newly-established populations had higher within-habitat patch mobility than females from old populations. I showed that dispersal rate is heritable and that flight activity is related to variation in a gene encoding the glycolytic enzyme phosphoglucose isomerase. Both among-individual and among-population variation in dispersal are correlated with the reproductive performance of females, though I found no evidence for a trade-off between dispersal and fecundity in terms of lifetime egg production or clutch size. Instead, the results suggest that highly dispersive females from newly-established populations have a shorter lifespan than females from old populations, and that dispersive females may pay a cost in terms of reduced lifetime reproductive success due to increased time spent outside habitat patches. In summary, the results of this thesis show that genotype-dependent dispersal rate correlates with other life history traits in the Glanville fritillary, and that the rapid turnover of local populations (extinctions and re-colonisations) is likely to be the mechanism that maintains phenotypic variation in many life history traits at the metapopulation level.

The role of cyclase-associated protein (CAP) in actin dynamics during cell motility and morphogenesis

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.