Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Intermolecular Interactions of Noble-Gas-Containing Species

The importance of intermolecular interactions to chemistry, physics, and biology is difficult to overestimate. Without intermolecular forces, condensed phase matter could not form. The simplest way to categorize different types of intermolecular interactions is to describe them using van der Waals and hydrogen bonded (H-bonded) interactions. In the H-bond, the intermolecular interaction appears between a positively charged hydrogen atom and electronegative fragments and it originates from strong electrostatic interactions. H-bonding is important when considering the properties of condensed phase water and in many biological systems including the structure of DNA and proteins. Vibrational spectroscopy is a useful tool for studying complexes and the solvation of molecules. Vibrational frequency shift has been used to characterize complex formation. In an H-bonded system A∙∙∙H-X (A and X are acceptor and donor species, respectively), the vibrational frequency of the H-X stretching vibration usually decreases from its value in free H-X (red-shift). This frequency shift has been used as evidence for H-bond formation and the magnitude of the shift has been used as an indicator of the H-bonding strength. In contrast to this normal behavior are the blue-shifting H-bonds, in which the H-X vibrational frequency increases upon complex formation. In the last decade, there has been active discussion regarding these blue-shifting H-bonds. Noble-gases have been considered inert due to their limited reactivity with other elements. In the early 1930 s, Pauling predicted the stable noble-gas compounds XeF6 and KrF6. It was not until three decades later Neil Bartlett synthesized the first noble-gas compound, XePtF6, in 1962. A renaissance of noble-gas chemistry began in 1995 with the discovery of noble-gas hydride molecules at the University of Helsinki. The first hydrides were HXeCl, HXeBr, HXeI, HKrCl, and HXeH. These molecules have the general formula of HNgY, where H is a hydrogen atom, Ng is a noble-gas atom (Ar, Kr, or Xe), and Y is an electronegative fragment. At present, this class of molecules comprises 23 members including both inorganic and organic compounds. The first and only argon-containing neutral chemical compound HArF was synthesized in 2000 and its properties have since been investigated in a number of studies. A helium-containing chemical compound, HHeF, was predicted computationally, but its lifetime has been predicted to be severely limited by hydrogen tunneling. Helium and neon are the only elements in the periodic table that do not form neutral, ground state molecules. A noble-gas matrix is a useful medium in which to study unstable and reactive species including ions. A solvated proton forms a centrosymmetric NgHNg+ (Ng = Ar, Kr, and Xe) structure in a noble-gas matrix and this is probably the simplest example of a solvated proton. Interestingly, the hypothetical NeHNe+ cation is isoelectronic with the water-solvated proton H5O2+ (Zundel-ion). In addition to the NgHNg+ cations, the isoelectronic YHY- (Y = halogen atom or pseudohalogen fragment) anions have been studied with the matrix-isolation technique. These species have been known to exist in alkali metal salts (YHY)-M+ (M = alkali metal e.g. K or Na) for more than 80 years. Hydrated HF forms the FHF- structure in aqueous solutions, and these ions participate in several important chemical processes. In this thesis, studies of the intermolecular interactions of HNgY molecules and centrosymmetric ions with various species are presented. The HNgY complexes show unusual spectral features, e.g. large blue-shifts of the H-Ng stretching vibration upon complexation. It is suggested that the blue-shift is a normal effect for these molecules, and that originates from the enhanced (HNg)+Y- ion-pair character upon complexation. It is also found that the HNgY molecules are energetically stabilized in the complexed form, and this effect is computationally demonstrated for the HHeF molecule. The NgHNg+ and YHY- ions also show blue-shifts in their asymmetric stretching vibration upon complexation with nitrogen. Additionally, the matrix site structure and hindered rotation (libration) of the HNgY molecules were studied. The librational motion is a much-discussed solid state phenomenon, and the HNgY molecules embedded in noble-gas matrices are good model systems to study this effect. The formation mechanisms of the HNgY molecules and the decay mechanism of NgHNg+ cations are discussed. A new electron tunneling model for the decay of NgHNg+ absorptions in noble-gas matrices is proposed. Studies of the NgHNg+∙∙∙N2 complexes support this electron tunneling mechanism.

Sexual reproduction in chydorid cladocerans (Anomopoda, Chydoridae) in southern Finland - implications for paleolimnology

The relationship between sexual reproduction of littoral chydorid cladocerans (Anomopoda, Chydoridae) and environmental factors in aquatic ecosystems has been rarely studied, although the sexual behavior of some planktonic cladocerans is well documented. Ecological monitoring was used to study the relationship between climate-related and non-climatic environmental factors and chydorid sexual reproduction patterns in nine environmentally different lakes that were closely situated to each other in southern Finland. Furthermore, paleolimnological ephippium analysis was used to clarify how current sexual reproduction is reflected in surface sediments of the same nine lakes. Additionally, short sediment cores from two of the lakes were studied with ephippium analysis to examine how recent climate-related and non-climatic environmental changes were reflected in chydorid sexual reproduction. Ephippium analysis uses the subfossil shells of asexual individuals to represent asexual reproduction and the shells of sexual females, i.e. ephippia, to represent sexual reproduction. The relative proportion of ephippia of all chydorid species, i.e. total chydorid ephippia (TCE) indicates the relative proportion of sexual reproduction during the open-water season. This thesis is part of the EPHIPPIUM-project which aims to develop ephippium analysis towards a quantitative climate reconstruction tool. To be able to develop a valid climate model, the influence of the environmental stressors other than climate on contemporary sexual reproduction and its reflection in sediment assemblages must be clarified so they can be eliminated from the model. During contemporary monitoring a few sexual individuals were observed during summer, apparently forced to sexual reproduction by non-climatic local environmental factors, such as crowding or invertebrate predation. Monitoring also revealed that the autumnal chydorid sexual reproduction period was consistent between the different lakes and climate-related factors appeared to act as the main inducers and regulators of autumnal sexual reproduction. However, during autumn, chydorid species and populations among the lakes exhibited a wide variation in the intensity, induction time, and length of autumnal sexual reproduction. These variations apparently act as mechanisms for local adaptations due to the genetic variability provided by sexual reproduction that enhance the ecological flexibility of chydorid species, allowing them to inhabit a wide range of environments. A large variation was also detected in the abundance of parthenogenetic and gamogenetic individuals during the open-water season among the lakes. On the basis of surface sediment samples, the general level of the TCE is ca. 3-4% in southern Finland, reflecting an average proportion of sexual reproduction in this specific climate. The variation in the TCE was much lower than could be expected on the basis of the monitoring results. This suggests that some of the variation detected by monitoring may derive from differences between sampling sites and years smoothed out in the sediment samples, providing an average of the entire lake area and several years. The TCE is always connected to various ecological interactions in lake ecosystems and therefore is always lake-specific. Hypothetically, deterioration of climate conditions can be detected in the TCE as an increase in ephippia of all chydorid species, since a shortening open-water season is reflected in the relative proportions of the two reproduction modes. Such an increase was clearly detected for the time period of the Little Ice Age in a sediment core. The paleolimnological results also indicated that TCE can suddenly increase due to ephippia of one or two species, which suggests that at least some chydorids can somehow increase the production of resting eggs under local environmental stress. Thus, some environmental factors may act as species-specific environmental stressors. The actual mechanism of the increased sexual reproduction seen in sediments has been unknown but the present study suggests that the mechanism is probably the increased intensity of gamogenesis, i.e. that a larger proportion of individuals in autumnal populations reproduce sexually, which results in a larger proportion of ephippia in sediments and a higher TCE. The results of this thesis demonstrate the utility of ephippium analysis as a paleoclimatological method which may also detect paleolimnological changes by identifying species-specific environmental stressors. For a quantitative TCE-based climate reconstruction model, the natural variation in the TCE of surface sediments in different climates must be clarified with more extensive studies. In addition, it is important to recognize the lakes where the TCE is not only a reflection of the length of the open-water season, but is also non-climatically forced. The results of ephippium analysis should always be interpreted in a lake-specific manner and in the context of other paleoecological proxies.

Spatial variation of climate and the impact of disturbances on local climate and forest recovery in northern Finland

The structure and function of northern ecosystems are strongly influenced by climate change and variability and by human-induced disturbances. The projected global change is likely to have a pronounced effect on the distribution and productivity of different species, generating large changes in the equilibrium at the tree-line. In turn, movement of the tree-line and the redistribution of species produce feedback to both the local and the regional climate. This research was initiated with the objective of examining the influence of natural conditions on the small-scale spatial variation of climate in Finnish Lapland, and to study the interaction and feedback mechanisms in the climate-disturbances-vegetation system near the climatological border of boreal forest. The high (1 km) resolution spatial variation of climate parameters over northern Finland was determined by applying the Kriging interpolation method that takes into account the effect of external forcing variables, i.e., geographical coordinates, elevation, sea and lake coverage. Of all the natural factors shaping the climate, the geographical position, local topography and altitude proved to be the determining ones. Spatial analyses of temperature- and precipitation-derived parameters based on a 30-year dataset (1971-2000) provide a detailed description of the local climate. Maps of the mean, maximum and minimum temperatures, the frost-free period and the growing season indicate that the most favourable thermal conditions exist in the south-western part of Lapland, around large water bodies and in the Kemijoki basin, while the coldest regions are in highland and fell Lapland. The distribution of precipitation is predominantly longitudinally dependent but with the definite influence of local features. The impact of human-induced disturbances, i.e., forest fires, on local climate and its implication for forest recovery near the northern timberline was evaluated in the Tuntsa area of eastern Lapland, damaged by a widespread forest fire in 1960 and suffering repeatedly-failed vegetation recovery since that. Direct measurements of the local climate and simulated heat and water fluxes indicated the development of a more severe climate and physical conditions on the fire-disturbed site. Removal of the original, predominantly Norway spruce and downy birch vegetation and its substitution by tundra vegetation has generated increased wind velocity and reduced snow accumulation, associated with a large variation in soil temperature and moisture and deep soil frost. The changed structural parameters of the canopy have determined changes in energy fluxes by reducing the latter over the tundra vegetation. The altered surface and soil conditions, as well as the evolved severe local climate, have negatively affected seedling growth and survival, leading to more unfavourable conditions for the reproduction of boreal vegetation and thereby causing deviations in the regional position of the timberline. However it should be noted that other factors, such as an inadequate seed source or seedbed, the poor quality of the soil and the intensive logging of damaged trees could also exacerbate the poor tree regeneration. In spite of the failed forest recovery at Tunsta, the position and composition of the timberline and tree-line in Finnish Lapland may also benefit from present and future changes in climate. The already-observed and the projected increase in temperature, the prolonged growing season, as well as changes in the precipitation regime foster tree growth and new regeneration, resulting in an advance of the timberline and tree-line northward and upward. This shift in the distribution of vegetation might be decelerated or even halted by local topoclimatic conditions and by the expected increase in the frequency of disturbances.

Postglacial climate changes and vegetation responses in northern Europe

Postglacial climate changes and vegetation responses were studied using a combination of biological and physical indicators preserved in lake sediments. Low-frequency trends, high-frequency events and rapid shifts in temperature and moisture balance were probed using pollen-based quantitative temperature reconstructions and oxygen-isotopes from authigenic carbonate and aquatic cellulose, respectively. Pollen and plant macrofossils were employed to shed light on the presence and response rates of plant populations in response to climate changes, particularly focusing on common boreal and temperate tree species. Additional geochemical and isotopic tracers facilitated the interpretation of pollen- and oxygen-isotope data. The results show that the common boreal trees were present in the Baltic region (~55°N) during the Lateglacial, which contrasts with the traditional view of species refuge locations in the south-European peninsulas during the glacial/interglacial cycles. The findings of this work are in agreement with recent paleoecological and genetic evidence suggesting that scattered populations of tree species persisted at higher latitudes, and that these taxa were likely limited to boreal trees. Moreover, the results demonstrate that stepwise changes in plant communities took place in concert with major climate fluctuations of the glacial/interglacial transition. Postglacial climate trends in northern Europe were characterized by rise, maxima and fall in temperatures and related changes in moisture balance. Following the deglaciation of the Northern Hemisphere and the early Holocene reorganization of the ice-ocean-atmosphere system, the long-term temperature trends followed gradually decreasing summer insolation. The early Holocene (~11,700-8000 cal yr BP) was overall cool, moist and oceanic, although the earliest Holocene effective humidity may have been low particularly in the eastern part of northern Europe. The gradual warming trend was interrupted by a cold event ~8200 cal yr BP. The maximum temperatures, ~1.5-3.0°C above modern values, were attained ~8000-4000 cal yr BP. This mid-Holocene peak warmth was coupled with low lake levels, low effective humidity and summertime drought. The late Holocene (~4000 cal yr BP-present) was characterized by gradually decreasing temperatures, higher lake levels and higher effective humidity. Moreover, the gradual trends of the late Holocene were probably superimposed by higher-frequency variability. The spatial variability of the Holocene temperature and moisture balance patterns were tentatively attributed to the differing heat capacities of continents and oceans, changes in atmospheric circulation modes and position of sites and subregions with respect to large water bodies and topographic barriers. The combination of physical and biological proxy archives is a pivotal aspect of this work, because non-climatic factors, such as postglacial migration, disturbances and competitive interactions, can influence reshuffling of vegetation and hence, pollen-based climate reconstructions. The oxygen-isotope records and other physical proxies presented in this work manifest that postglacial climate changes were the main driver of the establishment and expansion of temperate and boreal tree populations, and hence, large-scale and long-term vegetation patterns were in dynamic equilibrium with climate. A notable exception to this pattern may be the postglacial invasion of Norway spruce and the related suppression of mid-Holocene temperate forest. This salient step in north-European vegetation history, the development of the modern boreal ecosystem, cannot be unambiguously explained by current evidence of postglacial climate changes. The results of this work highlight that plant populations, including long-lived trees, may be able to respond strikingly rapidly to changes in climate. Moreover, interannual and seasonal variation and extreme events can exert an important influence on vegetation reshuffling. Importantly, the studies imply that the presence of diffuse refuge populations or local stands among the prevailing vegetation may have provided the means for extraordinarily rapid vegetation responses. Hence, if scattered populations are not provided and tree populations are to migrate long distances, their capacity to keep up with predicted rates of future climate change may be lower than previously thought.

Ecological processes and large-scale climate relationships in northern coniferous forests

Ilmasto vaikuttaa ekologisiin prosesseihin eri tasoilla. Suuren mittakaavan ilmastoprosessit, yhdessä ilmakehän ja valtamerien kanssa, säätelevät paikallisia sääilmiöitä suurilla alueilla (mantereista pallopuoliskoihin). Tämä väistöskirja pyrkii selittämään kuinka suuren mittakaavan ilmasto on vaikuttanut tiettyihin ekologisiin prosesseihin pohjoisella havumetsäalueella. Valitut prosessit olivat puiden vuosilustojen kasvu, metsäpalojen esiintyminen ja vuoristomäntykovakuoriaisen aiheuttamat puukuolemat. Suuren mittakaavan ilmaston löydettiin vaikuttaneen näiden prosessien esiintymistiheyteen, kestoon ja levinneisyyteen keskeisten sään muuttujien välityksellä hyvin laajoilla alueilla. Tutkituilla prosesseilla oli vahva yhteys laajan mittakaavan ilmastoon. Yhteys on kuitenkin ollut hyvin dynaaminen ja muuttunut 1900-luvulla ilmastonmuutoksen aiheuttaessa muutoksia suuren mittakaavan ja alueellisten ilmastoprosessien välisiin sisäisiin suhteisiin.

Jumping numbers of a simple complete ideal in a two-dimensional regular local ring

The multiplier ideals of an ideal in a regular local ring form a family of ideals parametrized by non-negative rational numbers. As the rational number increases the corresponding multiplier ideal remains unchanged until at some point it gets strictly smaller. A rational number where this kind of diminishing occurs is called a jumping number of the ideal. In this manuscript we shall give an explicit formula for the jumping numbers of a simple complete ideal in a two dimensional regular local ring. In particular, we obtain a formula for the jumping numbers of an analytically irreducible plane curve. We then show that the jumping numbers determine the equisingularity class of the curve.

Computational Techniques for Haplotype Inference and for Local Alignment Significance

This thesis which consists of an introduction and four peer-reviewed original publications studies the problems of haplotype inference (haplotyping) and local alignment significance. The problems studied here belong to the broad area of bioinformatics and computational biology. The presented solutions are computationally fast and accurate, which makes them practical in high-throughput sequence data analysis. Haplotype inference is a computational problem where the goal is to estimate haplotypes from a sample of genotypes as accurately as possible. This problem is important as the direct measurement of haplotypes is difficult, whereas the genotypes are easier to quantify. Haplotypes are the key-players when studying for example the genetic causes of diseases. In this thesis, three methods are presented for the haplotype inference problem referred to as HaploParser, HIT, and BACH. HaploParser is based on a combinatorial mosaic model and hierarchical parsing that together mimic recombinations and point-mutations in a biologically plausible way. In this mosaic model, the current population is assumed to be evolved from a small founder population. Thus, the haplotypes of the current population are recombinations of the (implicit) founder haplotypes with some point--mutations. HIT (Haplotype Inference Technique) uses a hidden Markov model for haplotypes and efficient algorithms are presented to learn this model from genotype data. The model structure of HIT is analogous to the mosaic model of HaploParser with founder haplotypes. Therefore, it can be seen as a probabilistic model of recombinations and point-mutations. BACH (Bayesian Context-based Haplotyping) utilizes a context tree weighting algorithm to efficiently sum over all variable-length Markov chains to evaluate the posterior probability of a haplotype configuration. Algorithms are presented that find haplotype configurations with high posterior probability. BACH is the most accurate method presented in this thesis and has comparable performance to the best available software for haplotype inference. Local alignment significance is a computational problem where one is interested in whether the local similarities in two sequences are due to the fact that the sequences are related or just by chance. Similarity of sequences is measured by their best local alignment score and from that, a p-value is computed. This p-value is the probability of picking two sequences from the null model that have as good or better best local alignment score. Local alignment significance is used routinely for example in homology searches. In this thesis, a general framework is sketched that allows one to compute a tight upper bound for the p-value of a local pairwise alignment score. Unlike the previous methods, the presented framework is not affeced by so-called edge-effects and can handle gaps (deletions and insertions) without troublesome sampling and curve fitting.

Optimisation Problems in Wireless Sensor Networks : Local Algorithms and Local Graphs

This thesis studies optimisation problems related to modern large-scale distributed systems, such as wireless sensor networks and wireless ad-hoc networks. The concrete tasks that we use as motivating examples are the following: (i) maximising the lifetime of a battery-powered wireless sensor network, (ii) maximising the capacity of a wireless communication network, and (iii) minimising the number of sensors in a surveillance application. A sensor node consumes energy both when it is transmitting or forwarding data, and when it is performing measurements. Hence task (i), lifetime maximisation, can be approached from two different perspectives. First, we can seek for optimal data flows that make the most out of the energy resources available in the network; such optimisation problems are examples of so-called max-min linear programs. Second, we can conserve energy by putting redundant sensors into sleep mode; we arrive at the sleep scheduling problem, in which the objective is to find an optimal schedule that determines when each sensor node is asleep and when it is awake. In a wireless network simultaneous radio transmissions may interfere with each other. Task (ii), capacity maximisation, therefore gives rise to another scheduling problem, the activity scheduling problem, in which the objective is to find a minimum-length conflict-free schedule that satisfies the data transmission requirements of all wireless communication links. Task (iii), minimising the number of sensors, is related to the classical graph problem of finding a minimum dominating set. However, if we are not only interested in detecting an intruder but also locating the intruder, it is not sufficient to solve the dominating set problem; formulations such as minimum-size identifying codes and locating dominating codes are more appropriate. This thesis presents approximation algorithms for each of these optimisation problems, i.e., for max-min linear programs, sleep scheduling, activity scheduling, identifying codes, and locating dominating codes. Two complementary approaches are taken. The main focus is on local algorithms, which are constant-time distributed algorithms. The contributions include local approximation algorithms for max-min linear programs, sleep scheduling, and activity scheduling. In the case of max-min linear programs, tight upper and lower bounds are proved for the best possible approximation ratio that can be achieved by any local algorithm. The second approach is the study of centralised polynomial-time algorithms in local graphs these are geometric graphs whose structure exhibits spatial locality. Among other contributions, it is shown that while identifying codes and locating dominating codes are hard to approximate in general graphs, they admit a polynomial-time approximation scheme in local graphs.

Food intake, growth and social interactions of signal crayfish, Pacifastacus leniusculus (Dana)

The knowledge about the optimal rearing conditions, such as water temperature and quality, photoperiod and density, with the understanding of animal nutritional requirements forms the basis of economically stable aquaculture for freshwater crayfish. However, the shift from a natural environment to effective culture conditions induces several changes, not only at the population level, but also at the individual level. The social contacts between conspecifics increase with increasing animal density. The competition for limited resources (e.g. food, shelter, mates) is more severe with the presence of agonistic behaviour and may lead to unequal distribution of these. The objectives of this study were to: 1) study the distribution of a common food resource between communally reared signal crayfish (Pacifastacus leniusculus) and to assign potential feeding hierarchy on the basis of individual food intake measurements, 2) explore the possibilities of size distribution manipulations to affect population dynamics and food intake to improve growth and survival in culture and 3) study the effect of food ration and spatial distribution on food intake and to explore the effect of temperature and food ration on growth and body composition of freshwater crayfish. The feeding ranks between animals were assigned with a new method for individual food intake measurement of communally reared crayfish. This technique has a high feasibility and a great potential to be applied in crayfish aquaculture studies. In this study, signal crayfish showed high size-related variability in food consumption both among individuals within a group (inter-individual) and within individual day-to-day variation (intra-individual). Increased competition for food led to an unequal distribution of this resource and this may be a reason for large growth differences between animals. The consumption was significantly higher when reared individually in comparison with communal housing. These results suggest that communally housed crayfish form a feeding hierarchy and that the animal size is the major factor controlling the position in this hierarchy. The optimisation of the social environment ( social conditions ) was evaluated in this study as a new approach to crayfish aquaculture. The results showed that the absence of conspecifics (individual rearing vs. communal housing) affects growth rate, food intake and the proportion of injured animals, whereas size variation between animals influences the number and duration of agonistic encounters. In addition, animal size had a strong influence on the fighting success of signal crayfish reared in a social milieu with a wide size variation of conspecifics. Larger individuals initiated and won most of the competitions, which suggests size-based social hierarchy of P. leniusculus. This is further supported by the fact that the length and weight gain of smaller animals increased after size grading, maybe because of a better access to the food resource due to diminished social pressure. However, the high dominance index was not based on size under conditions of limited size variation, e.g. those characteristic of restocked natural populations and aquaculture, indicating the important role of behaviour on social hierarchy.

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

Communities of wood-inhabiting fungi : Ecological requirements and responses to forest management and fragmentation

Despite much research on forest biodiversity in Fennoscandia, the exact mechanisms of species declines in dead-wood dependent fungi are still poorly understood. In particular, there is only limited information on why certain fungal species have responded negatively to habitat loss and fragmentation, while others have not. Understanding the mechanisms behind species declines would be essential for the design and development of ecologically effective and scientifically informed conservation measures, and management practices that would promote biodiversity in production forests. In this thesis I study the ecology of polypores and their responses to forest management, with a particular focus on why some species have declined more than others. The data considered in the thesis comprise altogether 98,318 dead-wood objects, with 43,085 observations of 174 fungal species. Out of these, 1,964 observations represent 58 red-listed species. The data were collected from 496 sites, including woodland key habitats, clear-cuts with retention trees, mature managed forests, and natural or natural-like forests in southern Finland and Russian Karelia. I show that the most relevant way of measuring resource availability can differ to a great extent between species seemingly sharing the same resources. It is thus critical to measure the availability of resources in a way that takes into account the ecological requirements of the species. The results show that connectivity at the local, landscape and regional scales is important especially for the highly specialized species, many of which are also red-listed. Habitat loss and fragmentation affect not only species diversity but also the relative abundances of the species and, consequently, species interactions and fungal successional pathways. Changes in species distributions and abundances are likely to affect the food chains in which wood-inhabiting fungi are involved, and thus the functioning of the whole forest ecosystem. The findings of my thesis highlight the importance of protecting well-connected, large and high-quality forest areas to maintain forest biodiversity. Small habitat patches distributed across the landscape are likely to contribute only marginally to protection of red-listed species, especially if habitat quality is not substantially higher than in ordinary managed forest, as is the case with woodland key habitats. Key habitats might supplement the forest protection network if they were delineated larger and if harvesting of individual trees was prohibited in them. Taking the landscape perspective into account in the design and development of conservation measures is critical while striving to halt the decline of forest biodiversity in an ecologically effective manner.