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.

Passive vector turbulence

This thesis consists of three articles on passive vector fields in turbulence. The vector fields interact with a turbulent velocity field, which is described by the Kraichnan model. The effect of the Kraichnan model on the passive vectors is studied via an equation for the pair correlation function and its solutions. The first paper is concerned with the passive magnetohydrodynamic equations. Emphasis is placed on the so called "dynamo effect", which in the present context is understood as an unbounded growth of the pair correlation function. The exact analytical conditions for such growth are found in the cases of zero and infinite Prandtl numbers. The second paper contains an extensive study of a number of passive vector models. Emphasis is now on the properties of the (assumed) steady state, namely anomalous scaling, anisotropy and small and large scale behavior with different types of forcing or stirring. The third paper is in many ways a completion to the previous one in its study of the steady state existence problem. Conditions for the existence of the steady state are found in terms of the spatial roughness parameter of the turbulent velocity field.

Computational Methods for Reconstruction and Analysis of Genome-Scale Metabolic Networks

Metabolism is the cellular subsystem responsible for generation of energy from nutrients and production of building blocks for larger macromolecules. Computational and statistical modeling of metabolism is vital to many disciplines including bioengineering, the study of diseases, drug target identification, and understanding the evolution of metabolism. In this thesis, we propose efficient computational methods for metabolic modeling. The techniques presented are targeted particularly at the analysis of large metabolic models encompassing the whole metabolism of one or several organisms. We concentrate on three major themes of metabolic modeling: metabolic pathway analysis, metabolic reconstruction and the study of evolution of metabolism. In the first part of this thesis, we study metabolic pathway analysis. We propose a novel modeling framework called gapless modeling to study biochemically viable metabolic networks and pathways. In addition, we investigate the utilization of atom-level information on metabolism to improve the quality of pathway analyses. We describe efficient algorithms for discovering both gapless and atom-level metabolic pathways, and conduct experiments with large-scale metabolic networks. The presented gapless approach offers a compromise in terms of complexity and feasibility between the previous graph-theoretic and stoichiometric approaches to metabolic modeling. Gapless pathway analysis shows that microbial metabolic networks are not as robust to random damage as suggested by previous studies. Furthermore the amino acid biosynthesis pathways of the fungal species Trichoderma reesei discovered from atom-level data are shown to closely correspond to those of Saccharomyces cerevisiae. In the second part, we propose computational methods for metabolic reconstruction in the gapless modeling framework. We study the task of reconstructing a metabolic network that does not suffer from connectivity problems. Such problems often limit the usability of reconstructed models, and typically require a significant amount of manual postprocessing. We formulate gapless metabolic reconstruction as an optimization problem and propose an efficient divide-and-conquer strategy to solve it with real-world instances. We also describe computational techniques for solving problems stemming from ambiguities in metabolite naming. These techniques have been implemented in a web-based sofware ReMatch intended for reconstruction of models for 13C metabolic flux analysis. In the third part, we extend our scope from single to multiple metabolic networks and propose an algorithm for inferring gapless metabolic networks of ancestral species from phylogenetic data. Experimenting with 16 fungal species, we show that the method is able to generate results that are easily interpretable and that provide hypotheses about the evolution of metabolism.

Studies on the international effects of real shocks

This doctoral thesis addresses the macroeconomic effects of real shocks in open economies in flexible exchange rate regimes. The first study of this thesis analyses the welfare effects of fiscal policy in a small open economy, where private and government consumption are substitutes in terms of private utility. The main findings are as follows: fiscal policy raises output, bringing it closer to its efficient level, but is not welfare-improving even though government spending directly affects private utility. The main reason for this is that the introduction of useful government spending implies a larger crowding-out effect on private consumption, when compared with the `pure waste' case. Utility decreases since one unit of government consumption yields less utility than one unit of private consumption. The second study of this thesis analyses the question of how the macroeconomic effects of fiscal policy in a small open economy depend on optimal intertemporal behaviour. The key result is that the effects of fiscal policy depend on the size of the elasticity of substitution between traded and nontraded goods. In particular, the sign of the current account response to fiscal policy depends on the interplay between the intertemporal elasticity of aggregate consumption and the elasticity of substitution between traded and nontraded goods. The third study analyses the consequences of productive government spending on the international transmission of fiscal policy. A standard result in the New Open Economy Macroeconomics literature is that a fiscal shock depreciates the exchange rate. I demonstrate that the response of the exchange rate depends on the productivity of government spending. If productivity is sufficiently high, a fiscal shock appreciates the exchange rate. It is also shown that the introduction of productive government spending increases both domestic and foreign welfare, when compared with the case where government spending is wasted. The fourth study analyses the question of how the international transmission of technology shocks depends on the specification of nominal rigidities. A growing body of empirical evidence suggests that a positive technology shock leads to a temporary decline in employment. In this study, I demonstrate that the open economy dimension can enhance the ability of sticky price models to account for the evidence. The reasoning is as follows. An improvement in technology appreciates the nominal exchange rate. Under producer-currency pricing, the exchange rate appreciation shifts global demand toward foreign goods away from domestic goods. This causes a temporary decline in domestic employment.

Traditional Village Governance (Desa Adat) and Inter-ethnic Conflict in Bali : A Study of Conflict between Balinese and Migrants: Cases from Municipality of Denpasar

Regional autonomy in Indonesia was initially introduced as a means of pacifying regional disappointment at the central government. Not only did the Regional Autonomy Law of 1999 give the Balinese a chance to express grievance regarding the centralist policies of the Jakarta government but also provided an opportunity to return to the regional, exclusive, traditional village governance (desa adat). As a result, the problems faced by the island, particularly ethnic conflicts, are increasingly handled by the mechanism of this traditional type of governance. Traditional village governance with regard to ethnic conflicts (occurring) between Balinese and migrants has never been systematically analyzed. Existing analyses emphasized only the social context, but do not explain either the cause of conflicts and the ensuing problems entails or the virtues of traditional village governance mechanisms for mediating in the conflict. While some accounts provide snapshots, they lack both theoretical and conflict study perspective. The primary aim of this dissertation is to explore the expression and the causes of conflict between the Balinese and migrants and to advance the potential of traditional village governance as a means of conflict resolution with particular reference to the municipality of Denpasar. One conclusion of the study is that the conflict between the Balinese and migrants has been expressed on the level of situation/contradiction, attitudes, and behavior. Yet the driving forces behind the conflict itself consist of the following factors: absence of cooperation; incompatible position and perception; inability to communicate effectively; and problem of inequality and injustice, which comes to the surface as a social, cultural, and economic problem. This complex of factors fuels collective fear for the future of both groups. The study concludes that traditional village governance mechanisms as a means of conflict resolution have not yet been able to provide an enduring resolution for the conflict. Analysis shows that the practice of traditional village governance is unable to provide satisfactory mechanisms for the conflict as prescribed by conflict resolution theory. Traditional village governance, which is derived from the exclusive Hindu-Balinese culture, is accepted as more legitimate among the Balinese than the official governance policies. However, it is not generally accepted by most of the Muslim migrants. In addition, traditional village governance lacks access to economic instruments, which weakens its capacity to tackle the economic roots of the conflict. Thus the traditional mechanisms of migrant ordinance , as practiced by the traditional village governance have not yet been successful in penetrating all aspects of the conflict. Finally, one of the main challenges for traditional village governance s legal development is the creation of a regional legal system capable of accommodating rapid changes in line with the national and international legal practices. The framing of the new laws should be responsive to the aspirations of a changing society. It should not only protect the various Balinese communities interests, but also that of other ethnic groups, especially those of the minority. In other words, the main challenge to traditional village governance is its ability to develop flexibility and inclusiveness.

Ethics and Aesthetics : Intersections in Iris Murdoch's Philosophy

The study addresses the question concerning the relationship between ethics and aesthetics in the philosophy of Iris Murdoch. The main argument is that Murdoch s philosophy cannot be accurately understood without an understanding of the relationship she sees between the aesthetic experience and morality. Reading Murdoch s philosophy with this relationship in mind shows that it must be considered as a relevant alternative to the main forms of aesthetic-ethical theories. The study consists of seven previously published articles and a summary. It shows that Murdoch belongs to a tradition of philosophers who seek to broaden the scope of ethics by reference to aesthetic value and aesthetic experience. She sees an attitude responsible for aesthetic experiences as relevant for morality. However, she does not collapse morality into aesthetic experience. The two meet on the level of the subject s attitude towards its object, but there is a distinction between the experiences that accompany the attitudes. Aesthetic experiences can function as a clue to morals in that they present in a pleasing manner moral truths which otherwise might be psychologically too difficult to face. Murdoch equates the aesthetic attitude with virtuous love characterized by unselfish attention to its object. The primary object of such love is in Murdoch s account another human individual in her particularity. She compares the recognition of the other person as a particular existence to the experience of the Kantian sublime and offers her own version of the true sublime which is the experience of awe in the face of the infinity of the task of understanding others. One of the most central claims in Murdoch s philosophy is that human consciousness is evaluatively structured. This claim challenges the distinction between facts and values which has had an immense influence on modern moral philosophy. One argument with which Murdoch supports her claim is the nature of great literature. According to her, the standard of greatness in literature is the authors awareness of the independent existence of individuals in the particularity of their evaluative consciousnesses. The analysis of the standard of greatness in literature is also Murdoch s only argument for the claim that the primary object of the loving unselfish attention is the other particular individual. She is convinced that great literature reveals a deep truth about the human condition with its capacity to capture the particular. Abstract philo¬sophical discourse cannot compete with this capacity but it should take truths revealed by literature seriously in its theorising. Recognising this as Murdoch s stand on the question of the relation between philosophy and literature as forms of human discourse settles whether she is part of what has been called philosophy s turn to literature. The answer is yes.

Enzymes with radical tendencies: the PFL family

The first glycyl radical in an enzyme was described 20 years ago and since then the family of glycyl radical enzymes (GREs) has expanded to include enzymes catalysing five chemically distinct reactions. The type enzymes of the family, anaerobic ribonucleotide reductase (RNRIII) and pyruvate formate lyase (PFL) had been studied long before it was known that they are GREs. Spectroscopic measurements on the radical and an observation that exposure to oxygen irreversibly inactivates the enzymes by cleavage of the protein proved that the radical is located on a particular glycine residue, close to the C-terminus of the protein. Both anaerobic RNRIII and PFL, are important for many anaerobic and facultative anaerobic bacteria as RNRIII is responsible for the synthesis of DNA precursors and PFL catalyses a key metabolic reaction in glycolysis. The crystal structures of both were solved in 1999 and they revealed that, although the enzymes do not share significant sequence identity, they share a similar structure - the radical site and residues necessary for catalysis are buried inside a ten stranded $\ualpha $/$\ubeta $-barrel. GREs are synthesised in an inactive form and are post-translationally activated by an activating enzyme which uses S-adenosyl methionine and an iron-sulphur cluster to generate the radical. One of the goals of this thesis work was to crystallise the activating enzyme of PFL. This task is challenging as, like GREs, the activating component is inactivated by oxygen. The experiments were therefore carried out in an oxygen free atmosphere. This is the first report of a crystalline GRE activating enzyme. Recently several new GREs have been characterised, all sharing sequence similarity to PFL but not to RNRIII. Also, the genome sequencing projects have identified many PFL-like GREs of unknown function, usually annotated as PFLs. In the present thesis I describe the grouping of these PFL family enzymes based on the sequence similarity and analyse the conservation patterns when compared to the structure of E. coli PFL. Based on this information an activation route is proposed. I also report a crystal structure of one of the PFL-like enzymes with unknown function, PFL2 from Archaeoglobus fulgidus. As A. fulgidus is a hyperthermophilic organism, possible mechanisms stabilising the structure are discussed. The organisation of an active site of PFL2 suggests that the enzyme may be a dehydratase. Keywords: glycyl radical, enzyme, pyruvate formate lyase, x-ray crystallography, bioinformatics

Evaluation of methods and applications for behavioural profiling of transgenic mice

During the last 10-15 years interest in mouse behavioural analysis has evolved considerably. The driving force is development in molecular biological techniques that allow manipulation of the mouse genome by changing the expression of genes. Therefore, with some limitations it is possible to study how genes participate in regulation of physiological functions and to create models explaining genetic contribution to various pathological conditions. The first aim of our study was to establish a framework for behavioural phenotyping of genetically modified mice. We established comprehensive battery of tests for the initial screening of mutant mice. These included tests for exploratory and locomotor activity, emotional behaviour, sensory functions, and cognitive performance. Our interest was in the behavioural patterns of common background strains used for genetic manipulations in mice. Additionally we studied the behavioural effect of sex differences, test history, and individual housing. Our findings highlight the importance of careful consideration of genetic background for analysis of mutant mice. It was evident that some backgrounds may mask or modify the behavioural phenotype of mutants and thereby lead to false positive or negative findings. Moreover, there is no universal strain that is equally suitable for all tests, and using different backgrounds allows one to address possible phenotype modifying factors. We discovered that previous experience affected performance in several tasks. The most sensitive traits were the exploratory and emotional behaviour, as well as motor and nociceptive functions. Therefore, it may be essential to repeat some of the tests in naïve animals for assuring the phenotype. Social isolation for a long time period had strong effects on exploratory behaviour, but also on learning and memory. All experiments revealed significant interactions between strain and environmental factors (test history or housing condition) indicating genotype-dependent effects of environmental manipulations. Several mutant line analyses utilize this information. For example, we studied mice overexpressing as well as those lacking extracellular matrix protein heparin-binding growth-associated molecule (HB-GAM), and mice lacking N-syndecan (a receptor for HB-GAM). All mutant mice appeared to be fertile and healthy, without any apparent neurological or sensory defects. The lack of HB-GAM and N-syndecan, however, significantly reduced the learning capacity of the mice. On the other hand, overexpression of HB-GAM resulted in facilitated learning. Moreover, HB-GAM knockout mice displayed higher anxiety-like behaviour, whereas anxiety was reduced in HB-GAM overexpressing mice. Changes in hippocampal plasticity accompanied the behavioural phenotypes. We conclude that HB-GAM and N-syndecan are involved in the modulation of synaptic plasticity in hippocampus and play a role in regulation of anxiety- and learning-related behaviour.

Pathogenicity, functional significance and clinical phenotype of mismatch repair gene MSH2 variants found in cancer patients

DNA ja siinä sijaitsevat geenit ohjaavat kaikkea solujen toimintaa. DNA-molekyyleihin kuitenkin kertyy mutaatioita sekä ympäristön vaikutuksen, että solujen oman toiminnan tuloksena. Mikäli virheitä ei korjata, saattaa tuloksena olla solun muuttuminen syöpäsoluksi. Soluilla onkin käytössä useita DNA-virheiden korjausmekanismeja, joista yksi on ns. mismatch repair (MMR). MMR vastaa DNA:n kahdentumisessa syntyvien virheiden korjauksesta. Periytyvät mutaatiot geeneissä, jotka vastaavat MMR-proteiinien rakentamisesta, aiheuttavat ongelmia DNA:n korjauksessa ja altistavat kantajansa periytyvälle ei-polypoottiselle paksusuolisyöpäoireyhtymälle (hereditary nonpolyposis colorectal cancer, HNPCC). Yleisimmin mutatoituneet MMR-geenit ovat MLH1 ja MSH2. HNPCC periytyy vallitsevasti, eli jo toiselta vanhemmalta peritty geenivirhe altistaa syövälle. MMR-geenivirheen kantaja sairastuu syöpään elämänsä aikana suurella todennäköisyydellä, ja sairastumisikä on vain noin 40 vuotta. Syövälle altistavan geenivirheen löytäminen mutaation kantajilta on hyvin tärkeää, sillä säännöllinen seuranta mahdollistaa kehittymässä olevan kasvaimen havaitsemisen ja poistamisen jo aikaisessa vaiheessa. Tämän on osoitettu alentavan syöpäkuolleisuutta merkittävästi. Varma tieto altistuksen alkuperästä on tärkeä myös niille syöpäsuvun jäsenille, jotka eivät kanna kyseistä mutaatiota. Syövälle altistavien mutaatioiden ohella MMR-geeneistä löydetään säännöllisesti muutoksia, jotka ovat normaalia henkilöiden välistä geneettistä vaihtelua, eikä niiden oleteta lisäävän syöpäaltistusta. Altistavien mutaatioiden erottaminen näistä neutraaleista variaatioista on vaikeaa, mutta välttämätöntä altistuneiden tehokkaan seurannan varmistamiseksi. Tässä väitöskirjassa tutkittiin 18:a MSH2 -geenin mutaatiota. Mutaatiot oli löydetty perheistä, joissa esiintyi paljon syöpiä, mutta niiden vaikutus DNA:n korjaustehoon ja syöpäaltistukseen oli epäselvä. Työssä tutkittiin kunkin mutaation vaikutusta MSH2-proteiinin normaaliin toimintaan, ja tuloksia verrattiin potilaiden ja sukujen kliinisiin tietoihin. Tutkituista mutaatiosta 12 aiheutti puutteita MMR-korjauksessa. Nämä mutaatiot tulkittiin syövälle altistaviksi. Analyyseissä normaalisti toimineet 4 mutaatiota eivät todennäköisesti ole syynä syövän syntyyn kyseisillä perheillä. Tulkinta jätettiin avoimeksi 2 mutaation kohdalla. Tutkimuksesta hyötyivät suoraan kuvattujen mutaatioiden kantajaperheet, joiden geenivirheen syöpäaltistuksesta saatiin tietoa, mahdollistaen perinnöllisyysneuvonnan ja seurannan kohdentamisen sitä tarvitseville. Työ selvensi myös mekanismeja, joilla mutatoitunut MSH2-proteiini voi menettää toimintakykynsä.

Missing-In-Metastasis (MIM) regulates cell morphology by promoting plasma membrane and actin cytoskeleton dynamics

The cells of multicellular organisms have differentiated to carry out specific functions that are often accompanied by distinct cell morphology. The actin cytoskeleton is one of the key regulators of cell shape subsequently controlling multiple cellular events including cell migration, cell division, endo- and exocytosis. A large set of actin regulating proteins has evolved to achieve and tightly coordinate this wide range of functions. Some actin regulator proteins have so-called house keeping roles and are essential for all eukaryotic cells, but some have evolved to meet the requirements of more specialized cell-types found in higher organisms enabling complex functions of differentiated organs, such as liver, kidney and brain. Often processes mediated by the actin cytoskeleton, like formation of cellular protrusions during cell migration, are intimately linked to plasma membrane remodeling. Thus, a close cooperation between these two cellular compartments is necessary, yet not much is known about the underlying molecular mechanisms. This study focused on a vertebrate-specific protein called missing-in-metastasis (MIM), which was originally characterized as a metastasis suppressor of bladder cancer. We demonstrated that MIM regulates the dynamics of actin cytoskeleton via its WH2 domain, and is expressed in a cell-type specific manner. Interestingly, further examination showed that the IM-domain of MIM displays a novel membrane tubulation activity, which induces formation of filopodia in cells. Following studies demonstrated that this membrane deformation activity is crucial for cell protrusions driven by MIM. In mammals, there are five members of IM-domain protein family. Functions and expression patterns of these family members have remained poorly characterized. To understand the physiological functions of MIM, we generated MIM knockout mice. MIM-deficient mice display no apparent developmental defects, but instead suffer from progressive renal disease and increased susceptibility to tumors. This indicates that MIM plays a role in the maintenance of specific physiological functions associated with distinct cell morphologies. Taken together, these studies implicate MIM both in the regulation of the actin cytoskeleton and the plasma membrane. Our results thus suggest that members of MIM/IRSp53 protein family coordinate the actin cytoskeleton:plasma membrane interface to control cell and tissue morphogenesis in multicellular organisms.

Structural and functional roles of KCC2 in developing cortex

Cation chloride cotransporters (CCCs) are critical for controlling intracellular chloride homeostasis. The CCC family is composed of four isoforms of K-Cl cotransporters (KCC1-4), two isoforms of Na-K-2Cl cotransporters (NKCC1-2), one Na-Cl cotransporter (NCC) and two the structurally related proteins with unknown function, CCC8 also known as cation-chloride cotransporter interaction protein, CIP, and CCC9. KCC2 is a neuron-specific isoform, which plays a prominent role in controlling the intracellular Cl- concentration in neurons and is responsible for producing the negative shift of GABAA responses from depolarizing to hyperpolarizing during neuronal maturation. In the present studies we first used in situ hybridization to examine the developmental expression patterns of the cation-chloride cotransporters KCC1-4 and NKCC1. We found that they display complementary expression patterns during embryonic brain development. Most interestingly, KCC2 expression in the embryonic central nervous system strictly follows neuronal maturation. In vitro data obtained from primary and organotypic neuronal cultures support this finding and revealed a temporal correlation between the expression of KCC2 and synaptogenesis. We found that KCC2 is highly expressed in filopodia and mature spines as well as dendritic shaft and investigated the role of KCC2 in spine formation by analyzing KCC2-/- neurons in vitro. Our studies revealed that KCC2 is a key factor in the maturation of dendritic spines. Interestingly, the effect of KCC2 in spine formation is not due to Cl- transport activity, but mediated through the interaction between KCC2 C-terminal and intracellular protein associated with cytoskeleton. The interacting protein we found is protein 4.1N by immunoprecipitation. Our results indicate a structural role for KCC2 in the development of functional glutamatergic synapses and suggest KCC2 as a synchronizer for the functional development of glutamatergic and GABAergic synapses in neuronal network. Studies on the regulatory mechanisms of KCC2 expression during development and plasticity revealed that synaptic activity of both the glutamatergic and GABAergic system is not required for up-regulation of KCC2 during development, whereas in acute mature hippocampal slices which undergo continuous synchronous activity induced by the absence of Mg2+ solution, KCC2 mRNA and protein expression were down-regulated in CA1 pyramidal neurons subsequently leading to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF-TrkB down-stream cascades involving both Shc/FRS-2 and PLCγ-CREB signaling. BDNF mediated changes in KCC2 expression indicate that KCC2 is significantly involved in the complex mechanisms of neuronal plasticity during development and pathophysiological conditions.

Ecological consequences of genetic modifications - an invasion analysis approach

Biological invasions are considered as one of the greatest threats to biodiversity, as they may lead to disruption and homogenization of natural communities, and in the worst case, to native species extinctions. The introduction of gene modified organisms (GMOs) to agricultural, fisheries and forestry practices brings them into contact with natural populations. GMOs may appear as new invasive species if they are able to (1) invade into natural habitats or (2) hybridize with their wild relatives. The benefits of GMOs, such as increased yield or decreased use of insecticides or herbicides in cultivation, may thus be reduced due the potential risks they may cause. A careful ecological risk analysis therefore has to precede any responsible GMO introduction. In this thesis I study ecological invasion in relation to GMOs, and what kind of consequences invasion may have in natural populations. A set of theoretical models that combine life-history evolution, population dynamics, and population genetics were developed for the hazard identification part of ecological risks assessment of GMOs. In addition, the potential benefits of GMOs in management of an invasive pest were analyzed. In the first study I showed that a population that is fluctuating due to scramble-type density dependence (due to, e.g., nutrient competition in plants) may be invaded by a population that is relatively more limited by a resource (e.g., light in plants) that is a cause of contest-type density dependence. This result emphasises the higher risk of invasion in unstable environments. The next two studies focused on escape of a growth hormone (GH) transgenic fish into a natural population. The results showed that previous models may have given too pessimistic a view of the so called Trojan gene -effect, where the invading genotype is harmful for the population as a whole. The previously suggested population extinctions did not occur in my studies, since the changes in mating preferences caused by the GH-fish were be ameliorated by decreased level of competition. The GH-invaders may also have to exceed a threshold density before invasion can be successful. I also showed that the prevalence of mature parr (aka. sneaker) strategy among GH-fish may have clear effect on invasion outcome. The fourth study assessed the risks and developed methods against the invasion of the Colorado Potato Beetle (CPB, Leptinotarsa decemlineata). I showed that the eradication of CPB is most important for the prevention of their establishment, but the cultivation of transgenic Bt-potato could also be effective. In general, my results emphasise that invasion of transgenic species or genotypes to be possible under certain realistic conditions and resulting in competitive exclusion, population decline through outbreeding depression and genotypic displacement of native species. Ecological risk assessment should regard the decline and displacement of the wild genotype by an introduced one as a consequence that is as serious as the population extinction. It will also be crucial to take into account different kinds of behavioural differences among species when assessing the possible hazards that GMOs may cause if escaped. The benefits found of GMO crops effectiveness in pest management may also be too optimistic since CPB may evolve resistance to Bt-toxin. The models in this thesis could be further applied in case specific risk assessment of GMOs by supplementing them with detailed data of the species biology, the effect of the transgene introduced to the species, and also the characteristics of the populations or the environments in the risk of being invaded.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Luonnonsuojelu arjessa : Maanomistajien näkemyksiä ja kokemuksia yksityismaiden tilapäisestä luonnonsuojelusta ja sen uudistumisen prosessista

Nature conservation in everyday life: Private landowners perceptions of and experiences on temporary nature conservation and its renewing process This study explores the legitimacy of official nature conservation of private lands. It describes how temporary nature conservation became a part of Finnish nature conservation policy and how forest owners perceived this tool and its usage. In addition, the study analyses forest owners' attitudes on official nature conservation. The study combines individual and official perspectives, and presents a nature conservation politics of everyday life. The theoretical background of the study is learning processes of environmentally responsible participation, and especially empowerment. Main methods in gathering the material for the study have been interviews and a survey. In the 1990 s, Finnish landowners opposed the implementation of nature conservation, especially conservation of shores and the establishment of the European-wide network of conservation areas (Natura 2000). After negative experiences on these conservation efforts, some private landowners were disempowered and some even rejected conservation completely. The Nature Conservation Act of 1996 launched an option to conserve nature officially for fixed time-periods. Use of such a policy tool did not immediately become a widely-used and appreciated conservation practice. During the following decade, however, it attracted remarkable attention within Finnish nature conservation policy discussions. The perspective of landowners began to be emphasised when local and regional organisations for nature conservation and forestry together defined the ideas of natural values trading. Later, the national governance process of the committee defining the Forest Biodiversity Programme for Southern Finland (METSO) institutionalised these ideas in one of its pilot projects. Landowners participated in the project of natural values trading by offering their forests for conservation, which reflects the increased acceptance and legitimacy of nature conservation on private forests. The central elements producing the legitimacy of natural values trading have been voluntariness, temporariness, and dialogue between nature conservation and forestry. Natural values trading analysed in the study is an example of new environmental policy instruments and its creation process represents governance in the implementation of nature conservation. It has increased the legitimacy of nature conservation policy of private forests in Finland. The results reveal the importance of participation and learning processes in the implementation of nature conservation policy, and the need to also pay attention to these processes in the future.

Cooperation and conflict in conspecific brood parasitism : an alternative reproductive tactic

Interactions among individuals give rise to both cooperation and conflict. Individuals will behave selfishly or altruistically depending on which gives the higher payoff. The reproductive strategies of many animals are flexible and several alternative tactics may be present from which the most suitable one is applied. Generally, alternative reproductive tactics may be defined as a response to competition from individuals of the same sex. These alternative reproductive tactics are means by which individuals may fine-tune their fitness to the reigning circumstances and which are shaped by the environment individuals are occupying as well as by the behaviour of other individuals sharing the environment. By employing such alternative ways of achieving reproductive output, individuals may alleviate competition from others. Conspecific brood parasitism (CBP) is an alternative reproductive strategy found in several egg laying animal groups, and it is especially common among waterfowl. Within this alternative reproductive strategy, four reproductive options can be identified. These four options represent a continuum from low reproductive effort coupled with low fitness returns, to high reproductive effort and consequently high benefits. It may not be evident how individuals should allocate reproductive effort between eggs laid in their own nest vs. in nests of others, however. Limited fecundity will constrain the number of eggs donated by a parasite, but also the tendency for hosts to accept parasitic eggs may affect the allocation decision. Furthermore, kinship, individual quality and the costs of breeding may play a role in complicating the allocation decision. In this thesis, I view the seemingly paradoxical effects of kinship on conflict resolution in the context of alternative reproductive tactics, examining the resulting features of cooperation and conflict. Conspecific brood parasitism sets the stage for investigating these questions. By using both empirical and theoretical approaches, I examine the nature of CBP in a brood parasitic duck, the Barrow's goldeneye (Bucephala islandica). The theoretical chapter of this thesis gives rise to four main conclusions. Firstly, variation in individual quality plays a central role in shaping breeding strategies. Secondly, kinship plays a central role in the evolution of CBP. Thirdly, egg recognition ability may affect the prevalence of parasitism. If egg recognition is perfect, higher relatedness between host and parasite facilitates CBP. Finally, I show that the relative costs of egg laying and post-laying care play a so far underestimated role in determining the prevalence of parasitism. The costs of breeding may outweigh possible inclusive fitness benefits accrued from receiving eggs from relatives. Several of the patterns brought out by the theoretical work are then confirmed empirically in the following chapters. Findings include confirmation of the central role of relatedness in determining the extent of parasitism as well as inducing a counterintuitive host clutch reduction. Furthermore, I demonstrate a cost of CBP inflicted on hosts, as well as results suggesting that host age reflects individual quality, affecting the ability to overcome costs inflicted by CBP. In summary, I demonstrate both theoretically and empirically the presence of cooperation and conflict in the interactions between conspecific parasites and their hosts. The field of CBP research has traditionally been divided, but the first steps have now been taken toward the acceptance of the opposite side of the divide. Especially the theoretical findings of chapter 1 offer the possibility to view seemingly contrasting results of various studies within the same framework, and may direct future research toward more general features underlying differences in the patterns of CBP between populations or species.