Evolutionary Genomics of Prokaryotic Viruses

Evolutionary history of biological entities is recorded within their nucleic acid sequences and can (sometimes) be deciphered by thorough genomic analysis. In this study we sought to gain insights into the diversity and evolution of bacterial and archaeal viruses. Our primary interest was pointed towards those virus groups/families for which comprehensive genomic analysis was not previously possible due to the lack of sufficient amount of genomic data. During the course of this work twenty-five putative proviruses integrated into various prokaryotic genomes were identified, enabling us to undertake a comparative genomics approach. This analysis allowed us to test the previously formulated evolutionary hypotheses and also provided valuable information on the molecular mechanisms behind the genome evolution of the studied virus groups.

Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

Transposable elements in a marginal plant population

Background
How new forms arise in nature has engaged evolutionary biologists since Darwin's seminal treatise on the origin of species. Transposable elements (TEs) may be among the most important internal sources for intraspecific variability. Thus, we aimed to explore the temporal dynamics of several TEs in individual genotypes from a small, marginal population of Aegilops speltoides. A diploid cross-pollinated grass species, it is a wild relative of the various wheat species known for their large genome sizes contributed by an extraordinary number of TEs, particularly long terminal repeat (LTR) retrotransposons. The population is characterized by high heteromorphy and possesses a wide spectrum of chromosomal abnormalities including supernumerary chromosomes, heterozygosity for translocations, and variability in the chromosomal position or number of 45S and 5S ribosomal DNA (rDNA) sites. We propose that variability on the morphological and chromosomal levels may be linked to variability at the molecular level and particularly in TE proliferation.

Results
Significant temporal fluctuation in the copy number of TEs was detected when processes that take place in small, marginal populations were simulated. It is known that under critical external conditions, outcrossing plants very often transit to self-pollination. Thus, three morphologically different genotypes with chromosomal aberrations were taken from a wild population of Ae. speltoides, and the dynamics of the TE complex traced through three rounds of selfing. It was discovered that: (i) various families of TEs vary tremendously in copy number between individuals from the same population and the selfed progenies; (ii) the fluctuations in copy number are TE-family specific; (iii) there is a great difference in TE copy number expansion or contraction between gametophytes and sporophytes; and (iv) a small percentage of TEs that increase in copy number can actually insert at novel locations and could serve as a bona fide mutagen.

Conclusions
We hypothesize that TE dynamics could promote or intensify morphological and karyotypical changes, some of which may be potentially important for the process of microevolution, and allow species with plastic genomes to survive as new forms or even species in times of rapid climatic change.

Molecular genetics and evolution of Puumala hantavirus

Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome. Finland has the highest documented incidence of NE with around 1000 cases diagnosed annually. PUUV is also found in other Scandinavian countries, Central Europe and the European part of Russia. PUUV belongs to the genus Hantavirus in the family Bunyaviridae. Hantaviruses are rodent-borne viruses each carried by a specific host that is persistently and asymptomatically infected by the virus. PUUV is carried by the bank voles (Myodes glareolus, previously known as Clethrionomys glareolus). Hantaviruses have co-evolved with their carrier rodents for millions of years and these host animals are the evolutionary scene of hantaviruses. In this study, PUUV sequences were recovered from bank voles captured in Denmark and Russian Karelia to study the evolution of PUUV in Scandinavia. Phylogenetic analysis of these strains showed a geographical clustering of genetic variants following the presumable migration pattern of bank voles during the recolonization of Scandinavia after the last ice age approximately 10 000 years ago. The currently known PUUV genome sequences were subjected to in-depth phylogenetic analyses and the results showed that genetic drift seems to be the major mechanism of PUUV evolution. In general, PUUV seems to evolve quite slowly following a molecular clock. We also found evidence for recombination in the evolution of some genetic lineages of PUUV. Viral microevolution was studied in controlled virus transmission in colonized bank voles and changes in quasispecies dynamics were recorded as the virus was transmitted from one animal to another. We witnessed PUUV evolution in vivo, as one synonymous mutation became repeatedly fixed in the viral genome during the experiment. The detailed knowledge on the PUUV diversity was used to establish new sensitive and specific detection methods for this virus. Direct viral invasion of the hypophysis was demonstrated for the first time in a lethal case of NE. PUUV detection was done by immunohistochemistry, in situ hybridization and RT-nested-PCR of the autopsy tissue samples.

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.

Karl Popper's Philosophy of Science - And the Evolution of the Popperian Worlds

This doctoral thesis in theoretical philosophy is a systematic analysis of Karl Popper's philosophy of science and its relation to his theory of three worlds. The general aim is to study Popper's philosophy of science and to show that Popper's theory of three worlds was a restatement of his earlier positions. As a result, a new reading of Popper's philosophy and development is offered and the theory of three worlds is analysed in a new manner. It is suggested that the theory of three worlds is not purely an ontological theory, but has a profound epistemological motivation. In Part One, Popper's epistemology and philosophy of science is analysed. It is claimed that Popper's thinking was bifurcated: he held two profound positions without noticing the tension between them. Popper adopted the position called the theorist around 1930 and focused on the logical structure of scientific theories. In Logik der Forschung (1935), he attempted to build a logic of science on the grounds that scientific theories may be regarded as universal statements which are not verifiable but can be falsified. Later, Popper emphasized another position, called here the processionalist. Popper focused on the study of science as a process and held that a) philosophy of science should study the growth of knowledge and that b) all cognitive processes are constitutive. Moreover, the constitutive idea that we see the world in the searchlight of our theories was combined with the biological insight that knowledge grows by trial and error. In Part Two, the theory of three worlds is analysed systematically. The theory is discussed as a cluster of theories which originate from Popper's attempt to solve some internal problems in his thinking. Popper adhered to realism and wished to reconcile the theorist and the processionalist. He also stressed the real and active nature of the human mind, and the possibility of objective knowledge. Finally, he wished to create a scientific world view.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com

Evolution and detection of cyanobacterial hepatotoxin synthetase genes

Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are Microcystis, Anabaena, and Planktothrix. Nodularins are produced by strains of Nodularia spumigena in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (mcyE) and the orthologous nodularin synthetase gene F (ndaF) sequences. General primers were designed to amplify the mcyE/ndaF gene region from microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that mcy genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that nda genes evolved from mcy genes. The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general mcyE primers, and in 91% of the lakes with the three genus-specific mcyE primers. Potential microcystin-producing Microcystis were detected in 70%, Planktothrix in 63%, and Anabaena in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of mcyE genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. Genus-specific probe pairs for microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the mcyE/ndaF genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. Analysis of the mcy and nda gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of Microcystis, Planktothrix, and Anabaena, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.

Magmatic evolution of topaz-bearing granite stocks within the Wiborg rapakivi granite batholith

This study brings new insights into the magmatic evolution of natural F-enriched peraluminous granitic systems. The Artjärvi, Sääskjärvi and Kymi granite stocks within the 1.64 Ga Wiborg rapakivi granite batholith have been investigated by petrographic, geochemical, experimental and melt inclusion methods. These stocks represent late-stage leucocratic and weakly peraluminous intrusive phases typical of rapakivi granites worldwide. The Artjärvi and Sääskjärvi stocks are multiphase intrusions in which the most evolved phase is topaz granite. The Kymi stock contains topaz throughout and has a well-developed zoned structure, from the rim to the center: stockscheider pegmatite equigranular topaz granite porphyritic topaz granite. Geochemically the topaz granites are enriched in F, Li, Be, Ga, Rb, Sn and Nb and depleted in Mg, Fe, Ti, Ba, Sr, Zr and Eu. The anomalous geochemistry and mineralogy of the topaz granites are essentially magmatic in origin; postmagmatic reactions have only slightly modified the compositions. The Kymi equigranular topaz granite shows the most evolved character, and the topaz granites at Artjärvi and Sääskjärvi resemble the less evolved porphyritic topaz granite of the Kymi stock. Stockscheiders are found at the roof contacts of the Artjärvi and Kymi stocks. The stockscheider at Artjärvi is composed of biotite-rich schlieren and pegmatite layers parallel to the contact. The schlieren layering is considered to have formed by velocity-gradient sorting mechanism parallel to the flow, which led to the accumulation of mafic minerals along the upper contact of the topaz granite. Cooling and contraction of the topaz granite formed fractures parallel to the roof contact and residual pegmatite magmas were injected along the fractures and formed the pegmatite layers. The zoned structure of the Kymi stock is the result of intrusion of highly evolved residual melt from deeper parts of the magma chamber along the fractured contact between the porphyritic granite crystal mush and country rock. The equigranular topaz granite and marginal pegmatite (stockscheider) crystallized from this evolved melt. Phase relations of the Kymi equigranular topaz granite have been investigated utilizing crystallization experiments at 100 to 500 MPa as a function of water activity and F content. Fluorite and topaz can crystallize as liquidus phases in F-rich peraluminous systems, but the F content of the melt should exceed 2.5 - 3.0 wt % to facilitate crystallization of topaz. In peraluminous F-bearing melts containing more than 1 wt % F, topaz and muscovite are expected to be the first F-bearing phases to crystallize at high pressure, whereas fluorite and topaz should crystallize first at low pressure. Overall, the saturation of fluorite and topaz follows the reaction: CaAl2Si2O8 (plagioclase) + 2[AlF3]melt = CaF2 (fluorite) + 2Al2SiO4F2 (topaz). The obtained partition coefficient for F between biotite and glass D(F)Bt/glass is 1.89 to 0.80 (average 1.29) and can be used as an empirical fluormeter to determine the F content of coexisting melts. In order to study the magmatic evolution of the Kymi stock, crystallized melt inclusions in quartz and topaz grains in the porphyritic and the equigranular topaz granites and the marginal pegmatite were rehomogenized and analyzed. The homogenization conditions for the melt inclusions from the granites were 700 °C, 300 MPa, and 24 h, and for melt inclusions from the pegmatite, 700 °C, 100 MPa, and 24/96 h. The majority of the melt inclusions is chemically similar to the bulk rocks (excluding H2O content), but a few melt inclusions in the equigranular granite show clearly higher F and low K2O contents (on average 11.6 wt % F, 0.65 wt % K2O). The melt inclusion compositions indicate coexistence of two melt fractions, a prevailing peraluminous and a very volatile-rich, possibly peralkaline. Combined petrological, experimental and melt inclusion studies of the Kymi equigranular topaz granite indicate that plagioclase was the liquidus phase at nearly water-saturated (fluid-saturated) conditions and that the F content of the melt was at least 2 wt %. The early crystallization of biotite and the presence of muscovite in crystallization experiments at 200 MPa contrasts with the late-stage crystallization of biotite and the absence of muscovite in the equigranular granite, indicating that crystallization pressure may have been lower than 200 MPa for the granite.

Archaean crustal evolution in eastern Finland : New geochronological and geochemical constraints from the Kuhmo terrain and the Nurmes belt

This thesis summarises the results of four original papers concerning U-Pb geochronology and geochemical evolution of Archaean rocks from the Kuhmo terrain and the Nurmes belt, eastern Finland. The study area belongs to a typical Archaean granite-greenstone terrain, composed of metavolcanic and metasedimentary rocks in generally N-S trending greenstone belts as well as a granitoid-gneiss complex with intervening gneissic and migmatised supracrustal and plutonic rocks. U-Pb data on migmatite mesosomes indicate that the crust surrounding the Tipasjärvi-Kuhmo-Suomussalmi greenstone belt is of varying age. The oldest protolith detected for a migmatite mesosome from the granitoid-gneiss complex is 2.94 Ga, whereas the other dated migmatites protoliths have ages of 2.84 2.79 Ga. The latter protoliths are syngenetic with the majority of volcanic rocks in the adjacent Tipasjärvi-Kuhmo-Suomussalmi greenstone belt. This suggests that the genesis of some of the volcanic rocks within the greenstone belt and surrounding migmatite protoliths could be linked. Metamorphic zircon overgrowths with ages of 2.84 2.81 Ga were also obtained. The non-migmatised plutonic rocks in the Kuhmo terrain and in the Nurmes belt record secular geochemical evolution, typical of Archaean cratons. The studied tonalitic rocks have ages of 2.83 2.75 Ga and they have geochemical characteristics similar to low-Al and high-Al TTD (tonalite-trondhjemite-dacite). The granodiorites, diorites, and gabbros with high Mg/Fe and LILE-enriched characteristics were mostly emplaced between 2.74 2.70 Ga and they exhibit geochemical characteristics typical of Archaean sanukitoid suites. The latest identified plutonic episode took place at 2.70 2.68 Ga, when compositionally heterogeneous leucocratic granitoid rocks, with a variable crustal component, were emplaced. U-Pb data on migmatite leucosomes suggest that leucosome generation may have been coeval with this latest plutonic event. On the basis of available U-Pb and Sm-Nd isotopic data it appears that the plutonic rocks of the Kuhmo terrain and the Nurmes belt do not contain any significant input from Palaeoarchaean sources. A characteristic feature of the Nurmes belt is the presence of migmatised paragneisses, locally preserving primary edimentary structures, with sporadic amphibolite intercalations. U-Pb studies on zircons indicate that the precursors of the Nurmes paragneisses were graywackes that were deposited between 2.71 Ga and 2.69 Ga and that they had a prominent 2.75 2.70 Ga source. Nd isotopic and whole-rock geochemical data for the intercalated amphibolites imply MORB sources. U-Pb data on zircons from the plutonic rocks and paragneisses reveal that metamorphic zircon growth took place at 2.72 2.63 Ga. This was the last tectonothermal event related to cratonisation of the Archaean crust of eastern Finland.

Bayesian Inference for Retrospective Population Genetics Models Using Markov Chain Monte Carlo Methods

Genetics, the science of heredity and variation in living organisms, has a central role in medicine, in breeding crops and livestock, and in studying fundamental topics of biological sciences such as evolution and cell functioning. Currently the field of genetics is under a rapid development because of the recent advances in technologies by which molecular data can be obtained from living organisms. In order that most information from such data can be extracted, the analyses need to be carried out using statistical models that are tailored to take account of the particular genetic processes. In this thesis we formulate and analyze Bayesian models for genetic marker data of contemporary individuals. The major focus is on the modeling of the unobserved recent ancestry of the sampled individuals (say, for tens of generations or so), which is carried out by using explicit probabilistic reconstructions of the pedigree structures accompanied by the gene flows at the marker loci. For such a recent history, the recombination process is the major genetic force that shapes the genomes of the individuals, and it is included in the model by assuming that the recombination fractions between the adjacent markers are known. The posterior distribution of the unobserved history of the individuals is studied conditionally on the observed marker data by using a Markov chain Monte Carlo algorithm (MCMC). The example analyses consider estimation of the population structure, relatedness structure (both at the level of whole genomes as well as at each marker separately), and haplotype configurations. For situations where the pedigree structure is partially known, an algorithm to create an initial state for the MCMC algorithm is given. Furthermore, the thesis includes an extension of the model for the recent genetic history to situations where also a quantitative phenotype has been measured from the contemporary individuals. In that case the goal is to identify positions on the genome that affect the observed phenotypic values. This task is carried out within the Bayesian framework, where the number and the relative effects of the quantitative trait loci are treated as random variables whose posterior distribution is studied conditionally on the observed genetic and phenotypic data. In addition, the thesis contains an extension of a widely-used haplotyping method, the PHASE algorithm, to settings where genetic material from several individuals has been pooled together, and the allele frequencies of each pool are determined in a single genotyping.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

From Francovich to Köbler and beyond : The evolution of a State liability regime for the EC

The aim of this paper is to present the evolution of the Francovich doctrine within the European legal order. The first part deals with the gradual development of the ECJ's case law on State liability in damages for breach of EC law. Starting from the seminal Francovich and Brasserie du Pêcheur, the clarification of the criteria set by the Court is attempted with reference to subsequent case law, whereas issues concerning the extent and form of the compensation owned are also mentioned. The second part concerns one of the more recent developments in the field, namely State liability for breaches of Community law attributed to national judiciary. The Court's ruling in Köbler is examined in connection with two other recent judgments, namely Commission v. Italy of 2003 and Kühne & Heitz, as an attempt of the ECJ to reframe its relationships with national supreme courts and appropriate for itself the position of the Supreme Court in the European legal order. The implications on State liability claims by the ruling in Commission v. France of 1997 constitute the theme of the third part, where it is submitted that Member States can also be held liable for disregard of Community law by private individuals within their respected territories. To this extent, Schmidberger is viewed as a manifestation of this opinion, with fundamental rights acquiring a new dimension, being invoked by the States, contra the individuals as a shield to liability claims. Finally, the third part examines the relationship between the Francovich doctrine and the principle of legal certainty and concludes that the solutions employed by the ECJ have been both predictable and acceptable by the national legal orders. Keywords: State liability, damages, Francovich, Köbler, Schmidberger