Pathogenic Mechanisms of Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy (PLOSL)

PATHOGENIC MECHANISMS OF PLOSL Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease, is a recessively inherited disease of brain and bone. PLOSL manifests as early-onset progressive dementia and bone fractures. Mutations in the TYROBP (DAP12) and TREM2 genes have been identified as the primary cause of PLOSL. DAP12 and TREM2 encode important signalling molecules in cells of the innate immune system. The mechanism by which loss-of-function of the DAP12/TREM2 signalling complex leads to PLOSL is currently unknown. The aim of this thesis work was to gain insight into the pathogenic mechanisms behind PLOSL. To first identify the central nervous system (CNS) cell types that express both Dap12 and Trem2, the expression patterns of Dap12 and Trem2 in mouse CNS were analyzed. Dap12 and Trem2 expression was seen from embryonic stage to adulthood and microglial cells and oligodendrocytes were identified as the major Dap12/Trem2 producing cells of the CNS. To subsequently identify the pathways and biological processes associated with DAP12/TREM2 mediated signalling in human cells, genome wide transcript analysis of in vitro differentiated dendritic cells (DCs) of PLOSL patients representing functional knockouts of either DAP12 or TREM2 was performed. Both DAP12 and TREM2 deficient cells differentiated into DCs and responded to pathogenic stimuli. However, the DCs showed morphological differences compared to control cells due to defects in the actin filaments. Transcript profiles of the patient DCs showed differential expression of genes involved in immune response and for genes earlier associated with other disorders of the CNS as well as genes involved in the remodeling of bone, linking the findings with the tissue phenotype of PLOSL patients. To analyze the effect of Dap12 deficiency in the CNS, genome wide expression analysis of Dap12 deficient mouse brain and Dap12 deficient microglia as well as functional analysis of Dap12 deficient microglia was performed. Regulation of several pathways involved in synaptic function and transcripts coding for the myelin components was seen in Dap12 knockout mice. Decreased migration, morphological changes and shortened lifespan of the Dap12 knockout microglia was further observed. Taken together, this thesis work showed that both Dap12 and Trem2 are expressed by CNS microglia and that Dap12 deficiency results in functional defects of these cells. Lack of Dap12 in the CNS also leads to synaptic abnormalities even before pathological changes are seen in the tissue level.This work further showed that loss-of-function of DAP12 or TREM2 leads to changes in morphology and gene expression in human dendritic cells. These data underline the functional diversity of the molecules of the innate immune system and implies their significant contribution also in demyelinating CNS disorders, including those resulting in dementia.

Innate immunity and its control in the pathogenesis of inflammatory rheumatic diseases

The pathogenesis of inflammatory rheumatic diseases, including rheumatoid arthritis (RA) and spondyloarthropathies (SpAs) such as reactive arthritis (ReA), is incompletely understood. ReA is a sterile joint inflammation, which may follow a distal infection caused by Gram-negative bacteria that have lipopolysaccharide (LPS) in their outer membrane. The functions of innate immunity that may affect the pathogenesis, prognosis and treatment of these diseases were studied in this thesis. When compared with healthy controls, whole blood monocytes of healthy subjects with previous ReA showed enhanced capacity to produce TNF, an essential proinflammatory cytokine, in response to adherent conditions (mimicking vascular endothelium made adherent by inflammatory signals) and non-specific protein kinase C stimulation. Also, blood neutrophils of these subjects showed high levels of CD11b, an important adhesion molecule, in response to adherence or LPS. Thus, high responsiveness of monocytes and neutrophils when encountering inflammatory stimuli may play a role in the pathogenesis of ReA. The results also suggested that the known risk allele for SpAs, HLA-B27, may be an additive contributor to the observed differences. The promoter polymorphisms TNF 308A and CD14 (gene for an LPS receptor component) 159T were found not to increase the risk of acute arthritis. However, all female patients who developed chronic SpA had 159T and none of them had 308A, possibly reflecting an interplay between hormonal and inflammatory signals in the development of chronic SpA. Among subjects with early RA, those having the polymorphic TLR4 +896G allele (causing the Asp299Gly change in TLR4, another component of LPS receptor) required a combination of disease-modifying antirheumatic drugs to achieve remission. It is known that rapid treatment response is essential in order to maintain the patients work ability. Hence, +896G might be a candidate marker for identifying the patients who need combination treatment. The production of vascular endothelial growth factor (VEGF), which strongly promotes vascular permeability and angiogenesis that takes place e.g. early in rheumatic joints, was induced by LPS and inhibited by interferon (IFN)-alpha in peripheral blood mononuclear cells. These long-living cells might provide a source of VEGF when stimulated by LPS and migrating to inflamed joints, and the effect of IFN-alpha may contribute to the clinical efficacy of this cytokine in inhibiting joint inflammation.

U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

Human genetic variation in the Baltic Sea region: Features of population history and natural selection

In this thesis, the genetic variation of human populations from the Baltic Sea region was studied in order to elucidate population history as well as evolutionary adaptation in this region. The study provided novel understanding of how the complex population level processes of migration, genetic drift, and natural selection have shaped genetic variation in North European populations. Results from genome-wide, mitochondrial DNA and Y-chromosomal analyses suggested that the genetic background of the populations of the Baltic Sea region lies predominantly in Continental Europe, which is consistent with earlier studies and archaeological evidence. The late settlement of Fennoscandia after the Ice Age and the subsequent small population size have led to pronounced genetic drift, especially in Finland and Karelia but also in Sweden, evident especially in genome-wide and Y-chromosomal analyses. Consequently, these populations show striking genetic differentiation, as opposed to much more homogeneous pattern of variation in Central European populations. Additionally, the eastern side of the Baltic Sea was observed to have experienced eastern influence in the genome-wide data as well as in mitochondrial DNA and Y-chromosomal variation – consistent with linguistic connections. However, Slavic influence in the Baltic Sea populations appears minor on genetic level. While the genetic diversity of the Finnish population overall was low, genome-wide and Y-chromosomal results showed pronounced regional differences. The genetic distance between Western and Eastern Finland was larger than for many geographically distant population pairs, and provinces also showed genetic differences. This is probably mainly due to the late settlement of Eastern Finland and local isolation, although differences in ancestral migration waves may contribute to this, too. In contrast, mitochondrial DNA and Y-chromosomal analyses of the contemporary Swedish population revealed a much less pronounced population structure and a fusion of the traces of ancient admixture, genetic drift, and recent immigration. Genome-wide datasets also provide a resource for studying the adaptive evolution of human populations. This study revealed tens of loci with strong signs of recent positive selection in Northern Europe. These results provide interesting targets for future research on evolutionary adaptation, and may be important for understanding the background of disease-causing variants in human populations.

The genome packaging machinery of dsDNA bacteriophage PRD1

Viral genomes are encapsidated within protective protein shells. This encapsidation can be achieved either by a co-condensation reaction of the nucleic acid and coat proteins, or by first forming empty viral particles which are subsequently packaged with nucleic acid, the latter mechanism being typical for many dsDNA bacteriophages. Bacteriophage PRD1 is an icosahedral, non-tailed dsDNA virus that has an internal lipid membrane, the hallmark of the Tectiviridae family. Although PRD1 has been known to assemble empty particles into which the genome is subsequently packaged, the mechanism for this has been unknown, and there has been no evidence for a separate packaging vertex, similar to the portal structures used for packaging in the tailed bacteriophages and herpesviruses. In this study, a unique DNA packaging vertex was identified for PRD1, containing the packaging ATPase P9, packaging factor P6 and two small membrane proteins, P20 and P22, extending the packaging vertex to the internal membrane. Lack of small membrane protein P20 was shown to totally abolish packaging, making it an essential part of the PRD1 packaging mechanism. The minor capsid proteins P6 was shown to be an important packaging factor, its absence leading to greatly reduced packaging efficiency. An in vitro DNA packaging mechanism consisting of recombinant packaging ATPase P9, empty procapsids and mutant PRD1 DNA with a LacZ-insert was developed for the analysis of PRD1 packaging, the first such system ever for a virus containing an internal membrane. A new tectiviral sequence, a linear plasmid called pBClin15, was identified in Bacillus cereus, providing material for sequence analysis of the tectiviruses. Analysis of PRD1 P9 and other putative tectiviral ATPase sequences revealed several conserved sequence motifs, among them a new tectiviral packaging ATPase motif. Mutagenesis studies on PRD1 P9 were used to confirm the significance of the motifs. P9-type putative ATPase sequences carrying a similar sequence motif were identified in several other membrane containing dsDNA viruses of bacterial, archaeal and eukaryotic hosts, suggesting that these viruses may have similar packaging mechanisms. Interestingly, almost the same set of viruses that were found to have similar putative packaging ATPases had earlier been found to share similar coat protein folds and capsid structures, and a common origin for these viruses had been suggested. The finding in this study of similar packaging proteins further supports the idea that these viruses are descendants of a common ancestor.

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

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.

Multifunctional RNA silencing pathway polymerase QDE-1 of Neurospora crassa

Double-stranded RNA and associated proteins are known to regulate the gene expression of most eukaryotic organisms. These regulation pathways have different components, outcomes and distinct nomenclature depending on the model system, and often they are referred to collectively as RNA silencing. In many cases, RNA-dependent RNA polymerases (RdRPs) are found to be involved in the RNA silencing, but their targets, activities, interaction partners and reaction products remain enigmatic. In the filamentous fungus Neurospora crassa, the RdRP QDE-1 is critical for silencing of transgenes a phenomenon known as quelling. In this thesis the structure, biochemical activities and biological functions of QDE-1 were extensively studied. This dimeric RdRP was shown to possess five distinct catalytic in vitro activities that could be dissected by mutagenesis and by altering reaction conditions. The biochemical characterization implied that QDE-1 is actually an active DNA-dependent RNA polymerase that has additional RdRP activity. It also provided a structural explanation for the dimerization and suggested a biological framework for the functions of QDE-1 in vivo. (I) QDE-1 was also studied in a broader context along with the other components of the quelling pathway. It was shown that DNA damage in Neurospora causes a dramatic increase in the expression level of the Argonaute protein QDE-2 as well as the synthesis of a novel class of small RNAs known as qiRNAs. The accumulation of qiRNAs was shown to be dependent on several quelling components, and particularly to be derived from an aberrant ssRNA (aRNA) molecule that is synthesized by QDE-1 in the nucleus. The genomic distribution of qiRNA targets was analyzed and the possible biological significance of qiRNAs was studied. Importantly, qiRNAs are the first class of small RNAs that are induced by DNA damage. (II) After establishing that QDE-1 is a multifunctional RNA polymerase with several activities, template specificities and subcellular locations, the focus was turned onto its interaction partners. It had been previously known that QDE-1 associates with Replication Protein A (RPA), but the RecQ helicase QDE-3 was now shown to regulate this interaction. RPA was also observed to promote QDE-1 dependent dsRNA synthesis in vitro. By characterizing the interplay between QDE-1, QDE-3 and RPA, a working model of quelling and qiRNA pathways in Neurospora was presented. (III) This work sheds light on the complexity of the various RNA silencing pathways of a fungal model system. It shows how an RdRP can regulate gene expression on many levels, and suggests novel lines of research in other eukaryotic organisms.

Molecular details of phage phi6 RNA-dependent RNA synthesis

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.

Molecular epidemiology of human rhinoviruses

The first part of this work investigates the molecular epidemiology of a human enterovirus (HEV), echovirus 30 (E-30). This project is part of a series of studies performed in our research team analyzing the molecular epidemiology of HEV-B viruses. A total of 129 virus strains had been isolated in different parts of Europe. The sequence analysis was performed in three different genomic regions: 420 nucleotides (nt) in the VP4/VP2 capsid protein coding region, the entire VP1 capsid protein coding gene of 876 nt, and 150 nt in the VP1/2A junction region. The analysis revealed a succession of dominant sublineages within a major genotype. The temporally earlier genotypes had been replaced by a genetically homogenous lineage that has been circulating in Europe since the late 1970s. The same genotype was found by other research groups in North America and Australia. Globally, other cocirculating genetic lineages also exist. The prevalence of a dominant genotype makes E-30 different from other previously studied HEVs, such as polioviruses and coxsackieviruses B4 and B5, for which several coexisting genetic lineages have been reported. The second part of this work deals with molecular epidemiology of human rhinoviruses (HRVs). A total of 61 field isolates were studied in the 420-nt stretch in the capsid coding region of VP4/VP2. The isolates were collected from children under two years of age in Tampere, Finland. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades. Seasonal clustering was found. Also, several distinct serotype-like clusters were found to co-circulate during the same epidemic season. Reappearance of a cluster after disappearing for a season was observed. The molecular epidemiology of the analyzed strains turned out to be complex, and we decided to continue our studies of HRV. Only five previously published complete genome sequences of HRV prototype strains were available for analysis. Therefore, all designated HRV prototype strains (n=102) were sequenced in the VP4/VP2 region, and the possibility of genetic typing of HRV was evaluated. Seventy-six of the 102 prototype strains clustered in HRV genetic group A (HRV-A) and 25 in group B (HRV-B). Serotype 87 clustered separately from other HRVs with HEV species D. The field strains of HRV represented as many as 19 different genotypes, as judged with an approximate demarcation of a 20% nt difference in the VP4/VP2 region. The interserotypic differences of HRV were generally similar to those reported between different HEV serotypes (i.e. about 20%), but smaller differences, less than 10%, were also observed. Because some HRV serotypes are genetically so closely related, we suggest that the genetic typing be performed using the criterion "the closest prototype strain". This study is the first systematic genetic characterization of all known HRV prototype strains, providing a further taxonomic proposal for classification of HRV. We proposed to divide the genus Human rhinoviruses into HRV-A and HRV-B. The final part of the work comprises a phylogenetic analysis of a subset (48) of HRV prototype strains and field isolates (12) in the nonstructural part of the genome coding for the RNA-dependent RNA polymerase (3D). The proposed division of the HRV strains in the species HRV-A and HRV-B was also supported by 3D region. HRV-B clustered closer to HEV species B, C, and also to polioviruses than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D coding region than in the VP4/VP2 coding region, in contrast to HEV. Moreover, the diversity of HRV in 3D exceeded that of HEV. One group of HRV-A, designated HRV-A', formed a separate cluster outside other HRV-A in the 3D region. It formed a cluster also in the capsid region, but located within HRV-A. This may reflect a different evolutionary history of distinct genomic regions among HRV-A. Furthermore, the tree topology within HRV-A in the 3D region differed from that in the VP4/VP2, suggesting possible recombination events in the evolution of the strains. No conflicting phylogenies were observed in any of the 12 field isolates. Possible recombination was further studied using the Similarity and Bootscanning analyses of the complete genome sequences of HRV available in public databases. Evidence for recombination among HRV-A was found, as HRV2 and HRV39 showed higher similarity in the nonstructural part of the genome. Whether HRV2 and HRV39 strains - and perhaps also some other HRV-A strains not yet completely sequenced - are recombinants remains to be determined.

Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae

Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level. In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function. The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose. The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.