Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

The range expansion of the European map butterfly in Finland

Climate change will influence the living conditions of all life on Earth. For some species the change in the environmental conditions that has occurred so far has already increased the risk of extinction, and the extinction risk is predicted to increase for large numbers of species in the future. Some species may have time to adapt to the changing environmental conditions, but the rate and magnitude of the change are too great to allow many species to survive via evolutionary changes. Species responses to climate change have been documented for some decades. Some groups of species, like many insects, respond readily to changes in temperature conditions and have shifted their distributions northwards to new climatically suitable regions. Such range shifts have been well documented especially in temperate zones. In this context, butterflies have been studied more than any other group of species, partly for the reason that their past geographical ranges are well documented, which facilitates species-climate modelling and other analyses. The aim of the modelling studies is to examine to what extent shifts in species distributions can be explained by climatic and other factors. Models can also be used to predict the future distributions of species. In this thesis, I have studied the response to climate change of one species of butterfly within one geographically restricted area. The study species, the European map butterfly (Araschnia levana), has expanded rapidly northwards in Finland during the last two decades. I used statistical and dynamic modelling approaches in combination with field studies to analyse the effects of climate warming and landscape structure on the expansion. I studied possible role of molecular variation in phosphoglucose isomerase (PGI), a glycolytic enzyme affecting flight metabolism and thereby flight performance, in the observed expansion of the map butterfly at two separate expansion fronts in Finland. The expansion rate of the map butterfly was shown to be correlated with the frequency of warmer than average summers during the study period. The result is in line with the greater probability of occurrence of the second generation during warm summers and previous results on this species showing greater mobility of the second than first generation individuals. The results of a field study in this thesis indicated low mobility of the first generation butterflies. Climatic variables alone were not sufficient to explain the observed expansion in Finland. There are also problems in transferring the climate model to new regions from the ones from which data were available to construct the model. The climate model predicted a wider distribution in the south-western part of Finland than what has been observed. Dynamic modelling of the expansion in response to landscape structure suggested that habitat and landscape structure influence the rate of expansion. In southern Finland the landscape structure may have slowed down the expansion rate. The results on PGI suggested that allelic variation in this enzyme may influence flight performance and thereby the rate of expansion. Genetic differences of the populations at the two expansion fronts may explain at least partly the observed differences in the rate of expansion. Individuals with the genotype associated with high flight metabolic rate were most frequent in eastern Finland, where the rate of range expansion has been highest.

Effects of oxygen provision on the physiology of baker's yeast Saccharomyces cerevisiae

The availability of oxygen has a major effect on all organisms. The yeast Saccharomyces cerevisiae is able to adapt its metabolism for growth in different conditions of oxygen provision, and to grow even under complete lack of oxygen. Although the physiology of S. cerevisiae has mainly been studied under fully aerobic and anaerobic conditions, less is known of metabolism under oxygen-limited conditions and of the adaptation to changing conditions of oxygen provision. This study compared the physiology of S. cerevisiae in conditions of five levels of oxygen provision (0, 0.5, 1.0, 2.8 and 20.9% O2 in feed gas) by using measurements on metabolite, transcriptome and proteome levels. On the transcriptional level, the main differences were observed between the three level groups, 0, 0.5 2.8 and 20.9% O2 which led to fully fermentative, respiro-fermentative and fully respiratory modes of metabolism, respectively. However, proteome analysis suggested post-transcriptional regulation at the level of 0.5 O2. The analysis of metabolite and transcript levels of central carbon metabolism also suggested post-transcriptional regulation especially in glycolysis. Further, a global upregulation of genes related to respiratory pathways was observed in the oxygen-limited conditions and the same trend was seen in the proteome analysis and in the activities of enzymes of the TCA cycle. The responses of intracellular metabolites related to central carbon metabolism and transcriptional responses to change in oxygen availability were studied. As a response to sudden oxygen depletion, concentrations of the metabolites of central carbon metabolism responded faster than the corresponding levels of gene expression. In general, the genome-wide transcriptional responses to oxygen depletion were highly similar when two different initial conditions of oxygen provision (20.9 and 1.0% O2) were compared. The genes related to growth and cell proliferation were transiently downregulated whereas the genes related to protein degradation and phosphate uptake were transiently upregulated. In the cultures initially receiving 1.0% O2, a transient upregulation of genes related to fatty acid oxidation, peroxisomal biogenesis, response to oxidative stress and pentose phosphate pathway was observed. Additionally, this work analysed the effect of oxygen on transcription of genes belonging to the hexose transporter gene family. Although the specific glucose uptake rate was highest in fully anaerobic conditions, none of the hxt genes showed highest expression in anaerobic conditions. However, the expression of genes encoding the moderately low affinity transporters decreased with the decreasing oxygen level. Thus it was concluded that there is a relative increase in high affinity transport in anaerobic conditions supporting the high uptake rate.

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 high- and low-activity forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays a crucial role in high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT). It mediates the generation of pre-beta-HDL particles, enhances the cholesterol efflux from peripheral cells to pre-beta-HDL, and metabolically maintains the plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. In addition to the antiatherogenic properties, recent findings indicate that PLTP has also proatherogenic characteristics, and that these opposite characteristics of PLTP are dependent on the site of PLTP expression and action. In human plasma, PLTP exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP), which are associated with macromolecular complexes of different size and composition. The aims of this thesis were to isolate the two PLTP forms from human plasma, to characterize the molecular complexes in which the HA- and LA-PLTP reside, and to study the interactions of the PLTP forms with apolipoproteins (apo) and the ability of apolipoproteins to regulate PLTP activity. In addition, we aimed to study the distribution of the two PLTP forms in a Finnish population sample as well as to find possible regulatory factors for PLTP by investigating the influence of lipid and glucose metabolism on the balance between the HA- and LA-PLTP. For these purposes, an enzyme-linked immunosorbent assay (ELISA) capable of determining the serum total PLTP concentration and quantitating the two PLTP forms separately was developed. In this thesis, it was demonstrated that the HA-PLTP isolated from human plasma copurified with apoE, whereas the LA-PLTP formed a complex with apoA-I. The separation of these two PLTP forms was carried out by a dextran sulfate (DxSO4)-CaCl2 precipitation of plasma samples before the mass determination. A similar immunoreactivity of the two PLTP forms in the ELISA could be reached after a partial sample denaturation by SDS. Among normolipidemic Finnish individuals, the mean PLTP mass was 6.6 +/- 1.5 mg/l and the mean PLTP activity 6.6 +/- 1.7 umol/ml/h. Of the serum PLTP concentration, almost 50% represented HA-PLTP. The results indicate that plasma HDL levels could regulate PLTP concentration, while PLTP activity could be regulated by plasma triglyceride-rich very low-density lipoprotein (VLDL) concentration. Furthermore, new evidence is presented that PLTP could also play a role in glucose metabolism. Finally, both PLTP forms were found to interact with apoA-I, apoA-IV, and apoE. In addition, both apoE and apoA-IV, but not apoA-I, were capable of activating the LA-PLTP. These findings suggest that the distribution of the HA- and LA-PLTP in human plasma is subject to dynamic regulation by apolipoproteins.

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

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

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

The biological functions of mouse twinfilin isoforms

The actin cytoskeleton is essential for many cellular processes, including motility, morphogenesis, endocytosis and signal transduction. Actin can exist in monomeric (G-actin) or filamentous (F-actin) form. Actin filaments are considered to be the functional form of actin, generating the protrusive forces characteristic for the actin cytoskeleton. The structure and dynamics of the actin filament and monomer pools are regulated by a large number of actin-binding proteins in eukaryotic cells. Twinfilin is an evolutionarily conserved small actin monomer binding protein. Twinfilin is composed of two ADF/cofilin-like domains, separated by a short linker and followed by a C-terminal tail. Twinfilin forms a stable, high affinity complex with ADP-G-actin, inhibits the nucleotide exchange on actin monomers, and prevents their assembly into filament ends. Twinfilin was originally identified from yeast and has since then been found from all organisms studied except plants. Not much was known about the role of twinfilin in the actin dynamics in mammalian cells before this study. We set out to unravel the mysteries still covering twinfilins functions using biochemistry, cell biology, and genetics. We identified and characterized two mouse isoforms for the previously identified mouse twinfilin-1. The new isoforms, twinfilin-2a and -2b, are generated from the same gene through alternative promoter usage. The three isoforms have distinctive expression patterns, but are similar biochemically. Twinfilin-1 is the major isoform during development and is expressed in high levels in almost all tissues examined. Twinfilin-2a is also expressed almost ubiquitously, but at lower levels. Twinfilin-2b turned out to be a muscle-specific isoform, with very high expression in heart and skeletal muscle. It seems all mouse tissues express at least two twinfilin isoforms, indicating that twinfilins are important regulators of actin dynamics in all cell and tissue types. A knockout mouse line was generated for twinfilin-2a. The mice homozygous for this knockout were viable and developed normally, indicating that twinfilin-2a is dispensable for mouse development. However, it is important to note that twinfilin-2a shows similar expression pattern to twinfilin-1, suggesting that these proteins play redundant roles in mice. All mouse isoforms were shown to be able to sequester actin filaments and have higher affinity for ADP-G-actin than ATP-G-actin. They are also able to directly interact with heterodimeric capping protein and PI(4,5)P2 similar to yeast twinfilin. In this study we also uncovered a novel function for mouse twinfilins; capping actin filament barbed ends. All mouse twinfilin isoforms were shown to possess this function, while yeast and Drosophila twinfilin were not able to cap filament barbed ends. Twinfilins localize to the cytoplasm but also to actin-rich regions in mammalian cells. The subcellular localizations of the isoforms are regulated differently, indicating that even though twinfilins biochemical functions in vitro are very similar, in vivo they can play different roles through different regulatory pathways. Together, this study show that twinfilins regulate actin filament assembly both by sequestering actin monomers and by capping filament barbed ends, and that mammals have three biochemically similar twinfilin isoforms with partially overlapping expression patterns.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

Ecological communities in variable environments : dynamics and diversity under coloured environmental stochasticity

While environmental variation is an ubiquitous phenomenon in the natural world which has for long been appreciated by the scientific community recent changes in global climatic conditions have begun to raise consciousness about the economical, political and sociological ramifications of global climate change. Climate warming has already resulted in documented changes in ecosystem functioning, with direct repercussions on ecosystem services. While predicting the influence of ecosystem changes on vital ecosystem services can be extremely difficult, knowledge of the organisation of ecological interactions within natural communities can help us better understand climate driven changes in ecosystems. The role of environmental variation as an agent mediating population extinctions is likely to become increasingly important in the future. In previous studies population extinction risk in stochastic environmental conditions has been tied to an interaction between population density dependence and the temporal autocorrelation of environmental fluctuations. When populations interact with each other, forming ecological communities, the response of such species assemblages to environmental stochasticity can depend, e.g., on trophic structure in the food web and the similarity in species-specific responses to environmental conditions. The results presented in this thesis indicate that variation in the correlation structure between species-specific environmental responses (environmental correlation) can have important qualitative and quantitative effects on community persistence and biomass stability in autocorrelated (coloured) environments. In addition, reddened environmental stochasticity and ecological drift processes (such as demographic stochasticity and dispersal limitation) have important implications for patterns in species relative abundances and community dynamics over time and space. Our understanding of patterns in biodiversity at local and global scale can be enhanced by considering the relevance of different drift processes for community organisation and dynamics. Although the results laid out in this thesis are based on mathematical simulation models, they can be valuable in planning effective empirical studies as well as in interpreting existing empirical results. Most of the metrics considered here are directly applicable to empirical data.

GDNF Receptors: Veterans and Novices

Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.

Structural basis of cytoskeletal regulation by twinfilin

The actin cytoskeleton is required, in all eukaryotic organisms, for several key cellular functions such as cell motility, cytokinesis, and endocytosis. In cells, actin exists either in a monomeric state (G-actin) or in a filamentous form (F-actin). F-actin is the functional form, which can assemble into various structures and produce direct pushing forces that are required for different motile processes. The assembly of actin monomers into complicated three-dimensional structures is tightly regulated by a large number of actin regulating proteins. One central actin regulating protein is twinfilin. Twinfilin consists of two actin depolymerizing-factor homology (ADF-H) domains, which are capable of binding actin, and is conserved from yeast to mammals. Previously it has been shown that twinfilin binds to and sequesters G-actin, and interacts with the heterodimeric capping protein. More recently it has been found that twinfilin also binds to the fast growing actin filament ends and prevents their growth. However, the cellular role of twinfilin and the molecular mechanisms of these interactions have remained unclear. In this study we characterized the molecular mechanisms behind the functions of twinfilin. We demonstrated that twinfilin forms a high-affinity complex with ADP-bound actin monomers (ADP-G-actin). Both ADF-H domains are capable of binding G-actin, but the C-terminal domain contains the high-affinity binding site. Our biochemical analyses identified twinfilin s C-terminal tail region as the interaction site for capping protein. Contrary to G-actin binding, both ADF-H domains of twinfilin are required for the actin filament barbed end capping activity. The C-terminal domain is structurally homologous to ADF/cofilin and binds to filament sides in a similar manner, providing the main affinity for F-actin during barbed end capping. The structure of the N-terminal domain is more distant from ADF/cofilin, and thus it can only associate with G-actin or the terminal actin monomer at the filament barbed end, where it regulates twinfilin s affinity for barbed ends. These data suggest that the mechanism of barbed end capping is similar for twinfilin and gelsolin family proteins. Taken together, these studies revealed how twinfilin interacts with G-actin, filament barbed ends, and capping protein, and also provide a model for how these activities evolved through a duplication of an ancient ADF/cofilin-like domain.

Environmental factors and uncertainty in fisheries management in the northern Baltic Sea

The Baltic Sea is a geologically young, large brackish water basin, and few of the species living there have fully adapted to its special conditions. Many of the species live on the edge of their distribution range in terms of one or more environmental variables such as salinity or temperature. Environmental fluctuations are know to cause fluctuations in populations abundance, and this effect is especially strong near the edges of the distribution range, where even small changes in an environmental variable can be critical to the success of a species. This thesis examines which environmental factors are the most important in relation to the success of various commercially exploited fish species in the northern Baltic Sea. It also examines the uncertainties related to fish stocks current and potential status as well as to their relationship with their environment. The aim is to quantify the uncertainties related to fisheries and environmental management, to find potential management strategies that can be used to reduce uncertainty in management results and to develop methodology related to uncertainty estimation in natural resources management. Bayesian statistical methods are utilized due to their ability to treat uncertainty explicitly in all parts of the statistical model. The results show that uncertainty about important parameters of even the most intensively studied fish species such as salmon (Salmo salar L.) and Baltic herring (Clupea harengus membras L.) is large. On the other hand, management approaches that reduce uncertainty can be found. These include utilising information about ecological similarity of fish stocks and species, and using management variables that are directly related to stock parameters that can be measured easily and without extrapolations or assumptions.

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.