Bio- ja ympäristötieteellinen tiedekunta


Recent Submissions

  • Broberg, Martin (Helsingin yliopisto, 2015)
    The interactions between phytopathogenic bacteria and their host plants can be characterized as an intricate web of signals and appropriate responses. Phytopathogenic soft rot bacteria occur globally, causing disease in Solanum tuberosum (potato) and other tubular staple foods in both the field and storage. One widely studied soft rot bacterium is Pectobacterium wasabiae, which has been identified in Eutrema wasabi (wasabi) plants in Japan and in potatoes in Finland. Generally, the interactions between this type of bacterium and host plants are characterized by maceration of plant tissue, due to the actions of secreted plant cell wall degrading enzymes (PCWDE), and the induction of phytohormone dependent defenses in the plants. The maceration of plant tissue involves the release of pectic oligogalacturonides (OGs) from plant cell walls. OGs have been identified as important signaling compounds, inducing the expression of a variety of defense-related genes. As the bacterial infection advances, the bacteria coordinate the production of virulence factors by utilizing regulatory proteins that modulate the transcriptome. Transcriptomic analyses have been used extensively in past studies to identify regulatory networks and signaling pathways, and these studies have provided insights into the processes underlying plant-pathogen interactions. The novel scientific results of this dissertation are derived from a combination of transcriptomic, genomic, genetic, and phenotypic analyses. This study analyzed various aspects of plant-pathogen interactions. The central bacterial model used was P. wasabiae, and the model plant of interest was Arabidopsis thaliana. This study characterized the genome of P. wasabiae via sequencing and bioinformatics analysis. Various virulence associated genes and operons, such as two distinct type 6 secretion systems, were identified and annotated. The bacterium was found to in fact be more related to P. wasabiae than Pectobacterium carotovorum, which the strain originally had been named after. Furthermore, a combination of functional genetics and transcriptomic methods, such as reverse transcription quantitative PCR (RT-qPCR) and microarrays, were used to determine the regulons controlled by the proteins ExpA and RsmA in P. wasabiae. These two proteins have been identified as important for the virulence of several γ-proteobacterial pathogens. This study analyzed the regulons via the use of three mutants: expA, rsmA, and an expA rsmA double mutant (DM). Overlapping and independently regulated targets were identified between ExpA and RsmA. Phenotypic assays for motility, growth, PCWDE activity, and virulence confirmed the transcriptomic data for the mutant strains. Novel findings included reduction of swimming motility in agar medium for P. wasabiae expA and rsmA mutants. In addition, the DM exhibited enhanced virulence and fitness in planta compared to either single mutant. Via analysis of transcriptomic data, a subset of genes was identified as affected in expression by an expA mutation independently of the presence of rsmA. The relatively unexplored role of short OGs (with a degree of polymerization (DP) less than 10) in damage-associated molecular pattern (DAMP) signaling in A. thaliana was characterized in this study. Comparative gene expression profiling based on RNA sequencing and RT-qPCR was performed on RNA harvested from plants treated with short OGs or with a mock suspension. Phenotypic assays confirmed the gene expression data. In a meta-data analysis, the resulting RNA sequencing and RT-qPCR data were compared with gene expression data from previous studies, in which long OGs (DP more than 10) were used to treat plants. This work demonstrated that short and long OGs induce genes and genesets associated with pathogen defense and phytohormone signaling, whereas reducing plant growth and development. The transcriptomic data of this study suggests that plant treatment with a mixture of short or long OGs yields a more pronounced and varied modulation of global gene expression, compared to treatment with only trimeric OGs. The regulation of the virulence of P. wasabiae, and the DAMP signaling triggered by plant cell wall damage in A. thaliana, are elements of the interactions between the plant and pathogen. The studies presented in this dissertation provide novel information about these two biological processes and highlights their connection.
  • Turja, Raisa (Helsingin yliopisto, 2015)
    Biomarkers measured in organisms are sensitive molecular, cellular or individual level biological effects, which can be applied as early-warning signals of environmental contamination before damage occurs at population, community or ecosystem levels. In this thesis, a suite of biomarkers and tissue concentrations of chemicals were measured in mussels (Mytilus trossulus) as indicators of environmental pollution. The mussels were transplanted in specially made cages in coastal areas of the northern Baltic Sea influenced by different types of contamination and environmental factors. The aims of the research were to apply the biomarker approach to (1) assess the impact of contaminants on the health status of mussels, (2) investigate the effects of seasonal variability in biotic and abiotic factors, and low salinity, and (3) validate the usefulness of the mussel caging method for biomonitoring of chemical contamination in the northern Baltic Sea. The results showed marked biomarker responses coinciding with higher concentrations of contaminants, such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), organotins and trace metals, in mussels caged at the most contaminated study sites. At the contaminated sites, for example the enzymatic biomark¬ers of the antioxidant defense system indicated higher stress level, which was closely linked to elevated level of DNA-damage, increased biotransformation activity and decreased general health status of the cells. Biomarker responses and tissue contaminant concentrations were also related to the seasonal variability in growth and especially in soft tissue weight of the mussels. In the northern Baltic Sea the mussels almost fully deplete their energy stores during the winter; in the spring mussels efficiently feed on the fresh phytoplankton, which forms the main source of energy for their reproduction, energy metabolism and growth. Natural environmental factors caused less variation in the biomarker responses in the late summer and autumn suggesting that this time period is the most suitable for studying contaminant induced effects in mussels. More pronounced effects were observed in mussels exposed jointly to low salinity and chemical contamination, indicating that increased environmental stress reduces the tolerance of mussels towards anthropogenic pressures. This work showed that the mussel caging approach is an efficient biomonitoring method to assess biological effects and tissue accumulation of complex mixtures of contaminants as long as the effects of seasonal variability and low salinity are taken into account.
  • Holopainen-Mantila, Ulla (Helsingin yliopisto, 2015)
    Barley (Hordeum vulgare L.) is a globally important grain crop. The composition and structure of barley grain is under genotypic and environmental control during grain development, when storage compounds (mainly starch and protein), are accumulated. Grain structure plays a significant role in malting and feed- and food-processing quality of barley. Hordeins, the major storage proteins in barley grains, are centrally located in the endosperm forming a matrix surrounding starch granules, but their role in the structural properties of barley grain is not completely understood. Thus, the main aim of the current study was to demonstrate the role of hordeins in barley grain structure. The dependence of the grain structure on the growth environment, in particular with respect to day-length and sulphur application relevant to northern growing conditions, was studied. The effects of the grain structure on end use properties in milling as well as in hydration and modification during malting were characterized. The longer photoperiod typical to latitudes in Southern Finland resulted in a C hordein fraction, entrapped by aggregated B and D hordeins, being more deeply located in the endosperm of barley cultivar Barke. Thus the impact of the growing environment on hordein deposition during grain filling was observed both at the tissue and subcellular level. However, the mechanism behind the differential accumulation of C hordein remains unclear. The deeper localization of entrapped C hordein was linked to improved hydration of grains during malting in three barley cultivars. Thus, the role of the subaleurone region in barley grain was found to be significant with respect to end use quality. Moreover, the results suggest that the growing environment affects the end-use properties of barley and that especially the northern growing conditions have a positive impact on barley processing quality. The influence of sulphur application on hordein composition in the Northern European growing conditions was demonstrated for the first time. Asparagine and C hordein served as nitrogen storage pools when the S application rate was lower than 20 mg S / kg soil, whereas total hordein and B hordein contents increased with higher S application rates. The current study also showed that even when sulphur is sufficiently available in field conditions, the hordein composition may react to sulphur application. The observed sulphur responses were in accordance with those reported earlier for hordein composition. This indicates that the more intensive growth rhythm induced in northern growing conditions does not alter greatly the effect of sulphur on grain composition. The current study confirmed that the main grain components: starch, protein and β-glucan, influence grain processing properties including milling, hydration and endosperm modification. However, their influence on endosperm texture (hardness or steeliness), which also affects the performance of barley grains in these processes, cannot be directly derived or estimated on the basis of the grain composition. The results obtained suggest that hordeins should also be taken into account in the evaluation of the processing behaviour of barley grains.
  • Gabryelczyk, Bartosz (Helsingin yliopisto, 2015)
    The possibility of controlling interactions at interfaces and surfaces of solid materials is highly interesting for a wide range of materials-related nanotechnological applications, for example, colloidal systems, adhesives, biosensors, biomimetic composite and biomedical materials. In Nature, many proteins and peptides possess the ability to recognize, specifically bind, and modify the surfaces of solid materials through sophisticated mechanism of molecular recognition. These properties have been developed during evolution via successive cycles of random mutations and selection. The natural evolution processes can be mimicked in the laboratory scale with the use of a directed evolution approach, for instance, based on the selection of short material-specific peptides from the combinatorial libraries displayed on the surface of bacteriophages or bacterial cells. Selected from billions of different variants, material-specific peptides can be studied by experimental and computational methods to define their sequence, structure, and binding properties. Subsequently, they can be engineered in order to improve their binding affinity and tailor their function for practical applications. The studies presented in this thesis show how phage display was used to identify peptides binding to diamond-like carbon (DLC). DLC is an amorphous form of carbon, with chemical and physical properties resembling natural diamond. It is used as a coating material in many industrial and biomedical applications. Peptides binding to DLC were selected form a combinatorial phage display library. Their binding and molecular basis of the function were investigated in different molecular contexts (when displayed on the phage surface, forming fusion proteins, or present in free soluble form), using multiple independent methods. It was also demonstrated that the peptides can be used in nanotechnological applications, i.e., as a self-assembling coating on the DLC surface, and for controlling properties of a colloidal form of DLC. Besides finding and characterizing peptides binding to DLC, the thesis also highlights different challenges of the directed evolution techniques, for example, selection of target unrelated peptides during biopanning, and the necessity of multiple independent ways of analyzing the functionality of selected peptides.
  • Pulkkinen, Henni (Helsingin yliopisto, 2015)
    Overfishing and environmental changes impose high risks on the wellbeing of the world's fish stocks. It is commonly acknowledged that fisheries management should be risk averse, following the principles of the precautionary approach, but unfortunately the statistical stock assessment methods often lack the ability to estimate the uncertainties related to their results. Further challenges arise from the fact that stocks which are in the most desperate need of a stock assessment are often data poor and resources to gather new data from them are scarce. Bayesian statistical inference can be utilized to conduct stock assessments cost-efficiently since these methods provide a formal way to combine information from various sources including databases, literature and expert knowledge. Bayesian inference is essentially a learning process where existing information is combined into a prior distribution, which is further updated with the most recent data. The result, a posterior distribution, expresses the best available knowledge about the phenomenon, including the related uncertainty. Furthermore, Bayesian hierarchical models enable learning between similar units, for example, stocks of the same or related species. This thesis consists of four studies that use Bayesian hierarchical models to improve knowledge in the fisheries stock assessment. Correlations between biological parameters, arising from different life history strategies among species, are utilized in paper [I] so that a data rich set of length-weight parameters can reduce the uncertainty of length-fecundity parameters for a data poor species. In paper [II], similarities between stocks of Atlantic salmon are used to estimate the stock specific key parameters of eggs-to-smolts relationship. A predictive distribution of this key parameter is also estimated, and could be used as an informative prior in a subsequent study of another Atlantic salmon stock. In addition, a hierarchical model is built to study structural uncertainty, estimating posterior probabilities of competing functional forms that describe the eggs-to-smolts survival at different juvenile densities. The Bayesian approach makes it possible to conduct analyses sequentially, as in the case of Baltic salmon stock assessment reviewed in paper [III]. Sequential analysis is useful if a stock assessment is complex and computational power does not enable analysis of all observation models at the same time. Thus, the Bayesian methods permit the creation of complex model frameworks, where incentives for structural choices arise rather from the biological process, than from the requirements or limitations of the datasets. The analysis of acoustic survey information to estimate herring resources at Bothnian Sea (paper [IV]) is another example of a computationally intensive model that has been built on the basis of available background information. This includes on one hand, the technical knowledge about the survey, and on the other hand, the biological knowledge about the spatial distribution of the fish.
  • Isomaa, Marleena (Helsingin yliopisto, 2015)
    The purpose of this thesis was to increase knowledge of heavily harvested cod stock dynamics and attain information for more suitable and sustainable management strategies. Achieving sustainable management strategies requires sufficient knowledge about the population dynamics. One tool for research this is to introduce stochastic variation models in investigated species stock dynamics. As a model species we use eastern Baltic Sea cod (Gadus morhua callarias), where the population dynamics are strongly influenced by environmental factors and fishing. We consider the stock dynamics and recovery potential under three different harvest strategies (proportional, threshold) and environmental noise scenarios. We developed age-structured discrete-time population models that include interactions with the environmental factors, e.g. salinity, fishing and natural mortality. Our results show that under current environmental conditions and threshold harvest methods cod fishing is at a sustainable level. Precautionary management ensures the best economical incomes and sustainable stocks in the long run, despite environmental fluctuations or moratoria. Results also reveal how the unpredictability of stock size, catch, and recovery increase with increasing environmental autocorrelation. Initial age structure has a strong impact on population recovery capacity in the short run, especially in over-harvested populations and under strongly fluctuating environmental conditions. Protection of younger age groups will substantially increase the recovery potential and will increase the maximum sustainable yield considerably. As a whole, precautionary harvesting strategies will increase the stock resilience against adverse environmental conditions and harvesting, guaranteeing sustainable yields in the future.
  • Voutilainen, Maria (Helsingin yliopisto, 2015)
    Mammary gland development begins during embryogenesis with the formation of species-typical number of mammary placodes that emerge along the flanks of the embryo at conserved positions. By birth, the mammary primordium has undergone branching morphogenesis and displays a small ductal tree with several branches. The organ development and growth continues throughout postnatal life and the mammary gland matures to functional form only during pregnancy and following lactation. Ectodysplasin (Eda), a member of the tumour necrosis factor family, is one of the key regulators of epithelial appendage development in all vertebrates. In humans, mutations in the Eda gene, or in other components of the signalling pathway, cause hypohidrotic ectodermal dysplasia (HED), a disorder characterized by sparse hair, missing teeth, and defects in several exocrine glands including the breast. Previous studies have shown that transgenic overexpression of Eda (K14-Eda mice) in the developing ectoderm leads to formation of ectopic mammary placodes, which give rise to supernumerary glands in the adult mice. Otherwise, effects of Eda signalling in the mammary gland have been fairly unknown. Here I have analysed the role of Eda in prepubertal mammary gland development. Characterization of the mammary glands of Eda gain- (K14-Eda) and loss-of-function (Eda−/−) mice revealed that the branching morphogenesis of the organs correlated with Eda levels. Overexpression of Eda induced precocious and accelerated branching whereas lack of Eda reduced number of ductal tips. Furthermore, Eda induced supernumerary mammary placode formation not only on the flank but also in the neck region. Analysis of the mouse line with suppressed NF-kappaB signaling (IκBαΔN mice) revealed that the transcription factor is a major mediator of Eda in the mammary gland. NF-kappaB activity was shown to be necessary for the ability of Eda to induce supernumerary mammary primordia and to accelerate branching morphogenesis. With a candidate gene approach and genome wide-profiling several potent Eda target genes were identified in the mammary gland. Among them were members of the Wnt/beta-cat pathway. The obtained results suggest that Eda promotes mammary cell fate by enhancing canonical Wnt pathway activity and other effects of Eda are cooperatively mediated by certain Wnt family members in addition to other factors. To study mammary placode formation and branching morphogenesis and to assess roles of individual downstream factors or pathways, ex vivo culture systems were developed and utilized in this thesis work.
  • Budria, Alexandre (Helsingin yliopisto, 2015)
    Recent research has strongly linked disease emergence and resurgence with human-induced environmental change. Habitat alteration, biodiversity loss, pollution, climate change and introduced species are nowadays considered potential threats to human and wildlife health. However, the impacts of these changes are not always so clear. Recent findings indicate that anthropogenic disturbances may favour the transmission of some parasites, but can also be disadvantageous to others. The aim of this thesis is to determine how human-induced environmental change affects host-parasite interactions by using anthropogenic eutrophication of the habitat of the threespine stickleback, Gasterosteus aculeatus, and its macroparasites as a model system. The different parts of the thesis focus on the influence of anthropogenic disturbances on epidemics, and on the ecological mechanisms underlying the observed patterns. After reviewing current knowledge about the influence of human-induced environmental change on host-parasites interactions, I focus on epidemiological patterns in fish populations inhabiting eutrophied waters, which vary in the extent of eutrophication. By analysing the data in respect to the degree of environmental disturbance and the knowledge about the biology of the parasites and their hosts, I develop hypotheses regarding the different mechanisms explaining the observed patterns. To test these hypotheses, I perform statistical modelling and experimental work focusing on the two most prevalent endoparasites in the studied stickleback populations, i.e. the eye fluke Diplostomum spp. and the tapeworm Schistocephalus solidus. The different approaches used in my thesis reflect the complexity of host-parasite systems and the need to mix theory, monitoring and experimental work to estimate the impact of human-induced environmental change on host-parasite interactions. Finally I point out areas that are in need of further investigation in order to elucidate the consequences of human activities for ecosystems and to build a predictive framework for how global change can affect parasitic diseases.
  • Heikkilä, Maria (Helsingin yliopisto, 2014)
    Lepidoptera (butterflies and moths) are one of the most diverse and species-rich groups of organisms. Currently, the number of described species is close to 160.000, but it is estimated that the true number of species could be as high as half a million. Nearly 99% of all Lepidoptera belong to the more advanced subclade Ditrysia. The focus of this thesis is the evolutionary history of the enormous ditrysian clade. The relatedness between the 30 ditrysian superfamilies has been a mystery that has only recently begun to unfold. The aim of the thesis is to find new information on the evolutionary relationships between these superfamilies, and within two of the largest of them, Gelechioidea and Papilionoidea, both with over 18.000 described species. Evidence on the evolutionary relationships is sought from both morphological and genetic data. The morphological datasets are based on characters coded from larval, pupal and adult stages, and are thus far the largest in terms of the number of coded characters and of the number of exemplar species. In the analyses of chapters II-IV, morphological data are combined to molecular data from eight gene regions sequenced from corresponding species or genera. Morphological characters supporting phylogenetic affinities are discussed in detail. Characters described in the articles of this thesis can be used in the identification of extant and fossil taxa, and in future studies focusing on the evolution of specific morphological traits. Chapter I elucidates the evolutionary affinities of the mysterious family Lypusidae, the phylogenetic position of which has not been known. It is demonstrated that this family belongs within the megadiverse superfamily Gelechioidea. Chapter II focuses on the phylogenetic relationships between the families of the superfamily Papilionoidea (Papilionidae, Pieridae, Nymphalidae, Lycaenidae, Riodinidae, Hesperiidae and Hedylidae). The results show that the traditional concept of butterflies, i.e. including only the first five above-mentioned families is paraphyletic. Hesperiidae and Hedylidae are sister-groups and are internested within the rest of the butterflies. A divergence time analysis indicates that this superfamily originated in the Cretaceous and diversified in the Tertiary. Chapter III explores the evolutionary relationships within one of the largest but least studied groups of Lepidoptera, Gelechioidea. Based on the results, a new classification into 16 families is proposed. The aim of chapter IV is to find morphological evidence on the relationships between the ditrysian superfamilies. Several morphological characters claimed to define subgroups of Ditrysia are tested. New characters supporting evolutionary affinities of clades are described and discussed. By combining morphological and sequence data, some taxa that have been unstable in analyses based on either type of data only, find a stable position, e.g. Epipyropiidae and Cyclotornidae, Urodidoidea + Schreckensteinioidea.
  • Miettinen, Juho (Helsingin yliopisto, 2014)
    Innate immunity provides the first line of defence against invading pathogens, and can also be activated by endogenous danger signals released upon tissue damage or injury. Macrophages play an important role in innate immunity. They perform the immune surveillance of their immediate surroundings seeking out tissue damage and/or invading pathogens via pattern recognition receptors. Macrophages are activated upon detection of an invading pathogen or tissue damage, leading to the expression, and secretion of the proteins required for an efficient innate immune response. Herpes simplex virus-1 (HSV-1) is a common human pathogen. It is a master of evading the host immune response leading to its ability to cause a life-long infection in its host. In most cases, HSV-1 does not cause any symptoms in the host, or causes nothing more than an occasional painful blister in the orofacial region. However, in certain cases when the host immune system is compromised, it can cause severe, even lethal, infections. Uric acid can act as an endogenous danger signal that cells can produce and release into the extracellular space after encountering a stress, including that caused by certain viral infections. A high amount of uric acid in cells can also be attributed to certain dietary habits. In the extracellular space if present at high enough concentrations and in the presence of sodium, uric acid can become crystallized into monosodium urate (MSU). MSU is the causative of gouty inflammation in the joints and is known to be phagocytosed by macrophages. In this work, human monocyte-derived macrophages infected with HSV-1, or stimulated with MSU were studied using several proteomic methods combined with functional assays. The main goals were: to characterize the protein secretion pathways and the proteins being secreted from macrophages upon (1) HSV-1 infection, and (2) MSU stimulation; and (3) to characterize the functional mechanisms of HSV-1 infected cell protein 27 (ICP27) in the inhibition of the interferon (IFN) response and also inhibition of apoptosis during HSV-1 infection. The results revealed that robust extracellular vesicle-mediated unconventional protein secretion is induced by IFN-beta priming in HSV-1-infected, and by MSU stimulation in Toll-like receptor 4 ligand, bacterial cell wall component lipopolysaccharide-primed human macrophages. The secreted proteins included endogenous danger signal proteins, and interferon-stimulated gene-encoded proteins. In addition, HSV-1 ICP27 protein requires both the nuclear localization sequence (NLS) and nuclear export sequence (NES) in order to inhibit the IFN response, whereas the NES, but not NLS, is required for the inhibition of apoptosis. The results also demonstrate that pattern recognition receptors RIG-I and/or MDA5 are involved in the HSV-1-induced activation of apoptosis. To conclude, this thesis provides new information on the innate immune response induced by HSV-1 infection, and MSU stimulation, and also on the evasion mechanisms deployed by HSV-1 to avoid the innate immune response in human macrophages. These new results presented in this thesis may be exploited in several ways; in the development of new therapies against viral infections, and in the treatment of autoinflammatory diseases.
  • Cui, Fuqiang (Hansaprint, 2014)
    To face the constant challenges from numerous pathogens in the environment, sophisticated defense systems have evolved in plants. Reactive oxygen species (ROS) and phytohormones are important cellular compounds that regulate plant defense systems to overcome biotic stresses from different pathogens. Against biotrophic pathogens, which require living host cells, hypersensitive cell death response (HR), a type of programed cell death mediated by ROS and salicylic acid (SA), is effective for immunity. However, to necrotrophic pathogens, which take host cell death as a hallmark of a successful colonization, the roles of ROS and phytohormones in the manipulation of cell death during plant defense are more complex. In this work, we utilized the model necrotrophic pathogen Botrytis cinerea (Botrytis; grey mold) and the model plant Arabidopsis thaliana (Arabidopsis), using mutants in reverse genetic screens, especially radical-induced cell death1 (rcd1) and botrytis susceptible1 (bos1), were used to study the functions of ROS and phytohormones in plant-Botrytis interactions. It was found that Botrytis-triggered signaling in Arabidopsis mostly overlapped with the signaling triggered by apoplastic ROS but not intracellular ROS. However, rcd1 and bos1 exhibited opposite symptoms in response to Botrytis and apoplastic ROS. This suggested that the resistance signaling regulated by RCD1 or BOS1 were distinct from a more common signaling programs induced by Botrytis and apoplastic ROS. Further study revealed that RCD1 negatively regulated Botrytis resistance independent of stress-hormones. RCD1 positively regulated Botrytis-toxin sensitivity and brassinosteroid (BR) signaling, which was demonstrated to negatively regulate plant resistance to Botrytis. In the BOS1 study, suppression of abscisic acid (ABA)-elicited cell death and control of cell death spread were identified as pivotal functions of BOS1 in its regulation of host resistance to Botrytis. This work emphasized the negative roles of both BR and ABA in response to Botrytis infection. Considering the established facts that: 1) ABA promoted plant cell death, 2) BR deficiency leads to delayed senescence, and 3) the ROS burst causes damage to both host and Botrytis; this work supports the view that cell death control plays a pivotal role in plant-Botrytis interactions, where defense combined with less cell death confers plants with an advantage in the battle against Botrytis.
  • Koskinen, Mikko (Helsingin yliopisto, 2014)
    Dendritic spines are small bulbous protrusions extending from dendritic shafts of neurons. These compartments house most of the postsynaptic terminals of excitatory synapses in the mammalian central nervous system. Dendritic spines are formed during early development and their density and morphology undergoes significant changes during maturation. After maturation dendritic spines are not static structures but display constant changes in their morphology and stability. The shape and size of dendritic spines have been linked to synaptic transmission, coupling the form of spines to neuron function. Several neurological diseases and disabilities are characterized by abnormal spine density and morphology. The main structural component of the dendritic spines is the actin filament, F-actin. Actin filaments are dynamic polymers of the monomeric protein actin. The filaments are constantly turning over and reorganizing. Both processes are regulated by actin binding proteins. All structural changes and the maintenance of dendritic spines are dependent on actin dynamics. Current research indicates that the dynamics of actin filaments do not change following spine maturation. Maturation does lead to a decrease in the movement of spines and an increase in stability, indicating changes in F-actin dynamics. In this study I have shown that the dynamics of F-actin do change during maturation. The stable pool of F-actin increases in size and the turnover of the dynamic pool increases. One of the actin binding proteins with a potential to regulate actin stabilization is myosin IIb, a motor protein with capabilities to bind F-actin and to introduce contractility into the filament network. Myosin IIb has been shown to regulate dendritic spine development, size and shape and play a role in memory consolidation. In this study I have shown that myosin IIb regulates dendritic spine F-actin via two distinct mechanisms. Myosin IIb can bind F-actin and stabilize it without affecting the turnover of the dynamic filaments. Myosin IIb-mediated contractility on the other hand can facilitate the turnover of the dynamic filaments. These findings help us to understand the molecular mechanism behind dendritic spine structure regulation and possibly in the future how it is related to synaptic transmission and different pathological states. Due to their small size, dendritic spines pose unique challenges for the study of actin dynamics. Most of the available methods are based on advanced fluorescence microscopy. In this study I have made a critical evaluation of the methods used to measure F-actin turnover in dendritic spines and the analysis of the data. I have also developed a novel approach to use fluorescence anisotropy to measure the level of actin bundling. The method has been previously applied to measure actin polymerization. My findings have led to the conclusion that in actin-dense compartments, such as the dendritic spines, fluorescence anisotropy reflects actin bundling rather than polymerization and that conclusions based on earlier research using similar techniques should be re-evaluated.
  • Stratoulias, Vassilis (Helsingin yliopisto, 2014)
    Developmental biology studies how a single cell will give rise to a fully developed, highly ordered and reproducible living organism. In order for this to occur, the fertilized egg needs to grow, divide and differentiate to produce different cell types. Subsequently, these different cell types need to organize into predefined arrays of committed cell groups to produce specialized tissues and organs, which constitute the living multicellular organism. Although the final product seems enormously complex, the multicellular organism is composed of cells that they all have the same set of genes. Cells differentiate by switching on and off the expression of different molecules and they produce and respond to signals which result in cells growing, dividing and dying in a strict spatiotemporal manner. In this work I aimed to address questions on the molecular mechanisms during development by using the fruit fly Drosophila melanogaster as a model system. The first aim of my studies was to characterize and investigate the role of Manf during Drosophila pupation and adulthood. MANF is an evolutionarily conserved neurotrophic factor, which has previously been reported to protect and restore dopaminergic neurons in mammals. In Drosophila embryos DmMANF has been shown to be specifically expressed in glial populations, while DmMANF null mutants die early during development while they exhibit specific and significant reduction of dopaminergic neurites. Our data reveal that in pupae and adults, DmMANF has a much wider expression pattern and it is localized both in glia and neurons. This analysis led to the identification of an unusual phenotype in the Drosophila pupal brain. We showed when Manf is silenced, or either autophagy or immunity is induced in glia, macrophage-like cells appear in areas of the pupal brain that are normally devoid of cell bodies. We identified molecular markers and pathways that are activated in these cells, as well as some of the unique subcellular features they possess. This study brings new and elegant data in several aspects of glial biology with exciting perspectives for studying brain plasticity and repair. The second project was to characterize the expression of Gfrl receptor, the Drosophila homolog of the mammalian GFRα receptor in the Drosophila adult brain. The results showed that although the Gfrl receptor is widely expressed in the fly adult brain, this expression is in surprising contrast to the missing expression of dRet, the Drosophila Ret homolog. This data provide novel insights into further elucidation of the Ret-dependent and Ret-independent -GFRα signaling complexes, as well as to further understand the several aspects of invertebrate brain development and function. The third aim of this study was to investigate the role of the conserved pleiotropic factor Lin28 during Drosophila development. Previous studies have shown that Lin28 is one of the factors sufficient to reprogram human somatic cells into induced pluripotent stem cells. By constructing Lin-28 null mutants, our data reveal that Lin-28 mutant files were viable but sub-fertile, exhibiting oogenesis defects. These results will help us improve our understanding of Lin28 role in stem cell maintenance and differentiation.
  • Ursache, Robertas (Helsingin yliopisto, 2014)
    Plant vascular tissues are supporting and conductive tissues composed of two major components, xylem and phloem. These tissues transport water, food, hormones and minerals within the plant. In my thesis work, I used the Arabidopsis root as a model system to study vascular tissue formation. The first part of my thesis work is focused on the formation of xylem, the water transporting tissue. In the Arabidopsis root, the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. It has been previously reported that high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. In this work, we provide evidence that auxin biosynthesis promotes HD-ZIP III expression and metaxylem formation. We observed that plants with mutations in auxin biosynthesis genes, such as trp2-12, wei8 tar2, or the quintuple yucca mutant, as well as plants treated with a pharmacological inhibitor of auxin biosynthesis, show reduced expression of the HD-ZIP III genes accompanied by specific defects in metaxylem formation. We were able to induce a partial rescue of the metaxylem defects by introducing an endogenous auxin supply. In addition, some of the patterning defects can be suppressed by synthetically elevating HD-ZIP III expression in the stele of the Arabidopsis root. The second part of my thesis work is focused on phloem tissue formation. Phloem is the tissue responsible for long-distance molecular transport and signaling. The conductive components of the phloem, the sieve elements, rely on specific junctions between the conducting cells in the form of highly perforated sieve areas. We identified mutations in the CHER1 (CHOLINE TRANSPORTER LIKE 1) locus of Arabidopsis which result in altered phloem conductivity, reduced sieve pore density, and defects in sieve pore formation. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We provide data showing that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and is associated with the late stage of phragmoplast formation during cytokinesis. Interestingly, CHER1 has a sustained, polar localization in forming sieve plates, which is consistent with its function in the elaboration of the sieve areas.
  • Susi, Hanna (Helsingin yliopisto, 2014)
    At the very core of the evolution of living organisms lie interactions with other species. Between two coevolving species, a change in one species may generate selection for a change in the other species. In host-pathogen coevolution the central dilemma is to understand how infectivity and virulence evolve. Infectivity is the ability to infect a given host while virulence is the harm the pathogen causes to its host, and therefore they determine the outcome of the interaction between the host and the pathogen. The emergence of new highly virulent pathogen species (e.g. Ash dieback pathogen Hymenoscyphus pseudoalbidus) and single pathogen strains (e.g. Ug99 of wheat stem rust pathogen Puccinia graminis f. sp. tritici) underline the urgent need for a deeper understanding of how virulence evolves. The aim of my thesis is to understand how life-history trade-offs and coinfection where two or more strains of the same pathogen are infecting the same host - are driving host-pathogen coevolution, and how these evolutionary trajectories translate to ecological dynamics in a metapopulation context using the Plantago lanceolata Podosphaera plantaginis interaction as a model system. The study approach ranged from the molecular level to population and metapopulation levels. I studied natural populations of P. lanceolata and P. plantaginis in the Åland islands to measure prevalence of coinfection and its consequences for disease epidemics in the wild. I also investigated variation in resistance in the natural host populations as well as the efficiency and costs of different plant resistance strategies in a common garden setting. Context dependence of evolutionary trade-offs were investigated by accounting for some of the spatial and temporal complexity of the natural pathogen metapopulation. Pathogen life-history trade-offs were studied in the context of local adaptation and costs of resistance in the perennial host were measured across multiple seasons. The pathogen s host exploitation versus transmission strategies were examined on relevant epidemiological time scales to understand factors creating heterogeneity in transmission dynamics. Key findings of the thesis include detection of high, yet variable levels of coinfection across the pathogen metapopulation, with more devastating epidemics measured in populations with higher levels of coinfection. This suggests a major role for coinfection in driving disease dynamics in natural populations. In the dynamic pathogen metapopulation, local adaptation mediates pathogen life history trade-offs and resistance polymorphism can be maintained through costs of resistance and changes in resource allocation under infection. In conclusion, this work contributes to our understanding of the drivers of evolution and maintenance of variation in the host and pathogen populations by linking evolutionary theory with empirical findings.