Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Rosti, Katja Marjukka (Helsingin yliopisto, 2016)
    This thesis work comprises the characterization of proteins from two different neuronal membrane receptor protein families: the growth factor receptor -type (GFRa) of protein, growth arrest specific-1 (GAS1) and the synaptic leucine rich repeat adhesion proteins, leucine-rich-repeat transmembrane-2 (LRRTM2), and synaptic adhesion-like molecules 1 and 5 (SALM1 and SALM5). The GAS1 project has focused on the structural characterization of the recombinant human GAS1 protein, and on the possible interaction and effect of GAS1 on the tyrosine kinase receptor protein, re-arranged during transfection (RET), signaling. GAS1 has two different types of interactions, GAS1-RET signaling participates in neuronal survival and maintenance and GAS1-Patched1-Sonic hedgehog (SHH) signaling is needed both for cell survival and in the development of the enteric nervous system during early development. As a conclusion GAS1 has very different structure from other members of the protein family and can interact directly with RET unlike the other GFRa co-receptors. Indicating a different biological role. The study of leucine-rich-repeat transmembrane proteins (LRRTMs), and the synaptic adhesion-like molecules (SALMs) concentrated characterization of an engineered variant of LRRTM2, SALM1 and SALM5. These proteins are involved in neurite outgrowth, branching and synapse formation. They are mainly expressed in brain, and their malfunction is connected to familial schizophrenia, bipolar disorder and autism. We developed a new strategy for protein structure solution and utilized a statistical sequence-based protein engineering method for generation of a stabilized protein for structural studies. We believe this method will have wider use in structural studies of difficult proteins. The goals of this thesis work were to produce these proteins, solve their structures, test their interactions with ligands and do functional characterization studies using a variety of methods. The results will contribute to a better understanding of the molecular structure and the roles these proteins in neuronal tissue, and possibly generate new research into the cellular phenomena, including diseases, linked to these proteins, and aid in future drug development.
  • Havula, Essi (Helsingin yliopisto, 2017)
    SUMMARY Sugars, amino acids and lipids provide the energy and building blocks for growth and maintenance in all animals. However, animal species display great variation in their dietary preferences. The optimal diet of even closely related species can vary tremendously. Also human populations and individuals vary in their dietary behavior and physiological responses to dietary interventions. Moreover, the high amounts of refined sugars in the modern human diet are suggested to contribute to the development of metabolic diseases such as metabolic syndrome and type 2 diabetes. Multicellular animals sense and control their energy homeostasis continuously by integrating nutritional, hormonal and neuronal inputs from their internal and external environment. For example, the counteracting hormones Insulin and Glucagon maintain the levels of circulating glucose constant during fluctuating nutritional conditions. At the cellular level, macronutrients are sensed by distinct mechanisms. The nutrient sensors and their downstream signaling pathways are activated in response to specific nutrients, and they ensure the metabolic homeostasis. Sugars are sensed by highly conserved transcription factor paralogs ChREBP (Carbohydrate-responsive element-binding protein) and MondoA, which share the same heterodimerization partner Mlx. ChREBP/MondoA-Mlx heterodimers regulate the majority of sugar-induced transcription in mammals, including genes of the glycolytic and lipogenic pathways. Dysregulation of ChREBP has been associated with the development of type 2 diabetes, and polymorphisms of ChREBP with circulating triglyceride levels and increased risk of coronary artery disease. The Drosophila genome encodes single orthologues for ChREBP/MondoA and Mlx called Mondo and Mlx, respectively. The function of the mammalian ChREBP/MondoA-Mlx has been largely studied in vitro. The aim of this thesis was to characterize the in vivo function of Drosophila Mondo-Mlx and to identify its target genes and their roles in regulating sugar metabolism. Due to the lack of genetic redundancy and with the extensive toolkit available, the Drosophila provides an optimal in vivo model for studying the role of Mondo-Mlx in nutrient sensing and metabolism. In this thesis, I demonstrate the physiological importance of Mondo-Mlx for organismal sugar tolerance. The mlx null mutant animals display severe sugar intolerance and a gene expression profile that confirms the role of Mondo-Mlx as a key regulator of glycolytic and lipogenic genes also in Drosophila. Furthermore, we expand the role of Mondo-Mlx as a metabolic regulator by showing that it directly controls the expression of several key enzymes of lipid storage, pentose phosphate pathway and amino acid metabolism in response sugars. We also show that Mondo-Mlx is a master regulator of a gene regulatory network composed of a secondary tier of transcriptional effectors including GLI similar transcription factor Sugarbabe and Krüppel-like factor Cabut. The metabolic profiling of the mlx null mutant animals revealed that in addition to being hyperglycaemic, the mutants show signs of amino acid catabolism and elevated ceramide levels that indicate lipotoxicity. This thesis demonstrates the use of Drosophila in studying the genetic basis of dietary sugar tolerance and metabolism. It reveals a number of new metabolic pathways and downstream effectors regulated by Mondo-Mlx, broadening its role as a master regulator of sugar-induced transcription.
  • Shirokova, Vera (Helsingin yliopisto, 2016)
    Development of the organ and its regeneration in adulthood are highly related events and are often guided by similar molecular cues. The hair follicle undergoes repetitive cycles of growth and represents an ideal model for studying both development and regeneration. Hair follicle is an ectodermally-derived organ formed trough interaction of embryonic epithelium and mesenchyme. Once morphogenesis is accomplished, hair follicle undergoes subsequent cycles of growth, regression and rest. Hair regeneration is ensured by the population of hair follicle stem cells which are set aside early during embryonic development. The most quiescent stem cells reside in the bulge, while the stem cells fated for the new growth are in the hair germ. Transcription factors control gene expression and regulate the variety of cell fate choices. Transcription factor Foxi3 is a causative gene for hairless phenotype in several dog breeds, but little else is known about its function in the hair follicle. In this thesis work, I have studied the role of Foxi3 in hair follicle development and postnatal regeneration. To address this question, several Foxi3-deficient mouse lines were used, as well as gene expression analysis, tissue culture techniques and molecular biology methods. Foxi3 is expressed in the developing hair follicle and in the hair germ, exclusively in the activated stem cells. Foxi3 is essential for specification of the hair follicle stem cells and for their activation at the beginning of the hair growth. Foxi3-deficient mice have delayed hair follicle downgrowth during embryogenesis. In the absence of Foxi3, postnatal natural hair regeneration and hair regrowth after depilation are both impaired due to poor activation of stem cells and decrease in their number. Genome-wide profiling, quantitative PCR and immunostaining showed downregulation of several stem cell associated genes upon Foxi3 loss. Thus, Foxi3 is a novel regulator of hair follicle stem cell specification, maintenance and activation.
  • Li, Ma (Helsingin yliopisto, 2016)
    Interactions between the brain and the immune system provide a complex microenvironment for brain development. Furthermore, these interactions cause behavioral changes in rodents and humans. Multiple molecules of the immune system in the brain can regulate neural development. Microglia, the resident phagocytes in the central nervous system (CNS), dynamically survey their microenvironment to maintain brain homeostasis. Microglia also actively respond to neuroinflammation induced by insults such as stress, injury, or infection. A dysfunctional immune system in the brain may cause aberrant brain development and adulthood behavior. Dysfunctional immune regulation has also been implicated in the pathophysiological progression of neuropsychiatric disorders in rodents and humans. The major goal of my thesis is to characterize brain immune genes, gene networks, and neuropeptides in inbred mouse strains. These factors might contribute to mouse anxiety- and sociability-like behavior related to symptoms in generalized anxiety disorder and schizophrenia, as inbred strains such as C57BL/6J(N) (C57BL/6J and C57BL/6N) and DBA/2J differ in their anxiety-, sociability- and sensorimotor gating-related behaviors. In this thesis, we utilized eight inbred mouse strains 129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, and SJL/J to investigate the contributions of brain immune genes and gene networks to social behavioral phenotypes. We first analyzed brain transcriptomics from eight inbred strains to detect differentially expressed immune genes. By correlation analysis, we then predicted the associations of these genes of interest with the animal behavior and brain morphology. Compared to female C57BL/6N mice, we observed high expression of cortical Il1b and Il6 and reduced expression of Cx3cl1 in female DBA/2J mice. Furthermore, male DBA/2J mice had lower levels of C1qb and H2-d1 in the brain compared to male C57BL/6J mice. Interestingly, the hippocampal mRNA level of C1qb was positively correlated with the time spent in social interaction processes in male DBA/2J mice but not C57BL/6J mice. We also investigated differences in cytokine expression and microglial signature gene (for M1-M2 polarization) expression among male C57BL/6J, FVB/N, DBA/2J and 129S2/Sv mice following systemic LPS challenge. Il1b, Il6, and Tnf were highly expressed in the hypothalamus of DBA/2J mice without LPS stimulation. After LPS challenge, the microglial proinflammatory M1-type signature gene Nos2 was highly expressed in the hypothalamus of DBA/2J mice compared to the mouse strains FVB/N, DBA/2J, and 129S2/Sv. We further found that social stress modulated anxiety-like and social behavior in female C57BL/6N and DBA/2J mice in a different manner. The experimental mice were divided into three groups: separate-housed C57BL/6N mice, separate-housed DBA/2J mice, and mix-housed C57BL/6N and DBA/2J mice. Mixed housing made socially active C57BL/6N mice more vulnerable to anxiety and social deficits compared to socially withdrawn DBA/2J mice. Furthermore, expression of glucocorticoid receptor Nr3c1 was attenuated in the hippocampus and cortex of mix-housed C57BL/6N mice compared to the separate-housed C57BL/6N mice. Mix-housed C57BL/6N mice also had a higher level of cortical Avpr1a, but a lower level of hippocampal Oxtr than mix-housed DBA/2J mice. Separate-housed DBA/2J mice had a higher level of hypothalamic Oxtr compared to the separate-housed C57BL/6N mice. These results imply a significant differential impact of social intervention on anxiety-like and social behavior between C57BL/6N mice and DBA/2J mice. In conclusion, my thesis revealed that brain immune genes and neuropeptides are associated with neuropsychiatric-like (anxiety-like and social deficits) behavior in mice. These observations may provide insight on the potential pathogenic molecular mechanisms of neuropsychiatric disorders.
  • Salvador-Martínez, Irepan (Helsingin yliopisto, 2016)
    Embryonic development has amazed scientists for centuries. Many reasons have been suggested for the perceivable increase in complexity in development, during which a single cell transforms itself into a larva or an adult. At the level of gene expression, it its assumed that genes change from being expressed in large spatial domains of the embryo in early development to spatially restricted domains (e.g., tissues, cells) in late development. For many developmental genes, the spatio-temporal expression dynamics have been thoroughly described. It is not clear however, if the global dynamics are similar, or if there are differences between types of genes or between species. Adaptive reasons have been also said to be the cause for the increase in complexity. Adaptations could be estimated with molecular evolution methods based on the analysis of genes expressed in different developmental stages or regions in the embryo. These methods estimate adaptive changes at the DNA sequence level assuming that a positive selected site would show less variance than other sites evolving neutrally. Different developmental stages might show distinct levels of positive or stabilizing selection, that could be related to inter-specific divergence patterns proposed by the von Baer's laws or the Hourglass model. The former states that the development of two species of a phylogenetic group would be very similar in early stages and increasingly divergent insubsequent stages. In contrast, the latter states that development is less divergent (more conserved) at mid development. In here, I analysed gene expression information to estimate both complexity and adaptation in the embryo using a statistical approach. To measure complexity, I developed quantitative measures of spatial complexity and used them in publicly available gene expression data (thousands of in situ hybridization experiments) in Drosophila melanogaster and Ciona intestinalis from the BDGP/FlyExpress and ANISEED databases respectively. To estimate adaptation, I combined diverse D. melanogaster gene expression data (modENCODE, in situ images from the BDGP/FlyExpress and gene expression data based on a controlled vocabulary of the embryo anatomy) with population genomic data (from the DGRP project). Using the DFE-alpha method (which uses coding-region polymorphism and divergence to estimate the proportion of adaptive changes), I charted a spatial map on adaptation of the fruit y embryo's anatomy. Finally, I analysed the pattern of positive selection on genomic coding regions of genes expressed through the entire life cycle of D. melanogaster and how it correlated with specific genomic determinants (e.g., gene structure, codon bias). Briefly, I found that Drosophila and Ciona complexity increases non-linearly with the major change in complexity being before and after gastrulation, respectively. In both species, transcription factors and signalling molecules showed an earlier compartmentalization, consistent with their proposed leading role in pattern formation. In Drosophila, gonads and head showed high adaptation during embryogenesis, although pupa and adult male stages exhibit the highest levels of adaptive change, and mid and late embryonic stages show high conservation, showing an HG pattern. Furthermore, I propose that the Hourglass model can be predicted by specific genetic and genomic features.
  • Chapman, Hugh (Helsingin yliopisto, 2016)
    The human ether á-go-go related gene (hERG1 or KCNH2) encodes the pore forming subunit of the cardiac delayed rectifier potassium (IKr) channel. Its unique kinetics result in a resurgent current crucial for the repolarisation of the cardiac action potential and a capability to suppress premature excitation. hERG1 is widely expressed with roles e.g. in neuronal firing, intestinal and uterine contractility, and insulin secretion. Furthermore overexpression and ectopic expression of hERG1 occurs in cancer where it is involved in proliferation, migration, chemotherapy resistance etc. The long QT syndrome (LQTS) often presents as sudden cardiac death in children and young adults. LQTS is characterised by electrocardiogram abnormalities with arrhythmia that can lead to palpitations, syncope, seizure, cardiac arrest and death. Underlying the congenital form of LQTS are mutations in ion channel proteins (including hERG1, the loss-of-function of which gives rise to LQT2) and their interacting proteins. Carriers of a particular mutation may be symptomatic (to varying extents) or asymptomatic, with the deleterious effects only emerging due to the presence of other factors. This is analogous to drug-induced LQTS where arrhythmia may occur in 1 of 120,000 users of certain non-cardiac drugs. Virtually all drug-induced LQTS is caused by inhibition of hERG1. Consequently in the field of safety pharmacology the hERG1 channel has for the last 20 years and continues to have a huge impact as the primary in vitro predictor of the proarrhythmic risk for a drug. Various aspects of the hERG1 channel are investigated in the studies presented in this thesis. The effect of prucalopride, a gastrointestinal prokinetic drug, on hERG1 was examined. Prucalopride exhibited rapid state and concentration dependent inhibition of hERG1 however, at therapeutic concentrations block is insignificant (hERG safety margin of ≥300). This in vitro prediction has translated to the clinical studies of this drug and the market. The heterogeneous phenotype associated with LQTS may arise from genetic modifiers such polymorphisms and mutations. Heterologous expression of the prevalent hERG1 K897T polymorphism identified a reduced hERG1 current density as the primary difference from wild-type, a result of decreased protein expression. Additionally a slowing of deactivation and alteration of inactivation was evident. Also studied but using induced pluripotent stem cell (iPSC) derived cardiomyocytes was hERG1 R176W. Unlike previous LQT2 iPSC models the origin here was a relatively asymptomatic individual. The phenotypic characteristics of LQT2 were however still reproduced in vitro (i.e. a decrease in IKr and action potential prolongation) though as a milder version. Finally the effect of ceramide, a sphingolipid which accumulates in heart failure and is involved in lipotoxicity, on hERG1 was investigated. Ceramide was found to reduce hERG1 current in a time dependent manner through tagging (ubiquitination) of the cell surface protein for internalisation and targeting to lysosomes.
  • Svirskaite, Julija (Helsingin yliopisto, 2016)
    Archaea were discovered only 4-5 decades ago. The majority of archaea and their viruses originate from extreme environments many of which are characterized by thriving in extreme salinities. The number of isolated archaeal viruses is just a small fraction of the known viruses. Such a lack of knowledge warrants further studies on archaeal viruses and their life cycles. The exit of mature progeny viruses from the archaeal cell is the focus of this study. The viruses used in this study represent all known haloarchaeal virus morphotypes: icosahedral-tailed (HHTV-1), icosahedral-tailless (SH1, HCIV-1), spindle-shaped (His1) and pleomorphic (His2). To describe the virion and its life cycle (by using cell culture turbidity and external virus concentration measurements) are established technologies. Factors associated with membrane integrity, the binding of lipophilic anion phenyldicarbaunundecaborane (PCB), oxygen consumption and adenosine triphosphate (ATP) levels were used to extend the traditional methods for the life cycle studies. These technologies were then utilized in the life cycle studies of HCIV-1 a recently isolated haloarchaeal virus with 12 virion structural proteins and an inner membrane. The internal membrane vesicle encloses a linear double-stranded DNA (dsDNA) genome of 31,314 bp. The genome sequence and its organization express a high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. A rapid cell culture turbidity drop and increase of virus concentration in the cell culture medium took place when SH1, HHTV-1 and HCIV-1 exited the cell. The data also demonstrated the simultaneously binding of lipophilic PCB anions to cell debris, a lethal decrease in respiration and ATP leakage. All the measured properties support the conclusion that these three viruses have a lytic life cycle. However, His1 and His2 virus release did not affect significantly cell physiology suggesting that these haloarchaeal viruses cross the plasma membrane without depolarizing the cell. These results provide insights into the enigmatic and unique release mechanisms of haloarchaeal viruses and highlights the step forward in our understanding of archaeal viruses and their interactions with their host cell.
  • Simpanen, Suvi (Helsingin yliopisto, 2016)
    Soil contamination is a result of human activities that allow hazardous substances to accumulate in soil and thereby to increase the risk to the environment or to human health. There is an estimate of over 2.5 million contaminated sites in Europe and nearly 24 000 of these are in Finland. The most common soil contaminants are oil hydrocarbons and metals. The main anthropogenic activities that contribute to soil contamination include fuel distribution and storage, industrial activity, waste treatment, shooting ranges as well as sawmill and impregnation areas. Soil restoration has mainly been done by excavating and treating or placing the contaminated soil elsewhere (ex situ -treatment). An alternative method is to treat the soil in the contaminated area, i.e. in situ, without excavation, which reduce both environmental effects and remediation costs. Various in situ remediation methods were evaluated in this PhD project by using laboratory-, pilot- and field scale experiments and comparing their usefulness and effectiveness in treating soils contaminated with fuels (diesel, gasoline) or wood preservatives (creosote, chlorophenols). The indigenous microbes in soil are capable of degrading organic contaminants. Degradation can be enhanced by increasing the soil nutrient- and oxygen concentration (biostimulation), and this was found to be applicable as an initial remediation method to decrease easily degradable, organic contaminants from the soil. Biostimulation of fresh oil-contaminated soil also reduced oil leakage through the soil, which indicated that it can help prevent contaminant migration into the groundwater. Chemical oxidation with hydrogen peroxide was not effective for fresh, oil-contaminated soil but it increased the oil migration through the soil. The method also requires large amounts of reagents and an effective infiltration system. Surface-active compound (methyl-β-cyclodextrin) was found to be useful in the solubilisation, and it enhanced biodegradation of contaminants which are strongly adsorbed onto soil particles (creosote components). Organic forest soil was found to have a natural chlorophenol degradation capacity and the addition of this soil on top of chlorophenol contaminated mineral soil increased chlorophenol degradation. An electric field that was applied into the dense and wet soil caused soil dewatering. After this the soil vapor extraction was effective for the removal of volatile organic contaminants (gasoline compounds) from soil. The combination of different methods and their sequential use can remove or decrease the need for excavation. Decisions about the remediation should be done early enough to make the use of the in situ methods feasible.
  • Laine, Merjo (Helsingin yliopisto, 2016)
    Anthropogenic actions and climate change greatly affect e.g. carbon (C) and nitrogen (N) cycles in soils. The consequences can differ in various soil types. The changes in soil C and N cycles may also have an effect on adjacent aquatic systems and in the atmosphere. Dissolved organic carbon (DOC), N, and phosphorus (P) loads to aquatic ecosystems in general have caused concern. In this thesis, I mostly discuss soil C and N cycles. To a minor extent I also cover C and N in aquatic ecosystems and in the atmosphere, as they are connected to soil cycles. My study involved two separate experimental areas, a peatland and an agroecosystem. In my peat soil mesocosm experiment, I studied peat profiles taken from a complex with a pristine and a forestry-drained peatland. My focus was on changes in DOC, dissolved organic nitrogen (DON), and ammonium (NH4+) concentrations in soil water as a response to hydrological manipulation. In my in situ mineral soil agroecosystem experiment I quantified gross N transformation process rates in no-till and moldboard-ploughed soils after harvesting. Hydrology remarkably impacted the element concentrations in pristine peat soil water. An increase in DON and NH4+ concentrations was seen as a response to hydrology, while DOC concentrations were not affected in comparison to control concentrations. However, DOC production in pristine peat, followed by its release into water, was also high enough to compensate the dilution caused by water additions to the mesocosms. These compounds were produced during the drought in the aerated soil layer and released to the added water by physicochemical processes when the mesocosms were rewetted. In drained peat mesocosms, the hydrological manipulation decreased the DOC concentrations, and the DON and NH4+ concentrations did not change significantly. My agricultural experiment results give some environmental support for no-till over ploughing. NH4+ is a substrate for nitrification, and nitrate (NO3 ) can easily leach into aquatic ecosystems, where it may cause eutrophication. Therefore, the observed higher gross immobilization rate, lower nitrification rate, and lower NO3 loss flux rate in no-till supported this practice when assessing the post-harvest leaching risk. In addition, a lower nitrification / immobilization ratio in the no-till indicated a decreased NO3 leaching risk. Higher post-harvest immobilization rate further supports no-till because it may be beneficial for crop growth during the following growing season.
  • Varghese, Finny Symon (Helsingin yliopisto, 2016)
    Chikungunya virus (CHIKV) is an arbovirus spread by the Aedes sp. of mosquitoes. Chikungunya fever results in a sudden onset of a febrile disease with headache, nausea and maculopapular rash. Additionally, a large proportion of the affected individuals experience persistent arthralgia months after all other signs of the disease have vanished. Originally discovered in Tanzania in 1952, it re-emerged with a massive outbreak in several islands of the Indian Ocean in 2004 and spilled over onto the Indian sub-continent and South-east Asia. Later CHIKV invaded Southern Europe and since the last two years has ventured into the western hemisphere, causing more than 1 million suspected infections in the Caribbean islands, Central and Latin America. The explosive nature of these outbreaks has led to a tremendous strain on the public health system of many of the affected countries already burdened with the endemically circulating Dengue virus. So far, no licensed vaccines or antivirals exist to counter this virus. Besides, it is paramount to have an in-depth understanding of the replication mechanisms of this re-emerging pathogen in order to come up with novel and effective therapeutic measures. A previously characterized CHIKV replicon cell line was used to conduct a high-throughput screen of ~3000 bioactive compounds, which are in clinical use or in clinical trials against other diseases. This led to the discovery of abamectin, ivermectin and berberine as novel antivirals effective at low micromolar concentrations and having broad-spectrum anti-alphaviral activity. Deciphering the mode of action of berberine led to the discovery that CHIKV infection robustly activates the three main branches of the mitogen-activated protein kinase (MAPK) signaling extracellular signal regulated kinase (ERK), p38 MAPK and c-Jun NH2-terminal kinase (JNK). Berberine was shown to reduce this virus-induced MAPK activation and also suppressed virus-independent ERK activation. These pathways were shown to be important for CHIKV replication, as specific inhibitors of the ERK and JNK pathways significantly reduced the viral progeny release. Most importantly, berberine reduced CHIKV-induced inflammatory disease in a mouse model and is one of the few compounds reported to show in vivo efficacy. Exploring the antiviral mechanism of obatoclax, an anticancer compound previously reported to be active against different viruses, including influenza A virus and Sindbis virus, revealed the compound to be active against other alphaviruses, including SFV and CHIKV. Further characterization showed that obatoclax inhibits viral fusion by rapidly neutralizing the acidic environment of endolysosomal organelles. Additionally, characterization of escape mutants showed that a single mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. This study has unearthed effective candidate antivirals against alphaviruses, which have served as useful tools to help us gain further insight into alphavirus biology when characterizing their modes of action.
  • Demina, Tatiana (Helsingin yliopisto, 2016)
    Viruses are significant biological, ecological, and evolutionary players, which are present virtually in all types of environments where cellular life is found. It has been suggested that one way to systematize the enormous virus diversity is to group viruses based on their virion architectural principles. Indeed, in spite of the overwhelming genetic diversity, all the known viruses display a limited number of morphotypes, and thus may be grouped into a limited number of structure-based lineages. To test how far virus structural conservation extends, new virus-host systems are needed to be isolated and characterized. Hypersaline environments, where halophilic archaea often dominate, are a rich source of new viruses. Currently, viruses that infect archaea are very little studied, as only ~100 archaeal viruses have been isolated. The aim of this thesis work was to isolate new archaeal viruses from hypersaline environments. In total, 36 new viruses were isolated from salt samples collected from the Samut Sakhon saltern (Thailand¬). These isolates displayed four previously known morphotypes: myovirus-like (27 isolates), siphovirus-like (4), tailless icosahedral (1), and pleomorphic (4). Among the obtained viruses, myoviruses had the widest host ranges (up to 14 hosts), infecting archaeal strains isolated from the same location or other hypersaline environments. Two of the isolated viruses, Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica pleomorphic virus 3 (HHPV3), were characterized in molecular detail. The characterization included studies on virus infectivity under various conditions, virus life cycle, virion structural components (proteins, lipids, and nucleic acids), and virus genome sequencing and annotation. HCIV-1 is closely related to the other tailless icosahedral viruses SH1, PH1, and HHIV-2 that infect halophilic archaea. The core virion components are highly conserved in these viruses and determine their place in the PRD1-adenovirus structural lineage. This lineage comprises viruses which are distributed world-wide and infect hosts from all three domains of cellular life. HHPV3 belongs to the group of archaeal pleomorphic viruses, which share virion organization and gene synteny, but may have different genome types (circular single-stranded DNA or circular/linear double-stranded DNA) and low similarity at sequence level. Pleolipoviruses also originate from distant geographical locations. This thesis work significantly increased the number of known archaeal viruses, from ~100 to ~140, and provided insights into molecular details of the two new halophilic archaeal viruses. The limited number of observed virus morphotypes and the conserved architectural principles revealed in HCIV-1 and HHPV3 highlight that similar virus architectures are found from all over the planet, supporting the idea of viral structural lineages. Future sampling of various environments and more detailed studies on the currently available virus isolates will help to portray the true viral diversity.
  • Noreikiene, Kristina (Helsingin yliopisto, 2016)
    Understanding the causes and consequences of life-history variation is one of the fundamental goals in evolutionary biology. A fairly recent discovery is that even individuals of the same species may differ in their life-histories strategies. However, we still have only a rudimentary understanding of how state and the environment jointly shape such individual life-history strategies within species. Glucocorticoid stress hormones (GCs) may serve as key endocrine modulators integrating information about the internal and external environment. However, we are still lacking detailed knowledge about how GCs may be modulating individually variable life-histories. In this thesis I set out to explore proximate and ultimate causes of intraspecific variation in life-history strategies, with a particular emphasis on GCs as potential drivers of life-history trade-offs. I have conducted this project on long-lived eider (Somateria mollissima) females and their ducklings. Female eiders invest heavily into reproduction which is linked to decreased investment into self-maintenance and survival. Moreover, eiders show repeatable individual differences in stress responsiveness and behavioural strategies, thus serving as a suitable system for studying life-histories in the wild. The results showed that eiders trade-off self-maintenance for reproduction, but that the magnitude of this trade-off depends on the state (e.g., telomere length, age, body condition) of the individual. The state of the individual is also important in making reproductive decisions in the face of predation risk. In light of my findings, GCs may serve as the link between reproductive investment decisions and reproductive success in the face of danger in eider females. Rather than having uniformly negative effects on reproductive decisions and reproductive success in female eiders, the effects of GCs are context-dependent. The effects of elevated GCs are advantageous in areas of high predation risk by allowing to better prepare for predatory attacks or to shorten incubation time, but may decrease net reproductive output in safer areas. The effects of GCs are also modulated by nest concealment so that females with elevated GCs breed more successfully in open areas with increased perceived predation risk while the opposite is observed in females with low GCs concentrations, having highest reproductive success in well-protected nesting habitats. These result suggest that the contrasting effects GCs may have on fitness are modulated by the environment. Lastly, maternal GCs are important predictors of both stress exposure in ovo and early-life telomere length in eider ducklings. The association between maternal GCs depended on offspring sex so that stress exposure increased with maternal GCs in daughters, but decreased in sons. Overall, the results presented in this thesis highlight the importance of physiological stress in driving life-history decisions. Further, it proposes that GCs may have both beneficial and negative effects on organismal fitness depending on the intrinsic and extrinsic environmental context, be it predation risk or intrinsic environment differences associated with sex of individual. However, GCs are a part of the complex endocrine system and are not acting in isolation. Thus, future studies should address how the concerted interactions between multiple hormones affect life-history decisions in different environmental contexts. Nonetheless, my results suggest that GCs are important elements driving individually variable life-history strategies in the wild. Moreover, the context-dependent association between fitness and GCs levels revealed by this thesis may be part of the mechanism maintaining individual variation in stress responsiveness in the population.
  • Fleming-Lehtinen, Vivi (Helsingin yliopisto, 2016)
    Secchi depth, a proxy of water clarity, is widely applied as an indicator of eutrophication or water quality both in open-sea- and coastal areas. In optically complex waters, such as the Baltic Sea, Secchi depth is known to respond to several components yet its performance, or possible restrictions, have not been explored. In this study, I investigated long-term changes in Secchi depth. I also explored the structure, scientific basis and use of Secchi depth as an indicator of eutrophication in the Baltic Sea. Secchi depth decreased in the open Baltic Sea during the last century (Paper I). The decrease was especially intense in the northern areas, amounting to 3.3 4.0 m (averaging 0.033 0.040 m y-1), when comparing summer time averages in 2005 2009 to those observed one hundred years earlier. The decrease was proposed to be strongly linked with documented simultaneous increase in chlorophyll-a concentration (Papers I, III). A closer look at the Finnish coastal areas, where a national monitoring program has taken place since 1970, revealed clear decreasing trends only in the Archipelago Sea accompanied by opposing trends in chlorophyll-a (Paper II). Contradictory to this, and to the development in adjacent open sea areas, Secchi depth was observed to increase in the coastal Bothnian Sea, Quark and Bothnian Bay. I suggest the increase was at least partly a consequence of decreased concentrations of dissolved iron in the surface waters near the coast. The relationship between Secchi depth and total organic carbon (TOC) was tested, but a significant relationship was not found indirectly indicating that a large part of organic carbon was colorless. Unfortunately, the long-term coastal dataset did not allow comparison to suspended inorganic matter, leaving the possible effect of potentially important coastal constituent unrevealed. The effect of the main optical constituents on light attenuation in the open sea were investigated through a bio-optical model setup, in order to resolve how the Secchi depth indicator should be applied in different parts of the Baltic Sea (Paper III). Secchi depth was shown to be highly sensitive to variation in both phytoplankton (by chlorophyll-a as proxy) and colorful dissolved organic matter (CDOM). As expected, based on the high spatial gradients in both optical constituents, the evaluation against monitoring data called for sub-basin-wise adjustments to the model outcome. Secchi depth is often applied together with other indicators, including chlorophyll-a. The modelling exercise revealed, that the environmental targets for Secchi depth, set by the Baltic Sea coastal states via their collaboration through the Baltic Marine Environment Protection Commission (HELCOM), were stricter than those set for chlorophyll-a. To facilitate future management use of the Secchi depth indicator, I made an effort to characterize it in relation to indicators in general. Secchi depth is a commonly applied and well established indicator of eutrophication and water quality in the Baltic Sea. It is technically relatively advanced: quantitative, regularly monitored, and includes ecological targets as well as documented methodology. It is also easily understood by the public. On the other hand, though simple to associate, it is a composite indicator, which requires case-specific analysis before its role in the eutrophication process can be accurately defined. Finally, Secchi depth was applied in the Baltic Sea eutrophication status assessment (Paper IV), and alternative ways to apply the indicator were explored. According to the assessment 2007-2011, all open-sea areas of the Baltic Sea were severely affected by eutrophication. Due to the deteriorated status of all indicators, variation in the construction of the assessment did not affect the general outcome. Secchi depth on its own expressed deteriorated status in most areas, meeting its environmental target only in the Bothnian Bay. The strong relationship between Secchi depth and chlorophyll-a motivates the use of Secchi depth as a eutrophication indicator throughout the open Baltic Sea. The strong association to CDOM, however, presents a combination of possible additional autochthonous as well as allochthonous signals. The sensitivity of Secchi depth to the main optical constituents varies between open-sea areas, and furthermore, needs to be addressed separately in the coastal zone, where inorganic constituents are expected to be significant. Being a composite indicator, Secchi depth was found suitable for expressing eutrophication together with other indicators; relying on Secchi depth alone would introduce a risk of misinterpretations, especially when the role of water clarity in the ecosystem is not solved area-specifically. On the other hand, Secchi depth may turn to be valuable in reflecting signals not currently captured by other indicators.
  • Yrjölä, Rauno (Helsingin yliopisto, 2016)
    Urbanization in general and the spread of industrial construction into natural areas can seriously reduce animal populations. In this thesis, I used breeding birds to investigate the impacts of the construction of Vuosaari harbour, ship channel and road connections on bird populations of nearby area. We monitored bird populations in Natura 2000 area and seabird breeding islets during the construction of the new Vuosaari harbour (Helsinki, Finland) in 2001 2011. The harbour was built less than 300 m from Natura 2000 area. We compared the bird populations before, during and after construction to evaluate whether the changes observed were linked with the construction work in the nearby harbour area. Our results indicate that in the Österängen agricultural area the number of species and territories increased during and after construction, with more territories located closer to the road than before the road was constructed. Thus, road construction resulted in at least a momentary positive impact on bird populations. New grass and bush areas and even large rocky outcrops clearly benefited some species such as the threatened northern wheatear. The bird populations declined slightly several years later, probably the as a result of habitat succession. In the forest areas, the population trends of 23 boreal forest bird species were significantly positively correlated with population changes in wider areas in southern Finland. Although the population trends were slightly weaker in the Natura 2000 sites compared with the general situation of southern Finland, this difference was not statistically significant. In the Natura 2000 wetlands he numbers of species and territories varied between years and subareas, but we found no clear impacts of harbour construction on bird population trends. The bird population changes in nearest bay, Porvarinlahti, were not more negative in comparison with those of the two other bays. We found that the waterfowl reproduction success was very poor, and that the number of small predators may be reason for that. The more predators were hunted, the less waterfowl nestlings were observed at the same year. The archipelago seabird study revealed that the population trends of most species showed no substantial differences between the monitored islets near the construction area and the reference islets. In conclusion, the numbers of species and territories varied between years and subareas, but we found no clear impacts of harbour construction on bird population trends. Although the population trends were slightly lower in the Natura 2000 sites compared with the general situation of southern Finland, this difference was not statistically significant. Our results indicate that despite the construction work demolished bird populations at the construction site, the impact of the harbour construction work had only limited impact on population trends of birds in the Natura 2000 site. Perhaps other more general factors such as climate and changes in the non-breeding grounds were likely a more important driver of local bird dynamics. This monitoring project has shown how difficult it is to estimate possible negative impacts caused by a single construction project. Before the project started, biologists said that severe negative impacts are possible, and according to the so called precautionary principle construction of Vuosaari harbour should not be allowed. Monitoring lasted ten years, and we couldn t see any clear significant negative impacts caused by construction.
  • Lõhmus, Andres (Helsingin yliopisto, 2016)
    Potato virus A (PVA), a positive-strand RNA ([+]RNA) virus, belongs to the genus Potyvirus, which is the largest RNA virus group in plants. Like all (+)RNA viruses of eukaryotes, potyviruses replicate in association with cellular endomembranes, incorporating host proteins to their cellular multiplication processes. These host proteins could be potential targets for engineering resistant crops, which is why studying the molecular interactions during virus infection is important. In this study the molecular processes of PVA translation and replication were investigated. The focus was on two viral proteins involved in these processes: the viral coat protein (CP) and helper-component proteinase (HCpro). Furthermore, the protein composition of PVA replication complexes was studied. The results obtained here confirm that the viral CP is required for PVA replication and suggest that it could be involved in the formation of the viral replication complex (VRC). Moreover, we show that CP turnover is regulated by phosphorylation and targeted proteasomal degradation, involving the host proteins coat protein interacting protein (CPIP), heat-shock protein 70 (HSP70) and carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase. Altogether, tight control over CP interaction with viral RNA is required for efficient PVA infection. This study also reports the discovery of PVA-induced granules (PGs). PGs are ribonucleoprotein complexes that are induced by HCpro and contain viral RNA and host proteins involved in RNA translation and processing. PG formation is counteracted by viral genome-linked protein (VPg)-assisted PVA translation, suggesting that the components of PGs are involved in the regulation of PVA translation. Moreover, we demonstrate that HCpro acts synergistically with VPg, enhancing PVA gene expression and RNA stability. The presence of argonaute 1 (AGO1) in PGs and the inability of silencing-suppression defective HCpro to induce PGs suggests that PGs may have a role in local silencing suppression. PGs often associate with VRCs, pointing to a close relationship between viral replication and HCpro-mediated functions. An affinity-purification method coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used to study the protein composition of PVA replication complexes. Viral replication-associated proteins were abundantly present in the VRCs, validating the VRC purification approach. The presence of ribosomal and translation-related proteins in PVA VRCs is in line with the notion of closely coupled viral replication and translation. Moreover, the abundance of HSP70 and other chaperones in the VRCs supports their important role in PVA replication. Lastly, the proteome data has provided several interesting candidate proteins that can be studied further in relation to PVA infection.