Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Yan, Xu (Helsingin yliopisto, 2017)
    Progressive development of pathology in neuroanatomically connected brain regions is a common feature of many neurodegenerative diseases. The spread of disease pathology is suggested to be dependent on the transmissibility of disease-associated proteins, particularly soluble aggregates of misfolded proteins. Emerging evidence suggests that many disease-associated proteins such as α-synuclein (aSyn) and tau, in certain misfolded and aggregated states convert from physiologically normal proteins into forms that lead to progression of disease pathology in a template-dependent manner, which is also known as seeding . The propagation and the proteinopathy have been suggested to occur via cell-to-cell transmission. The exact mechanisms involved in the seeding and spreading process are incompletely understood. In this thesis work, three critical steps of the seeding pathway (a process involves multiple steps), the intracellular aggregation, cellular release and uptake of aSyn and tau, were carefully studied primarily via a newly developed platform based on protein-fragment complementation assay. The main findings of this thesis are: a)Prolyl oligopeptidase (PREP) is a serine peptidase that was previously known to accelerate the process of aSyn aggregation and suppress autophagy clearance in cells and transgenic aSyn mice. The results of this thesis show that PREP directly interacts with aSyn in neuro2A cells and cell-free environment, and enhances aSyn dimerization, which is an early event in aSyn aggregation pathway. In addition, the PREP-mediated aSyn dimerization can be antagonized by KYP-2047, a small-molecule PREP inhibitor. b) Late-onset Alzheimer s disease (LOAD) susceptibility genes affect the individual risk of developing Alzheimer s disease, which is one of the common tauopathies. In this work, the functional connection between selected LOAD susceptibility genes and cell-to-cell transmission of tau was studied in vitro. We observed that RNAi knockdown of CD2AP and FRMD4A reduced tau secretion, and knockdown of APOE reduced tau uptake in HEK293T cells. Further mechanistic studies revealed that FRMD4A modulates tau secretion via the FRMD4A-cytohesin-Arf6 signalling pathway and the Par6/aPKC polarity signalling complex. This data, for the first time, demonstrates a functional connection between LOAD risk genes and cell-to-cell propagation of tau. c) Following internalization, extracellular, hyperphosphorylated tau was found to be recruited to stress granules, transient non-membraneous cytosolic structures composed of RNA and self-aggregating RNA-binding proteins. Tau recruitment was dependent on TIA-1, an RNA-binding stress granule protein. Importantly, the stress granules induced by and containing internalized tau were resistant to normal clearance and associated with increased sensitivity of cells to other stresses. This data describe a previously unrecognized mechanism and pathological consequence of cell-to-cell propagation of tau-mediated by stress granules, which have previously been associated with the pathophysiology of various neurodegenerative diseases. Overall, the work described in this thesis provides several novel findings that improve our understanding of cellular mechanisms underlying the development and spreading of aSyn and tau-related neurodegenerative pathologies. These pieces of knowledge may be potential avenues towards the development of crucial therapeutics against aSyn and tau-related neurodegenerative diseases.
  • Kulashekhar, Shrikanth (2017)
    Working memory is used to maintain information for cognitive operations, and its deficits are associated with several neuropsychological disorders. Human functional magnetic resonance imaging (fMRI) f isolated key brain areas associated with the maintenance of sensory and duration information. However, the systems-level mechanisms coordinating the collective neuronal activity in these brain areas have remained elusive. It has been suggested that synchronized oscillations could regulate communication in neuronal networks and could hence serve such coordination, but their role in the maintenance of sensory and duration information has remained largely unknown. In this thesis, combined magnetoencephalography (MEG) and electroencephalography (EEG) together with minimum norm estimate (MNE) based source modelling was used to study the oscillatory dynamics underlying visual and temporal working memory. In Publication I, we developed a neuro-informatics approach to understand the anatomical and dynamic structures of network synchrony supporting visual working memory (VWM). VWM was associated with a sustained and stable inter-areal phase synchrony among frontoparietal and visual areas in alpha- (10 13 Hz), beta- (18 24 Hz), and gamma- (30 40 Hz) frequency bands. In this study, the subjects' individual behavioural VWM capacity was predicted by synchrony in a network in which the intraparietal sulcus was the most central hub. In Publication II, we characterised the oscillatory amplitude dynamics associated with the VWM maintenance. Increasing VWM load was associated with strengthened oscillation amplitudes in the occipital and occipitotemporal cortical areas, in the alpha (8 14 Hz) beta- (15 30 Hz), gamma- (30-50 Hz), and high-gamma- (50 150 Hz) frequency bands. In Publication III, we addressed the functional significance of local neuronal synchronization, as indexed by the amplitudes of cortical oscillations, in the estimation and maintenance of duration information. The estimation of durations in the seconds range was associated with stronger beta-band (14 30 Hz) oscillations in cortical regions that have earlier been associated with temporal processing. The encoding of duration information was associated with strengthened gamma- (30 120 Hz), and the retrieval and comparison with alpha-band (8 14 Hz) oscillations. Further, the maintenance of stimulus duration was associated with stronger theta- and alpha-band (5 14Hz) frequencies. These data suggested that both local and large-scale phase synchrony in the alpha-, beta-, and gamma-frequency bands in the frontoparietal and visual regions could be a systems level mechanism for coordinating and regulating the maintenance of visual information in VWM. In addition, it suggested that beta-band oscillations may provide a mechanism for estimating short temporal durations, while gamma, alpha and theta-alpha oscillations support their encoding, retrieval, and maintenance in working memory, respectively.
  • von Ossowski, Lotta (Helsingin yliopisto, 2017)
    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are glutamate-gated cation channels and mediators of fast excitatory neurotransmission in the mammalian central nervous system. Trafficking and functional regulation of AMPA receptors GluA1-4 is carried out through numerous intracellular protein interactions and post-translational modifications. The aim of this thesis work was to study the selective interaction between AMPA receptor subunit GluA1 and synapse-associated protein 97 (SAP97), a protein scaffold belonging to the protein family of membrane associated guanylate kinase homologs. The interaction between SAP97 and GluA1 has been implicated in AMPA receptor trafficking, neuronal development and synaptic plasticity, while disturbances in normal levels of both GluA1 and SAP97 have been linked to neuropathologies such as Alzheimer s disease and schizophrenia. In the present study, a combination of biochemical and structural work was employed to gain detailed information on the selective interaction of GluA1 with SAP97 identifying molecular determinants involved in and regulating the interaction. X-ray crystallization screens of the second PDZ domain of SAP97 (SAP97PDZ2) yielded well-diffracting crystals both for the apo and ligand bound form. The solved crystal structure of the SAP97PDZ2-GluA1 peptide complex conformed to a conventional class I PDZ interaction with hydrogen bonds forming between the carboxylate group of the ultimate C-terminal residue of the peptide and main chain nitrogens in the carboxylate binding loop of the PDZ domain, and a hydrogen bond between the antepenultimate residue of the peptide and a conserved histidine in the αB helix lining the peptide binding groove. Beside these typical PDZ interactions, as a novel finding we observed contacts within the PDZ domain reorganizing upon peptide binding leading to a slight opening of the peptide binding groove facilitating better accommodation of the ligand. In vitro binding analysis of isolated PDZ domains and short GluA1 peptides showed that, in addition to the prototypic PDZ interaction, a C-terminal cysteine, C893 located upstream from the short PDZ binding motif on GluA1 participated in the interaction through a disulfide bond formed with cysteine C378 of SAP97 under in vitro conditions. Streptavidin pull-down experiments with full-length molecules expressed in cultured cells showed that the C893S mutation leads to a substantial reduction in binding of GluA1 to SAP97, confirming the involvement of C893 in the regulation of the interaction in live cells. Reactive cysteines, like C893, can in addition to disulfide bonds participate in other thiol modifications. In our work we constructed several deletion and cysteine replacement mutants of GluA1 and tested their sensitivity to S-nitrosylating agents nitrosoglutathione and nitrosocysteine. Out of the three C-terminal cysteine residues in GluA1, we identified C893 as the sole cysteine residue sensitive to a post-translational modification by nitric oxide (NO). Furthermore, we found evidence of a physical link between GluA1 and the NO generating neuronal enzyme nitric oxide synthase nNOS via SAP97. The results of the present study provide, for the first time, detailed structural information on the interaction between GluA1 AMPA receptor and SAP97. In addition to a canonical PDZ interaction, the association with SAP97 involves a reactive cysteine residue, C893, in GluA1 C-terminal tail, a potential regulatory target for nitric oxide and oxidative conditions.
  • Andreevskaya, Margarita (Helsingin yliopisto, 2017)
    Lactic acid bacteria (LAB) play a dual role in food manufacturing. While being indispensable for food fermentations and preservation, they are also involved in spoilage of foods and beverages, and some food-borne LAB are pathogens. Particularly, they became the main spoilage organisms in the cold-stored modified atmosphere packaged (MAP) foods. LAB species composition and their relative abundances depend on the nature of food products and preservation technology. However, two LAB species, Leuconostoc gelidum and Lactococcus piscium, have frequently been predominating at the end of shelf life in a variety of packaged and refrigerated foods of animal and plant origin. Besides the predominant species, spoilage LAB communities contain less abundant and slower growing species, such as Lactobacillus oligofermentans, the role of which in food spoilage is unclear. Taking into account the increased popularity of MAP technology combined with cold storage for preservation of minimally processed fresh foods, the need to obtain more information on the metabolism, genomics, ecology and interactions of psychrotrophic food-spoilage-associated LAB is clear. In this thesis a genomic approach was used to study these LAB. In order to characterize spoilage community members, we sequenced and annotated genomes of Lc. piscium MKFS47 and Lb. oligofermentans LMG 22743T, both isolated from broiler meat, and seven strains of Le. gelidum, isolated from vegetable-based foods. The analysis of their gene contents and their comparison with gene repertoire of other close related species allowed us to predict putative factors that might facilitate their survival in their habitats and increase competitiveness in the spoilage microbial communities. No major differences in the gene contents of the vegetable and meat Le. gelidum strains were observed that would suggest niche-specificity, therefore, indicating that the absence of strain dissemination between vegetable- and meat-processing chains is a more likely factor responsible for the reported strain segregation between vegetable and meat-based products. Lc. piscium MKFS47 was identified as an efficient producer of buttery off-odors compounds from glucose under aerobic conditions, which is in agreement with the previous inoculation studies. Time course glucose catabolism-based transcriptome profiles revealed the presence of classical carbon catabolite repression mechanism for the regulation of carbohydrate catabolism, which was relieved along with decreasing concentration of glucose. During the same time, the shift from homolactic to heterolactic fermentation mode was observed. For Lb. oligofermentans, a pentose-preferring obligate heterofermentative LAB, the induction of efficient utilization of hexoses was confirmed indicating that it has flexible carbohydrate catabolism, which can be adjusted depending on the carbohydrate sources available in the environment. Unexpectedly, transcriptome responses of Lb. oligofermentans during growth on glucose and xylose were more alike than during fermentation of ribose in the early exponential growth phase. In addition, cross induction of glucose and xylose catabolic genes by either glucose or xylose was observed. These phenomena could be governed by the CcpA transcriptional regulator, the regulation mechanism of which remains to be determined. Transcriptome-based study of interspecies interactions between three above mentioned LAB species revealed their different survival strategies to cope with competition for the common resources. Le. gelidum was shown to enhance its nutrient- (mainly carbohydrates) scavenging and growth capabilities under glucose limitation conditions when competing with the other LAB species, while the opposite was observed for Lc. piscium and Lb. oligofermentans. Such behavior might explain the competitive success and, hence, the predominance of Le. gelidum in spoilage microbial communities. Peculiarly, interspecies interactions activated expression of prophages and restriction modification systems in Lc. piscium and Lb. oligofermentans, but not in Le. gelidum. The downregulation of stress protection-related genes in all the LAB at the early growth stage was unexpected, and it requires further studies. Finally, overexpression of the numerous putative adhesins in Lb. oligofermentans during growth with other LAB could be one of the factors explaining its survival in actively growing communities in meat.
  • 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.