Bio- ja ympäristötieteellinen tiedekunta


Recent Submissions

  • Virtanen, Laura (Helsingin yliopisto, 2015)
    Water quality in streams usually changes fast, and sensitive biological indicators are essential for monitoring these changes. Diatoms are widely used in biological stream quality assessments. However, there are temporal and spatial variations in diatom communities that may result in unreliable quality assessments. Under different kinds of environmental stressors, diatoms can produce deformed frustules. Heavy metals are among the most common causes of these teratological forms. In addition to eutrophication and organic pollution, diatoms could potentially be used for indicators of heavy metal enrichment. The aim here was to examine the interannual and intra-annual turnover of stream diatom communities. The focus of this study was on whether specific species traits and species local abundance and regional distribution affected temporal occurrence of diatom species. The study also included an examination of how stable diatom inferred stream classifications are in time and if pure spatial variation in diatoms should be considered when assessing stream water quality based on diatoms. The final aspect of the study was to determine which heavy metals, and to what extent, trigger the occurrence of deformations in diatom Achnanthidium minutissimum. Our results imply that abundant and widely distributed species with larger niches and the ability to attach to the substratum sustain persistent populations in varying environmental conditions typical for streams. The most persistent species are thus perhaps the most reliable species to be used as indicators of water quality. The stream classification based on diatoms resulted in temporally stable and statistically distinct community types. Thus, the results suggest that sampling of diatoms in every three years seems to be a reliable procedure to assess biological water quality. The investigation also revealed that study regions differ in their diatom species composition more than in their environmental features indicating that diatoms are structured not only by the local environment but also by large-scale processes, possibly related to climate, dispersal and history. As diatom species composition varies between regions, future bioassessments would benefit from regional stratification. Finally, study results indicated that two metals, copper and zinc, and a metalloid, antimony, were the most likely triggers of A. minutissimum deformations. The definition of their morphological and quantitative characteristics, along with a better taxonomic circumscription of the affected species, should allow the use of deformations as a reliable indicator of heavy metal enrichment in freshwater habitats.
  • Xu, Enjun (Helsingin yliopisto, 2015)
    Regulation of cellular homeostasis is crucial for proper development, survival, defense responses, programmed cell death and ultimately survival. Maintaining cellular homeostasis requires tight regulation of multiple highly interactive signaling pathways. The apoplast lies at the frontier between the cell and the environment, where the plant perceives environmental cues. Since the apoplast is also a site for cell-to-cell communication, it has an important role in mediating plant-environment interactions. Reactive oxygen species (ROS) are known as both toxic agents and indispensable signaling molecules in all aerobic organisms. A ROS burst in the apoplast is one of the first measurable events produced in response to different biotic and abiotic stresses, eventually leading to the initiation of signal transduction pathways and altered gene expression. Apoplastic ROS signaling is well known to dynamically coordinate multiple signaling pathways in the activation of defense responses in plants. Dissection of the signaling crosstalk within such a signaling network could therefore reveal the molecular mechanisms underlying defense responses. Treatments with ozone (O3) have been adopted as an efficient tool to study apoplastic ROS signaling. Plants exposed to O3 trigger a ROS burst in the apoplast and induce extensive changes in gene expression and alteration of defense hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene. Genetic variation in O3 sensitivity among Arabidopsis thaliana accessions or mutants highlights the complex genetic architecture of plant responses to ROS. To gain insight into the genetic basis of apoplastic ROS signaling, a recombinant inbred line (RIL) population from a reciprocal cross between two Arabidopsis accessions C24 (O3 tolerant) and Tenela (O3 sensitive) was used for quantitative trait loci (QTL) mapping. Through a combination of QTL mapping and transcriptomic analyses in the response to apoplastic-ROS treatment, three QTL regions containing several potential candidate genes were identified in this study. In addition, multiple mutants with varying O3-sensitivities were employed to dissect the signaling components involved in the early apoplastic ROS signaling and O3-triggered cell death. A combination of global and targeted gene expression profiling, genetic analysis, and cell death assays was performed to dissect the contribution of hormone signaling and various transcription factors to the regulation of apoplastic ROS-triggered gene expression and cell death. The contributions of SA, JA and ethylene were assessed through analysis of mutants deficient in these hormones, mutants with constitutively activated hormone signaling and the exogenous application of hormones. Plants with elevated SA levels were found to be associated with an attenuated O3 response, whereas simultaneous elimination of SA-dependent and SA- independent signaling components enhanced the response to apoplastic ROS treatment. JA could act as both a positive and negative modifier of apoplastic ROS signaling, which was enhanced when ethylene signaling was also impaired. However, transcriptome analysis of a triple mutant deficient in SA, JA and ethylene revealed that these hormones signaling only contributed part (about 30%) of early-apoplastic ROS-triggered changes in gene expression, suggesting multiple signaling pathways could be required to regulate the apoplastic ROS response via combinatorial or overlapping mechanisms.
  • Ruppel, Meri M. (Helsingin yliopisto, 2015)
    The Arctic has been warming twice as fast as the rest of the world during the last decades of global warming. Reasons for the amplified Arctic warming are thought to partly relate to positive feedbacks affecting the radiative budget of the area. Black carbon (BC) is a light-absorbing particulate produced by incomplete combustion of biomass and fossil fuels. BC strongly warms the atmosphere, and its climate effects are amplified in the Arctic where its deposition on light surfaces decreases their reflectivity, resulting in elevated heat absorption and further hastening melt of snow and ice. Globally, BC is estimated to be the second most important climate-warming agent after carbon dioxide. Historical information on BC deposition plays a significant role in the assessment of long-term climate effects of BC, but scarce data on this past variability has been available from the Arctic. Historical BC records can be attained from environmental archives, such as ice cores, peat deposits and marine and lake sediments, which store direct evidence of past BC deposition in chronological order. The objective of this thesis is to collect new spatial and temporal data on BC deposition in the European Arctic from the preindustrial to the present (i.e., the last ca. 300 years), and assess BC sources and climatic implications, by analysing five lake sediment cores from Arctic Finland and an ice core from Svalbard. No standard method exists to determine BC, and the precise definition of BC depends on the methodology used for its quantification. Here, three different analytical methods were used to quantify different components of BC. Spheroidal Carbonaceous Particles (SCP) and soot-BC (SBC) were analysed from the Arctic Finland lake sediments by SCP analysis and chemothermal oxidation at 375 °C, respectively, and elemental carbon (EC) from the Svalbard ice core with a thermal optical method. The results suggest temporal variation in past BC trends, both between study sites and between methods. While SCPs show a marked trend with fluxes peaking around 1980 and declining afterwards, they represent only a minor fraction of total BC. SBC and EC are better suited to indicate general historical BC trends. SBC fluxes vary between sites but some regional patterns are noticeable. The two northernmost lakes indicate generally decreasing SBC fluxes during the latter half of the 20th century which is in line with previous data from Greenland ice cores, modelling studies and atmospheric measurements. However, two other lake sites indicate increasing SBC fluxes from ca. 1970 to the end of the records, likely caused by local emissions from the Kola Peninsula, Russia. Moreover, an increasing EC deposition trend from ca. 1970 to 2004 is also recorded in the Svalbard ice core. This increasing trend is unexpected and unparalleled among available arctic records. The observed increase in the ice core is likely caused by flaring emissions from northern Russia. The fact that a similar trend in BC fluxes and deposition is recorded in two separate environmental archives analysed with different methods and receiving atmospheric transport from mostly different source areas, highlights the plausibility of such a trend, and implies that it may also be observable at other locations in the Arctic. In such a case, BC may have exerted a significant impact on the radiative forcing and thereby past climatic warming of the Arctic, in the most recent three or four decades. However, the present study indicates that local results cannot necessarily be extrapolated over wider areas, and therefore further studies are required to establish regional BC deposition trends within the Arctic. In addition, the study highlights that BC results obtained using different analytical methods should be compared with caution.
  • Anttila, Jani (Helsingin yliopisto, 2015)
    Environmental opportunist pathogens are a class of organisms that are able to both infect multicellular hosts and grow in the outside-host environment as free-living organisms. Environmental opportunism differs from obligate parasitism in that direct host-to-host contact is not necessary for disease transmission and that there are environmental pathogen reservoirs which in suitable conditions act as sources of infection. Because of this, environmental opportunist pathogens form a persistent threat to human health, livestock, and wildlife, and cannot be eradicated by treating hosts. Three well-known examples of pathogens of this class are Vibrio cholerae, Flavobacterium columnare, and Bacillus anthracis, all of which cause sporadic outbreaks. Between infections, these pathogens are subject to multiple biotic and abiotic environmental pressures in the outside-host environment. While environmental opportunist pathogens are not dependent on live hosts for transmission and thus benefit from increased virulence, balancing between the two environments, within-host and outside-host, might incur trade-offs and thus limitations to their spread. In this thesis I have developed mathematical models of environmental opportunist pathogen dynamics and studied the effects of environmental variation and outsidehost interactions on patterns of pathogen outbreaks. The studies included in the thesis address (i) the origin of a sigmoidal dose-dependent infectivity response, (ii) the effect of competition in the outside-host environment on opportunist pathogen outbreaks, (iii) the effect of environmental variation on environmental opportunist dynamics, and (iiii) how environmental variation enables invasions of emerging opportunist pathogen strains. The modelling approach has enabled identification of factors such as alleviation of competitive pressure and certain kinds of environmental variation as outside-host environmental factors that promote outbreaks. Additionally, modelling results can be used to suggest control strategies to reduce the probability of environmental opportunist pathogen outbreaks.
  • Pukkila, Veera (Helsingin yliopisto, 2015)
    The worldwide use of pesticides (herbicides, insecticides, and fungisides) currently amounts to 2.4 billion kilos. Only a small proportion of pesticides actually reach the target organism, whereas the majority becomes a potent contaminant that threatens the environment and humans. Microbes, present everywhere in the environment, have the ability to degrade many kinds of man-made chemical compounds, xenobiotics. By studying the degrading microbes and the optimal conditions for microbial degradation, bioremediation techniques may be developed to clean contaminated sites. A metabolite of the herbicide 2,6-dichlorobenzonitrile (dichlobenil), is 2,6-dichlorobenzamide (BAM). BAM is frequently detected in groundwater worldwide, and thus the use of dichlobenil is nowadays banned in the EU. Dichlobenil is degraded in soil relatively quickly, but BAM is much more persistent. Due to its high water solubility and low sorption affinity, BAM easily leaches down to deeper soil layers and even to groundwater where it is considered stable. This study focused on the degradation of dichlobenil and BAM in Finnish groundwater sedimentary deposits and topsoil. The biotic and abiotic factors associated with effective dichlobenil or BAM degradation were studied. The aim was to examine how the presence of microbes and oxygen, and the chemical characteristics of soil and groundwater deposits, affect the degradation rates of dichlobenil and BAM. In addition, the indigenous microbes degrading these compounds were enumerated, and some were isolated and identified. Dichlobenil was degraded in all studied groundwater sedimentary deposits and topsoil. The presence of microbes and oxygen, and high carbon and nitrogen contents enhanced dichlobenil degradation. As expected, BAM was more resilient to microbial degradation than dichlobenil. Significant aerobic microbial degradation of BAM was detected only in one out of five deposits, and in another weak biodegradation was observed. In these two deposits the concentrations of the elements manganese, zinc, cobalt, lead, and nickel were high. Aerobic bacterial strains growing in the presence of dichlobenil or BAM were isolated from all studied groundwater sedimentary deposits and topsoil. The isolates belonged to the phyla Proteobacteria, Actinobacteria, and Bacteroidetes, Gammaproteobacteria being the largest group of isolates. The dichlobenil or BAM degradation capacity of the isolates was rather low (5-46%) and not demonstrated for all isolates. In conclusion, the diversity of dichlobenil and BAM degrading aerobic microbes in Finnish groundwater sedimentary deposits and topsoil was relatively high. Especially the high number and diversity of isolated BAM degrading strains was unexpected, as only few BAM degrading strains have been reported earlier. Due to their low degradation capacity the potential of these isolates in bioremediation is not considered high, but they could be used e.g. to identify the genes and enzymes involved in the degradation of dichlobenil and BAM.
  • Gu, Yuexi (Helsingin yliopisto, 2015)
    Women with germline mutations of BRCA1 gene are predisposed to the development of basal-like breast cancer, which is characterized by the absence of the hormonal and growth factor receptors, ER, PR and HER2. Mutation of BRCA1 predicts increased sensitivity to certain DNA-damaging agents, e.g. PARP inhibitors and cisplatin. Yet in clinical trials, some BRCA1-mutant breast tumors show resistance to these drugs. Therefore, it is necessary to understand the molecular mechanisms of the acquired resistance and identify novel therapeutic targets for the treatment of those tumors. In order to better understand the biology of breast cancers caused by BRCA1 mutations, we collected and characterized four BRCA1-mutant breast cancer cell lines as surrogates of BRCA1-mutant tumors. Together with several cell lines expressing wildtype BRCA1, we tested their sensitivities to a panel of DNA-damaging agents. We also carried out a high-throughput chemical compound screen on the BRCA1-mutant and BRCA1-wildtype cell lines, and performed a proteome profiling assay to test the kinase activities of those cell lines. Our results reveal extensive heterogeneity among the BRCA1-mutant breast cancer cell lines, which showed resistance to DNA-damaging agents. Then we created isogenic MDA-MB-231 cells with or without BRCA1 depletion by siRNA transfection, and carried out a high-throughput chemical compound screen on the pair of cells in order to identify potential targets that are synthetically lethal with BRCA1 deficiency. Two proteasome inhibitors, bortezomib and carfilzomib, were found to be able to selectively kill BRCA1-depleted cells. Further studies on mechanisms demonstrate that the proteasome inhibitor bortezomib does not induce DNA damage; rather it inactivates G1 cell cycle checkpoint in BRCA1-deficient cells and leads to the accumulation of these cells at G2/M phase. This is caused by inactivation of the retinoblastoma protein (Rb) through its hyperphosphorylation, which in turn activates its downstream transcription factor E2F1. In addition, bortezomib causes compromised G2/M cell cycle checkpoint in BRCA1-deficient cells, which drives cells to enter mitosis and leads to apoptosis due to uncontrolled cell division. In order to investigate the mechanisms underlying the consistent basal-like phenotype of breast tumors associated with BRCA1 mutations, we established an in vitro assay to study the transition of mouse mammary epithelial cells from luminal to basal lineages. Our results showed that loss of BRCA1 promotes basal-like differentiation by sustaining ΔNp63 activity. In luminal cells, ΔNp63 is not expressed or remains inactive through localization in nucleoli. Depletion of BRCA1 leads to translocation of ΔNp63 into nucleoplasm and promotes transition of luminal cells into a basal state. This study provides a potential link between BRCA1 loss and the basal-like differentiation, which may help to explain why BRCA1-mutant breast cancer tends to bear a basal-like phenotype.
  • Hirvenkari, Lotta (Helsingin yliopisto, 2015)
    Social interaction consists of events of different modalities that unfold on a subsecond timescale and are usually influenced by all involved participants. Therefore, social interaction is difficult to be simulated in laboratory, as simple, static, and unidirectional stimuli and tasks do not cover its properties accurately enough. However, moving towards more natural experimental setups in brain imaging, e.g. in magnetoencephalography (MEG), means giving up many traditional ways of analysis, such as signal averaging on the basis of pre-classified well-controlled stimuli. Thus, in addition to developing naturalistic experimental setups, new ways are needed to analyse the data and to classify the events of interest. In this thesis, ecologically valid experimental setups for brain imaging of social interaction were developed and tested in three MEG and two behavioural experiments. Of the MEG studies, the first study presented in this thesis introduced a free-viewing paradigm for MEG and showed different responses to congruent and incongruent audiovisual stimuli in the auditory cortex. In the second MEG study auditory cortex was shown to respond differently to the anticipation of emotional and neutral sounds. The third MEG study presented a setup for simultaneous MEG measurements of two interacting persons, validating its feasibility by showing reproducible and similar auditory responses in both subjects to stimuli delivered from the two measurement sites. The two behavioural studies of this thesis concentrated on turn taking behaviour in conversation. The first of them showed that the organization of turn-taking guides the gaze of an external viewer of the conversation. The latter study demonstrated that speech is a strong inducer of behavioural entrainment as speakers mutually adapted their speaking rhythms when producing sentences with a partner.
  • Kislin, Mikhail (Helsingin yliopisto, 2015)
    Acute brain trauma and ischemia are severe injuries that have no adequate treatment to date. In vivo two-photon microscopy allows studying longitudinally the process of injury development and brain recovery. This thesis summarizes the work on: i) animal model of acute brain injury and role of extracellular matrix in neuronal recovery and plasticity, ii) investigation of mitochondria during physiological/pathological calcium elevations in vitro and further implementation of quantitative microscopic analysis of neuronal mitochondrial morphology in vivo, iii) mitochondrial damage and recovery in animal models of acute neurodegenerative disorders in the neocortex of anesthetized mice, iv) a novel approach for awake head-fixed recordings. The results described in this work provide novel approaches for intravital morphological analysis of neurons and of their mitochondria, increase our understanding of pathogenesis after traumatic and ischemic injury in neocortex of rodents, and enable the development of novel therapies for CNS injuries.
  • Karjalainen, Katja (Helsingin yliopisto, 2015)
    Acute myeloid leukemia (AML), although rare, is highly malignant neoplasms that account for a majority of leukemia-associated deaths. AML results from an over-growth of immature myeloid cells in the bone marrow. These cells are functionally abnormal and interfere with the production of normal hematopoietic cells. Despite apparent phenotypic uniformity, AML is a heterogeneous group of myeloid malignancies, in which multiple genetic and epigenetic aberrations accumulate in hematopoietic stem or progenitor cells leading to disturbed cellular growth, proliferation, and differentiation. The nature and number of these AML-associated molecular abnormalities can vary widely among patients. Accordingly, the survival rate also fluctuates greatly, with an average ~25% overall survival rate. Chemotherapy is currently the mainstay treatment option for the patient with AML. However, although required to induce initial remission, chemotherapy contributes to new mutations and clonal evolution, which often leads to disease relapse. Consequently, targeted therapies are urgently needed to eradicate AML cells. One of the major challenges in clinical oncology today is that therapeutic agents cannot be selectively delivered to tumor site without causing toxicity to rest of the body. In addition, tumor microenvironment has a key role in mediating drug efficacy and resistance, which hampers the discovery of clinically relevant drugs. Therefore, functional screening platforms that can identify cancer-specific targets as well as assess the therapeutic relevance of drug candidates within the appropriate disease microenvironment are fundamental in identification of novel targets and therapeutic agents with better attributes than conventional chemotherapy drugs. In our studies, we have used two discovery approaches: (i) a phage display technology that allows the identification of ligands binding to physiologically relevant targets, and (ii) a novel ex vivo screening assay, which allows identification of candidate drugs against leukemia that are effective in the presence of human blood and bone marrow components. Specifically, our studies have elucidated the function of the leukemia invadosome a supramolecular complex containing certain β2 integrins and matrix metalloproteinases in the context of extramedullary leukemia. We show that this complex is essential for extravasation of leukemia cells as well as for leukemia cell growth, and that blocking the function of this complex possesses potent anti-leukemia and anti-invasion effects. We also demonstrate new roles for neuropilin-1 and interleukin-11 receptors in the pathogenesis of leukemia. We show that these membrane-associated proteins are highly expressed in leukemia cell lines as well as in bone marrow samples from leukemia patients. In addition, we show that the ligands binding to these receptors can be utilized in targeted drug delivery. Finally, we developed a new functional ex vivo screening assay to identify candidate anti-leukemia agents in the presence of human blood or bone marrow under hypoxic conditions. Under these conditions, leukemic cells deplete oxygen faster than normal cells causing a shift in the hemoglobin oxygenation state. This shift, detected by measuring the optical density at 600 nm (OD600) after an appropriate incubation time, directly correlates with leukemic cell counts. Thereby, the oxygenation state of native hemoglobin serves as a reliable and reproducible built-in indicator of leukemia cell growth and/or viability. Our study showed that this assay is highly amenable for high-throughput screening against leukemia. Our results support the idea that this new methodology could be a tool for the prediction of drug efficacy and/or response in leukemia patients.
  • Heiskanen, Jouni (Helsingin yliopisto, 2015)
    Greenhouse gas (GHG) emissions from lakes result from processes between the watershed, lake characteristics and the atmosphere. The organic matter loading from the watershed both provides carbon for the lake biota and in major part defines water clarity, which, in addition to wind and heat flux, is essential in thermocline formation. Thermal stratification suppresses the wind-induced momentum input to the surface water preventing effective mixing of gases throughout the water column. The lake biota process the organic matter, producing carbon dioxide (CO2) in oxic surface water and also methane (CH4) if the near-bottom water becomes anoxic, and thus influence the chemical properties of the water column. Finally, air-water GHG exchange occurs over a thin layer at the water surface. Lakes are typically supersaturated with CO2 and CH4 in relation to average atmospheric mixing ratios causing fluxes of these gases to the atmosphere. Even though lakes cover only 2 % of the world s land surface, it has been estimated that lakes release about 10 % of the carbon fixed annually by the terrestrial ecosystems back to the atmosphere. A critical parameter in the gas exchange estimates is the gas transfer velocity (k), which is governed by turbulence. The implementation of direct flux measurement using the eddy covariance (EC) technique allows the detailed measurements needed to estimate k. However, on lakes, the EC method is a novel subject and as of yet, there has been no published estimates of the error related to these measurements nor fully established set of accepted procedures. The aim of this thesis was to assess the current global CO2 evasion estimates from lakes to the atmosphere by comparing parameterizations for k and the significance of wind and heat flux to the gas transfer in small lakes. To improve future predictions of gas evasion from lakes, we focused on the changes in water clarity and how they affect water column physics and processes in the air-water interface. We used the EC method for the high precision data needed, and therefore also aimed to improve the EC methodology on lakes. The air-water gas transfer was related to both wind and heat loss during times of seasonal stratification, but only to wind during autumn overturn, and the mean value for k of CO2 was 6.0 cm h-1 in Lake Kuivajärvi. When wind-induced thermocline tilting and resulting spatial variability in surface water CO2 concentrations was accounted for, k derived from the measurements dropped to 5.2 cm h-1. This was still over twice the estimate (2.2 cm h-1) calculated with a widely used model for k in lakes suggesting that the global estimates of gas evasion from lakes might be underestimations. Unsolved question is that how important factor the thermocline tilting is in other lakes in defining spatial variability? Our results showed that k for CH4 was higher than for CO2, a result which has been reported in some other studies, but as of yet, no solid explanation has been found. Water clarity was a significant parameter defining the thermal stratification of the lake: a change from clear to dark water would lead to shorter stratification period and lower water column temperatures in small lakes and therefore have significant impact on the lake-atmosphere exchange processes. Important question is how changes is climate will affect lake water clarity e.g. via precipitation and runoff related DOC loading. We concluded that the EC method produces reliable results even in a small lake after rigorous data processing. After these procedures, about half of the CO2 and turbulent heat flux data were of good quality with relative random errors of 10 % and 26 % for heat and CO2 fluxes, respectively. When measuring GHG fluxes accurately from lakes, methods that integrate over time and space are a necessity. These will provide more detailed knowledge on the complex processes that contribute to the gas transfer, from large scale physical phenomena such as thermocline tilting to small scale such as near-surface turbulence. Since most of the world s lakes are small and in northern latitudes, our studies have wide implications even to the global level. Better understanding of the lake biogeochemistry will allow us to make more accurate estimates of GHG evasion from lakes in different regions as well as predictions of how the climate change will affect the lake-atmosphere GHG fluxes.
  • Kysenius, Kai (Helsingin yliopisto, 2015)
    Aging-related increase of neuronal stress may promote the development of sporadic late-onset Alzheimer s disease (LOAD) and other forms of dementia. LOAD risk is also increased by genetic factors such as ApoE4 and diseases such as type 2 diabetes (T2D). Early LOAD pathology is characterized by alterations in brain lipoprotein receptor expression and neuronal hypometabolism. Multifunctional lipoprotein receptors regulate neuronal plasticity, cholesterol and metabolic homeostasis. Lipoprotein receptors apolipoprotein receptor 2 (ApoER2) and very-low density lipoprotein receptor (VLDLR) bind ApoE, but also interact with proteins centrally involved in LOAD pathogenesis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and the nutraceutical berberine modulate lipoprotein receptor levels in vivo. PCSK9 inhibitors and berberine have recently surfaced as promising treatment options for hypercholesterolemia and T2D, respectively. Additionally, PCSK9 and berberine are implicated in pathways modulating neuronal viability, suggesting they may hold therapeutic potential against neurodegenerative diseases. However, the effects of PCSK9 and berberine on neuronal cell death and lipoprotein receptors are currently poorly understood. The objective of this study was to elucidate the role of PCSK9 and berberine as modulators of lipoprotein receptors and cell death in neurons. The effects of RNAi-mediated PCSK9 downregulation and berberine on neuronal viability were studied in mouse and rat primary neuron cultures. Mechanistic basis of effects were further studied in combination with lentiviral RNA interference, kinase inhibitors and various inducers of cellular stress and cell death. Cell viabilities were assessed by immunofluorescence, Western blotting, and cell toxicity and mitochondrial assays. The main conclusions of this study are: (1) reducing endogenous PCSK9 levels genetically by lentiviral-mediated RNAi protects neurons against apoptotic cell death in an ApoER2-dependent fashion; (2) a potential PCSK9 inhibitor and a widely used nutraceutical berberine causes mitochondria- and NMDA receptor-dependent neuronal cell death at micromolar concentrations; (3) at subtoxic nanomolar concentrations, berberine sensitizes neurons to rotenone and glutamate toxicity calling for caution in berberine dosing and chronic use; and (4) subtoxic stress, including berberine, increase neuronal VLDLR expression, associated with a biphasic effect on the stabilization of the transcription factors hypoxia-inducible factor 1α and β-catenin. To conclude, ApoER2, VLDLR and their modulators PCSK9 and berberine contribute to the regulation of neuronal cell death via multiple mechanisms, suggesting a potential role in neurodegenerative disease pathogenesis at the interface of metabolism and survival signaling.
  • Kulesskiy, Evgeny (Helsingin yliopisto, 2015)
    Syndecans are cell surface heparan sulfate proteoglycans which are present in all tissues and cell types and have distinct temporal and spatial expression patterns. They play important roles in embryonic development of the organism and control relocation and alteration of extracellular matrix components. Syndecans regulate cell migration, adhesion and proliferation and are engaged in tissue injury, inflammation processes, pathogenesis of infectious diseases and tumor biology. This thesis summarizes the results of studies on one of the syndecan family receptors syndecan-3 (also known as N-syndecan). This proteoglycan is abundantly expressed in developing brain. Syndecan-3 acts as a signaling receptor upon binding of its ligand, heparin-binding growth associated molecule (HB-GAM; also known as pleiotrophin), which activates the cortactin c-Src signaling pathway. This leads to rapid neurite extension in neuronal cells, which makes syndecan-3 an interesting transmembrane receptor in neuronal development and regeneration. However, little is known about the signaling mechanism of syndecan-3. Here I show formation of ligand-syndecan-3 signaling complexes at the cell surface using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET). Ligand binding leads to dimerization of syndecan-3 at the cell surface. The dimerized syndecan-3 colocalizes with actin in the filopodia of cells. Lysine 383 in the juxtamembrane (ERKE) sequence and G392 and G396 from GXXXG canonical motif are shown to be important for the ligand-induced dimerization, whereas the cytosolic domain are not required for the dimerization. In addition to acting as a signaling receptor, syndecan-3 acts as a co-receptor in epidermal growth factor receptor (EGFR) ligand binding. FRET analysis suggests that interactions of syndecan-3 and EGFR depend on a shared ligand such as heparin-binding EGF-like growth factor (HB-EGF). Furthermore, it was shown that syndecan-3 may act as a receptor for other ligands, like glial cell line-derived neurotrophic factor (GDNF). In addition, I have found a new receptor for HB-GAM glypican-2 which may be involved in regulation of HB-GAM signaling by competing with syndecan-3 for ligand binding.
  • Keceli, Mehmet Ali (Helsingin yliopisto, 2015)
    In order to protect themselves against several biotic and abiotic stresses, plants are equipped with an array of defense mechanisms. Induced defenses and stress responses play a major role in plant disease resistance and are regulated by a network of interconnected signal transduction pathways with the plant hormones ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) as the crucial mediators. These specific hormone-mediated signaling cascades trigger the expression of distinct sets of stress-related genes leading to tolerance or resistance. F-box proteins are important components in mediating stress responses. They act as members of Skp1-Cullin-F-box (SCF) protein complexes, which target substrate proteins for modification and degradation and thereby allow plants to respond rapidly to environmental stress factors. Arabidopsis genome encodes roughly 700 F-box proteins involved in various processes including plant metabolism, hormonal responses as well as responses to environmental stresses. The amount of F-box proteins in plants is significantly higher than in other eukaryotes indicating SCF-dependent ubiquitination to be a major route for selective protein degradation in plants. In this study we screened a group of Arabidopsis F-box T-DNA lines for their tolerance to several abiotic and biotic stresses. To do this, we employed a reverse genetic screening approach and characterized the phenotypes of the T-DNA mutant lines. Among the tested lines, two T-DNA insertion lines with insertions in genes encoding MAX2 and AFB4 demonstrated altered response to ozone and pathogens, respectively, and were therefore chosen for further characterization. In the last part of the thesis, the role of EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), a negative regulator of abscisic acid responses in Arabidopsis was further characterized concerning its role in biotic stress responses in Arabidopsis.
  • Juuri, Juuso (Helsingin yliopisto, 2015)
    Kainate-type of ionotropic glutamate (KA) receptors are associated with the modulation of neuronal excitability, synaptic transmission, and activity of neuronal networks. They are believed to have an important role in the development of neuronal connections. In this thesis, the role of KA receptors in the early brain development was assessed by conducting in vitro electrophysiological recordings from individual neurons at CA3 region in acute slices of neonatal rodent hippocampi. It was found that activation of separate KA receptor populations promoted action potential firing in both glutamatergic pyramidal neurons and GABAergic interneurons. The receptors in pyramidal neurons displayed a high affinity for agonist kainate, appeared to lack subunit GluK1, and promoted spontaneous firing of pyramidal neurons without depolarizing them. The receptors in interneurons contained subunit GluK1 and their activation suppressed afterhyperpolarizing current of medium duration (ImAHP). Receptors in both neuron types appeared to be activated tonically by ambient glutamate, suggesting that their physiological role may be to act as a modulatory mechanism sensitive to changes in extracellular glutamate concentration. Changes in activity of neurons at CA3 by activation of KA receptors were reflected on the network level. Promotion of pyramidal cell firing by pharmacological activation of high-affinity KA receptors lead to enhanced glutamatergic drive and generation of network bursts in the CA3 region. The ImAHP in interneurons was also suppressed by apamin, a blocker of SK potassium channels that mediate majority of this current, and apamin enhanced generation of network bursts. This suggests that also KA receptor mediated regulation of ImAHP may modulate network activity. It was also found that there was an interaction between KA receptors and ethanol in the modulation of hippocampal network: ethanol decreased the occurrence of the network bursts at postnatal days 1 (P1) and P10, whereas it increased bursting at P5. The network effects of ethanol were partially or completely counteracted by specific pharmacological block of GluK1 subunit-containing KA receptors. The findings disclose that via regulation of activity of individual neurons, KA receptors are capable of robust modulation of network activity in immature hippocampus. Additionally, exogenous agents affecting KA receptors may perturb activity dependent developmental processes that are central for the synaptic development. The results shed light on the mechanisms underlying development of hippocampal connectivity, and may help to understand early pathologies of the brain that have developmental origins.
  • Kankainen, Matti (Helsingin yliopisto, 2015)
    Lactobacilli are generally harmless gram-positive lactic acid bacteria and well known for their broad spectrum of beneficial effects on human health and usage in food production. However, relatively little is known at the molecular level about the relationships between lactobacilli and humans and about their food processing abilities. The aim of this thesis was to establish bioinformatics approaches for classifying proteins involved in the health effects and food production abilities of lactobacilli and to elucidate the functional potential of two biomedically important Lactobacillus species using whole-genome sequencing. To facilitate the genome-based analysis of lactobacilli, two new bioinformatics approaches were developed for the systematic analysis of protein function. The first approach, called LOCP, fulfilled the need for accurate genome-wide annotation of putative pilus operons in gram-positive bacteria, whereas the second approach, BLANNOTATOR, represented an improved homology-based solution for general function annotation of bacterial proteins. Importantly, both approaches showed superior accuracy in evaluation tests and proved to be useful in finding information ignored by other homology-search methods, illustrating their added value to the current repertoire of function classification systems. Their application also led to the discovery of several putative pilus operons and new potential effector molecules in lactobacilli, including many of the key findings of this thesis work. Lactobacillus rhamnosus GG is one of the clinically best-studied Lactobacillus strains and has a long history of safe use in the food industry. The whole-genome sequencing of the strain GG and a closely related dairy strain L. rhamnosus LC705 revealed two almost identical genomes, despite the physiological differences between the strains. Nevertheless of the extensive genomic similarity, present only in GG was a genomic region containing genes for three pilin subunits and a pilin-dedicated sortase. The presence of these pili on the cell surface of L. rhamnosus GG was also confirmed, and one of the GG-specific pilins was demonstrated to be central for the mucus interaction of strain GG. These discoveries established the presence of gram-positive pilus structures also in non-pathogenic bacteria and provided a long-awaited explanation for the highly efficient adhesion of the strain GG to the intestinal mucosa. The other Lactobacillus species investigated in this thesis was Lactobacillus crispatus. To gain insights into its physiology and to identify components by which this important constituent of the healthy human vagina may promote urogenital health, the genome of a representative L. crispatus strain was sequenced and compared to those of nine others. These analyses provided an accurate account of features associated with vaginal health and revealed a set of 1,224 gene families that were universally conserved across all the ten strains, and, most likely, also across the entire L. crispatus species. Importantly, this set of genes was shown to contain adhesion genes involved in the displacement of the bacterial vaginosis-associated Gardnerella vaginalis from vaginal cells and provided a molecular explanation for the inverse association between L. crispatus and G. vaginalis colonisation in the vagina. Taken together, the present study demonstrates the power of whole-genome sequencing and computer-assisted genome annotation in identifying genes that are involved in host-interactions and have industrial value. The discovery of gram-positive pili in L. rhamnosus GG and the mechanism by which L. crispatus excludes G. vaginalis from vaginal cells are both major steps forward in understanding the interaction between lactobacilli and host. We envisage that these findings together with the developed bioinformatics methods will aid the improvement of probiotic products and human health in the future.