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  • Yu, Liying (Helsingin yliopisto, 2009)
    Programed cell death (PCD) is a fundamental biological process that is as essential for the development and tissue homeostasis as cell proliferation, differentiation and adaptation. The main mode of PCD - apoptosis - occurs via specifi c pathways, such as mitochondrial or death receptor pathway. In the developing nervous system, programed death broadly occurs, mainly triggered by the defi ciency of different survival-promoting neurotrophic factors, but the respective death pathways are poorly studied. In one of the best-characterized models, sympathetic neurons deprived of nerve growth factor (NGF) die via the classical mitochondrial apoptotic pathway. The main aim of this study was to describe the death programs activated in these and other neuronal populations by using neuronal cultures deprived of other neurotrophic factors. First, this study showed that the cultured sympathetic neurons deprived of glial cell line-derived neurotrophic factor (GDNF) die via a novel non-classical death pathway, in which mitochondria and death receptors are not involved. Indeed, cytochrome c was not released into the cytosol, Bax, caspase-9, and caspase-3 were not involved, and Bcl-xL overexpression did not prevent the death. This pathway involved activation of mixed lineage kinases and c-jun, and crucially requires caspase-2 and -7. Second, it was shown that deprivation of neurotrophin-3 (NT-3) from cultured sensory neurons of the dorsal root ganglia kills them via a dependence receptor pathway, including cleavage of the NT- 3 receptor TrkC and liberation of a pro-apoptotic dependence domain. Indeed, death of NT-3-deprived neurons was blocked by a dominant-negative construct interfering with TrkC cleavage. Also, the uncleavable mutant of TrkC, replacing the siRNA-silenced endogeneous TrkC, was not able to trigger death upon NT-3 removal. Such a pathway was not activated in another subpopulation of sensory neurons deprived of NGF. Third, it was shown that cultured midbrain dopaminergic neurons deprived of GDNF or brainderived neurotrophic factor (BDNF) kills them by still a different pathway, in which death receptors and caspases, but not mitochondria, are activated. Indeed, cytochrome c was not released into the cytosol, Bax was not activated, and Bcl-xL did not block the death, but caspases were necessary for the death of these neurons. Blocking the components of the death receptor pathway - caspase-8, FADD, or Fas - blocked the death, whereas activation of Fas accelerated it. The activity of Fas in the dopaminergic neurons could be controlled by the apoptosis inhibitory molecule FAIML. For these studies we developed a novel assay to study apoptosis in the transfected dopaminergic neurons. Thus, a novel death pathway, characteristic for the dopaminergic neurons was described. The study suggests death receptors as possible targets for the treatment of Parkinson s disease, which is caused by the degeneration of dopaminergic neurons.
  • 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.
  • Haansuu, Johannes Pasi (Helsingin yliopisto, 2002)
  • Joensuu, Merja (Helsingin yliopisto, 2014)
    The boundaries of the cell and the intracellular organelles are created by biological membranes consisting of lipids, proteins and sugars. These membranes are used for a number of purposes, of which, probably the most important is the compartmentalization of the cell s functions. Understanding the mechanisms to create/ maintain the characteristic shape of organelles, and identifying the players involved, allows us to connect the structure of an organelle to its functions or dynamics and, importantly, to the disorders associated with malfunctioning organelle. How these processes in endoplasmic reticulum (ER) are coupled, has remained unclear. In this thesis, the structure and dynamics of ER and the connection to some of the ER s functions were analysed. By using live cell imaging and 3D-electron microscopy, biochemical approaches, and novel quantitative image analysis, we demonstrated the great variation in interphase ER network organization and sheet structures among several cultured mammalian cell lines. We described interphase ER sheet dynamics and showed that sheets were static and persistent structures. Our work revealed that a specific subset of actin filaments have a role in ER sheet persistence and the ER network organization. Furthermore, we discovered a novel role for molecular motor myosin 1c in regulating the ER-associated actin filament arrays. Moreover, we learned that in mitosis ER undergoes a progressive spatial reorganization and a structural transformation towards more fenestrated sheets and tubular forms. We showed that ER remains continuous during mitosis and that the partition of nuclear envelope was subordinate to ER, which, in addition to structural transformations of ER into smaller subunits, favours the stochastic model of inheritance of ER. Importantly, we showed that the natural increase of ER fenestrations and tubulation in mitosis correlated with the reduced number of membrane-bound ribosomes, and that the structural transformation could be mimicked by dissociating the membrane-bound ribosomes from the interphase ER by a drug treatment, suggesting that ribosomes and the associated luminal translation machinery have a role stabilizing the sheet structures. Collectively, this work describes the significant plasticity of ER morphology and organization in several common cell culture cells in interphase and upon inheritance of ER. Importantly, this work also demonstrates the dynamic rearrangements of ER in mitosis and interphase cells and provides novel information about the role of ribosomes and actin on ER sheets and the role of myosin 1c on the ER-associated actin arrays, serving as an opening for further studies on variety of regulatory possibilities of the interplay between ER subdomains and the identified player involved.
  • Vesikansa, Aino (Helsingin yliopisto, 2013)
    The development of glutamatergic transmission in the brain occurs gradually during the first postnatal weeks. During this critical period, nascent synaptic connections are finely tuned to form networks reliably transmitting and processing information. In the hippocampus, kainate receptors (KARs) are heavily expressed during early development and suggested to have an instrumental role in the activity-dependent development of neuronal connectivity. KARs are composed of various combinations of five subunits, GluK1-GluK5. Additional structural and functional diversity of receptors is achieved by alternative splicing and RNA editing of the subunits. The function of KARs differs from the other types of ionotropic glutamate receptors (iGluRs) in two essential respects: first, their primary role is not to mediate but to modulate transmission, and second, KARs use a non-canonical metabotropic signaling mechanism in addition to the classical ionotropic action. The diverse functional roles of KARs are reflected in their highly polarized subcellular localization, which is regulated in a subunit- and cell-specific manner. Despite the increasing number of roles characterized for KARs, their function during development is poorly understood. The aim of this study was to clarify the physiological roles of KARs in the developing glutamatergic connectivity in area CA1 of the rat hippocampus. First, we present a novel, developmentally restricted type of endogenous KAR activity, which has major influence on glutamatergic transmission in the immature hippocampus. During early development, high affinity G-protein-coupled presynaptic KARs are shown to be tonically activated by ambient glutamate to maintain a low probability of glutamate release in a subpopulation of CA3-CA1 synapses. This KAR-mediated presynaptic silencing has a critical impact on the transmission of glutamatergic information; KARs filter out sporadic low-frequency activity and promote transmission during highfrequency bursts representing natural-type of activiy within the immature hippocampal network. Next, we demonstrate that the GluK1c splice variant plays a pivotal role in immature-type KAR activity. The developmental and cell-type specific expression pattern of GluK1c mRNA corresponds to the tonic KAR activity. Furthermore, the presynaptic expression of GluK1c is shown to directly suppress glutamatergic transmission in cell-pairs in vitro and to mimic tonic KAR activity at CA3-CA1 synapses in vivo at a developmental stage when the immature-type KAR activity is already downregulated. Thus, the developmental downregulation of tonic KAR activation can be fully explained by the loss the GluK1c expression in CA3 pyramidal cells. We further show that activity-dependent mechanisms, such as the experimental induction of LTP, can rapidly downregulate tonic KAR activity and switch immature, labile synapses to mature ones. This involves a modification of the receptor per se, leading to the loss of high-affinity KARs. Finally, we show the critical involvement of tonic KAR activity in the formation /stabilization of glutamatergic connections in the hippocampal slice cultures. Mimicking tonic KAR activity by pharmacological activation of GluK1 containing KARs resulted in significant and permanent increase in the number of functional glutamatergic synapses. The essential role of endogenous KAR activity was indicated by the finding that blocking KARs during the period of intense synaptogenesis led to dramatic decrease in glutamatergic connectivity later in development. In summary, the novel findings of this work demonstrate that endogenous KAR activity has crucial role in modulating the glutamatergic transmission and connectivity in the developing hippocampus. This not only broadens our view of the activity-dependent mechanisms underlying the development of synaptic connectivity in the brain, but also provides a basis for understanding the pathophysiological functions of KARs in neurodevelopmental disorders.
  • Luchkina, Natalia V. (Helsingin yliopisto, 2015)
    Activity-dependent synaptic plasticity, and long-term potentiation in particular, represents the predominant model of memory and learning at the cellular level. In addition, synaptic plasticity plays a critical role in the activity-dependent refinement and fine-tuning of neuronal circuits during development by maintaining and stabilising certain synaptic connections and eliminating others. The main goal of this project was to increase our understanding of the molecular mechanisms underlying activity-dependent synaptic plasticity in the developing brain, with particular emphasis on the mechanisms that are specific to early postnatal development. First, we characterise in detail the properties of developmentally restricted neonatal presynaptic long-term potentiation (LTP) in CA1 area of the hippocampus and demonstrate its susceptibility to regulation via protein kinase C (PKC) signalling. Next, we explore the physiological functions of GluA4 subunit-containing AMPA type glutamate receptors, predominantly expressed at developing CA3 CA1 synapses. We show that GluA4 expression is necessary for protein kinase A (PKA)-dependent LTP at immature synapses. Further, the loss of GluA4 expression in parallel with circuit maturation explains the developmental switch in LTP signalling requirements from PKA- to Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent. Further, we also explore the role of GluA4 C-terminal interaction partners in synaptic trafficking of GluA4-containing AMPA receptors and its importance for synapse maturation. We confirm a critical role for the membrane proximal region of GluA4 C-terminal domain in trafficking and identify a novel mechanism for activity-dependent synaptic delivery of GluA4 by the extreme C-terminal region. Finally, we show an important role of the GluA4 subunit in strengthening of AMPA receptor-mediated transmission, observed during early postnatal development. In summary, we provide novel information on the pre- and postsynaptic plasticity mechanisms operating at hippocampal CA3 CA1 synapses during the critical period of activity-dependent maturation of glutamatergic neuronal circuitry in rodents. This expands our knowledge on the cellular mechanisms guiding development of synaptic connectivity in the brain. Dysfunction of such mechanisms may play fundamental roles in the underlying pathophysiological causes of various neurodevelopmental disorders.
  • Sakha, Prasanna (Helsingin yliopisto, 2016)
    Neurons have distinctive polar morphology with distinctive subcellular features comprising of cell soma, axons and dendrites. Primary objective of this study was to develop a novel microfluidic device for spatial isolation of axons from the somatodendritic compartment of cultured hippocampal neurons. A new method was developed for asymmetrical genetic manipulation improving specificity in studies of how individual proteins affect axonal morphology, presynaptic development and function. Subsequently, the microfluidic culture system was used to study the signaling events involved in synaptogenesis, focusing on the roles of kainate type of glutamate receptors (KARs). Functional studies have shown that KARs are present in axons and may regulate presynaptic function. However, the molecular composition and detailed subcellular localization of axonal KARs as well as their roles in presynaptic differentiation are largely unknown. The results show that axonal KARs promote early stages of synaptogenesis. Expression of low (GluK1-3) and high affinity (GluK4-5) KAR subunits promoted filopodiogenesis function at the isolated axons. In addition, axonal low affinity subunits enhanced clustering of synaptic vesicles and transmission efficacy at nascent glutamatergic synapses, an effect which was associated with widening of presynaptic active zone. High affinity KAR subunits had no effect on synaptic vesicle clustering, nor presynaptic transmission efficacy. However their heteromerization with low affinity subunits completely prevented the synapse promoting effects and instead lead to strong inhibition of presynaptic transmission efficacy. The presynaptic effects of GluK1-3 on synaptic vesicle clustering involved both PKA and PKC pathways. GluK1 expression was developmentally regulated in neonatal and juvenile hippocampus and heteromeric combination of GluK1c with high affinity subunits suppressed glutamatergic synaptic transmission. KARs are linked to various neurological and neuropsychiatric disorders. Our observations and previous findings strongly suggest that KARs are involved in morphological maturation of neurons and in refinement of neuronal circuitry in the brain. The present results provide novel insights into the involvement of different types of KAR subunits in synaptic development and morphological differentiation. Hence, they are potential therapeutic targets in various developmentally originating neurological disorders.
  • Mäntylahti, Sampo (Helsingin yliopisto, 2014)
    In the field of bioscience there is an ongoing explosive growth in discovery and information. Novel means in biotechnology as well as in medicines are introduced at an unseen rate. One of the aspects contributing to this development is the increased understanding of protein function and structure. Proteins have a role in almost every biological process. The function and structure of proteins are linked. Recent studies have discovered that the understanding of the protein structure has been biased. Namely, the studies have unearthed a previously dismissed protein structure state: intrinsically disordered proteins (IDPs). In this highly dynamic state a protein is without a globular fold, but does not meet the requirements of a random coil either. Rapid transition between folds renders most of the established research techniques to be poor methods to study the IDPs. Nuclear magnetic resonance (NMR) is a spectroscopy method, which enables the study of molecules at atomic resolution. The technique is based upon manipulation of the nuclear spins in specifically produced sample under strong magnetic field. In this method, spins of the system generate quantum coherence state(s), which is utilized to obtain information about the system. NMR is suitable for studying samples in solid and liquid mediums, but in case of biomolecules, water solution is preferable as it resembles in vivo environment. Highly mobile structure and chemical composition of IDPs cause many established NMR experiments to fail. Development of NMR pulse sequences is an obvious approach to solve the problem. This thesis presents a number of NMR pulse sequences, which are designed to improve acquisition of information from highly mobile sections of proteins. The key aspect is to utilize H atom instead of HN in coherence transfer. Additional improvements include limited residue specific identification and novel coherence transfer pathways. Articles I, II, and III present triple resonance experiments, which correlate protein backbone atoms. Combination of the spectra enables full sequential assignment. Article IV introduces an improved pulse sequence for measuring J couplings between nitrogen and amide proton. The experiments were subjected to experimental verification. Comparisons were drawn between established pulse sequences. In both globular proteins and IDPs the results show improvement over established pulse sequences. The proposed sequences yielded improved assignment coverage, resolution and sensitivity enhancement.
  • Gabryelczyk, Bartosz (Helsingin yliopisto, 2015)
    The possibility of controlling interactions at interfaces and surfaces of solid materials is highly interesting for a wide range of materials-related nanotechnological applications, for example, colloidal systems, adhesives, biosensors, biomimetic composite and biomedical materials. In Nature, many proteins and peptides possess the ability to recognize, specifically bind, and modify the surfaces of solid materials through sophisticated mechanism of molecular recognition. These properties have been developed during evolution via successive cycles of random mutations and selection. The natural evolution processes can be mimicked in the laboratory scale with the use of a directed evolution approach, for instance, based on the selection of short material-specific peptides from the combinatorial libraries displayed on the surface of bacteriophages or bacterial cells. Selected from billions of different variants, material-specific peptides can be studied by experimental and computational methods to define their sequence, structure, and binding properties. Subsequently, they can be engineered in order to improve their binding affinity and tailor their function for practical applications. The studies presented in this thesis show how phage display was used to identify peptides binding to diamond-like carbon (DLC). DLC is an amorphous form of carbon, with chemical and physical properties resembling natural diamond. It is used as a coating material in many industrial and biomedical applications. Peptides binding to DLC were selected form a combinatorial phage display library. Their binding and molecular basis of the function were investigated in different molecular contexts (when displayed on the phage surface, forming fusion proteins, or present in free soluble form), using multiple independent methods. It was also demonstrated that the peptides can be used in nanotechnological applications, i.e., as a self-assembling coating on the DLC surface, and for controlling properties of a colloidal form of DLC. Besides finding and characterizing peptides binding to DLC, the thesis also highlights different challenges of the directed evolution techniques, for example, selection of target unrelated peptides during biopanning, and the necessity of multiple independent ways of analyzing the functionality of selected peptides.
  • Karppinen, Sirpa (Helsingin yliopisto, 2003)
  • Yang, Ying (Helsingin yliopisto, 2014)
    Cyclin-dependent kinases (Cdks) are an evolutionary conserved group of serine/threonine protein kinases involved in critical cellular processes such as cell cycle and transcription. Cdk7 and CCRK (cell cycle related kinase; also known as Cdk20) form a separate branch together in a phylogenetic alignment of Cdks family. This study here has identified distinct cellular functions of these two kinases and does not support overlapping functions as suggested by orthologs in yeast. Cdk7 together with cyclin H and Mat1 forming the kinase subcomplex of TFIIH basal transcription factor complex is proposed to regulate RNA polymerase II (Pol II) mediated mRNA synthesis by phosphorylating the serine-5 (Ser5) residues of POL II large subunit C-terminal domain (CTD). Investigations in the genetic systems generated here allowing acute depletion of the Cdk7 subcomplex demonstrate that Cdk7 is the mammalian Ser5 phosphorylating kinase and is required for general transcription noted by analysis of newly transcribed RNAs. The analysis also reveals a requirement of Cdk7 for RNA polymerase I mediated rRNA synthesis. The reduced transcription following Cdk7 disruption is associated with changes on chromatin but not reflected in steady-state RNA levels due to increased RNA stability. These results also reveal a coupled regulation of transcription and RNA degradation. A tissue-specific function of the Cdk7 subcomplex is identified as a physiological roadblock to adipogenesis by phosphorylating the master transcription factor of adipogenic program-peroxisome proliferator-activated receptor gamma (PPARγ). The observation that the Cdk7 subcomplex is absent from adipose tissues indicates the so-called basal transcription machinery has very diverse composition in differentiated cells. CCRK is involved in regulating formation of primary cilium, a sensory organelle acting as a signaling hub in the cell. CCRK promotes cell cycle progression by inhibiting ciliogenesis. In glioblastoma cells, reducing the deregulated high level of CCRK or two related substrate kinases of CCRK restores cilia, leading to decreased glioblastoma cell proliferation. Here is identified the first kinase cascade used by tumor cells to disrupt cilia for a growth advantage and offered new therapeutic possibilities.
  • Tervonen, Topi (Helsingin yliopisto, 2008)
    Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.
  • Pertola, Sari (Helsingin yliopisto, 2006)
    In this thesis the role played by expansive and introduced species in the phytoplankton ecology of the Baltic Sea was investigated. The aims were threefold. First, the studies investigated the resting stages of dinoflagellates, which were transported into the Baltic Sea via shipping and were able to germinate under the ambient, nutrient-rich, brackish water conditions. The studies also estimated which factors favoured the occurrence and spread of P. minimum in the Baltic Sea and discussed the identification of this morphologically variable species. In addition, the classification of phytoplankton species recently observed in the Baltic Sea was discussed. Incubation of sediments from four Finnish ports and 10 ships ballast tanks revealed that the sediments act as sources of living dinoflagellates and other phytoplankton. Dinoflagellates germinated from all ports detected and from 90% of ballast tanks. The concentrations of cells germinating from ballast tank sediments were mostly low compared with the acceptable cell concentrations set by the International Maritime Organization s (IMO s) International Convention for the Control and Management of Ships Ballast Water and Sediments. However, the IMO allows such high concentrations of small cells in the discharged ballast water that the total number of cells in large ballast water tanks can be very high. Prorocentrum minimum occurred in the Baltic Sea annually but with no obvious trend in the 10-year timespan from 1993 to 2002. The species occurred under wide ranges of temperatures and salinities and the abundance of the species was positively related especially to the presence of organic nitrogen and phosphorus. This indicated that the species was favoured by increased organic nutrient loading and runoff from land and rivers. The cell shape of P. minimum varied from triangular to oval-round, but morphological fine details indicated that only one morphospecies was present. P. minimum also is, according to present knowledge, the only potentially harmful phytoplankton species that has recently expanded widely into new areas of the Baltic Sea.
  • Howlader, Mohammad Sajid Ali (Helsingin yliopisto, 2016)
    Amphibians are the most threatened class of vertebrates. About 48% of the known amphibian species are threatened by extinction, and many species still remain undescribed, especially from tropical and sub-tropical countries such as Bangladesh. In contrast to India and Sri Lanka, amphibian diversity in Bangladesh is poorly known, and little effort has been put towards documenting the species diversity and resolving evolutionary affinities among amphibian taxa in this country. Hence, the actual diversity of amphibians in Bangladesh remains unknown. The aim of this dissertation work was to improve our knowledge of amphibian diversity in Bangladesh by identifying and describing new amphibian species and investigating their evolutionary relationships with closely related taxa. I used morphological and molecular phylogenetic methods to identify and describe one new genus and five new species from different genera. In addition to using traditional morphological comparisons, I also utilized mitochondrial gene fragments to estimate phylogenetic affinities among the studied taxa, with Maximum-likelihood and Bayesian methods. The first two chapters of the thesis focus on the amphibian genera Fejervarya and Zakerana, the latter which was previously embedded within Fejervarya. These chapters also include descriptions of two new species Fejervarya asmati sp. nov. (now Zakerana asmati), as well as Fejervarya burigangaensis sp. nov. , respectively. In the third chapter, a new species (Zakerana dhaka sp. nov.) is described from the urban core of Dhaka, the capital of Bangladesh and one of the most densely populated mega cities in the world. In the fourth chapter, I describe Euphlyctis kalasgramensis sp. nov., which was earlier recognized as E. cyanophlyctis, and show that it is genetically highly divergent from the E. cyanophlyctis described from southern India. In the last and fifth chapter, I describe Microhyla nilphamariensis sp. nov. as a new species. It is a member of a highly genetically heterogeneous group of frogs that have been recognized as M. ornata for the past 173 years. In general, the results of the studies included in this dissertation advance our understanding of amphibian diversity in Bangladesh and adjacent regions, and show that discovery and description of new amphibian species from this region is still fairly easy. Consequently, it seems likely that more thorough sampling and further investigations in this region can uncover additional new amphibian species to science. Such studies, together with the discoveries described in this thesis, should also provide useful information for understanding and conserving the amphibian biodiversity in this poorly studied region.
  • Faccio, Greta (Helsingin yliopisto, 2011)
    Enzymes offer many advantages in industrial processes, such as high specificity, mild treatment conditions and low energy requirements. Therefore, the industry has exploited them in many sectors including food processing. Enzymes can modify food properties by acting on small molecules or on polymers such as carbohydrates or proteins. Crosslinking enzymes such as tyrosinases and sulfhydryl oxidases catalyse the formation of novel covalent bonds between specific residues in proteins and/or peptides, thus forming or modifying the protein network of food. In this study, novel secreted fungal proteins with sequence features typical of tyrosinases and sulfhydryl oxidases were iden-tified through a genome mining study. Representatives of both of these enzyme families were selected for heterologous produc-tion in the filamentous fungus Trichoderma reesei and biochemical characterisation. Firstly, a novel family of putative tyrosinases carrying a shorter sequence than the previously characterised tyrosinases was discovered. These proteins lacked the whole linker and C-terminal domain that possibly play a role in cofactor incorporation, folding or protein activity. One of these proteins, AoCO4 from Aspergillus oryzae, was produced in T. reesei with a production level of about 1.5 g/l. The enzyme AoCO4 was correctly folded and bound the copper cofactors with a type-3 copper centre. However, the enzyme had only a low level of activity with the phenolic substrates tested. Highest activity was obtained with 4-tert-butylcatechol. Since tyrosine was not a substrate for AoCO4, the enzyme was classified as catechol oxidase. Secondly, the genome analysis for secreted proteins with sequence features typical of flavin-dependent sulfhydryl oxidases pinpointed two previously uncharacterised proteins AoSOX1 and AoSOX2 from A. oryzae. These two novel sulfhydryl oxidases were produced in T. reesei with production levels of 70 and 180 mg/l, respectively, in shake flask cultivations. AoSOX1 and AoSOX2 were FAD-dependent enzymes with a dimeric tertiary structure and they both showed activity on small sulfhydryl compounds such as glutathione and dithiothreitol, and were drastically inhibited by zinc sulphate. AoSOX2 showed good stabil-ity to thermal and chemical denaturation, being superior to AoSOX1 in this respect. Thirdly, the suitability of AoSOX1 as a possible baking improver was elucidated. The effect of AoSOX1, alone and in combi-nation with the widely used improver ascorbic acid was tested on yeasted wheat dough, both fresh and frozen, and on fresh water-flour dough. In all cases, AoSOX1 had no effect on the fermentation properties of fresh yeasted dough. AoSOX1 nega-tively affected the fermentation properties of frozen doughs and accelerated the damaging effects of the frozen storage, i.e. giving a softer dough with poorer gas retention abilities than the control. In combination with ascorbic acid, AoSOX1 gave harder doughs. In accordance, rheological studies in yeast-free dough showed that the presence of only AoSOX1 resulted in weaker and more extensible dough whereas a dough with opposite properties was obtained if ascorbic acid was also used. Doughs containing ascorbic acid and increasing amounts of AoSOX1 were harder in a dose-dependent manner. Sulfhydryl oxidase AoSOX1 had an enhancing effect on the dough hardening mechanism of ascorbic acid. This was ascribed mainly to the produc-tion of hydrogen peroxide in the SOX reaction which is able to convert the ascorbic acid to the actual improver dehydroascorbic acid. In addition, AoSOX1 could possibly oxidise the free glutathione in the dough and thus prevent the loss of dough strength caused by the spontaneous reduction of the disulfide bonds constituting the dough protein network. Sulfhydryl oxidase AoSOX1 is therefore able to enhance the action of ascorbic acid in wheat dough and could potentially be applied in wheat dough baking.
  • Pietilä, Maija (Helsingin yliopisto, 2013)
    Extremophiles are found in all three domains of cellular life but especially archaea are able to withstand harsh conditions. Halophilic archaea thrive in hypersaline environments like salt lakes and salterns which have been shown to contain high abundance of virus-like particles. So far, head-tailed viruses are the most common isolates infecting haloarchaea, which is in contrast to a variety of morphologies described for the viruses of hyper-thermophilic archaea. Altogether, approximately 100 archaeal viruses have been isolated but only a fraction of them has been subjected to detailed structural analyses. In this thesis, a novel haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), was isolated from a solar saltern. This virus was shown to have a flexible, pleomorphic vesicle-like virion devoid of a rigid protein capsid. The genome analyses revealed that HRPV-1 is the first archaeal virus to be isolated which does not have a double-stranded but a single-stranded DNA genome. A genomic region of HRPV-1 showed similarity to the genome of another haloarchaeal virus, Haloarcula hispanica virus 2 (His2), as well as to the genome of Haloarcula marismortui and Natronomonas pharaonis indicating that HRPV-1-like elements are widespread. Consistent with this, pleomorphic viruses resembling HRPV-1 and infecting haloarchaea of the genera Haloarcula, Halorubrum and Halogeometricum have subsequently been isolated from geographically distant locations, and this study was extended to altogether seven viruses. All these viruses were sensitive to lowered ionic strength confirming their halophilic nature. Based on the virion properties, these haloviruses were defined as pleolipoviruses. Life-cycle studies showed that the pleolipoviruses are nonlytic and progeny virions are produced continuously resulting in host growth retardation. The most likely exit mechanism is budding which is consistent with the observation that the pleolipoviruses acquire their lipids unselectively from the host lipid pool. All pleolipoviruses have two major structural protein species, and biochemical dissociation studies showed that the larger-sized proteins form spike-like protrusions on the virion surface and the smaller-sized proteins are embedded in the inner surface of the membrane vesicle. The three-dimensional virion structure of HRPV-1 revealed that the spike structures are randomly distributed on the virion surface. The genome of the pleolipoviruses is enclosed in a lipid vesicle without associated nucleoproteins. Although the pleolipoviruses have different genome types, single- or double-stranded, circular or linear DNA, the membrane vesicle-based virion architecture is conserved. This work introduced a novel group of pleomorphic viruses infecting extremely halophilic archaea and showed that vesicle-like virion architecture is common in hypersaline environments. Interestingly, the archaeal pleolipoviruses were observed to share several similarities with a bacterial mycoplasmavirus indicating that these viruses may form a viral lineage with an ancient origin.
  • Merikanto, Ilona (Helsingin yliopisto, 2016)
    Many existing and emerging microbial infectious diseases are caused by environmentally growing opportunist pathogens. These pathogens are, contrary to obligatory pathogens, able to survive and replicate in the outside-host environment as free-living microbes that use within-host growth as an alternative replication strategy. This disease class has eco-evolutionary implications in natural populations and causes a serious health and economical threat to humans, our food production and to wildlife. Because of the ability of environmentally growing opportunists to survive and replicate independently of hosts, these diseases are hard to eradicate with conventional methods. The conditions that favor or disfavor environmental opportunism are still poorly understood. Better understanding of the dynamics of these diseases is needed in order to develop proper control methods against them. In this thesis I have developed novel epidemiological models to describe the disease dynamics of environmentally growing pathogens. These models modify the traditional Susceptible-Infected host (SI-model) framework by combining it to the outside-host community of an environmentally growing pathogen. I have considered how the environmental growth of the pathogens and the antagonistic ecological interactions these pathogens face in the outside-host environment, such as competition, predation and parasitism, affect the disease dynamics, invasion of novel pathogens and biological control of environmentally growing infectious diseases. The analyses show that the disease dynamics of environmentally growing pathogens differ from obligatory pathogens. Importantly, ability to grow in the outside-host environment promotes disease outbreaks and can lead to the extinction of the host, which is untypical in the case of obligatory pathogens. Antagonistic interactions the pathogen faces in the outside-host environment can on the other hand limit disease outbreaks and prevent extinction of the hosts that would otherwise occur due to the disease. I conclude that the eradication can be accomplished 1) by increasing the outside-host competition, 2) through predation of pathogens, or 3) through viral infections in pathogens.