Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Wang, Kai (Helsingin yliopisto, 2019)
    The plant phyllosphere environment offers a habitat for multiple kinds of microbes, including bacteria, fungi, yeast, etc. Microbes can be beneficial, pathogenic, or mostly neutral to plants. Increasingly the interaction patterns and related plant immunity signaling pathways against bacteria and filamentous fungi have been extensively studied. However, the interaction between plants and yeast or yeast-like fungi is largely unclear. Phyllosphere yeast-like fungi from wild Arabidopsis were isolated and characterized in this study. Around a hundred yeast isolates, including ascomycete Protomyces species, were identified and cultured. Protomyces species have been described as pathogens of plants in the Umbelliferae and Compositae families, however, with questionable phylogeny and little genomic information. We isolated and investigated the interaction of a strain Protomyces sp. SC29 (SC29) with Arabidopsis. SC29 can persist in the Arabidopsis phylloplane, and activate Arabidopsis immune responses with MAPKs (mitogen-activated protein kinases) activation and upregulation of salicylic acid signaling and camalexin biosynthesis marker genes. Additionally, indolic compounds produced by Protomyces species are able to activate plant auxin responses. The genomes of SC29 and all currently available Protomyces species were sequenced, assembled, and annotated. Comparative genomic analysis revealed genomic characters of SC29 related to adaptation to the phyllosphere environment. Genomic insights into the pathogenesis of Protomyces species were also discovered. The phylogenetic relationships of both the genus Protomyces and the subphylum Taphrinomycotina were re-constructed with genome-wide single-copy protein sequences. Small secreted proteins from the genomes of Protomyces spp. were analyzed as candidate effectors. Physiological, phylogenetic, and genomic evidence supported SC29 to be a novel species distinct from currently accepted Protomyces species. Thus, the study of SC29 and its interaction with Arabidopsis represents a new model system for the exploration of the genetics of plant interactions with phyllosphere resident yeasts.
  • Hunter, Kerri (Helsingin yliopisto, 2019)
    In order to maintain health, growth, and productivity, plants must be able to adapt to increasingly variable environmental conditions. Plants are continuously flooded with information from their surrounding environment, which must be sensed, incorporated, and responded to accordingly. Much of the communication between plant cells and the extracellular environment is carried out by the receptor-like protein kinases (RLKs), including the cysteine-rich receptor-like kinase (CRK) subfamily. Despite the large size of the CRK gene family, their physiological roles and functions on a biochemical and cellular level remain largely uncharacterized. We performed large scale phenotyping of a crk T-DNA mutant collection in Arabidopsis thaliana (Arabidopsis), which suggested roles for the CRKs in several developmental processes, as well as during abiotic and biotic stress responses. CRK2 emerged as an important CRK, with several strong loss-of-function phenotypes and a notable phylogenetic position. We established that CRK2 enhances salt tolerance through the regulation of callose synthase 1 (CALS1) dependent callose deposition at plasmodesmata. This revealed a previously uncharacterized role for callose deposition in response to high salinity. We showed that this callose deposition has an effect on plasmodesmal permeability, and therefore a potential impact on intercellular signalling. Additionally, CRK2 was found to regulate the formation of an unknown vesicle type during salt stress, which could possibly be involved in cell-to-cell signalling as well. We have described how CRK2 regulates ROS production during immunity by regulation of RBOHD via C-terminal phosphorylation. We observed highly specific changes in the subcellular localization of CRK2 in response to various stress treatments, and demonstrated that these localization patterns are critical for protein function and interactions. The subcellular localization and many of the cellular functions of CRK2 were dependent on phospholipase D alpha 1 (PLDɑ1) activity, and PLDɑ1 was consistently identified as one of the top proteins to interact with CRK2. Thus, we propose that CRK2 is a fundamental CRK, which acts in connection with PLDɑ1 to regulate several cellular processes during the response to environmental stimuli.
  • Winkel, Frederike (Helsingin yliopisto, 2019)
    Structural brain plasticity is an essential process to adjust maladapted networks, but it dramatically declines after closure of the critical periods during early postnatal life. Growing evidence suggests, however, that certain interventions, such as environmental enrichment and antidepressant treatment, can reinstate a network plasticity that is similar to that observed during the critical periods. Chronic treatment with the antidepressant fluoxetine, for example, can reopen visual cortex plasticity when combined with monocular deprivation. Further, it promotes the erasure of previously acquired fear memory when combined with extinction training. Fluoxetine can bind to and activate the neurotrophic TrkB receptor and can therefore regulate the downstream pathway to induce synaptic plasticity. Considering that TrkB receptors are expressed in essentially all neurons, the question to be answered is through which neuronal subpopulation are the plasticity effects regulated within these two circuitries. Visual cortex plasticity is tightly regulated by the inhibitory Parvalbumin (PV)-specific GABAergic network, which highly expresses TrkB receptors. During the critical periods TrkB’s ligand Brain- Derived Neurotrophic Factor (BDNF) promotes the maturation of PV interneurons, thereby stimulating a precocious onset and closure of critical periods. Hence, our first aim was to understand TrkB actions specifically in PV interneurons and their subsequent effects on visual cortex plasticity during adulthood. We used optically activated TrkB (optoTrkB) expressed only in PV interneurons of the visual cortex and found that optoTrkB activation by light combined with monocular deprivation is sufficient to induce ocular dominance plasticity. Strikingly, optoTrkB activation rapidly induces LTP in layer II/III of the visual cortex after theta burst stimulation (TBS). This potentiation in excitatory transmission is mediated by rapid decreases in the intrinsic excitability of PV regulated by reduced expressions of Kv3.1 and Kv3.2 mRNA. In addition, optoTrkB activation promotes the removal of perineuronal nets (PNNs) and shifts the PV and PNN networks into a plastic, immature configuration. Conversely, deleting TrkB from PV interneurons and using chronic fluoxetine treatment to pharmacologically induce plasticity prevented the effects of fluoxetine treatment. Our second aim was to identify the effects of optoTrkB activation expressed specifically in pyramidal neurons of the ventral hippocampus on the fear circuitry. We therefore directed the expression of optoTrkB to pyramidal neurons of the ventral hippocampus. During fear extinction optoTrkB was activated with light, and spontaneous recovery and fear renewal were tested one and three (remote memory) weeks after extinction training. We found that optoTrkB activation during extinction training promoted the erasure of remote fear memory. This effect was accompanied by increased LTP expression after brief TBS stimulation. Finally, fluoxetine and methylmercury (MeHg) are a common intervention and stressor, respectively, in our society, and exposure to either during pregnancy is known to impact brain development and functioning. An altered critical period can result in impairments that are retained into adulthood. Our aim was to understand how perinatal exposure to fluoxetine or MeHg affects the development of PV and PNNs, two well-established markers for the time course of critical periods, in the hippocampus and basolateral amygdala. We found that upon closure of the normal critical periods (P24) the number of PV and PNNs, and PV cell intensity increase. Perinatal fluoxetine treatment resulted in reduced expression of PNNs throughout critical periods, indicating a delayed closure. In contrast, perinatal MeHg exposure impaired the development of PV interneurons and PV expression at the onset of critical periods (P17), which were, however, restored upon critical period closure (P24), suggesting a delayed onset. Our results provide new evidence that TrkB activation in PV interneurons rapidly orchestrates cortical networks by reducing the intrinsic excitability of PV cells regulated by decreased expression of Kv3.1 and Kv3.2 channels, subsequently promoting excitatory transmission. In contrast, TrkB activation in pyramidal neurons of the ventral hippocampus also potentiates excitatory transmission. These opposite findings demonstrate that TrkB employs different mechanisms to increase the excitability of the neuronal network to induce plasticity. We propose that TrkB is a promising therapeutic target for the treatment of neuropsychiatric diseases that benefit from high plasticity modes. We further shed light on the effects of fluoxetine and MeHg exposure during pregnancy on the time course of the critical periods, which can help in developing better guidelines for the use and consumption of both during pregnancy.
  • Pulliainen, Unni (Helsingin yliopisto, 2019)
    Ants are an ecologically dominant group often acting as keystone species. The key to their success is their social lifestyle. Living in a society means that it is vital to distinguish a friend from a foe, in order to protect the colony and its resources from exploitation by unwanted quests. Recognition in ants is based on detecting a mixture of hydrocarbons found on the surface of other individuals. Typical colony intruders are adult individuals, and thus recognition among adults is very well studied. However, brood is an important part of these societies, representing the reproductive efforts of the whole society, and accurate recognition of brood, and by brood, could be important in many different contexts, such as when a colony gets usurped by a social parasite. In this thesis, I investigated brood recognition in two Formica ant species from different points of view. I used behavioural assays to study brood discrimination of adults and larvae and further explored the mechanisms underlying recognition behaviour. I found that brood discrimination can be affected by caste, colony and species of brood, and that brood discrimination behavior follows inclusive fitness predictions. I also characterized the surface chemistry of pupae, showing how a species with a simple chemical profile in adults, can have complex brood profiles, which potentially carry cues for recognition. Furthermore, I studied the so far completely unknown chemosensory biology of ant larvae, and showed that larvae have the molecular machinery to perceive their social environment and react to information gathered from their surroundings. This thesis adds to the accumulating knowledge that immature stages of social insects are not merely passive bystanders, by suggesting that ant larvae may take part in colony defense against social parasites. I also take the first steps in figuring out how ant larvae sense the world around them, and provide a basis for more detailed studies on the sensory biology of developing social insects. By describing the surface chemistry of immature ants, I furthermore help advance our understanding of the information ants use to recognize each other, highlight the context dependency of brood discrimination, and suggest new avenues of exploration in the field of chemical ecology.
  • Göös, Helka (Helsingin yliopisto, 2019)
    Transcription factors (TFs) are one of the most important groups of proteins for the development and differentiation of cells. They control the gene expression of all cells in all stages of development. Defects in TF signalling may lead to severely altered development and diseases. However, while TF DNA binding has been widely studied, we are still lacking a systems-level understanding of human TF signalling. TFs’ action in gene expression regulation is highly dependent on their interactions with multiple proteins, such as cofactors, dimerization partners, chromatin modulating proteins, enzymes, inhibitory proteins and general TFs. Therefore, the aim of this study is to shed light on TF protein-protein interactions and, more specifically, to examine the effect of TF mutations found in primary immunodeficiency patients. A comprehensive interactome analysis of 110 TFs revealed over 7,000 TF protein-protein interactions, most of which are nuclear and play a role in transcriptional regulation (I). The large number of TF interactions discovered in this study enabled us to conduct a systems-level analysis that revealed groups of TFs with specific biological functions, such as actin and myosin signalling and RNA splicing. Interestingly, 54 of the TFs studied interacted with the nuclear factor family of TFs. Nuclear factors are known to control a number of genes in development; for instance, they are essential for central nervous system, tooth, brain, skeletal, lung and muscle development. In addition, they are linked to several cancer types. Our data suggest that transcription control by NFIs may be regulated by nuclear factor interactions with other TFs. A219H mutation in the C/EBPε TF was found in a Finnish family with immunodeficiency and autoinflammatory syndrome (II). A data-driven multiomics study of the mutation revealed a novel TF-related disease mechanism; mutation decreased association with transcriptional repressors, increased chromatin binding and widely dysregulated transcription. These changes resulted in disturbed non-canonical inflammasome activation due to the increased expression of NLRP3 and constitutively expressed CASP5. Three different damaging mutations in NFKB1 resulted in diverse immunological phenotypes due to different mechanisms (III): H67R led to decreased nuclear entry, reduced association with RelB and decreased transcriptional activity; I553M led to decreased phosphorylation of S893 and p907 and enhanced p105 subunit degradation upon TNF treatment; and R157X led to an almost total loss of NFKB1 subunits due to proteasome-mediated dominant negative degradation. This study provides valuable information on TF protein-protein interactions at systems level (I). In addition, this study provides examples of how single TF mutation may affect TF signalling on many levels, such as in protein interactions, DNA binding and transcription (II) and how different mutations in the same TF can have different outcomes (III). TFs are downstream players of many signalling cascades and targeting TF protein interactions can offer a high degree of specificity in future therapeutics applications.
  • Orav, Ester (Helsingin yliopisto, 2019)
    Neural circuits emerge when neurons become connected by synaptic contacts. In rodents, this process begins already before birth and continues during the first postnatal weeks. The initial steps of synapse formation are guided by intrinsic molecular cues. The emerging synaptic contacts are then refined and fine-tuned by activity-dependent mechanisms. Kainate-type glutamate receptors (KARs) are involved in synapse formation and refinement during this “critical period” by regulating neurotransmitter release and neuronal excitability in both principal cells and interneurons in a developmentally restricted manner. Functional KAR tetramers are assembled from various combinations of five core subunits GluK1-5 and supplemented with auxiliary subunits, Neuropilin and tolloid-like proteins (NETO) 1 and 2 that are not part of the pore-forming receptor. KAR interaction with NETOs affects multiple aspects of KARs like subcellular localization of the receptor complex, receptor gating and current kinetics, and even KAR affinity to main agonists, kainate and glutamate. Despite the accumulating evidence emphasizing the functional significance of NETOs in regulating KAR functions in the adult brain, the role of NETO/KAR complex in the immature brain remains elusive. The main aim of this study was to clarify the physiological significance of NETO/KAR complex in the maturation of hippocampal circuitry. First, we found that NETO1 is an important regulator of physiologically relevant KAR activity at immature glutamatergic synapses. NETO1 deficiency significantly reduced axonal delivery of KARs resulting in loss of presynaptic KAR function and delayed maturation of CA3-CA1 synapses. At the network level, NETO1 deficiency caused impaired synchronization between areas CA3 and CA1 of the hippocampus. This phenotype was fully rescued by GluK1c expression at CA3 principal neurons, emphasizing the role of NETO1 and axonal GluK1-containing immature-type KARs in the development of CA3-CA1 synapses. Next, we showed that NETO1 is necessary for the dendritic delivery of KAR subunits and for formation of KAR-containing synapses in cultured GABAergic neurons. In CA3 interneurons, loss of NETO1 disrupted postsynaptic and metabotropic KAR signaling, while a subpopulation of ionotropic KARs in the somatodendritic compartment remained functional. NETO1 was not necessary to maintain the excitability of the immature CA3 network at physiological levels. However, kainate-dependent modulation of network bursts and GABAergic transmission in the developing hippocampus was significantly impaired in the absence of NETO1. In conclusion, these new findings elucidate the cellular mechanisms and physiological significance of NETO/KAR interaction in hippocampal principal cells and interneurons during the first week of postnatal development. This early period of neural network development is extremely sensitive to external stimuli. Accordingly, disturbances in circuit structure or activity patterns that take place during this “critical period” could predispose to neuropsychiatric disorders later in life.
  • Moliterno de Camargo, Ulisses (Helsingin yliopisto, 2019)
    Modern technologies for the automated acoustic monitoring of animal communities enable species surveys that yield data in unprecedented volumes. Interpretation of these data bring new challenges related to the need of automated species identification. Coupling automated audio recording with automated species identification has enormous potential for biodiversity assessment studies, but it has posed many challenges to the effective use of techniques in real-world situations. This thesis develops new methods in the field of bioacoustics applied to automated monitoring of vocal species in terrestrial environments. Specifically, I developed automated methods to classify acoustic ecological data generated under the two most common contexts used in ecology: identification of vocalization data stored in acoustic libraries of sounds and identification of vocalizations in audio data collected from the field, through e.g., acoustic monitoring programs. The methods bring key developments across the entire pipeline for automated acoustical identification, connecting techniques from the data acquisition in the field to the ecological modelling of data identified utilizing automated classification methods. I show the performance of methods over huge datasets, compare them with alternative cutting-edge techniques and provide an ample study case of Amazonian bird communities to show the tools in practice. The methods in this thesis are available as open source and ready-to-use software capable to work directly on field data collected from acoustic monitoring efforts.
  • Vesala, Risto (Helsingin yliopisto, 2019)
    Fungus-growing termites are ecologically important animals in tropical Africa and Asia. Especially in dry savannas, they contribute to local carbon and mineral recycling and alter soil physical properties, thus facilitating the success of many plant species. This, in turn, has indirect impacts also on animals that may e.g. benefit from improved food supply and quality. The success and ecological significance of fungus-growing termites arise from their exosymbiotic relationship with the fungal genus Termitomyces. Termites cultivate fungal symbionts within specialized compost structures in their underground nests where the mycelium assists in degradation of plant matter collected by the termites, thus providing a constant food supply for the large termite colonies. Symbiotic food processing is especially advanced in the termite genus Macrotermes which construct large above-ground soil structures – termite mounds – to enhance ventilation of the below-ground nests and to provide a favorable microclimate for fungal growth even in arid savanna environments. The aim of this thesis was to study interactions between Macrotermes termites and their Termitomyces symbionts in the semiarid Tsavo Ecosystem in Southern Kenya. We assessed the local diversity of the host insects and their fungal symbionts and produced an up-to-date phylogeny of the fungal symbionts based both on our new results and previously published DNA data. We found that the Macrotermes–Termitomyces diversity in the Tsavo Ecosystem involves two host species and three symbiont species that occur in different combinations, and the frequencies of different associations vary over the landscape. Studies on mound architecture and symbiont diversity revealed correlations between the size and type of above-ground mounds and specific host-symbiont combinations. These were linked to architecturally induced differences in nest temperatures, suggesting that different Termitomyces species may differ in their ranges of tolerable growth temperatures. Stable isotope studies provided important new information on the nutritional role of Termitomyces for Macrotermes colonies. Termitomyces promotes the nutrition of the host insects directly, as highly nitrogenous food for queen and young larvae, and indirectly, by decomposing plant matter that is eaten by workers, soldiers, and developing alates. Thereby, the fungal symbiont does not have a single universal role in the nutrition of a termite colony, but instead, different termite castes depend on the symbiosis in different ways. The isotopic imbalance of nitrogen also implied that, although the nutrition of fungus-growing termites is facilitated by the fungal symbionts, also bacterial nitrogen fixing may provide an essential complementary nitrogen source for termite colonies.
  • Steinzeig, Anna (Helsingin yliopisto, 2019)
    Visual system is a well-established model for studying developmental plasticity in the cerebral cortex. Monocular deprivation – occluding of one eye – causes an ocular dominance shift in favour of the open eye in the binocular primary visual cortex. It was long thought that monocular deprivation can cause the shift in ocular dominance only during the critical period – a brief period of elevated plasticity in early postnatal development. However, recent studies showed that numerous treatments, including chronic antidepressant fluoxetine treatment, can reinstate juvenile-like form of plasticity in adult brain. Impaired plasticity is implicated in pathophysiology of various neurological diseases including depression – a highly debilitating mental disorder that brings on a serious socio-economic burden. Indeed, stress – a known trigger for mood disorders – deteriorates brain plasticity, in particular decreasing BDNF-TrkB signalling. In turn, antidepressants were recently shown to increase plasticity in the brain, via elevation of BDNF levels and enhancement of TrkB signalling. Brain-derived neurotrophic factor BDNF and its receptor TrkB play an important role in brain plasticity, however, how exactly they interact with antidepressants remain obscure. In this thesis, we used visual cortex as a model to study the action of antidepressants on brain plasticity. The first aim of this thesis was to find an appropriate approach to assess plasticity in the mouse visual cortex. Thus, we have described and tested a skull preparation technique called the “transparent skull” method for optical imaging experiments. We also described the temporal patterns of fluoxetine-induced plasticity. In addition, we tested rapid-acting antidepressants such as ketamine and its active metabolite 2R,6R – hydroxynorketamine in our plasticity assay, to reveal whether they are also able to influence plasticity . Finally, we aimed to determine which neuronal subtypes express TrkB important for plasticity in the visual cortex. To address this issue, we developed conditional knockout mice with a specific deletion of TrkB from parvalbumin and serotonergic neurons. Overall, our findings broaden the understanding of the mechanisms underlying the action of conventional and rapid-acting antidepressants and implicated an important role of the TrkB receptor in antidepressant-induced plasticity.
  • Sun, Yu (Helsingin yliopisto, 2019)
    Fossil evidence suggests that liverworts may have existed already in the Ordovician. The long-standing hypothesis, that widely disjunct geographic ranges of various extant liverwort groups have been largely resulted from vicariance events from fragmentation of widespread ancestors through tectonic plate movements, has been challenged by the recent studies based on molecular data. In this thesis, two groups of the leafy liverworts in the order Jungermanniales were investigated in order to better understand the phylogenetic relationship and biogeography of liverworts. They are the family Schistochilaceae ranging mostly in the Southern Hemisphere, and the cosmopolitan genus Herbertus of family Herbertaceae. Both families possess a diverse morphology and wide disjunct distribution. The phylogeny and biogeographic history of Schistochilaceae and Herbertus were studied by using DNA sequence data of chloroplast and nuclear gene regions. Morphological characters used for species delimitation were also studied and re-evaluated. Schistochilaceae is resolved as monophyletic being one of the early derived leafy liverwort groups. The phylogenetic position of the Chilean endemic Pleurocladopsis simulans is resolved within the genus Schistochila and the new combination Schistochila simulans (C. Massal.) Xiao L. He & Yu Sun is made. Schistochilaceae is inferred to have originated in the Late Cretaceous at c.100 Mya, in an ancestral area including southern South America, West Antarctica and New Zealand. New Zealand was recognized as the early divergence and dispersal center, most dispersals were transoceanic. Herbertus is resolved as a monophyletic group with one clade consisting of the southern hemispheric taxa Herbertus runcinatus, H. oldfieldianus, and H. juniperoideus, and the other including species of H. sendtneri complex and of H. aduncus complex, mostly belonging to the Northern Hemisphere. H. armitanus and H. circinatus were synonymized with H. sendtneri. H. borealis, H. buchii, H. delavayi, H. dicranus, H. kurzii, H. longifissus, H. norenus and H. stramineus were synonymized with H. aduncus. Herbertus is inferred to have originated in the Cenozoic era about 51 Mya, in an ancestral area including southern South America, the Neotropics, Oceania, and Southeast Asia, supporting its Gondwanan origin. In the Southern Hemisphere the distribution pattern of Herbertus is characterized by in situ persistence and did not show further dispersal until the uplift of the Andean Cordillera. Herbertus in the Northern Hemisphere showed more recent diversifications, wide range expansions both north- and southward and repeated recolonizations, and the range shifts had occurred more frequently since the late Miocene. Long-distance dispersal has played an important role in the formation of the global distribution pattern of the genus. Our obtained phylogenies of liverworts all resolved the New Zealand endemic Herzogianthus vaginatus as the first diverged lineage in the Jungermanniales, not supporting its current placement in the Ptilidiales. Our study supports the southern temperate origins for both Schistochilaceae and Herbertus, and most of the diversification occurred in the Cenozoic, a pattern that has been shown to occur also in other leafy liverwort groups. Our results suggest that species with separate sexes do not constrain long-distance dispersal. The causes for the formation of current geographical ranges of liverworts seem to be manifold, likely linked with ecophysiology, lineage age, dispersibility and diversification rate.
  • Kumar, Ashwini (Helsingin yliopisto, 2019)
    This thesis is comprised of three studies demonstrating the application of different statistical and bioinformatic approaches to address distinct challenges of implementing precision medicine strategies for hematological malignancies. The approaches focus on the analysis of next-generation sequencing data, including both genomic and transcriptomics, to deconvolute disease biology and underlying mechanisms of drug sensitivities and resistance. The outcomes of the studies have clinical implications for advancing current diagnosis and treatment paradigms in patients with hematological diseases. Study I, RNA sequencing has not been widely adopted in a clinical diagnostic setting due to continuous development and lack of standardization. Here, the aim was to evaluate the efficiency of two different RNA-seq library preparation protocols applied to cells collected from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. The poly-A-tailed mRNA selection (PA) and ribo- depletion (RD) based RNA-seq library preparation protocols were compared and evaluated for detection of gene fusions, variant calling and gene expression profiling. Overall, both protocols produced broadly consistent results and similar outcomes. However, the PA protocol was more efficient in quantifying expression of leukemia marker genes and drug targets. It also provided higher sensitivity and specificity for expression-based classification of leukemia. In contrast, the RD protocol was more suitable for gene fusion detection and captured a greater number of transcripts. Importantly, high technical variations were observed in samples from two leukemia patient cases suggesting further development of strategies for transcriptomic quantification and data analysis. Study II, the BCL-2 inhibitor venetoclax is an approved and effective agent in combination with hypomethylating agents or low dose cytarabine for AML patients, unfit for intensive induction chemotherapy. However, a limited number of patients responding to venetoclax and development of resistance to the treatment presents a challenge for using the drug to benefit the majority of the AML patients. The aim was to investigate genomic and transcriptomic biomarkers for venetoclax sensitivity and enable identification of the patients who are most responsive to venetoclax treatment. We found that venetoclax sensitive samples are enriched with WT1 and IDH1/IDH2 mutations. Intriguingly, HOX family genes, including HOXB9, HOXA5, HOXB3, HOXB4, were found to be significantly overexpressed in venetoclax sensitive patients. Thus, these HOX-cluster genes expression biomarkers can be explored in a clinical trial setting to stratify AML patients responding to venetoclax based therapies. Study III, venetoclax treatment does not benefit all AML patients that demands identifying biomarkers to exclude the patients from venetoclax based therapies. The aim was to investigate transcriptomic biomarkers for ex vivo venetoclax resistance in AML patients. The correlation of ex vivo venetoclax response with gene expression profiles using a machine learning approach revealed significant overexpression of S100 family genes, S100A8 and S100A9. Moreover, high expression ofS100A9was found to be associated with birabresib (BET inhibitor) sensitivity. The overexpression of S100A8 and S100A9 could potentially be used to detect and monitor venetoclax resistance. The combination of BCL-2 and BET inhibitors may sensitize AML cells to venetoclax upon BET inhibition and block leukemic cell survival.
  • Wang, Liang (Helsingin yliopisto, 2019)
    Mitochondria are a powerhouse for cells and a hub for numerous signaling pathways. Mitochondria are highly dynamic organelles, frequently changing their shape by shifting the balance of fusion and fission. Dysregulation in mitochondrial function or dynamics causes many human diseases. The sub-mitochondrial localization and biological function of 13 mtDNA-encoded proteins have been clearly characterized, while many novel nuclear-encoded mitochondrial proteins are yet to be discovered. To comprehensively determine the mechanisms of mitochondrial-related diseases, it is imperative to reveal cellular functions of key mitochondrial proteins essential for mitochondrial biogenesis, structure, function, and dynamics. In this thesis, we have identified two novel nuclear-encoded mitochondrial proteins in mammalian cells: the Bin-Amphiphysin-Rvs (BAR) protein FAM92A1 essential for regulation of mitochondrial membrane ultrastructure, and the GTP-binding protein GTPBP8 required for mitochondrial translation. Due to the critical roles of mitochondria in cell signaling and cell survival, mitochondria, especially mitochondrial proteins, are the potential drug targets for treatment of a wide spectrum of diseases. Thus, prickly zinc-doped copper oxide (Zn-CuO) nanoparticles (prickly NPs) are designed and synthesized for cancer therapy. In paper I, FAM92A1 localizes to the matrix side of the mitochondrial inner membrane through an N-terminal mitochondrial targeting sequence. Loss of FAM92A1 causes a severe disruption to mitochondrial morphology, ultrastructure, and membrane dynamics, impairing organelle bioenergetics. Furthermore, the purified recombinant FAM92A1 protein binds to model membranes through preferential binding to negatively charged phospholipids. After insertion into a lipid bilayer, FAM92A1 transforms spherical liposomes into narrow tubules. Importantly, the aberrant mitochondrial morphology and function caused by depletion of FAM92A1 can be rescued by wild-type FAM92A1, but only partially rescued by the FAM92A1 mutants with defects in membrane binding and remodeling activity. In paper III, GTPBP8 localizes to the mitochondrial matrix associated with the mitochondrial inner membrane. The N-terminal 46aa is indispensable for the mitochondrial localization of GTPBP8. Importantly, GTPBP8 exclusively interacts with the large subunit of mitochondrial ribosome. Genetic knockdown of GTPBP8 causes a significant reduction in the level of mitoribosomes, inducing defects in mitochondrial translation and mitochondrial bioenergetics. In paper II, the prickly NPs are very effective in inducing cell death of 3T3 and MCF-7 cancer cell lines. The prickly NPs efficiently accumulate in mitochondria, resulting in severe mechanic disruption to mitochondria and inducing cell apoptosis. Collectively, these studies reveal the biological functions of two previously uncharacterized mitochondrial proteins and an efficient nanoparticle cancer treatment by targeting mitochondria. These new findings elucidate the crucial relation between mitochondrial form and function, and hence, contribute to establishing the concept of membrane-mediated signaling in mitochondria. Furthermore, mitochondrial gene expression is critical for maintaining cellular homoeostasis. Therefore, the mitochondrial proteins and mitochondria per se are valuable potential drug targets for overcoming many mitochondrial diseases.
  • Jokinen, Maarit (Helsingin yliopisto, 2019)
    Research and evaluation are crucial components of evidence-based policy, decision-making and effective conservation management. As research consumes the same limited resources that could be used for conservation, it should provide as valuable and useful information as possible. This requires framing research questions to be relevant to both researchers and users of the information. A general problem is that researchers may be detached from conservation policy and practice, and thus do not recognize information needs. Furthermore, measuring and predicting effects of conservation is not an easy task. The spatial scale considered, and whether the costs and side effects are taken into account, may affect the conclusions. Effective conservation has population-level effects, but accurately measuring population change requires adequate resources and both ecological and statistical expertise. In addition, as societal actions and change in human behaviour are needed to bring about the desired changes, interdisciplinary approaches are needed for finding solutions to conservation problems. In this thesis, I use the conservation of the Siberian flying squirrel (Pteromys volans) in Finland as a model case for developing more effective conservation research. As an arboreal species, the flying squirrel is threatened by forest habitat loss and degradation. I evaluate the ecological effectiveness of species protection regulation along with the side effects it has had on forest owner attitudes toward the species. I use species distribution modelling (SDM) to predict the occurrence probability of the species in Finland. I also evaluate the methods and results of the species monitoring scheme by using information from the SDM and modelled relationship between the species observed occurrence and abundance. I found that the effectiveness of the prior approval system for forest management on flying squirrel nest sites was low but made the species a symbol of broader socio-political disputes. Conservation ineffectiveness is partly due to insufficient restrictions for forest management, but also due to lacking occurrence information. I found that species occurrence probability is explained by several environmental variables, but the ability of SDM to predict occurrence at specific sites and years remains limited with available data. I also found that the design of the population monitoring scheme does not allow reliable inference of the change in population size from the collected occurrence data. To conclude, the effectiveness of both conservation measures and research related to the evaluation and monitoring of the effects of conservation − including the population monitoring scheme – have suffered from shortcomings. Certain relevant study questions have been ignored, the monitoring scheme suffers from methodological problems, available data are not analysed or synthetized, or results have not been provided in usable form. In addition, the connection between available information and conservation policy and practice is very weak: even the most usable and objectively relevant information may not have substantial instrumental value. Much stronger partnerships between policymakers, managers and researchers of various disciplines is needed to increase the effectiveness of conservation and conservation research.
  • Vainio, Elisa (Helsingin yliopisto, 2019)
    Methane (CH4) is a strong greenhouse gas, and its ecosystematmosphere exchange depends on the consumption and production rates. The boreal zone includes nearly one third of the world’s forests, and boreal forest soil is the largest carbon stock among different ecosystem types. Upland soils are a globally important sink of CH4 due to microbes oxidizing atmospheric CH4. During the last decades, the understanding of the CH4 dynamics of forests has been reshaped and increased substantially, as the trees have been shown to contribute to the CH4 exchange. The newly-found aerobic CH4 emissions from plants have also revealed the existence of previously unknown processes. Meanwhile, the ecosystem-scale studies on CH4 exchange have shown that forests may occasionally be net sources of CH4. In this thesis, the objective was to quantify the CH4 exchange in a boreal pine forest, regarding the contributions of soil, ground vegetation and trees. The effects of soil water conditions and the CH4-consuming and -producing microbes were also studied. The research included the most abundant boreal tree species: Scots pine, downy birch and Norway spruce. The effect of ground vegetation on the forest floor CH4 flux was studied by classifying the vegetation into four groups, and by measuring the CH4 fluxes of three common shrubs (bilberry, lingonberry, and heather) in the laboratory. The forest floor CH4 flux was upscaled to the whole research site from topography-modelled soil moisture. The results demonstrated that the CH4 flux of the forest floor is strongly dependent on the soil moisture. All the studied tree species emitted CH4 from the stems and the branches, and the stem-emissions were significantly higher from trees growing at wet soil compared to drier soil. The ground vegetation species and soil moisture are strongly connected, and based on the results, both affect the CH4 flux. In the laboratory, heather shoots resulted in mean CH4 emissions, while bilberry and lingonberry shoots indicated uptake. Thus, the studied shrub species seem to have different CH4 dynamics. In addition, the shrubs increased the amount of CH4-consuming microbes and thus CH4 uptake in the soil. While the forest floor at the site was on average a sink of CH4 throughout the growing season, the upscaled forest floor CH4 flux revealed high spatial variation and CH4-emission patches at the area. The size and CH4 flux of these patches was related to temporal variation in the soil moisture.
  • Lindström, Stafva (Helsingin yliopisto, 2019)
    Biotic and abiotic characteristics shape the microbial communities in the soil environment. These characteristics vary both spatially and temporally, depending on variations in temperature, water availability and plant coverage, which on a larger scale are driven by topography, soil type, land use, vegetation and the ruling climate. In a sub-arctic climate, the seasonal fluctuations in temperature, precipitation and plant cover force the microbes in the top soil to adapt or decline. For many organisms trying to survive the cold winters, a nest can provide protection from the harsh environment. For example, the mound building wood ants, such as the Formica exsecta, selectively choose both the spot, and the building material for their nest mounds, in which they overwinter. These ants are also able to generate extra heat by using their lipid reserves during early spring, to secure the brood development. The higher and steadier temperature inside the nest is further enhanced by the decomposing process, intensified by the constant addition of organic plant material carried out by the ants, and the availability of nutrients. This creates a unique microclimate inside the nest mounds, in which the microbes can stay largely protected from the environmental drivers of the microbial communities in the surrounding top soil. I hypothesized, that the nest environment would promote the formation of unique bacterial and fungal communities in the nests, compared with the surrounding soil. I also hypothesized that the nest communities are stable in time, and that they would show less temporal than spatial variation. Furthermore, I expected to see nest-characteristic, temporally stable taxa, which could be identified as the core microbiome of the F. exsecta nests. However, to ensure a robust methodological approach, I first tested the combination of Next Generation Sequencing (NGS) and a classical community fingerprinting method, Terminal Restriction Fragment Length Polymorphism (T-RFLP). I sampled nests of the mound-building ant F. exsecta, and generated NGS (Illumina MiSeq), and T-RFLP data of the bacterial and fungal communities. I used ordination techniques and network analysis to disclose the community structures in the nests, and compared them with the ones in the reference soil. I assessed the variation in diversity, evenness and enrichment of taxa in the nests, and evaluated their spatial and temporal variations. I also identified significantly nest characteristic and temporally stable taxa, which represent a potential core microbiome of the nests. The results show that the bacterial and fungal communities, in the rigorously curated nest environment, are significantly different from those in the reference soils. I demonstrate that the nests represent a niche, where microbial species can adapt and diverge from the communities in the surrounding soils. The findings in my thesis work contribute to our understanding of the composition, function and adaptation of microbial communities in patchy, secluded habitats, and open up for further studies in a multilayered ecological system.
  • Ciuba, Katarzyna (Helsingin yliopisto, 2019)
    Fundamental processes of essentially each cell of the human body depend on structures built of actin. This relatively small (MW=42 kDa) protein is a central building block of the cytoskeleton. Actin-based structures contribute to diversified processes, including cell migration, morphogenesis, and intracellular transport. Above-mentioned activities are crucial in multiple basic physiological events, for example embryonal development, wound healing and immunological responses. Moreover, abnormal regulation of actin networks has implications in various diseases, such as cancer. Although extensively studied over the years, the field of actin-based structures still lacks answers for many important questions. For example, contractile actomyosin bundles of non-muscle cells, stress fibers, are essential for cell morphogenesis, adhesion and mechanosensing. However, the precise mechanisms regulating their assembly and function remain unclear. Studies included in this thesis reveal novel aspects underlying regulation of contractile structures in non-muscle cells. Assembly and organization of stress fibers depend not only on biochemical signals, but also on mechanical ones. Study I identifies mechanosensitive Ca2+-dependent CaMKK2-activation as an upstream regulator of AMPK-VASP pathway, which is crucial for contractile stress fiber maturation. Studies II and III unravel how two distinct proteins, calponin-3 and caldesmon, contribute to regulation of stress fiber organization and contractility in non-muscle osteosarcoma cells. We show, that calponin-3 is a dynamic component of all stress fiber subtypes. Moreover, its presence is essential to manage their proper contractility, as calponin-3 deficiency leads to uncontrolled behavior and breakage of stress fibers due to increased tension. We also show for the first time that non-muscle caldesmon is present exclusively in contractile stress fibers, where it colocalizes with myosin II motor domains. Moreover, we demonstrate an important role for caldesmon in regulation of stress fiber contractility, as lack of this protein leads to lower number of thick contractile bundles and reduced traction forces. Together, these studies broaden our knowledge on the mechanisms underlying assembly and regulation of contractile stress fibers in non-muscle cells.
  • Laitila, Jenni (Helsingin yliopisto, 2019)
    The nemaline myopathies (NM) are a group of genetic muscle disorders, ranging in severity from severe, early lethal forms to milder muscle disorders with onset in childhood, sometimes presenting in adulthood. Nemaline myopathies present with usually non-progressive or slowly progressive generalised muscle weakness and are pathologically characterised by the presence of cytoplasmic inclusions called nemaline bodies or rods. So far, mutations in twelve genes have been identified underlying the disorder. This Thesis aimed at elucidating the expression of the nebulin gene (NEB) and the function of the protein, in order to gain insight into the pathogenetic mechanisms leading to NM. Approximately half of NM cases are caused by mutations in NEB, and the vast majority of these are inherited recessively. NEB consists of 183 exons, giving rise to transcripts up to 26 kb in length. Nebulin is an enormous protein, with important roles in maintaining the structure and function of skeletal muscle. It is localised in the thin filament of the muscle sarcomere, and one nebulin protein spans the entire length of the filament. Nebulin structure is highly modular, consisting of actin-binding simple repeats, and super repeats formed by seven simple repeats. In NEB, more than 240 different pathogenic variants have been published, causing myopathies with no cure currently available. Furthermore, most NEB-NM patients have a compound heterozygous genotype, with two private mutations anywhere along the gene, which further hampers the discovery of any genotype-phenotype correlations. Many of the NEB exons are alternatively spliced. In this study, we investigated NEB expression at the RNA level, in 21 different skeletal muscles and in brain. We demonstrated co-expression of several different nebulin isoforms in all the muscles studied, but no isoforms specific for certain muscles were identified. The results also indicated that all 183 NEB exons are expressed in adult brain, and that the expression levels were comparable with expression in skeletal muscle. Further studies are warranted to elucidate the expression in brain. We however, went on to further investigate the expression and function of NEB in skeletal muscle. Exons 143 and 144 are mutually exclusive, encoding the super repeats S21a and S21b, respectively. The amino acid sequences of the alternative region differ in both charge and hydrophobicity. We developed specific antibodies for these two isoforms, enabling studies of their expression at the protein level. Our results showed differences in the usage of these isoforms between various myofibres, and during muscle development. S21b was the only isoform present in early myotubes, whereas regulated expression of S21a appeared later, mainly in fast myofibres. These results form a basis for studying the functional differences between the isoforms, and add to the basic knowledge of normal muscle function. The size of nebulin complicates studies at the protein level as well as genetic studies. As the super-repeat region encompasses nearly the entire protein, we constructed a panel of 26 mini-genes, corresponding to all the different nebulin super repeats, to enable protein interaction studies. The studies covering the entire nebulin super-repeat region revealed differences in actin binding between the seemingly similar super repeats, depending on their location in the protein. Actin binding was significantly stronger towards the end of the protein, while the entire central region bound actin weakly. This finding constitutes an important step in understanding nebulin function, and may facilitate the interpretation of the effects of various NEB mutations, depending on their location along the protein. Furthermore, the super-repeat panel can be utilised for studying mutational effects functionally at the protein level. To shed light on the pathogenetic mechanisms underlying NM, and to enable the study of nebulin function in normal and defective muscle, we developed the first murine model of NM with compound heterozygous Neb mutations. The aim was to produce a model recapitulating the most common form of NM, i.e. the typical congenital form. By studying the new compound heterozygous model, and the parental lines, we have gained valuable insight into the effects of Neb mutations on muscle function. The mouse models will be useful in deciphering the pathogenetic mechanisms of NEB-NM, and will be suitable for the assessment of potential therapeutic approaches. The results of this Thesis shed light on the expression and function of nebulin in health and disease. This brings us closer to understanding the pathogenetic mechanisms of NM, and how the disorder evolves, which in turn will enable providing more accurate diagnosis and prognosis for NM patients. Understanding the pathogenetic mechanisms underlying NM is also a prerequisite for developing effective therapies for NM in the future.
  • Jokinen, Mikko (Helsingin yliopisto, 2019)
    Väitöskirja Lapin ympäristökiistojen kulttuuriset tekijät tarkastelee millaisia kulttuurisia merkityksiä Lapin luonto ja sen käyttö saa paikallisten ihmisten ja maankäyttöä ohjaavan hallinnon parissa. Sosiaalinen ja kulttuurinen kestävyys sekä konfliktinhallinta ovat väitöskirjan läpäiseviä peruskäsitteitä ja teemoja, joiden toteutumista tarkastellaan useiden eri tapaustutkimusten kautta. Tutkimuksen maantieteellinen pääpaino on saamelaisten kotiseutualueella Ylä-Lapissa, ei kuitenkaan tiukasti sinne rajautuen. Väitöskirja koostuu seitsemästä erillisartikkelista, jotka on julkaistu kotimaisissa ja ulkomaisissa tieteellisissä sarjoissa. Artikkeleiden teoreettinen viitekehys ankkuroituu luonnonvarojen ja ympäristökiistojen hallintaa koskeviin sekä kulttuurintutkimuksen jaettujen merkitysten teorioihin. Käytetyt aineistot ovat kysely- ja haastattelututkimusaineistoja – aineistot sekä analyysimenetelmät ovat siten määrällisiä ja laadullisia. Tutkimus asettuu yhteiskuntatieteellisen ympäristötutkimuksen kenttään. Tutkimuksen keskeisiä havaintoja on, että Lappiin sijoittuvia ympäristökiistoja ja niiden hallintaa luonnehtii toimijoiden moninaisuus ja kohtaamattomuus. Osapuolten roolit ja motiivit ympäristökiistojen käsittelyssä ovat usein epäselvät tai ongelmalliset, eikä jaettua ymmärrystä kiistan luonteesta, syistä ja mahdollisista seurauksista useinkaan synny. Suomalaiselta yhteiskunnalta ja valtion maita hallinnoivalta Metsähallitukselta on puuttunut sellaisia institutionaalisia rakenteita ja toimintatapoja, jotka ovat kulttuurisensitiivisiä ja kykenevät sosiaalisesti kestävästi hallinnoimaan Lapin luonnonvaroja ja niitä koskevia kiistoja. Uusia aiempaa parempia toimintatapoja on kuitenkin viime vuosina otettu käyttöön. Lapissa korostuu perinteisten elinkeinojen kuten poronhoidon ja luonnonvarojen kotitarvekäytön merkitys ja arvostus, mutta niiden saamia kulttuurisia sisältöjä ei aina tunnisteta. Väitöskirjan teesi on, että luonnonkäytön ja ympäristökiistojen hallinnassa tarvitaan kulttuurisensitiivistä otetta. Nopean kulttuurimuutoksen läpikäyneessä Ylä-Lapissa menneisyys on vahvasti läsnä ja selittää paikallisten ihmisten luonnonkäyttöön liittämiä arvostuksia ja merkityksiä. Luonnonsuojelu on varsinkin Ylä-Lapissa merkittävä maankäyttömuoto, jonka toteuttaminen kaipaa kulttuuriset tekijät tunnistavia ja avoimempia lähestymistapoja saavuttaakseen paikallisväestön hyväksynnän ja tuen. Luonnonsuojeluhankkeissa kannattaa jatkossa hyödyntää luontoon ja sen käyttöön liittyvää kulttuurista tietoa yhdessä ekologisen tiedon kanssa, jotta hankkeet saavat myönteisen vastaanoton. Avainsanat: Lappi, ympäristökiista, kulttuurinen kestävyys, luonnonsuojelu, luonnonvaro­jen hallinta, saamelaiset.
  • Lyytinen, Outi (Helsingin yliopisto, 2019)
    Viruses are obligate parasites infecting the cells from all the three domains of life: Bacteria, Archaea and Eukarya. Ribonucleic acid (RNA) viruses contain ribonucleic acid as their genomic element instead of deoxyribonucleic acid (DNA). There are three main types of RNA viruses: positive-sense single-stranded [(+)ssRNA], negative-sense single-stranded [(-)ssRNA] and double-stranded RNA (dsRNA) viruses. This Thesis is focused on revealing molecular details of replication and assembly of two dsRNA viruses: Pseudomonas phage phi6 (phi6) and human picobirnavirus (hPBV). Double-stranded RNA viruses need to carry an RNA-dependent RNA polymerase (RdRp) inside their virion to the host in order to be able to replicate their genome. We characterized the hPBV RdRp enzymatically and structurally and revealed the similarities of this RdRp to the other known small dsRNA virus RdRps like phi6 RdRp, which has been extensively studied. We showed that hPBV RdRp has a canonical cupped right-hand polymerase structure, it can replicate and transcribe homologous and heterologous template RNA in the absence of capsid proteins and it also possesses terminal nucleotidyl transferase activity. This is only the second dsRNA virus RdRp reported with this activity. The assembly of these two viruses is relatively different due to the differences in their structures. Phi6 has three layers and a lipid-protein envelope as its outermost layer whereas hPBV does not have any lipids in its structure and is composed of only one layer of capsid proteins surrounding the dsRNA genome. The assembly of the inner protein layers of phi6 is very well-known whereas as the envelope formation and the assembly of hPBV capsid layer is largely uncharacterized. Our results suggest that hPBV might use a co-assembly of its capsid proteins and genomic RNA precursors as its assembly strategy. The envelope assembly of phi6 was studied expressing phi6 membrane proteins in Escherichia coli bacteria. Our results revealed that only one small membrane protein P9 can induce phi6-specific vesicle formation in E. coli cells. Also, heterologous green fluorescent protein can be added to the vesicles by co-expressing non-structural P12 protein. This study reveals interesting molecular details about the genome replication and assembly of hPBV, a relatively unknown opportunistic human pathogen, and the envelopment process of phi6. These results are biologically interesting and may have also biotechnological applications in the future.
  • Hlushchenko, Iryna (Helsingin yliopisto, 2019)
    This thesis explores the role of several actin-binding proteins in the regulation of brain physiology with a focus on dendritic spines. Dendritic spines are considered the plausible physical substrate for learning and memory, as their morphology allows for modulating incoming signals. Disruptions in spine density and morphology are also often associated with neuropsychiatric disorders. The two cellular processes representing neuronal learning are long-term potentiation (LTP) and long-term depression (LTD). Here, I show that the actin-severing protein gelsolin transiently relocates to dendritic spines upon LTD induction, but not LTP induction or spontaneous neuronal activity. It is plausible that the modest – but relatively long-lasting – LTD-induced elevation of Ca2+ concentration increases the affinity of gelsolin to F-actin, thus inducing the relocalization of gelsolin to dendritic spines. Proper spine regulation is crucial for learning in live animals. MIM is an I-BAR containing membrane curving protein, shown to be involved in dendritic spine initiation and dendritic branching in Purkinje cells in the cerebellum. Behavioral analysis of MIM knock-out (KO) mice revealed defects in both learning and reverse-learning, alterations in anxiety levels and reduced dominant behavior, and confirmed the previously described deficiency in motor coordination and pre-pulse inhibition. Anatomically, we observed a decreased density of thin dendritic protrusions, enlarged brain ventricles and decreased cortical volume. Genetic studies have pointed out that genes often disturbed in neuropsychiatric disorders encode synaptic actin regulators. We selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. These mutations induced changes in the localization of α-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only α-actinin-4 expression caused a significant change in dendritic spine morphology by increasing mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in α-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density with mutations in myosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.

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