Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Hakanpää, Laura (Helsingin yliopisto, 2020)
    Vascular integrity is essential for proper vessel function, and for the maintenance of tissue and organ homeostasis. Endothelial cells (ECs) in the inner lining of the blood vessels form a barrier that dynamically regulates permeability across the vessel wall. Permeability via EC-EC junctions is transiently increased during inflammation, whereas abnormally or persistently elevated EC permeability promotes disease pathogenesis. For example, in sepsis, systemic capillary leakage compromises blood perfusion, and may lead to hypovolemic shock and multiorgan failure. Despite the significant amount of research on the mechanisms that control the EC barrier, no targeted therapies currently exist to seal the leaky vessels and maintain tissue perfusion. The aim of this study was to investigate how vascular permeability is controlled via an EC-derived growth factor angiopoietin-2 (ANGPT2), which is upregulated in various human diseases, including sepsis. ANGPT2 was found to signal via 1-integrin, and therefore the function of endothelial 1-integrin in vascular permeability was investigated. The results identified a novel signaling pathway, where ANGPT2–1-integrin signaling promotes EC permeability. 1-integrin was found to play a previously uncharacterized role in inflammation-induced vascular permeability, and an antibody against 1-integrin inhibited vascular leakage, improved EC junction integrity and protected from cardiac failure in LPS-induced murine endotoxemia. Earlier studies have shown that ANGPT2 destabilizes blood vessel integrity in a context-dependent manner via its classical receptor TEK receptor tyrosine kinase (TIE2) on ECs. These studies have raised interest on ANGPT2 as a potential target in various diseases, including cancer and ocular neovascular diseases. This study revealed that ANGPT2 can promote EC destabilization independently of TIE2, which is downregulated during inflammation. These results suggest that a better understanding of the signaling function of ANGPT2 is necessary, in order to optimally target ANGPT2 in disease. This study also highlights the crucial role of endothelial 1-integrin in controlling inflammation-induced EC permeability. The results showed that various inflammatory agents induced EC monolayer destabilization via 1-integrin, manifested by the loss of junctional VE-cadherin, the formation of actin stress fibers, and altered EC-extracellular matrix (ECM) adhesions. The EC-ECM adhesions that formed in inflammation were elongated fibrillar adhesions that can be distinguished from focal adhesions by the presence of the adapter protein tensin-1. Furthermore, 1-integrin promoted inflammation-induced EC contractility and reduced the EC barrier function. Importantly, targeting 1-integrin using a monoclonal antibody, or via a heterozygous genetic deletion in the endothelium of gene-targeted mice decreased vascular leakage in LPS-induced murine endotoxemia. Notably, the 1-integrin antibody was effective both as a prophylactic and as an intervention therapy, administered after the onset of systemic inflammation and vascular leakage, and its mechanism of action was independent of attenuating systemic inflammation, and of the vascular stabilizing function of TIE receptors. In summary, this thesis provides new knowledge on the mechanisms that lead to vascular leakage via ANGPT2 and 1-integrin. 1-integrin was identified as a potentially universal regulator of EC permeability. A major finding was that targeting the EC 1-integrin in a preclinical model of sepsis decreased vascular leakage, thereby improving cardiac function. The results of this thesis call for further studies in evaluating the translational potential of 1-integrin mediated vascular permeability.
  • Kalha, Solja (Helsingin yliopisto, 2020)
    Cornea is the outermost surface of the eye that refracts light to the lens and protects the sensitive ocular machinery. The cornea is divided to three cellular compartments; epithelium, stroma and endothelium. Our work focuses on the corneal epithelium, which is located closest to the tear film and, together with the film, forms a physiological barrier to pathogens and small particles from the environment. We followed the maturation of the mouse corneal epithelium from birth to adulthood and discovered a novel marker, Krt19, in this process. Krt19 expression gradually restricted from the central cornea to the limbus, concomitantly with eyelid opening and epithelial stratification, which are the hallmarks of postnatal maturation of the murine cornea. Corneal epithelium is renewed continuously throughout life by stem cells. Previous studies demonstrated that the limbus, located in the periphery of the cornea, houses the corneal stem cells. Immediate progeny of the stem cells, the progenitor cells, localize to the limbus, peripheral, and central cornea. We identified the gene Bmi1 in the corneal, epithelial progenitor cells. By lineage tracing of the Bmi1+ cells, we followed renewal dynamics in the central cornea and estimated the turnover of the epithelium to be 2-8 weeks in adult mice. However, we noticed a decrease in renewal rate with older animals. This is in line with evidence from renewal studies of the limbal stem cells, suggesting a general decrease of corneal epithelial renewal upon aging. We optimized a method to perform in vivo epithelial abrasion injury on mouse cornea. The development of this assay was instrumental for the experiments that followed. Using the abrasion model, we showed that the Bmi1+, central, corneal progenitor cells do not contribute to wound healing. Instead, the wound closed by rearrangement and migration of the remaining epithelial cells. We extended our analysis of the corneal barrier to encompass an accessory organ of the eye, the lacrimal gland (LG, tear gland). LG produces and secretes the aqueous part of the tear film, which is the largest portion of the film. The tear film provides another layer of protection to the ocular surface, because it contains anti-inflammatory and antimicrobial components as well as assists eyelid movements. We studied the role of Ectodysplasin-A (Eda) gene in the LG. Eda is critical in the development of ectodermal appendages, however LG development was not affected by the loss-of-function mutation in Eda. Instead, lack of EDA resulted in modulation of LG secretion and the development of a dry eye disease (DED). Furthermore, we discovered that Eda signalling activity was inhibited in response to corneal injury and suggest that this is necessary for the production of reflex tears that are released in ocular insult. In this assay, we shed light on the cooperation between cornea and the LG in homeostasis and injury. Our work is part of the research that aims to understand maturation and homeostatic maintenance of the anterior segment of the eye, cornea and the LG. This work provides new information regarding the development of Eda-linked DED. This is of importance, because the DED affects a large part of the population. Furthermore, we call for further studies on the mechanisms of how these two tissues communicate, as they are intricately linked and dependent of each other.
  • Kalha, Solja (Helsingin yliopisto, 2020)
    Cornea is the outermost surface of the eye that refracts light to the lens and protects the sensitive ocular machinery. The cornea is divided to three cellular compartments; epithelium, stroma and endothelium. Our work focuses on the corneal epithelium, which is located closest to the tear film and, together with the film, forms a physiological barrier to pathogens and small particles from the environment. We followed the maturation of the mouse corneal epithelium from birth to adulthood and discovered a novel marker, Krt19, in this process. Krt19 expression gradually restricted from the central cornea to the limbus, concomitantly with eyelid opening and epithelial stratification, which are the hallmarks of postnatal maturation of the murine cornea. Corneal epithelium is renewed continuously throughout life by stem cells. Previous studies demonstrated that the limbus, located in the periphery of the cornea, houses the corneal stem cells. Immediate progeny of the stem cells, the progenitor cells, localize to the limbus, peripheral, and central cornea. We identified the gene Bmi1 in the corneal, epithelial progenitor cells. By lineage tracing of the Bmi1+ cells, we followed renewal dynamics in the central cornea and estimated the turnover of the epithelium to be 2-8 weeks in adult mice. However, we noticed a decrease in renewal rate with older animals. This is in line with evidence from renewal studies of the limbal stem cells, suggesting a general decrease of corneal epithelial renewal upon aging. We optimized a method to perform in vivo epithelial abrasion injury on mouse cornea. The development of this assay was instrumental for the experiments that followed. Using the abrasion model, we showed that the Bmi1+, central, corneal progenitor cells do not contribute to wound healing. Instead, the wound closed by rearrangement and migration of the remaining epithelial cells. We extended our analysis of the corneal barrier to encompass an accessory organ of the eye, the lacrimal gland (LG, tear gland). LG produces and secretes the aqueous part of the tear film, which is the largest portion of the film. The tear film provides another layer of protection to the ocular surface, because it contains anti-inflammatory and antimicrobial components as well as assists eyelid movements. We studied the role of Ectodysplasin-A (Eda) gene in the LG. Eda is critical in the development of ectodermal appendages, however LG development was not affected by the loss-of-function mutation in Eda. Instead, lack of EDA resulted in modulation of LG secretion and the development of a dry eye disease (DED). Furthermore, we discovered that Eda signalling activity was inhibited in response to corneal injury and suggest that this is necessary for the production of reflex tears that are released in ocular insult. In this assay, we shed light on the cooperation between cornea and the LG in homeostasis and injury. Our work is part of the research that aims to understand maturation and homeostatic maintenance of the anterior segment of the eye, cornea and the LG. This work provides new information regarding the development of Eda-linked DED. This is of importance, because the DED affects a large part of the population. Furthermore, we call for further studies on the mechanisms of how these two tissues communicate, as they are intricately linked and dependent of each other.
  • Dhaygude, Kishor Uttam (Helsingin yliopisto, 2019)
    The majority of the planet's biological diversity comprises of diverse microorganisms, including large communities of insects. It is only through symbiotic, pathogenic and vectoring association, a diverse relationship between the microorganisms and the insects can be established. In spite of having an independent interaction, microorganisms are expected to fulfill the important roles of insect nutrition, reproduction, development, as well as behavioral resistance to pathogen colonization. So to understand the molecular diversity, population structure, and ecological importance of the majority of microorganisms, it is very essential to discover and characterize these microbial communities. The multi-omics approaches have the potential of in-depth screening of microorganisms as well as answering some fundamental microbial ecology questions. So, multi-omics approaches and bioinformatic analysis are considered as the powerful tool to study the non-model microbes and ultimately to study the composition and function of dynamic microbial communities. In spite of these, the microbial community largely remains unknown to the domain of social insects. This thesis majorly utilizes the multi-omics approaches for demonstrating the dynamic interplay between host and microbes. On the basis of the observational study it has been found that pathogenic and natural microbial community are associated with ant Formica exsecta. The findings included members of several endogenous bacterial phyla, such as Wolbachia, two obligate endogenous and possibly entomopathogenic fungi, as well as complete genomes of three novel RNA viruses belonging to the classes of Iflaviridae, Dicistroviridae and Mononegavirales. In this thesis, RNA sequencing data for the ant F. exsecta constructed from the samples of several life stages of both sexes as well as female castes of queens and workers to maximize the representation of expressed genes. Additionally, for the first time the horizontal gene transfer is demonstrated in this thesis from Wolbachia endosymbiont to host F. exsecta ant genome and at the same time the process of releasing of the first genome of Wolbachia endosymbiont from ant species. Moreover, the focus of thesis is on genome organization and molecular characterization of the three F. exsecta viruses and at the same time explaining the viral transmission in other related ant species. By adopting the advantages of the power of genomic technologies, this thesis tries to provide new insights into the host and microbe interactions, and the evolution of host-parasite genomes in a more general framework. However, in general the studies of this thesis provide useful information, guidelines and resources for social insects and genomics research.
  • Ehrnsten, Eva (Helsingin yliopisto, 2020)
    Benthic macrofauna is an important component linking pelagic and benthic ecosystems, especially in productive coastal seas. Through their metabolism and behaviour, benthic animals affect biogeochemical fluxes between the sediment and water column. Mechanistic models that quantify these benthic-pelagic links are crucial for understanding the functioning of coastal ecosystems and their responses to anthropogenic pressures, such as climate change and eutrophication. In this thesis the flows of carbon through functional groups of benthic macrofauna and their sediment food sources were explored using a new mechanistic model, called the Benthic Macrofauna model. The model was coupled to the hydrodynamic-biogeochemical BALTSEM model and used to simulate past, present and future biomass and metabolic carbon processing of aphotic soft-sediment communities of macrofauna in the Baltic Sea. The aims of this thesis were to identify the main drivers of macrofaunal biomass and community composition and to quantify the effects of environmental change on macrofaunal communities and their contribution to benthic carbon processing. Sedimentation of particulate organic carbon as a food source was identified as a main driver of macrofaunal biomass in two coastal areas as well as in the four largest basins of the Baltic Sea. Together with results of a food-web model of the central Baltic Sea, these results indicate that eutrophication has led to increased biomass in most parts of the ecosystem through increased productivity and sedimentation, except where counteracted by associated expanding hypoxia. Hypoxia has severe local effects on community biomass and composition, but on the scale of the Baltic Sea, biomass gains in oxic areas seem to exceed biomass losses due to hypoxia during past eutrophication. Increasing the bottom water temperature had a relatively small negative effect on community biomass in comparison to the other tested drivers, but the indirect effect of increasing surface water temperature through intensification of pelagic recycling and reduction of organic matter input to the sediment was substantial. Macrofaunal metabolism can contribute substantially to benthic carbon and nutrient processing, especially in shallow, productive coastal areas where biomass is primarily food-limited. For example, in a coastal area of the Gulf of Finland in the early 2000s, the benthic macrofauna was estimated to process up to 80% of simulated carbon input through ingestion and mineralize 40% through respiration. On the scale of the Baltic Sea, the benthic macrofauna was estimated to mineralize about 20% of organic carbon input to the sediments. These results together with a literature review suggest that the role of benthic macrofauna needs to be considered in models of coastal and global carbon and nutrient cycling. Simulations combining changes in climate and nutrient loads resulted in large reductions in benthic macrofaunal biomass and carbon processing capacity by the end of the 21st century if nutrient loads to the Baltic Sea are reduced according to the Baltic Sea Action Plan, but also if loads are kept at present levels. With increased nutrient loads, climate change counteracted the effects of increased productivity, also leading to a decrease in organic matter sedimentation, macrofaunal biomass and carbon processing capacity in the second half of the century.
  • Valkonen, Sami (Helsingin yliopisto, 2019)
    Extracellular vesicles (EV) are nanosized lipid bilayered particles produced by all cells. Due to their remarkable capacity to transport molecular cargo, EVs are considered important mediators of intercellular signalling, making EVs major contributors to cellular functions in health and disease. Recent technological development has improved the analytical techniques of assessment of EV composition and functionality, which has resulted in the interest to apply EVs to diagnostics and therapeutics. Currently, one notable shortcoming of EV studies is the lack of standardisation and comparability of analytical methods. To provide more EV-like alternative for currently used synthetic reference materials that do not resemble EVs, biological reference material was produced from disrupted red blood cells. The produced particles, nanoerythrosomes, were shown to be in many aspects similar to red blood cell EVs but with an additional advantage of mass producibility, nanoerythrosomes are a compelling option for widely distributed reference material enabling improved comparability of EV studies. The second part of the research examined the EVs of platelet concentrate as a potential blood product quality markers and a factor influencing platelet concentrate functionality. In the present study, the EV count of platelet concentrates was shown to be exploitable as a sensitive marker for platelet activation, when compared to other activation markers. Further, platelet-derived EVs in platelet concentrates were shown to contain molecular attributes critical for lipid signalling, e.g., specific phospholipid profile, enzymes important for lipid signalling, and bioactive lipid mediators. Taken together, the results of this thesis show that blood products are a source for nanoerythrosomes with noteworthy potential to improve the comparability of EV studies and, on the other hand, EVs that help to understand the blood product functionality better.
  • Anwar, Tahira (Helsingin yliopisto, 2019)
    Autophagy is an evolutionarily conserved pathway used by cells to degrade cargoes that are larger than individual proteins in lysosomes. It maintains cellular homeostasis and degrades aggregate-prone proteins and/or whole organelles. Impaired autophagy plays a crucial role in human diseases like neurodegeneration, cancer and infections. During autophagy, a double-membrane structure, named phagophore, forms around the cytoplasmic cargo. The phagophore grows until it forms a double-membrane vesicle called the autophagosome. The autophagosome fuses with a lysosome to gain degradative properties, forming a structure named autolysosome. Within the autolysosome, the cargo is degraded, and the resulting building blocks are recycled back to the cytoplasm for energy production or biosynthetic reactions. Many aspects of the molecular mechanisms regulating autophagosome biogenesis in starvation-induced and selective autophagy are still unresolved. Moreover, the origin of the phagophore still holds many questions. We studied the role of the protein TRIM17 in selective autophagy. We found that TRIM17 inhibits selective autophagy of various targets while targeting midbodies for autophagic degradation. The protein Mcl-1 regulates autophagy-inducing or -inhibiting functions of TRIM17. The formation of the TRIM17-Beclin 1-Mcl-1 complex inhibits selective autophagy. TRIM17-dependent autophagic degradation of midbodies is induced upon dissociation of Mcl-1 from the Beclin 1-TRIM17 complex. We next revealed that Lagotto Romagnolo dogs carrying an ATG4D missense mutation exhibit altered basal autophagy and abnormal cytoplasmic vacuolization. Our study also confirmed normal lysosomal degradation in affected dogs, excluding a link between the ATG4D mutation and lysosomal storage diseases. Lastly, we showed that upon starvation, Beclin 1 targeted to the endoplasmic reticulum (ER) partially rescued autophagosome formation in cells lacking the ULK1 and ULK2 kinases. The autophagy flux was impaired in these cells and this defect was not rescued by expression of Beclin 1 targeted to the ER. These results suggest a regulatory role for the ULK kinases in autophagosome maturation in addition to autophagosome biogenesis. Moreover, our study demonstrated that the ULK1 and UK2 kinases might play a role in regulating Beclin 1 enrichment in the ER under nutrient-rich conditions.
  • Salgado Maldonado, Ana Lucia (Helsingin yliopisto, 2019)
    The life-history and performance of insect herbivores can be significantly affected by environmental stress, and the responses may vary across the life cycle of the insect. This variability in the responses depends on the specific demands of different life stages, but also on their potential to adjust behaviourally by seeking better environmental conditions. Drought is one of the most dominant environmental stressors that can affect natural populations. The increased prevalence and severity of droughts during the last decades have generated radical changes in ecosystems with further consequences for biotic interactions. Therefore, it is necessary to study the effects imposed by drought on trophic interactions. Insect herbivores are intimately dependent on their host plants, which means that the effects of drought on host plant may impact life-history traits and behaviour of the insect herbivore. Drought may, for example, alter the nutritional quality of the host plant with subsequent impacts on the herbivores acquiring resources from these plants. This PhD thesis investigates the responses of the Glanville fritillary butterfly (Melitaea cinxia) to variation in host plant quality, by exposing one of its prominent host plants (Plantago lanceolata) to drought. In my research, I combined lab experiments with detailed field measurements and existing long-term monitoring data. The lab experiments demonstrated that drought-exposed host plants are nutritionally richer than well-watered host plants, and that the larval responses to host plant quality changes imposed by drought are developmental stage-dependent. Furthermore, I show that post-diapause larvae and adult females each preferred host plants that enhanced their growth rate or survival of their offspring, respectively. Using long-term monitoring data and detailed information of the habitat conditions from the field, I found that maternal oviposition preference was directed towards drought-exposed microhabitats with high host plant abundance. This preference was correlated with higher overwinter survival of the offspring on normal years. However, as females did not show an ability to adjust their oviposition preferences in a year of severe drought, this led to very few surviving offspring. These data suggest that the metapopulation may be at risk under increasing drought conditions in the future. The results from this dissertation highlight that both the host plant and the insect herbivore are somewhat adapted to live under variable environmental conditions. The host plants show ability to respond to drought conditions and also the insect herbivore. The insect herbivore is, to some extent, also able to adjust its behaviour in response to variation in host plant quality induced by drought. Additionally, my results show that microhabitat conditions are important in determining insect herbivores’ performance. However, if conditions become even drier in the future this may impose risks. In conclusion, to fully understand the potential impacts of anthropogenic climate change, we need to understand their impacts on the interactions of species, such as host plants and insect herbivores.
  • 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 BrainDerived Neurotrophic Factor (BDNF) promotes the maturation of PV interneurons, thereby stimulating a precocious onset 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.

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