Bio- ja ympäristötieteellinen tiedekunta

 

Recent Submissions

  • Hakala, Sanja Maria (Helsingin yliopisto, 2020)
    There are intricate links between the evolution of sociality and the spatial population structures created by dispersal. In ants, the evolution of the most complex societies, supercolonies, is strongly connected to limited dispersal. The supercolonies consist of hundreds of interconnected nests where thousands of queens and their workers cooperate over large areas. Supercolonies arise from simpler family units when large proportions of daughter queens are philopatric and stay in their natal colony as additional reproducing queens instead of dispersing by wing. This allows the colonies to grow quickly and colonize large areas, but also increases social conflicts due to very low local relatedness in these societies. In this thesis I inspect the evolution and maintenance of ant supercolonies, by focusing on dispersal traits and the consequences of dispersal in socially polymorphic Formica ants. Formica have both simple family-based monodomous colonies and complex supercolonies, and some species have also intraspecific variation. To lay a sound theoretical background for my work, I begun by synthesizing current knowledge of dispersal evolution in ants. In my literature review I showed how eco-evolutionary feedbacks link the evolution of ant dispersal strategies and social organization, and pinpointed the most relevant future research directions. To be able to inspect some of the hypotheses formulated in the review, I clarified the species identification of one of my intended study species, Formica fennica with molecular methods, and concluded that the populations I study should be treated as conspecific to Formica exsecta. I analyzed the individual dispersal traits of six socially variable species of Formica ants to assess whether dispersal ability varies between monodomous and supercolonial societies in accordance to the observed behavioral difference. According to my results the dispersal behavior of these species is likely not restricted by their morphology or resources the individuals get from their natal colonies. Overall the traits of all species and both sexes indicate good flight ability, with overall male bias and large variation both among and within species. The increased philopatry in supercolonial species and populations is more a behavior change: the queens are philopatric even though the society provides them resources for dispersal. However, I observe a small decrease of male flight muscle ratio in supercolonial species, which indicates strong coevolution of the sexes. In order to better understand the outcomes of limited dispersal in supercolonial Formica, I analyzed the behavioral and genetic structure of a dense supercolonial population of Formica pressilabris. The population is genetically viscous at a small spatial scale, but still not genetically structured by location on a larger spatial scale. This indicates that although dispersal is limited within the population, a large enough proportion of individuals do disperse to keep the local populations connected. Interestingly, when analyzing worker behavior among the polydomous nests, the observed aggression pattern indicates that they are not a single cooperative unit – but also not clearly separate colonies. The sensitive behavioral assay developed for this study shows that workers allow a proportion of individuals from outside nests to enter their own nest material, but are slightly more aggressive towards individuals from further away. This indicates the population is only partially cooperative over the whole nest aggregation, and shows that the inner structures of Formica supercolonies should be analyzed in more detail. This thesis sets light on ants’ dispersal ability and behavior, and demonstrates the crucial role of dispersal in the evolution of their different social structures. My results also raise new questions about possible conflicts over dispersal in ant societies. 
  • Björklund, Mia (Helsingin yliopisto, 2020)
    The basic unit of all life forms is a cell, and all multicellular organisms are the result of programmed cell divisions of a single fertilised oocyte. This intricately controlled process, where a cell duplicates its DNA, grows and divides into two daughter cells, is called the cell cycle. Maintaining the appropriate control of the cycle is critical for the homeostasis of an organism. When the control fails, it may result in uncontrolled cell proliferation, one of the hallmarks of cancer. Cell cycle can be promoted or inhibited by signalling pathways induced by intra- or extracellular cues. Signals such as growth factors and hormones can bind cell surface or nuclear receptors causing signalling events and/or gene transcription resulting in positive or negative cell cycle regulation. External environment such as nutrient availability, or changes in energy metabolism can also signal to the cell cycle machinery, preventing cell division under high energy expenditure or external stress conditions. Central proteins in cell cycle regulation are the Cyclin-dependent-kinases, CDKs, and their cyclin partners. They phosphorylate other proteins causing changes in their activity or abundance, eventually leading to events required for cell duplication, such as DNA synthesis and chromosome segregation. CDK activity is in turn regulated by phosphorylation and cyclin abundance as well as the presence and binding of CDK-inhibitor proteins such as p27Kip1. P27Kip1 belongs to the Ink4-family of inhibitors, and can also function as an assembly factor for some CDK-cyclin complexes. We identified a form of p27Kip1, which was not bound to CDKs (p27NCDK) but was increased under growth restricting conditions such as TGF-β growth inhibition or serum starvation. On the other hand, inhibition of growth promoting pathways such as the PI3K-pathway was also found to increase the levels of p27NCDK. We also discovered that cellular stress induces the levels of p27NCDK, and appears to do so mainly via the AMP-activated protein kinase AMPK. AMPK is a kinase that is activated under energy stress conditions, like strenuous exercise. In this work we also investigated the crosstalk of these pathways and found points of convergence including p27NCDK and TGF-β pathway protein Ski. Ski negatively regulates TGF-β signalling pathway by binding the TGF-β signalling molecules. Ski is rapidly degraded upon TGF-β stimulation thereby allowing the signal to pass. We discovered that the PI3K-pathway intersects with TGF-β pathway as the PI3K-pathway kinase Akt phosphorylates Ski, thereby destabilising it. Destabilisation of Ski leads to an induction of a TGF-β target gene Smad7. Given that Smad7 is another negative regulator of TGF-β signalling, several genes such as the CDK-inhibitor p15 are downregulated. Concomitant activation of Akt and TGF-β therefore reduced induction of p15 and relaxed cell cycle controls. In addition the effects of external stress on cell cycle were investigated in a human prostate epithelial cells. Surprisingly, these cells did not undergo cell cycle arrest in response to ionising radiation. These cells were noted to have low or undetectable levels of Wee1 kinase, which normally exerts an inhibitory phosphorylation on CDK2 and CDK1 in response to external stress. Due to lack of inhibition, CDK2 activity remained high even after high doses of radiation. In this work we identified different points of cross talk between cell cycle promoting and inhibiting signalling pathways. These findings have significance in cancer and cancer treatment, which is often based on inhibiting the hyper activation of cell cycle promoting pathways by using hormones, radiation or small molecule compounds.
  • Pham Duc, Dan (Helsingin yliopisto, 2020)
    Lipid disturbances reflect the dysregulation of molecular pathways associated with metabolic syndromes and neurological diseases like Alzheimer’s disease and Huntington’s disease. Several factors underlay the pathogenesis of lipid disorders, including accumulation of lipids, disturbed cell signaling, inflammation, mitochondria dysfunctions, ER stress and activation of cell death pathways. Here we have studied the roles of the neurotrophin, NGF and pro-NGF, and their receptor, p75NTR, a member of the tumor necrosis factor (TNF) superfamily, in lipid regulation. NGF and p75NTR have been previously studied in the nervous system but they are also expressed in non-neuronal cells, particularly after tissue injury and inflammation. In our study, we have used various cell and molecular biology methods, as well as protein methods, to elucidate the function of p75NTR in hepatocytes and neuronal cells. We have further employed p75NTR gene-deleted mice, and CRISPR/Cas9-mediated gene inactivation in human hepatocyte cells to elucidate the mechanisms of p75NTR in LDLR regulation. We have thus identified a molecular pathway by which NGF and pro-NGF influence SREBP activation leading to altered LDLR regulation and LDL uptake involving caspase-2/caspase-3 actions in Huh7 hepatocyte cells. Furthermore, we provide insight into the influence of LDL particles on neurite outgrowth in neuronal cells. RNA-sequencing analysis of wild-type and p75NTR-KO mice has demonstrated further that lipogenic genes are altered in the absence of p75NTR. There was also a down-regulation of serum cholesterol in p75NTR-KO mice in comparison with controls. Our findings showed further that NGF and pro-NGF are elevated, together with an increase in LDLRs in ob/ob and db/db mouse livers. This work will add to our understanding of p75NTR in health and disease, and may open up new possibilities to identify novel therapeutics to combat fatty liver and various metabolic disorders.
  • Tikker, Laura (Helsingin yliopisto, 2020)
    The anterior hindbrain segment rhombomere 1 (r1) generates in its ventral (basal) region GABAergic and serotonergic neurons that give rise to various nuclei in the adult brainstem that participate in the modulation of mood and motivation. This thesis focuses on the function of specific transcription factors (TFs) and their co-regulators that control the differentiation of r1derived neurons and their subtypes. First, we characterized early ventral r1 development to determine which neurons are generated in this area. In general, the developing neural tube can be divided dorso-ventrally into multiple progenitor domains that have distinct gene expression patterns and give rise to diverse cell types. We found that ventral r1 contains at least three different progenitor domains. Using genetic fate mapping in the mouse, we determined that the two most ventral progenitor domains (Nkx2-2+ rp3 and rpvMN) produce serotonergic neurons and oligodendrocytes, whereas the more dorsal progenitor domain (Nkx6-1+ rp2) generates GABAergic and glutamatergic neurons. By combining single-cell RNA sequencing (scRNAseq) and expression analyses of subtype-specific TFs, we show that embryonic ventral r1 contains molecularly distinct populations of post-mitotic GABAergic and glutamatergic precursors. We further report that GABAergic neurons from ventral r1 give rise to multiple GABAergic nuclei in the anterior brainstem, such as the posterior substantia nigra pars reticulata (pSNpr), rostromedial tegmental nucleus (RMTg) and ventral tegmental nucleus (VTg), whereas GABAergic neurons of the dorsal tegmental nucleus (DTg) originate from progenitors located in dorsal region of r1. Second, we analysed the functions of GATA TFs and their regulators in the development of r1derived GABAergic neurons by conditional mouse mutagenesis. We showed that GATA2 and GATA3, together with their co-factor TAL1, act as neuron-type selectors in early post-mitotic precursors to promote a GABAergic over glutamatergic neuron fate. Analysis of these mutants during later developmental stages showed that GABAergic neurons in the pSNpr, RMTg and VTg were absent, while the number of glutamatergic neurons was increased in other nuclei such as the interpeduncular nucleus (IPN) and the laterodorsal tegmental nucleus (LDTg). We found that ZFPM1 and ZFPM2, two GATA cofactors, are also expressed in GABAergic neuron precursors in r1. However, ZFPM2 does not function as a neuron-type selector in these cells, but rather is required for the proper development of pSNpr, RMTg and VTg GABAergic neurons. Finally, we studied the role of GATA2, GATA3 and ZFPM1 in the development of dorsal raphe (DR) serotonergic neurons in r1. Conducting overexpression experiments in chicken embryos, we demonstrated that GATA2 and GATA3 guide the differentiation of serotonergic neurons in the absence of their TAL1 partner, which is vital for GABAergic differentiation. We determined that GATA2 acts as a neuron-type selector and is important for all post-mitotic serotonergic neuron precursors to acquire serotonergic identity, whereas GATA3 is required for the differentiation of specific subtype of serotonergic neurons from r1. In addition, GATA2 and GATA3 are necessary for the development of non-serotonergic glutamatergic neurons in the DR. It was further shown that the GATA cofactor ZFPM1 is essential for the correct development of serotonergic neurons in DR subregions DRVL (ventrolateral part of dorsal raphe) and DRD (dorsal part of dorsal raphe). Loss of ZFPM1 function resulted in increased anxiety-like behaviour and elevated contextual fear memory, a phenotype that was alleviated by chronic treatment with fluoxetine, a selective serotonin reuptake inhibitor (SSRI). In conclusion, this work reveals neuronal subtypes present in and mechanisms involved with anterior brainstem development and that are important in the determination of behavioural phenotypes. Furthermore, it demonstrates that a complex gene regulatory system, where functions of GATA family selector TFs are modulated by their cofactors, is employed to achieve cell diversity in the central nervous system, mechanisms of which share marked similarities with cell fate determination programs in other developing tissues, such as the hematopoietic system.
  • Miettinen, Heli (Helsingin yliopisto, 2020)
    Boreal freshwater ecosystems play an important role in landscape carbon (C) cycle. Streams connecting lakes form an extensive network, where terrestrially fixed C is transported, processed, stored, and released to the atmosphere before reaching the oceans. The terrestrial influence is most significant in headwater streams and lakes. Despite the close connection of terrestrial, lotic, and lentic ecosystems, the ecosystems are mainly studied separately, and the possible interactions between the ecosystems are lost. Also, the C dynamic models are often based on sparse measurements, or singular processes are investigated as snapshot studies. Thus, to reveal the C flux dynamics in the continuum, comprehensive studies based on detailed temporal data series over long periods are needed. In this thesis, dissolved organic carbon (DOC) concentrations and lateral transport in runoff from an upland catchment site was studied over 15 years (1998-2012). The annual and seasonal dynamics of carbon dioxide (CO2) and methane (CH4) concentrations, lateral fluxes, whole-lake storages and atmospheric release were explored through the aquatic continuum over three years study period (2011-2013). Besides, special attention was paid to CO2 and DOC concentrations and lateral fluxes during spring freshet periods by using automatic high-frequency measurements in the lake and its draining streams. In general, the continuum showed remarkable spatial and temporal variation in C concentrations and fluxes. The C fluxes in both terrestrial and aquatic ecosystems were seasonally controlled mainly by precipitation and local hydrological conditions. Also, fluxes, concentrations and whole-lake storage of CH4 were regulated by temperature and DOC runoff from upland catchment was regulated by previous year’s net ecosystem exchange and litter production. This study highlighted the importance of spring and autumn for lateral C transport and atmospheric release. The allochthonous C gas input of terrestrial origin plays an essential role in the temporal C dynamics of the lake. In spring, the laterally transported C gases accumulated under the ice cover during the last weeks of the ice cover period. This connection was confirmed with synchronous changes in concentrations and whole-lake storages in the lake and C gas transport peak in the streams. External input increased the whole-lake storages of C gases as well as the CO2 concentration in the upper water layer of the stratified lake. The atmospheric release at the ice-out was long-lasting, and fluxes were high in comparison to earlier studies. The external input covered up to 24 % of CO2 and 42 % of CH4 released during the first week after ice-out. Due to the transience of the C gas transport and atmospheric release, the lateral impact is easily missed with sparse sampling. The lateral transport of DOC from the upland catchment on mineral soil was small in comparison to the other ecosystem C fluxes, 0.32 % of the net ecosystem exchange (NEE). Considering the whole catchment, the atmospheric emission from the lake accounted for 9.3 % of the catchment NEE. However, these results shed light on the increasing importance of freshwaters in transporting and releasing C in the changing future climate. An increasing trend in DOC concentration was found in runoff water from the terrestrial upland catchment, which indicates higher terrestrial C load into freshwaters in the future, too. Warmer winters may result in changes in the seasonal pattern; the differences in snow accumulation did not influence the daily amount of C transported, but the C inputs into the lake took place earlier during the winter months instead of spring. When addressing the impacts of climate change on a catchment scale, it is crucial to consider aquatic and terrestrial ecosystems together to get precise estimates of C sinks and sources.
  • Saarela, Sanna-Riikka (Helsingin yliopisto, 2020)
    Environmental governance has a close relationship with scientific knowledge. Over decades, volumes of scientific knowledge have contributed to discovering, framing, understanding, monitoring and reconciling environmental problems. Quite recently, concerns about the use of scientific knowledge in decision-making and the ‘science-policy gap’ have emerged; and as a consequence, multi-way science-policy interaction (SPI) has been proposed. This thesis addresses SPI in Finnish public environmental governance by focusing on how SPI is constructed, managed and reflected upon by researchers and public authorities. I bring out challenges and successes that key actors have faced in their attempts to bridge the science-policy gap. I utilise key theoretical considerations from existing research on actors, knowledge-brokering processes, outcomes and context in SPI in analysing qualitatively four SPI cases. The results show that interaction between researchers and policymakers in Finnish environmental policy and planning cases has only recently started to evolve towards a more collaborative form. Although the actors still strongly subscribe to low-level interaction, they have also started to pay increasing attention to the various interaction means as well as to societal and personal benefits, constraints and driving forces associated with SPI. Path-dependency of an institutionalised SPI, competency and reward structures appear to be important in defining the nature of interaction. In addition, my results also demonstrate that strategic actions by researchers and policymakers may significantly affect SPI. The cases presented in this thesis show that careful and sensible co-development of concrete or abstract boundary objects have an important role in connecting people, issues and processes in SPI. Based on the results, it appears that the current structures and procedures do not yet adequately support mainstreaming SPI. For instance, institutionalised SPI may hinder the introduction of new interaction means, and researchers and policymakers appear not to have required competencies for taking on new roles as interactive actors in SPI. For those developing and advancing SPI in a national context, the practical lessons of this study emphasize careful design and preparation of SPI, a need to tailor interaction means according to the context and different phases of the SPI process, utilisation and co-development of a suitable boundary object in SPI. The inclusion of a knowledge broker in a multi-disciplinary SPI process, and ex post evaluation of outcomes of the SPI process. In the future, additional empirical research should be carried out on actualised SPI processes in order to achieve better understanding on what kind of interaction works in which policy and planning contexts.
  • Hakala, Markku (Helsingin yliopisto, 2020)
    Polymerization of actin filaments against cellular membranes and contraction of actomyosin fibers generate pushing and pulling forces for cell migration, endocytosis, cell division, and maintenance of cell morphology, as well as for intracellular motility and morphogenesis of organelles. Thus, the actin cytoskeleton is a fundamental cellular component in development, immune responses, and in several other aspects of physiology. Moreover, the actin cytoskeleton is hijacked by viruses and pathogens during the infection process. Owing to their central role in above-mentioned cellular processes, actin and actin-binding proteins have been in the limelight of cancer research. Actin is a globular protein, which can polymerize into filaments and depolymerize back to monomers. Dozens of actin binding proteins regulate actin dynamics in cells. Whereas regulation of actin filament nucleation and filament elongation are relatively well understood, the disassembly is far more enigmatic topic. ADF/cofilin is the key actin disassembly factor. It belongs to a family of six actin depolymerizing homology (ADF-H) domain proteins, which all interact with actin or actin-related proteins. However, apart from ADF/cofilin, biochemical and cellular functions of the members of this protein family have remained elusive. In this work, I studied the cellular and biochemical roles of two ADF-H domain proteins, glia maturation factor (GMF) and twinfilin. I show that they both promote the disassembly of dendritic actin networks in cells, but by distinct mechanisms. GMF, which binds actin-related proteins (Arp) in the Arp2/3 complex, debranches dendritic actin networks in vitro. The data presented here show that GMF regulates the dynamics of lamellipodial, dendritic actin network in Drosophila cells and promotes collective border cell migration in vivo. Moreover, Drosophila GMF display a strong genetic interaction in cells and in vivo with another actin-regulatory protein, actin-interacting protein 1 (Aip1), indicating that they facilitate actin disassembly in a synergistic manner. Twinfilin interacts with actin monomers and actin filament barbed ends to inhibit actin polymerization. Moreover, it binds heterodimeric Capping Protein (CP) and membrane phosphatidylinositol phosphates (PIPs), which inhibit the actin-binding function of twinfilin. However, the molecular mechanism of this interaction has remained unknown. Thus, in the second part of the thesis I utilized a combination of mutagenesis and biochemistry, supplemented with molecular dynamics simulations, to reveal how PIPs inhibit twinfilin. Interestingly, twinfilin interacts with PIPs with a two-step mechanism. First, the CP-interaction motif in the carboxy-terminal (C-terminal) tail of twinfilin anchors the protein to plasma membrane. Subsequently, the actin-binding interface interacts with lipids, leading to inhibition of both the CP- and actin-binding activities of twinfilin. Cellular functions of twinfilin have remained elusive despite extensive studies in past decades. In the third part of the thesis, I generated mouse twinfilin knockout cell lines and showed that twinfilin regulates both actin and CP turnover in lamellipodia. Surprisingly, twinfilin promotes CP dynamics in cells and in vitro by uncapping filament barbed ends, thus providing an explanation why the localization of CP in cells is restricted to the very distal edge of lamellipodia. Moreover, biochemical experiments demonstrated that twinfilin itself does not accelerate filament disassembly after uncapping, but instead allows filaments to disassemble after removal of CP from actin filament barbed ends. These findings explain the diminished actin filament disassembly rates in lamellipodia of twinfilin-deficient cells. Together, the work presented here highlights the important roles of twinfilin and GMF in regulation of lamellipodial actin networks. Their distinct roles in actin disassembly show that actin turnover in dendritic arrays is maintained by several functionally different proteins which, in concert, facilitate the turnover of branched actin filament networks in cells.
  • Salonen, Iines (Helsingin yliopisto, 2020)
    The marine benthic ecosystem, encompassing the sea floor and the sedimentary habitats within, plays a crucial role in major biogeochemical cycles and the functioning of the marine ecosystem. Despite its importance, many aspects of its ecology remain understudied, such as microbial community composition and bacteria–eukaryote interactions. In recent years, molecular ecology methods, in particular DNA metabarcoding, have provided us with new insights into benthic ecology. Compared with traditional methods, metabarcoding has the advantage of being applicable to various samples, generating large data sets and allowing reliable taxonomic identification without requiring morphological identification. This thesis explores the use of the DNA metabarcoding method in examining the benthic ecosystem from different angles and targeting both eukaryote and prokaryote communities. 18S rDNA metabarcoding was used to target and track temporal variation in eukaryote communities in coastal sediment of the northern Gulf of Finland. The results demonstrate that DNA metabarcoding can be used to study sediment eukaryote community composition and variation over time. The key factor shaping the sediment eukaryote community was time, firstly the year and then the season, whereas location played a smaller role in explaining the community variation. In addition, the changes in the community composition could be linked to larger environmental phenomena, such as the timing and duration of the ice season, which in turn influenced the phytoplankton bloom. The ability of DNA metabarcoding surveys to resolve sediment community response to environmental factors indicates a potential for applications in biomonitoring and environmental assessment. Nevertheless, some limitations remain, such as the lack of standardization in metabarcoding methods and data analysis, and deficiencies in the reference databases. In addition, 18S and 16S rDNA metabarcoding were employed to resolve trophic strategies and microbial interactions of a common benthic unicellular eukaryote, the foraminifera. Samples for these studies were collected from the intertidal mudflats of Texel Island in the Netherlands. Both intracellular bacteria and eukaryotes of foraminifera were targeted in these studies, as well as the foraminifera’s own DNA, which allowed reliable genus-level identification. The intracellular eukaryote operational taxonomic units (OTUs) of different foraminiferal species reflected their trophic preferences: Haynesina sp. (genotype S16) and Elphidium sp. (genotype S5), which are likely to prefer an algal diet and/or are known to have a tendency for kleptoplasty, had an intracellular eukaryote content dominated by diatoms. In contrast, Ammonia sp. (genotype T6) contained also metazoan OTUs, implying potential predatory behaviour in addition to an algal diet. Based on these results, DNA metabarcoding can provide a comprehensive tool for the investigation of life strategies and ecology of even unicellular organisms, such as the foraminifera. The intracellular bacterial OTUs of all foraminiferal species were enriched in sulphur-oxidizing and sulphate-reducing bacteria compared with the surrounding sediment bacterial community, where the relative abundancies of these bacteria were lower. The intracellular bacterial 16S OTUs of foraminifera were found to be species-specific, and the phylogenetic analysis of the sulphur-cycle related aprA OTUs showed that some of these intracellular bacteria were closely related to known endobionts of other organisms. Therefore, the results suggest that intertidal benthic foraminifera may have a previously overlooked role in the benthic sulphur cycle. Further research is needed to understand the exact role of the sulphur-cycle associated bacteria in foraminiferal ecology. For example, looking into the environmental conditions under which transcription of the sulphur-cycle genes takes place would enable assessment of their role and the potential foraminifera/endobiont contribution to the benthic sulphur cycle.
  • Junikka, Leo (Helsingin yliopisto, 2020)
    This dissertation deals with bark morphology and anatomy of the Neotropical members of the family Annonaceae, and formal taxonomy of the genus Oxandra. This mainly pantropical family comprises ca. 2440 species in 109 genera, which belong to the early divergent mangoliids. About one-third of these taxa occur in the Neotropics (few also in N-America), where they contribute a significant part of plant diversity, both in abundance of species and individuals. Members of the family are mainly trees, shrubs and lianas, which are growing predominantly in tropical and subtropical lowland rainforests. The family is monophyletic and can be recognised, in addition to its flowers and fruits, also by its wood and bark, which possess homogenous, distinctive features representative to the family. The first study deals with the macroscopical patterns seen in the outer structures and in the slash (i.e. tangential cut) of the bark and with the terminology used in the bark descriptions. Morphological terminology of barks is critically surveyed in the forest floras and the expert papers and terms are listed for a comparison of bark features. Suggestions are given for a standardised usage of the terms to stimulate a practice of pertinent field notes and to facilitate understanding of the descriptions. The second study concentrates on the structural anatomy of barks in 32 Neotropical genera of Annonaceae. Selected character states at the family and genus levels are scrutinised for identification purposes. Taxonomical and phylogenetic relevances of bark characters are discussed in view of the tentative molecular phylograms. Although the value of the bark characters, in the present understanding, proved to be negligible, some features may help to delineate some genera and even tribes as such. The systematically most distinctive features in bark structure are the sclerification of the phellem cells, the shape of fibre groups and the occurrence of crystals in bark components. A taxonomic revision based on morphological characters is presented for the Neotropical genus Oxandra (Annonaceae). Twenty-seven species are recognised in the genus, which has wide distribution in Central and South America. Two species occur in the Antilles, but the majority are growing in the lowland rainforests of the Amazonia and in the Atlantic rainforest. A few species are flourishing in drier habitats and in nutrient-poor soils. Altogether five species, i.e. O. aberrans, O. bolivarensis, O. rheophytica, O. saxicola and O. unibracteata, are described as new to the science. A special attempt is made to find supportive morphological characters in view of the possible division of the genus, because of the polyphyly uncovered by the earlier phylogenetic analyses (verified by plastid data) in the tribe Malmeeae, in which the genus belongs. Only few morphological characters, i.e. the midrib type of the leaves, the length of stipe of the apocarpous fruits and, less so, geographical distribution pattern of the species, corroborate the evidence of segregation of the genus in two clades found in various phylogenetic studies. However, last preliminary results of nuclear and mitochondrial sequence data show the genus to be monophyletic. More species and more resolutions are needed from plant genomic research to make definitive statements about the status of the genus.
  • Ravikumar, Balaguru (Helsingin yliopisto, 2020)
    Rational approaches to the traditional drug discovery process rely on high-throughput and lowthroughput bioactivity or phenotypic screening studies. Such profiling strategies have been the foremost step utilized when identifying or curating lead molecules, and more recently, when evaluating compound repositioning/repurposing opportunities. Although promising, such systematic approaches are inherently limited by their cost and labor constraints, and the necessity of specific screening equipment’s renders them outside the scope of many academic laboratories. Further, elucidation of any novel associations through such experimental techniques require exhaustive searches of compound libraries. Hence, many positive drug indications revealed, though rational, are often by the virtue of serendipity. The accumulation and standardization of existing compound profiling datasets has paved the way to the field of chemoinformatics. Wherein, data-driven computational approaches are employed as a pragmatic solution to challenge the inherent notion of serendipity in screening studies. Such in silico models serve as an efficient and cost-effective augmentation to the experimental screening approaches, circumventing the intrinsic limitations of current drug discovery methods. This study therefore was motivated towards identifying niches and limitations prevalent in the current pharmacological paradigm, where an effective computational framework could be utilized to complement and expedite the conventional drug discovery process. The various computational approaches proposed in this article-based thesis include exploratory web-tools, new data resources, and prediction models, that are introduced as supplements to extend the current computer-aided drug discovery process (CADD). Firstly, I developed a web-application, termed C-SPADE, a novel compound-centric chemoinformatic tool that facilitates interactive analysis and visualization of compound screening experiments using Compound-SPecific bioActivity DEndrograms. The tool employs compound-compound similarity metrics to estimate the diversity amidst compounds profiled in the screening panel and intuitively represents the chemical similarity space and the observed bioactivity values. The web-tool provides users an exploratory framework to perform pharmacological analysis and investigate novel compound associations employing diverse compound similarity clusters. Secondly, to address the heterogeneous and non-standardized bioactivity data in existing data resources, a comprehensive open-data platform, called Drug Target Commons (DTC), was developed. DTC feature tools for data annotation, standardization, curation to address intra-resource heterogeneity and provide users a one-stop resource for drug discovery and in silico model development endeavours. User-specific applications of both the above-mentioned resources have been demonstrated through several case studies and experimental validations. In addition to the tools and databases, I have also designed and implemented diverse machine learning models primarily to predict potent compound-kinase interactions and to fill the current experimental gaps in large-scale activity profiling studies. Firstly, through collaborative efforts, we employed the Kronecker kernel-based regularized least square regression (KronRLS) algorithm under different crossvalidation settings to predict both the uncharacterised binding measures in large-scale profiling studies and novel compound-target associations. As a case study, the off-target profile of an investigational VEGF receptor inhibitor tivozanib was predicted and experimentally validated. Secondly, I designed and implemented an efficient statistical model utilizing an ensemble SVM classifier to prioritize potent compound-kinase association for biochemical testing. The developed computational framework was termed Virtual Kinome Profiler (VKP) and was efficiently used in compound repositioning and lead identification studies, wherein 19 novel kinase-compound interactions spanning across different kinases were predicted and experimentally validated. Apart from elucidating the chemogenomic similarities prevalent among distinct kinase proteins, VKP with a positive prediction value (PPV) of 84% was shown to reduce the time and cost constraints related to traditional experimental screening process. Most of the computational frameworks proposed in this thesis are designed and deployed with an accompanying web-based graphic user interface (GUI). This in turn aids in the translatability of the platforms, overcoming the current prerequisites required when utilizing CADD models, including prior expertise in data analysis and scripting languages. These studies together exemplify new applications of computational models in diverse areas of the drug discovery process, subsequently making invaluable augmentations to a chemical biologist’s toolbox.
  • Abouelezz, Amr (Helsingin yliopisto, 2020)
    The axon initial segment (AIS) is the site of action potential initiation and plays an important role in maintaining neuronal polarity. Recent advances in super-resolution microscopy revealed the presence of an intricate membrane-associated periodic lattice in the AIS that contains sub-membranous actin rings periodically spaced ~190 nm and connected to a spectrin-ankyrin lattice. The precise function of these actin rings in unclear, as well as details of their structure and dynamics. The insensitivity of the AIS to actin-disrupting drugs led to the long-held view that actin is not a critical component of AIS structure. Here I show that the AIS contains a population of relatively stable, latrunculin-resistant actin filaments that are decorated by the tropomyosin isoform Tpm3.1. Disrupting these filaments through the perturbation of Tpm3.1 function led to the loss of accumulation of ankyrin G and other AIS markers, disruption of neuronal polarity, the loss of the clustering of voltage-gated sodium channels, and a rapid reduction in firing frequency. The findings I present in this thesis suggest that actin plays an important role in maintaining AIS structure and function, more important than previously appreciated.
  • Danilova, Tatiana (Helsingin yliopisto, 2020)
    Neurotrophic factors are small secretory proteins with essential roles in neuronal and non-neuronal tissues. Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) form a distinct family of unconventional neurotrophic factors. MANF and CDNF are endoplasmic reticulum (ER) located, but also secreted proteins. Initially, MANF was discovered as a trophic factor for dopamine neurons in vitro. Further studies revealed its protective and restorative properties in different animal disease models such as Parkinson´s disease, spinocerebellar ataxia, brain- and heart-ischemia. MANF is also identified as a protein upregulated in unfolded protein response (UPR) and protecting against ER stress-induced cell death. CDNF was identified based on its homology to MANF and characterized for its ability to protect and restore dopamine neurons in rodent models of Parkinson´s disease. However, the physiological roles of MANF and CDNF in mammals have remained unclear. The main objective of this thesis was, therefore, to study the biological roles of MANF in vivo by characterizing the phenotypes of MANF conventional and conditional knockout mice as well as analyzing MANF and CDNF expression in mouse tissues. Comprehensive expression analysis of MANF mRNA and protein revealed that MANF is widely expressed in most mouse tissues. It is highly expressed in neurons regulating energy homeostasis within the hypothalamus and neurons of other appetite-regulating areas including the brainstem structures and mesolimbic/mesocortical dopamine system. Exceptionally high levels of MANF was observed on peripheral mouse tissues with metabolic function, especially in cells with secretory functions within the endocrine and exocrine glands, suggesting essential roles for MANF in cells with high protein synthesis and secretion. Highest levels of CDNF protein was observed in tissues with high energy production and oxidative function including skeletal muscle, heart, testis and brown adipose tissue. In order to study the roles of MANF in mammals, we deloped MANF conventional knockout mice (Manf-/-), that showed severe growth retardation, poor survival and a progressive postnatal reduction of beta-cell mass resulting in severe insulin-deficient hyperglycemia and diabetes mellitus caused by decreased beta-cell proliferation and increased beta-cell apoptosis. In our further studies, we verified that diabetic phenotype of the Manf-/- mice was caused by a lack of MANF in the insulin-producing beta-cells in the pancreas and not in other organs by generating pancreas- and beta-cell-specific conditional Manf-/- mice. We found that pancreatic islets of conventional and conditional MANF deficient mice displayed chronic activation of the UPR, preceding downregulation of beta-cell markers, indicating unresolved ER stress as one possible cause of beta-cell failure in these mice. Thus, this work shows that MANF is an essential regulator of beta-cell maintenance and UPR in mice. We discovered that MANF protein increased beta-cell proliferation in vitro in islets isolated from young and even old mice. MANF protein also rescued mouse beta-cells from thapsigargin-induced apoptosis and ER stress-induced glucotoxicity in culture. Importantly, we found that MANF overexpression in the mouse pancreases mediated by adeno-associated virus vector was able to regenerate beta-cells in vivo in a mild low-dose streptozotocin mouse model of diabetes. Hence, these results indicate that MANF is a vital mitogen and protective protein for mouse beta-cells and can thus serve as a potential new regenerative drug for the diabetes therapy. Furthermore, we identified the decreased number of growth hormone (GH) and prolactin (PRL) expressing cells in the anterior pituitary gland of Manf-/- knockout mice associated with increased expression of UPR markers and decreased expression of Gh and Prl genes. Thus, reduced GH production could be one of the reasons for the growth retardation in Manf-/- mice. Taken together, the results in this thesis provide new critical biological functions of MANF in mouse in vivo which can be used to exploit the roles of MANF in human beta-cells and diabetes as well as in endocrine somatotropic cells and growth failure in human.
  • 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 beta1-integrin, and therefore the function of endothelial beta1-integrin in vascular permeability was investigated. The results identified a novel signaling pathway, where ANGPT2–beta1-integrin signaling promotes EC permeability. Beta1-integrin was found to play a previously uncharacterized role in inflammation-induced vascular permeability, and an antibody against beta1-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 beta1-integrin in controlling inflammation-induced EC permeability. The results showed that various inflammatory agents induced EC monolayer destabilization via beta1-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, beta1-integrin promoted inflammation-induced EC contractility and reduced the EC barrier function. Importantly, targeting beta1-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 beta1-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 beta1-integrin. Beta1-integrin was identified as a potentially universal regulator of EC permeability. A major finding was that targeting the EC beta1-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 beta1-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.

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