Faculty of Biological and Environmental Sciences

 

Recent Submissions

  • Järvenpää, Marja (Helsingin yliopisto, 2020)
    Environmental changes of anthropogenic origin cause serious challenges to animal populations. Animals often first respond to changes in the environment by altering their behaviour. This can have marked consequences for both individuals and populations, in particular when behavioural responses are related to survival or reproduction. In aquatic environments, a major anthropogenic change is eutrophication, with more than 400 eutrophicated coastal systems having been identified globally. Eutrophication, due to enhanced growth of planktonic algae, leads to increased turbidity, decreased visibility and a narrowing of the light spectrum in the water column, as well as to increased siltation of decomposing algae and alterations in oxygen levels. All these changes to the environment hold the potential to interfere with the behaviour of aquatic organisms in many ways. In this thesis, I experimentally studied the effects of increased abundance of planktonic algae and consequent turbidity on fish reproductive behaviour in the severely eutrophicated Baltic Sea. As a model system, I used the sand goby (Pomatoschistus minutus), a small marine fish species with a resource-defence mating system and male parental care. I conducted a series of laboratory studies in which I manipulated water turbidity using cultivated planktonic algae and explored the effects of it on various aspects of sand goby reproduction: mating system and sexual selection (Chapter I), parental care and offspring survival (Chapter II and III), male mating success (Chapter III) and reproductive decisions under predation risk (Chapter IV). In Chapter I, I focused on the effects of algal turbidity on the mating system and opportunity for sexual selection in the sand goby. Due to intense male-male competition and female choice, large sand goby males can typically monopolize multiple matings, whereas some males do not get a chance to mate at all. To study how these aspects of sand goby reproduction are influenced by algal turbidity, I allowed four males of different size to establish nests and sequentially introduced them four ripe females either in turbid or clear water. I found that in turbid conditions matings were more evenly distributed among males, opportunity for sexual selection was lower and mating success was less skewed towards large males than in clear water. These effects of turbidity may, for example, be due to a change in the preferences of the females or their ability to express these, or because of changes in male-male competition or male mate attraction effort. In Chapters II and III, I explored whether algal turbidity affects male parental care behaviour and egg survival. I allowed sand goby males to care for a single female egg clutch (Chapter II) or a large egg clutch spawned by multiple females (Chapter III) and monitored male parental behaviour and egg survival under turbid and clear water conditions. Males caring for a single female egg clutch fanned their eggs less and spent more time away from their nest in turbid than clear water in the beginning of the care period. This difference in parental behaviour disappeared later in the brood cycle. Despite decreased fanning early in the parental phase, egg survival was higher in turbid conditions. The time spent fanning early in the brood cycle did not affect egg survival in either clear or turbid water, suggesting that fanning early in the parental phase may function more as courtship than care. Decreased fanning in turbid water could be due to decreased efficacy of visual sexual signalling under low visibility. Sand goby females prefer males showing high fanning effort. Under low visibility, this advertisement component of care is likely to be less effective and may not be worth investing in. Instead, searching for females might be more beneficial for mating success, which is in accordance with my observation that males spent more time away from their nest when water was turbid rather than clear. Lesser fanning and higher egg survival under algal turbidity could also be explained by the conditions in turbid water being better for the developing eggs. Interestingly, when males were caring for a large egg clutch spawned by multiple females (Chapter III), neither parental care nor egg survival was affected by water turbidity. It may be that a large brood required a very high parental effort from the male in both clear and turbid water. Additionally, the high perceived female density may have decreased the need for mate search in turbid conditions. Lastly, males may perceive the value of a large egg mass they already have so highly that they rather invest in care than searching for additional mates. In Chapter III, I also examined whether turbidity affects male maximal mating success. I introduced five ripe females of random size to one nest-holding male in either clear or turbid water and observed how many mates and eggs the male received. When the fish were spawning in clear water, the number of eggs in the male’s nest increased with the total weight of the five females in his aquarium. When spawning took place in turbid water, however, there was no such relationship between the total weight of the females and the number of eggs laid, even though the number of females that spawned was the same as in clear water. It seems that this effect was due to a female decision to adapt spawning to water turbidity rather than a male decision to limit the number of eggs in his nest. In Chapter IV, I investigated the effects of algal turbidity and predation risk on one particular reproductive decision: the latency of gobies to spawn. I introduced one ripe female to a male either in the presence or absence of a predator in water that was either clear or turbid and recorded the latency to spawning. I found that gobies were more reluctant to spawn in the presence than in the absence of the predator. Interestingly, latency to spawning was unaffected by turbidity, suggesting that the gobies experienced the predation threat equally high in turbid as in clear water, for instance because they relied on non-visual (e.g. chemical) predator cues at least under turbid conditions. Taken together, the results of this thesis show that eutrophication can influence important aspects of fish reproductive behaviour: mating success, sexual selection and parental care were all affected by algal turbidity. Most importantly, I show that sexual selection is relaxed in turbid water. By hampering sexual selection and parental care, eutrophication holds a potential to influence, not only the fitness of individuals, but also population viability and community structure. My results emphasize that human-induced environmental changes can have severe consequences already through subtle alterations in animal behaviour.
  • Alonso-Serra, Juan (Helsingin yliopisto, 2020)
    Plant development takes place through continuous changes in the size and shape of organs. Along the organs’ morphogenic gradients, cells derived from the undifferentiated meristematic stem cells follow different regulatory pathways leading to a variety of developmental trajectories and cellular functions. In the developmental process called secondary growth, molecular factors and physical forces interact in the radial growth of stems and roots to produce their cylindrical shape. Cambium, the largest connected meristem in plants, is responsible for secondary growth. It produces vascular tissues with two essential functions: the transport of water, nutrients and photoassimilates, and the physical support of the plant. Recent years have seen an increasing number of studies focused on the regulation of cambial activity, primarily because this meristem produces a great part of the Earth’s woody biomass, thereby fixing a large quantity of carbon. The aim of this thesis is to explore aspects of radial growth which have thus far remained largely uncharacterized: the contribution of bark tissues, the role of mechanical forces, and the genetic robustness of cambial development.
  • Mishra, Pashupati (Helsingin yliopisto, 2020)
    Background: A wide range of diseases, normal variations in physiology and development of different species are caused by alterations in gene regulation. The study of gene expression is thus crucial for understanding both normal physiology and disease mechanisms. High-throughput mea- surement technologies allow the profiling of tens of thousands of genes simultaneously. However, the high volume of data thus generated poses methodological challenges in inferring biological consequences from gene expression changes. Traditional gene wise analysis of high dimensional data is overwhelming, prone to noise and unintuitive. The analysis of sets of genes (gene set analysis, GSA), solves the problem by boosting statistical power and biological interpretability. Despite more than a decade of research on gene set analysis, there are still serious limitations in the existing methods. Aims of the study: The objectives of this study were: (1) development of an efficient p-value estimation method for GSA; (2) development of an advanced permutation method for GSA of multi-group gene expression data with fewer replicates; and (3) implementation of the developed methods for the identification of novel smoking induced epigenetic signatures at biological pathway level. Materials and methods: The first study involved the assessment of four different statistical null models for modeling the distribution of gene set scores calculated with the Gene Set Z-score (GSZ) function from permuted gene expression data. A new GSA method - modified GSZ (mGSZ) - based on GSZ and the most optimal distribution model was developed. mGSZ was evaluated by comparing its results with seven other popular GSA methods using four different publicly available gene expression datasets. The second study involved the evaluation of six different permutation schemes for GSA of multi-group (more than two groups) datasets based on the identification of reference gene sets generated using a novel data splitting approach. A new GSA method based on a modification of mGSZ (mGSZm) was developed by implementing the best permutation method for the analysis of multi-group data with fewer than six replicates per group. mGSZm was evaluated by contrasting its performance with seven other state-of-the-art GSA methods suitable for multi-group data. The evaluation was based on three different publicly available multi-group datasets. The third study involved an implementation of mGSZ for GSA of genome-wide DNA methylation data from the Cardiovascular Risk in Young Finns study (YFS) cohort with gene sets downloaded from the Molecular Signature Database (MSigDB). Methylation measurements were done on a subset of 192 individuals from whole-blood samples from the 2011 follow-up study using Illumina Infinium HumanMethylation450 BeadChips. Results: Overall, efficient and robust GSA methods were developed (studies I-II) and implemented (study III). In study I, the results demonstrated a clear advantage of asymptotic p-value estimation over empirical methods. mGSZ, a GSA method based on asymptotic p-values, requires fewer permutations which speeds up the analysis process. mGSZ outperformed state-of-the-art methods based on three different evaluations with three different datasets. In study II, results from a novel evaluation approach with two different datasets suggested that the proposed advanced permutation method outperformed the naive permutation method in GSA of multi-group data with fewer than six replicates. Evaluation of mGSZm, a GSA method equipped with the advanced permutation method and asymptotic
  • Lipsewers, Tobias (Helsingin yliopisto, 2020)
    The Baltic Sea is a substantial brackish water system, and due to its shallow average depth, physicochemical dynamics affecting, for instance, phytoplankton growth, are controlled by atmospheric forces. This unique ecosystem is threatened by environmental changes, with implications for the water quality. Eutrophication remains the major challenge for the Baltic Sea ecosystem. The increases in nitrogen (N) and phosphorus (P) are not proportional to the carbon (C) input, which might cause imbalances in seston C:N:P stoichiometry and affect biogeochemical cycles. In turn, plankton growth, food web dynamics, and nutrient- / C-cycling, which plays a pivotal role in climate change, will be affected. The ongoing eutrophication supports the formation of high phytoplankton biomass, enhancing the spreading of anoxic bottom waters, affecting benthic communities, which might be worsened by an extended annual growing season caused by increasing sea surface temperatures (SST). Higher SST and effects of eutrophication are known to support harmful algal blooms such as the occurrence of toxic dinoflagellates and cyanobacteria in the Baltic Sea during summer. The spring bloom is the most productive period of the year (~50 % annual C-fixation), driven by high inorganic nutrient concentrations and improving light conditions. The co-existence of cold-water diatoms and dinoflagellates during this period is characteristic of the Baltic Sea. Heterotrophic bacteria will become more abundant due to increased concentrations of allochthonous organic matter, which could reduce the phototrophic contribution already in spring. The health of arctic and sub-arctic ecosystems is highly dependent on environmental factors such as temperature affecting, for example, sea ice conditions. Climatically driven changes have already caused variations in the plankton community structure during the Baltic Sea spring bloom. These alterations have led to lower diatom-dinoflagellate-proportions (DDP), affecting food web dynamics and biogeochemical cycles. Therefore, shifts in the community composition may have far-reaching ecosystem consequences. The dominant phytoplankton group defines the quantity and quality of bioavailable (labile) dissolved organic carbon (DOC), providing different niches for heterotrophic bacteria. Furthermore, environmental factors affect the abundances and community compositions of both phototrophic and heterotrophic plankton. Microzooplankton is particularly crucial in linking the microbial loop with higher trophic levels, and in the recycling of inorganic nutrients. For example, heterotrophic ciliates become more abundant at elevated temperatures and can significantly reduce the biomass of bacteria and phytoplankton. The Baltic Sea spring bloom is dominated by phytoplankton, while the grazer community is not well developed yet. Thus, considering the ongoing climatic changes, it can be expected that the share of microzooplankton and, in turn, the partially unaccounted biomass will be higher at elevated temperatures (e.g., in summer). Temporal and spatial shifts of zooplankton populations, affecting higher trophic levels, could be the consequence. The main objectives of this study: • The identification of environmental drivers shaping the nano- and microplankton community composition. • To study the effects of changing communities and bloom phases on seston nutrient stoichiometry. • The investigation of bacterioplankton responses to changing dominance patterns of diatoms and dinoflagellates. • To reveal the proportion of microzooplankton during the spring bloom. For this, data from research cruises (chapters 1 and 3) in the Baltic Sea and experiments carried out at Tvärminne Zoological Station (TVZ, University of Helsinki) on the south-west coast of Finland (chapter 2) were used. Surface water samples (z = 3 m) for the analysis of nutrients (inorganic and organic), biomass, community composition, and primary production amongst others were collected from seven sub-basins and different spring bloom phases (April / May) during four cruises on-board R/V Aranda (2013 to 2016). The Gulf of Finland (GOF) and the Baltic Proper were the main study areas. The two experiments (2012 and 2013) at TVZ (also GOF) took place in February / March. The water was collected from 20 m under the sea ice and featured low biomass and high inorganic nutrient concentrations (winter conditions). In addition to nutrients and phototrophic variables, samples were taken to determine bacterial abundances, production, and community composition (BCC). The main difference between these two approaches was the fact that the cruise data comprise natural mixed communities, and samples from different bloom phases represent an extract of the spring bloom. In contrast, dominance patterns were artificially shifted by adding cultured strains to the natural pre-bloom community for the experiments. Furthermore, the bacterial growth phase was induced by increasing the temperature, and the course of the bloom was followed in 20 L mesocosms. The determination of nutrient and chlorophyll a (Chl a) concentrations as well as the microscopy counting strategy (field data) were based on in-house protocols of the Finnish Environment Institute (SYKE), certified by the Finnish Accreditation Service (FINAS). Some of the main methods were explicitly chosen for the study area (Baltic Sea), the sampling season (spring bloom), and the database (Hertta, SYKE) that was used (plankton community composition, upper mixed layer depth, definition of bloom phases, bacterial production, and gross primary production). Various multivariate and other statistical analyses were applied to study different aspects of the presented studies. In addition to the different chapters, three cold-water dinoflagellates, usually not identified to species-level, were distinguished, and their biomass-contributions investigated (cruise data, unpublished). The corresponding species (Gymnodinium corollarium, Biecheleria baltica, and Apocalathium malmogiense) can co-occur during the Baltic Sea spring bloom but are not separable by traditional microscopy of Lugol-preserved samples due to their similar appearance. Thus, this group is referred to as DinoComplex throughout this summary. Two different methods for the identification and quantification of the three species were applied: inverted epifluorescence microscopy of stained (fluorescence brightener) cells and quantitative polymerase chain reaction (qPCR), using species-specific primers and fluorescent (Taqman®) probes. Environmental factors, mostly temperature and the concentration of inorganic nutrients, defined the nano- and microplankton community composition (phototrophs and heterotrophs) and affected the collective physiology as well as the biomass. Combined, Mesodinium rubrum, Peridiniella catenata, the DinoComplex, and heterotrophic ciliates dominated the biomass in the cruise data (n = 119). The experiments have shown that lower DDP´s reduce the bacterial activity (protein- and DNA-synthesis) and alter the BCC due to decreasing availability of diatom-derived dissolved organic matter (DIADOM). Additionally, a predominant diatom-species has shown specific effects on bacterial responses, i.e., Chaetoceros wighamii dominance yielded the highest bacterial activity. In the field data, the community composition explained 19 % (average) of shifts in seston C:N:P ratios, which were significantly different between some of the bloom phases. The presented findings highlight the importance of species-level identification (e.g., DinoComplex) and the introduction of a microzooplankton monitoring program. The fact that the most relevant diatom (Thalassiosira baltica) contributed <10 % to the biomass (relative contribution, n = 119), combined with high dinoflagellate-proportions throughout the bloom, agrees with previous findings on the increase of dinoflagellates over diatoms in several sub-basins. Not only variations in the community composition, but also different growth phases (physiology) of the bloom affected the interactions between phyto- and bacterioplankton. For instance, high proportions of T. baltica (equivalent to ~6 µg Chl a L-1) were associated with low primary production (field data, presented study) as well as low bacterial activity (field data, not shown). At the bloom peak, the cells are less active, but not decaying and excreting larger amounts of labile DOM, yet. Thus, a decreasing availability of DIADOM will cause a decrease in the energy efficiency of the microbial food web. Comparing the C:N:P ratios from the field study and the experiments revealed that the absolute dominance of either diatoms or dinoflagellates lead to more clear connections to the intracellular element composition. However, there was no significant relationship between the DDP and seston C:N:P ratios, and thus, it was concluded that the overall community activity supersedes the effect of different dominance-patterns on seston stoichiometry in mixed communities. Species-specific effects on bacterial responses and seston stoichiometry, with implications for biogeochemical cycles, are challenging to study in diverse communities. Seston ratios that were clearly affected by the community composition, namely Chl a:C (and its fixed range), C:Si, and N:Si, could be predicted with relatively high certainty for the spring bloom. The Chl a:C ratio can be used as a proxy for phototrophic and heterotrophic production and to study C-budgets. For example, C:Si indicates the diatom-proportion of the seston, which could be interesting for modeling studies. Due to a high inter-annual variability in seston ratios, season-specific findings should be combined to estimate future developments of, for example, the annual primary production and C-fluxes.
  • Gao, Yajing (Helsingin yliopisto, 2020)
    The tumor suppressor kinase, LKB1 (encoded by STK11), plays important functions in regulating diverse cell processes, including cell growth, metabolism, and polarity. As a bioenergetic sensor, LKB1 is required for metabolic balancing and maintenance of stem cell homeostasis in the haematopoietic system (Gurumurthy et al. 2010; Gan et al. 2010; Nakada, Saunders, and Morrison 2010) and in muscle (Shan et al. 2014). Intestinal stem cells (ISCs) are regulated by various cues from their niche-derived paracrine signals such as NOTCH and WNT (Clevers and Batlle 2013), and metabolic status (Rodríguez-Colman et al. 2017; Schell et al. 2017). Study I of this thesis aimed to investigate whether the metabolic regulator, LKB1, has a role in actively cycling ISCs, and identified it as a critical factor for maintaining ISC homeostasis. Mechanistically, LKB1 represses the transcription of the secretory lineage gatekeeper, Atoh1, via pyruvate dehydrogenase kinase 4 (PDK4) in ISCs and restricts ISC differentiation towards secretory cell lineages. These findings define LKB1 as an essential regulator of ISCs, and provide a connection between metabolism and fate determination of ISCs. Germline mutations inactivating LKB1 lead to gastrointestinal tumorigenesis in Peutz-Jeghers Syndrome (PJS) patients (Ylikorkala et al. 1999) and mouse models (Rossi et al. 2002; Bardeesy et al. 2002). However, little is known about the cell types and signaling pathways that underlie tumor formation, not much has been learnt about the progression of PJS polyposis neither. The upregulation of Cyclooxygenase-2 (COX-2) is a feature of PJS polyposis (Rossi et al. 2002; H. Takeda et al. 2004), and COX-2 inhibition reduces polyp growth in Lkb1+/− mice modelling PJS polyposis (Udd et al. 2004). Study II of this thesis evaluated the effect of the mutagenic carcinogen, N-methylnitrosourea (MNU), on gastrointestinal tumorigenesis in Lkb1+/− mice and concluded that MNU aggravates Peutz-Jeghers polyposis independently of COX-2. Study III of this thesis demonstrated that the loss of Lkb1 in mesenchymal progenitor or stromal fibroblasts leads to the clonal expansion of stromal cells and to the induction of an inflammatory program involving the IL-11–JAK/STAT3 pathway, which is critical for tumorigenesis. The findings from Studies II and III provide further understanding of the function of LKB1 in Peutz-Jeghers tumorigenesis, and suggest potential therapeutic avenues for related tumor diseases.
  • Angove, Charlotte (Helsingin yliopisto, 2020)
    Aquatic plant meadows provide a variety of global ecosystem services. Their populations are declining globally. To conserve and restore aquatic plant meadows and the services which they provide, it is necessary to understand their ecology. A key approach which allows us to explore plant ecology is to investigate the relationships between plant functional traits and ecosystem processes. By investigating plant functional traits, it is possible to develop insights about functional diversity and plant growth strategies. In this thesis, plant functional traits, functional diversity and plant growth strategies are used to investigate aquatic plant biomass production responses to the environment. A series of manipulative experiments were conducted in situ in submerged aquatic plant meadows of the northern Baltic Sea using SCUBA. Firstly, the role of plant traits, species identity and sediment porewater NH₄⁺ availability for plant nitrogen uptake rates were investigated using a short-term (3.5 h) nitrogen enrichment experiment (Chapter I). Secondly, a 15-week transplant experiment was conducted to explore plant functional trait and functional diversity relationships to productivity (Chapter II). Finally, a similar experiment with additions of the bivalve Limecola balthica (12 weeks) was conducted to investigate infauna effects to plant functional trait–productivity relationships (Chapter III). Chapter I showed that short-term nitrogen uptake rates from the sediment were driven by plant-biomass related demands. Similarly, results suggested that plants likely drained ammonium availability from their adjacent sediment porewater. Overall, Chapter I parameterised the possible unfulfilled potential for larger temperate aquatic plants to cycle nutrients. Chapter II results showed strong relationships between productivity and traits which enhanced light capture (height and leaf area). Leaf tissue δ¹³C and functional richness were also related to community productivity. The relationship between height and productivity was likely exacerbated by a competitive height interaction between the tallest and second tallest species. Overall, functional richness was related to community biomass production, likely by selecting for traits which enhanced light capture (selection effect) with potential consequences to carbon supply. Findings support inferences from previous studies of aquatic plant communities which showed that height is strongly related to aquatic plant productivity and trait identity may be more descriptive for primary production compared to functional diversity indices. Chapter III results showed Specific Root Length (SRL) exhibited the strongest relationship to productivity. Leaf area was also related to community production and Median Maximum Root Length (MMRL) exhibited a marginally non-significant relationship to productivity. SRL exhibited collinearity to species identity, therefore it was not possible to interpret SRL effects separately to other traits which may coincide with species identity. Community SRL was related to community shoot frequency, not aboveground biomass production. SRL and shoot proliferation both represent strategies to enhance nutrient absorption from the sediment. Relationships between plant leaf tissue nutrient concentrations (N (% DW), δ¹⁵N, δ¹³C) and L. balthica condition index suggested that L. balthica affected the sediment nutrient supply and enriched the plants with nutrients. Overall, Chapter III showed that infauna, common in aquatic plant meadows, can change aquatic plant trait-productivity relationships and thus arguably the drivers for submerged aquatic plant community growth. Findings of this thesis can be applied to a variety of other temperate submerged aquatic plant communities. Targeted research questions could contribute to further understanding of submerged aquatic plant ecosystem ecology, including the ecology of monocultures. This thesis summary suggests updating the current description of context-dependent seagrass biomass responses to sediment nutrient enrichment. It proposes a model which, once tested, would help to improve predictive modelling for submerged aquatic plant biomass responses to future change. Also, results of this thesis contribute towards increasing effectiveness of future management by providing insights to infauna effects on plant functioning. This is beneficial to current restoration development because infauna additions to submerged aquatic plant meadows are an option for increasing seagrass restoration success and seagrass resilience to future change. This thesis identifies that there is requirement for further research in seagrass meadows which form dense root-rhizome mattes, and it describes potential options for future research. It also recommends isotope-tracing experiments and compound-specific isotope tracing experiments to better understand mechanisms of nutrient exchange between infauna and temperate submerged aquatic plants. It has empirically shown current limitations of global plant trait syntheses and it identifies constructive steps forward to improve the global perspective of plant trait ecology. Finally, this thesis advocates the value of insights gained from data-rich functional diversity experiments and plant functional trait experiments. To conclude, this thesis has improved the collective understanding of temperate aquatic plant ecosystem functioning.
  • Pärnänen, Pirjo (Helsingin yliopisto, 2020)
    Our studies focused on using Candida glabrata (C. glabrata) as a model organism to isolate and investigate the role of C. glabrata cell wall proteases as host protein- degrading virulence factors and the inhibition of their action. The cell wall proteins of microbes are in the frontline of first contact with the host cells in oral mucosa. C. glabrata is the second most prominent Candida yeast, and it is commonly found in the normal oral microbial flora causing opportunistic yeast infections, particularly in hospitalized patients. It is considered innately azole- resistant and treatment is more difficult compared to the typical Candida albicans (C. albicans) infections. Azoles are the most commonly used antifungal agents used in candidosis. There is an urgent need of development of topical antimicrobial agents, and wild berries such as lingonberry, have been increasingly studied. Lingonberries are known to have antioxidant, anti-inflammatory, antimicrobial and anticancerous properties and are considered beneficial to health. To this background we studied in vitro and in vivo the effects of a patented, fermented lingonberry juice (Lingora®, from now on abbreviated as FLJ). It was specially developed to be used as a mouthwash on C. glabrata and other typical microbes of the oral flora related to yeast infections and caries. Our primary goal was to isolate, identify and characterize C. glabrata cell wall proteases with biochemical methods: enzymatic treatment of C. glabrata cells, MDPF-zymography, SDS-PAGE, 2D-PAGE and LC-MS/MS. These methods may be used to isolate and identify novel Candida cell wall proteases enabling their further characterization and inhibition studies. Further in vitro studies were conducted on the effect of FLJ on intracellular protein expression of C. glabrata with the 2D-DIGE method. The proteins were identified by LC-MS/MS. The inhibition of proliferation and invasion of two aggressive oral tongue squamous cell carcinoma (OTSCC) lines (HSC-3, SCC-25) with FLJ and curcumin were measured in vitro by colorimetric ELISA and three- dimensional Myogel spheroid assay. Finally, we conducted a clinical pilot study including oral examinations, microbial cultivations and measurements of active MMP-8 concentrations using PerioSafe® point-of-care test. FLJ was used as a mouthwash to see if it has also in vivo effects on three microbes of the oral microbiota. From the C. glabrata cell wall we identified a novel, uncharacterized 25 kDa serine protease, Cwp1.2., with an estimated pI of 7.6 and gelatinolytic activity. This activity was inhibited by PMSF, a known serine protease inhibitor. Certain C. glabrata intracellular protein expressions related to glycolysis, oxidative phosphorylation, oxidative stress and biofilm formation were significantly diminished after treatment with FLJ. These proteins include e.g. heat shock protein and redoxin, which are expressed by C. glabrata when predisposed to stress. Downregulation of these proteins causes C. glabrata cells to be more vulnerable to environmental stress and may cause lower virulence. FLJ showed to inhibit proliferation and invasion of two aggressive OTSCC cell lines similar to curcumin. FLJ is safe, has no known interactions with medications and could be studied to be used as an adjunctive therapy in management of OTSCC. The clinical mouthwash pilot study with FLJ results showed statistically significant reduction in Candida and S. mutans counts. Our in vitro studies also indicate growth inhibitory effect on the most common periodontitis- related bacteria. Bleeding on probing (BOP), visible plaque index (VPI) and trend of active matrix metalloprotease-8 (aMMP-8) values were also reduced during the FLJ mouthwash period. Lactobacilli counts increased during the mouthwash period. Although lactobacilli are thought to be related to caries the clinical parameters and clinical outcome indicate a balancing effect on the oral microbial flora from a dysbiotic to a symbiotic direction. This diminished microbial related inflammatory burden should be studied further in context with broader positive general health effects. The results show several beneficial aspects of FLJ in the oral environment. The methodology used in these studies might be applicable to other oral microbes in developing novel antimicrobial agents related to cell wall proteases of Candida. Combined in vitro and in vivo studies showed effects of FLJ on C. glabrata intracellular proteins, host cell derived proteins including anti-inflammatory effects, tongue carcinoma cells and oral microbiota.
  • Pieristè, Marta (2020)
    This dissertation focuses on the effect of sunlight on leaf litter decomposition. Sunlight can affect litter decomposition positively or negatively through the process known as photodegradation. Photodegradation is the ensemble of direct, indirect and mediated mechanisms. Short-wavelength solar radiation, carrying high energy, has the capacity to directly break down relatively stable components of plant tissues, such as lignin and cellulose, through photochemical mineralization causing the release of volatile carbon compounds into the atmosphere. Photochemical mineralization produces more-labile molecules, which can enhance the activity of microbial decomposers through a process known as photofacilitation or photopriming. Solar radiation has also the ability to indirectly alter decomposition through negative effects (photoinhibition) on both the activity and community composition of decomposer organisms. We examined the process of photodegradation under forest canopies in a temperate and a boreal environment. Through two field experiments, we tested the effects of photodegradation on mass loss and carbon content during leaf litter decomposition in each environment (I in France and II in Finland). We also studied these processes under controlled conditions in a filter experiment (II). In France, we performed an additional field experiment, in the same forest as the first, to analyse the effect of photodegradation on microbial assemblages colonizing the litter (III). In these experiments, we employed “photodegradation-litterbags”, bespoke litterbags adapted from classical litterbags used in litter decomposition studies incorporating different types of film filter-material, allowing us to manipulate the spectral composition of sunlight. Finally, we conducted a meta-analysis (IV) to summarise the effect of photodegradation driven by different spectral regions of solar radiation at the global scale, and across different biomes, and to test whether the photodegradation rate is modulated by initial litter traits. This dissertation highlights the importance of blue light as a major driver of photodegradation in a temperate mid-latitude forest understorey, with the potential to enhance both litter mass loss and carbon loss. However, at a higher latitude, the full spectrum of sunlight decreased mass loss, suggesting that the effect of photodegradation is specific to each biome. Forest canopies not only modify the amount of incoming solar radiation and its spectral composition, but also shape the microclimate of the understorey, producing unique combinations of temperature, moisture and snow-pack depth. Hence, each canopy generates novel interactions of solar radiation and other environmental factors which act on leaf litter to determine the photodegradation rate. At both boreal and temperate latitudes, our spectral manipulations revealed the effect of photodegradation to be litter species-specific, with recalcitrant litter experiencing higher rates of photodegradation. In terms of microbial decomposition, we highlighted how blue light, UV-A radiation and green light, act synergistically to shape the structure of microbial decomposer communities, with bacteria tending to dominate in sunlight and fungi in dark conditions. The results of our meta-analysis show that the direction and magnitude of photodegradation are dependent on the spectral region considered. We highlight the crucial role of blue light and UV-A radiation as drivers of photodegradation across biomes. Blue light has a positive effect in enhancing mass loss, while UV-A radiation has a negative effect. Moreover, our meta-analysis shows that the rate of photodegradation at the global level is modulated by climate and ecosystem type; whereby arid and semiarid ecosystems with low canopy cover experience the highest photodegradation rates. On the other hand, initial litter traits failed to predict the rate of photodegradation on the global scale, despite being important at the local level; suggesting that different traits could be important in different biomes. Photodegradation is known to have a role in the carbon cycle, as the process of photochemical mineralization causes the release of volatile carbon compounds into the atmosphere. Therefore, we can expect photodegradation to reduce the amount of carbon sequestered by ecosystems. However, further research is needed to estimate the actual contribution of photodegradation to the global carbon cycle. Moreover, this contribution is likely to be affected by climate change, which modifies environmental factors such as temperature and the amount and pattern of precipitation; these factors together with spectral irradiance determine the photodegradation rate. Overall, our results show that the process of photodegradation has an effect on litter decomposition in the understorey of mid- and high- latitude forests, despite the low irradiance to which litter in these ecosystems is exposed. Blue light appears to be more important than other spectral regions in driving photodegradation in these habitats. However, the photodegradation rate is modulated by both climate and ecosystem type.
  • Myllykangas, Jukka-Pekka (Helsingin yliopisto, 2020)
    Methane is a powerful greenhouse gas that contributes significantly to global warming. In aquatic systems, microbes in anoxic sediments are the main methane producers. However, due to effective oxidative filters in the sediments and the water column, most of the methane produced does not end up in the atmosphere. This study explores methane dynamics in the Baltic Sea from the open sea, to estuaries and specific microbial processes. Major inflow cycles control methane dynamics in the open Baltic by bringing oxygen to the deep basins, where methane typically accumulates in large amounts during stagnation. The introduction of oxygen during a major inflow in 2014–2015 caused the disappearance of methane from the deep basins due to a combination of oxidation and displacement. However, the effects of the inflow were short-lived and methane started accumulating again in less than a year after the inflow. The coastal areas were more dynamic, and the primary source of methane varied with distance offshore. Near the river mouth of the studied estuarine system, methane brought in by the river was the most important source, whereas further offshore sedimentary methanogenesis fuelled by a legacy of eutrophication was the primary source. Atmospheric fluxes of methane were highest near the river mouth and decreased seawards, while bathymetry was the main control of sedimentary fluxes. Seasonality had a strong effect on methane dynamics, with methane concentrations generally increasing towards winter. However, as in the open Baltic, displacement also played a role at times, removing large amounts of methane at a time. While aerobic methane oxidation in the water column was the primary sink offshore, in the coastal areas anaerobic oxidation of methane (AOM) was by far the most important sink. Offshore, sulphate mediated AOM is expected to be the most important type of AOM. However in this study, metal mediated AOM was an equally important sink in the sediments. Both rates of AOM, and microbial community abundances, were highest below the main sulfidic zone in the sediment, pointing towards non-sulphate AOM pathways. Overall, eutrophication has had a large impact on methane dynam ics in the Baltic Sea. The legacy of past eutrophication fuels methanogenesis in the coastal areas to this day, despite reductions in nutrient and organic matter input from land, leading to enhanced atmospheric flux of methane. In the future, climate change will likely exacerbate methane emissions from the Baltic Sea.
  • Wang, Fang (Helsingin yliopisto, 2020)
    Introduction Plants must maintain a balanced water budget to support fundamental processes. Light, on the other hand, is the ultimate energy source and therefore vital in the plants’ energy balance. To use light or to use water efficiently represents a trade-off that persists throughout a plant’s life, but is especially important during the establishment of a seedling. Control of gas exchange is required to attain the optimal balance between the fluxes of carbon dioxide and water vapour. This is achieved by regulating the degree of stomatal opening. Individual leaf gas-exchange measurements allow the assessment of the trade-off in stomatal conductance at a point in time while repeated measurements over an extended period can provide diurnal time-courses which are useful to evaluate the strategies that plants employ to control water use efficiency. Tree seedlings exposed to shade or sunlight require a strategy for water use that accommodates both light conditions. Under the current changing of climate, droughts in many regions increasingly threaten seedling survival, providing the context for the research in this thesis. Objectives The main objective is to improve our understanding of the effect of light quality and irradiance on water relations. To achieve this, it is necessary to link sensory and physiological mechanisms that control gas exchange to adaptation and acclimation of whole plants. Conclusions Light can affect plant water relations by regulating the rate and magnitude of stomatal movement. Blue-light photoreceptors play important roles in this movement under blue and green light, with their contributions to stomatal control changing through the photoperiod (I). When plants grow in shade, the response of stomatal conductance to long-term water-deficit may become more conservative (III). Under drought, related species within the same genus can have different water-use strategies, including the regulation of stomatal conductance (II). Differences in water use are correlated with local climate and help to explain the geographic distribution of related species (II) and variation among local populations (III). My findings broaden our understanding of the mechanisms behind maintenance of a favourable water balance in plants. I achieved this by connecting photoreceptor function to stomatal regulation at different times of the day, and stomatal regulation to the tradeoff between water use efficiency and maintenance of hydraulic conductivity. Much remains to be done before these connections can be validated over the full range environmental gradients that occur in nature. The roles of photoreceptors in diurnal regulation of water relations during a drought period are far from clear; neither are their roles in acclimation and adaptation of different species and populations to the dynamic light environments of natural habitats. Further research will be required to understand the roles played by photoreceptors in different water-use strategies and at different time scales spanning from days to seasons.
  • Höckerstedt, Layla (Helsingin yliopisto, 2020)
    Infectious diseases have a major influence on the demography of human, animal and agricultural populations. However, we rarely see extensive disease epidemics in the nature, where pathogens play an important role in maintaining biodiversity. Coevolution has the potential to drive the genetic composition of hosts and pathogens by linking their genomes through adaptation and counteradaptation, often leading to evolutionary arms race dynamics. Moreover, predicting the emergence, spread and evolution of pathogens within and among host populations requires understanding of how resistance diversity is maintained through varying migration rates in spatially structured systems. The main goal of this thesis is to understand how pathogen-imposed natural selection maintains diversity in host resistance, from genes to metapopulation. Theoretical and experimental evolution studies predict that migration influences coevolution between hosts and their pathogens, yet evidence of this phenomenon occurring in natural populations is scarce. Using the Plantago lanceolata – Podosphaera plantaginis model system, I revealed the evolutionary trajectories of resistance in spatially isolated and well-connected populations within a metapopulation framework. I then asked if pathogens may also shape host fitness and resistance traits through transgenerationally induced maternal priming mechanism. Finally, we characterized the resistance gene repertoire in P. lanceolata and tested whether any particular genes show signs of pathogen-imposed selection. I found that well-connected host populations support higher resistance and phenotypic resistance diversity than isolated host populations. In contrast to isolated populations, hosts in well- connected populations do not show local adaption to their own pathogens. Local adaptation patterns varied considerably among well-connected host populations, and resistance levels fluctuated over years. These fluctuations may result from either pathogen-imposed selection or from transgenerational maternal effects, as results suggest that pathogen infection may transcend to subsequent generation and hence create an additional layer determining phenotypic resistance. Finally, we found that different host genotypes activate unique genetic defense mechanisms in response to the same pathogen. Jointly, these results highlight that maintaining high levels of diversity in resistance is the key for hosts to cope with their pathogens. Once pathogens have successfully established, they have the upper hand in the coevolutionary arms race, especially in isolated host populations. In well-connected populations hosts may be better able to respond to pathogen-imposed selection. Altogether, this study brings the new perception into how landscape fragmentation and biodiversity loss may dramatically change host survival and the intensity of selection for host resistance in nature and offers insights for resistance studies in both natural and managed plant populations.
  • Punttila, Pekka (Helsingin yliopisto, 2020)
    In order to be able to safeguard the native forest species and habitats, and to develop ecologically sustainable management regimes for forestry, we need to understand how human impact has changed the natural successional patterns and structural characteristics of forest stands and landscape composition in boreal forests, and how these changes have affected the boreal biota. In this work, I tested hypotheses concerning ant-community succession in a patchwork of forest stands of different age and size using data gathered with pitfall trapping and nest-mound mapping mainly in southern Finland. Specifically, I aimed to evaluate the effects of old-forest fragmentation and age-class distribution changes caused by forest management, with a special focus on a group of territorial keystone ant species, the wood ants (Formica rufa group), and the strength of their impact on the forest ecosystem. Wood ants are considered keystone species in boreal forests because of the multitude and magnitude of the ecological interactions of these species both with other forest organisms and with their environment. The wood-ant dominated system of mature forests was found to disintegrate after severe disturbances such as large-scale clearcutting, and the subsequent ant-community succession followed rather deterministic pathways where nest-founding strategies and species interactions, especially competition and slave-making, played important roles. The occurrence of territorial wood-ant species matched quite closely the predictions developed based on the social strategies exhibited by the species, and the results suggest that changes in forest-disturbance regime, either natural or anthropogenic, may lead to remarkable changes in wood-ant worker population sizes and, consequently, in the strength of their impact on forest ecosystem processes. Wood-ant communities may experience complete species turnover from the dominance of monogynous (single-queened) species to the dominance of polygynous (multiple queened) ones, or vice versa, depending on the prevailing disturbance regime, and the accompanied changes in worker densities were shown to be much larger than could be predicted from mere nest densities. Such changes may have remarkable impacts on forest ecosystem processes.
  • Donner, Iikki (Helsingin yliopisto, 2020)
    Although most cancers are sporadic, a considerable proportion is due to genetic susceptibility. Many cancer types display moderate to high heritability, but predisposition genes have been established for only a few. In this thesis project, we have utilized the data in the Finnish Cancer Registry (FCR) and the Population Information System (PIS) databases to identify cancer patients with a potential genetic predisposition. For most of the patients in the FCR archival tissue material in the form of paraffin-embedded formalin-fixed (FFPE) blocks is available in the pathological department of their local hospital, which makes genetic studies on selected cases possible. The Finnish population is due to its demographic history more homogenous than most populations, and thus optimal for studies on germline predisposition to disease. This thesis includes studies on two cancer types currently lacking well-established susceptibility genes: lung adenocarcinoma (LUAD), and angioimmunoblastic T-cell lymphoma (AITL). In addition, my thesis work examines esophageal squamous cell carcinoma (ESCC) and hereditary diffuse gastric cancer (HDGC) susceptibility. A fraction of these cancers can be attributed to germline mutations in RHBDF2 and CDH1, respectively. We utilized exome sequencing in all studies with the aim to discover novel susceptibility genes for the different phenotypes. Gastric cancer is the second leading cause of cancer mortality. Three distinct hereditary gastric cancer syndromes are recognized: HDGC, familial intestinal gastric cancer, and gastric adenocarcinoma and proximal polyposis of the stomach. Up to one third of HDGC families have heterozygous germline mutations in CDH1, which encodes E-cadherin. Most cases showing familial aggregation do not, however, have a known genetic basis. The aim of the first study was to find the putative cancer-predisposing gene defect in an HDGC family that had previously been tested negative for CDH1 mutations. We identified three candidate variants: p.Glu1313Lys in INSR, p.Arg81Pro in FBXO24 and p.Pro1146Leu in DOT1L. INSR is of special interest as insulin signaling has been shown to modulate E-cadherin glycosylation and stability. ESCC represents the most common subtype of esophageal cancer, a common and often fatal malignancy. Although ESCC shows familial clustering and moderate to high heritability, few studies on moderate to high penetrance predisposition exist. We have previously found familial aggregation of classic Kaposi sarcoma by performing family name at birth and municipality at birth based clustering of all cancer cases in the FCR. In study II, we used the same clustering algorithm to find potential familial cases of ESCC. We managed to find a geographical population subset that showed enrichment of clustered ESCC patients. The finding may indicate that a susceptibility variant is enriched in this population subset. We collected FFPE tissue material and exome sequenced a total of 30 ESCC cases. Six variants passed filtering and analysis criteria, the most frequent being a nonsense mutation in DNAH9 detected in four unrelated patients. One patient's tumor showed loss of the wild type allele of DNAH9, suggesting a tumor-suppressive function. The other candidate genes GKAP1, BAG1, NFX1, FCSK, and DDOST harbored missense variants. A rare variant in EP300, a gene that has previously been implicated in ESCC carcinogenesis, was discovered to segregate in three affected individuals in a single family. Lung cancer is the most common cancer in the world and the leading cause of cancer death in both men and women. It is well established that the primary cause of the malignancy is cigarette smoke, however, other factors are involved as only approximately 15% of smokers develop lung cancer and an estimated 10-25% of all lung cancers occur in never smokers. Gender influences the risk of lung cancer and women are overrepresented among never smokers with the disease. Because of a possible difference in the molecular basis, we chose to study never-smoker women with LUAD in study III. We selected the youngest patients listed in the FCR, who are more likely to harbor a predisposing germline mutation. We managed to collect normal FFPE tissue material from 21 never-smoker women who had been diagnosed with lung adenocarcinoma before the age of 45. Potentially pathogenic variants were found in eight Cancer Gene Census germline genes: BRCA1, BRCA2, ERCC4, EXT1, HNF1 A, PTCH1, SMARCB1, and TP53. The variants in TP53, BRCA1, and BRCA2 are very likely to have contributed to the early-onset lung cancer in the respective patients. This supports the idea that lung adenocarcinoma can be a component of certain cancer predisposition syndromes. In addition, fifteen genes displayed potentially pathogenic mutations in at least two patients: ABCC10, ATP7B, CACNA1S, CFTR, CLIP4, COL6A1, COL6A6, GCN1, GJB6, RYR1, SCN7A, SEC24A, SP100, TTN, and USH2A. Some of these candidate genes may explain a subset of female lung adenocarcinoma. AITL is a subtype of peripheral T-cell lymphoma with a bleak prognosis. Somatic driver mutations have been found in TET2, IDH2, DNMT3A, RHOA, FYN, PLCG1, and CD28, whereas germline susceptibility to AITL has to our knowledge not been studied. In study IV we performed an exome-wide rare variant analysis in 23 AITL patients. No germline mutations were found in the driver genes, indicating that they are not frequently involved in genetic AITL predisposition. Potentially pathogenic variants present in at least two patients and showing significant enrichment in our sample set were found in ten genes: POLK, PRKCB, ZNF676, PRRC2B, PCDHGB6, GNL3L, TTC36, OTOG, OSGEPL1, and RASSF9. The most significantly enriched variants were p.Lys469Ter in a splice variant of POLK and p.Pro588His in PRKCB. These two are intriguing candidates, as Polk deficient mice display a spontaneous mutator phenotype and PRKCB has been found somatically mutated in 33% in adult T-cell lymphoma, another form of peripheral T-cell lymphoma. These studies show that rarely performed next-generation sequencing of DNA extracted from FFPE tissue samples is well suited for research and should be utilized to a greater extent. We found plausible candidate genes for the phenotypes under study. However, due to relatively small sample sets, our findings need to be genetically validated in independent sample series and through functional studies to prove causality.
  • Kasela, Mariann (Helsingin yliopisto, 2020)
    Lynch syndrome (LS, previously known as hereditary non-polyposis colorectal cancer, HNPCC) is an inherited cancer predisposition syndrome caused by DNA mismatch repair (MMR) malfunction. MMR mechanism is a post-replicative repair pathway. The mismatch in the DNA is recognised and bound by MutSα, heterodimer of the proteins MSH2 and MSH6, or less often by MutSβ (MSH2+MSH3), after which the recruitment of MutLα (MLH1+PMS2) heterodimer initiates the repair process. Up to 90% of LS causing mutations are found in MLH1, MSH2 and MSH6 genes, whereas PMS2 has been suggested to be only a low-risk LS susceptibility gene due to small number of families segregating a disease causing PMS2 mutation. Cancer predisposition in Lynch syndrome is inherited dominantly through one defective MMR gene allele and tumorigenesis starts only after the loss of the second allele, giving rise most commonly to early-onset colorectal and endometrial cancers, and more rarely cancers of the uterine, stomach, urinary tract, ovary, small intestine or bile tract. The risk of developing colorectal cancer is higher in MLH1 and MSH2 mutation carriers than in MSH6 and PMS2 mutation carriers. However, the risk of developing endometrial cancer seems to be the highest in MSH6 mutation carriers. Early diagnosis of LS families and MMR gene mutation carriers is extremely important, since risk-reducing clinical surveillance and prophylactic surgeries have been shown to reduce cancer-related mortality. LS diagnosis is generally based on cancer history of the family and on tumour studies, followed by genetic testing to determine a predisposing mutation. However, the atypical clinical phenotypes such as late age at onset, lower penetrance and different tumour spectrums associated with MSH6 and PMS2 families, as well as the increasing number of variants of uncertain significance (VUS) found in sequencing, complicate LS diagnosis and highlight the need for pathogenicity assessment. The present work aimed to study the functional effect of lowered MMR gene expression as indication of Lynch syndrome and to assess the pathogenicity of MMR gene variants of uncertain clinical significance. Here, we studied how decreased MLH1, MSH2, MSH6 and PMS2 gene expression levels affect MMR efficiency. Using a stable shRNA knockdown approach we generated and studied altogether eleven cell lines retaining 23%, 50% and 74% of MLH1, 26%, 47% and 68% of MSH2, 50% and 79% of MSH6, and 19%, 33% and 53% of PMS2 mRNA expression. The results of an in vitro MMR assay showed that the repair efficiency was not only associated with gene expression level but the expression decrease affected different genes differently. For MSH2 and MSH6 genes, an expression decrease to 75% already caused a significant decrease in MMR efficiency, while for PMS2 the repair capability decreased significantly near the mutation carrier level (~50%), and for MLH1 gene only at the lowest expression level (23%). Unexpectedly, 19% and 33% of PMS2 expression resulted in higher MMR efficiency than the carrier-like level, 53%, suggesting some kind of functional compensation for PMS2 repair activity in the cell. The functional significance of five MLH1 and four MSH2 VUSs found in suspected LS families was determined by the in vitro MMR assay. For that, nine recombinant MLH1 and MSH2 protein variants were used to complement MMR-deficient cancer cell lines lacking the normal respective protein. A MMR gene variant whose protein retained repair efficiency in the assay was determined MMR proficient, while variants that resulted in the lack of repair were determined MMR deficient. Here, the MLH1 variants p.Leu348Ser, p.Arg474Pro and p.Glu605Ala were shown to be MMR proficient and p.Gly101Ser and p.Leu260Arg deficient, while only one MSH2 variant p.Lys82Glu was MMR proficient and the variants p.Gly669Val, p.Phe694Ser and p.Pro696Leu deficient. Our results, together with the clinical and tumour data collected from the families, allowed the pathogenicity assessment of the MMR gene variants and Lynch syndrome diagnosis in the families. Findings from these studies provide new insights into the severity of the malfunction that decreased levels of different MMR genes expression may cause. Furthermore, the results show that the functional assessment of variants of uncertain significance considerably helps their pathogenicity assessment. Both of these findings may have an important impact on Lynch syndrome diagnosis in future.
  • Brelsford, Craig (Helsingin yliopisto, 2020)
    Light quality varies in space and time, and plants are able to detect and respond to these environmental cues. Plants must time when their leaves come out in spring and fall off in autumn, to maximise opportunities for photosynthesis whilst conditions are favourable. Similarly, they must optimise the amount of sun-screening pigments in their leaves, to minimise the harmful effects of ultraviolet radiation at high irradiance. Solar radiation reaching the Earth, as well as its composition, vary diurnally and seasonally with solar angle. During twilight, plants are able to detect changes in red:far-red light, and use this to help time their spring and autumn phenology. When forest canopies leaf out in spring, and cause canopy closure, the understorey becomes mostly covered in shade. This shade also causes a low red: far-red ratio, that plants are able to detect and increase their stem elongation. However, the amount of blue and UV radiation also varies in space and time, and we know considerably less about how plants respond to these changes in the blue-and-UV region. Using a combination of controlled indoor experiments, literature review, and manipulative field experiments, we set out four aims. 1) How do blue and UV-A radiation affect leaf pigments under controlled conditions? 2) How does blue light affect spring bud burst under controlled conditions? 3) How do blue and UV radiation affect leaf pigments and leaf phenology for understorey plant species? 4) How important is light quality as a phenological cue? We found that both under controlled conditions and in the field, blue light had a large positive effect on the accumulation of flavonoids, most likely governed by cryptochrome photoreceptors. Interestingly, the flavonols in more light-demanding species of plants were more responsive to changes in light quality, particularly blue light. Similarly, blue light advanced spring bud burst in tree species both in the lab and in the field. We also report that both blue light and UV radiation can advance autumn leaf senescence in understorey plants. Lastly, when critically comparing the effect sizes of light quality treatments on phenological responses in trees, we found that light quality effects on spring phenology are generally small. However, the effects reported on autumn phenology are much larger. This adds to the complexity of drivers affecting autumn phenology, and may be one reason why autumn phenology is typically much harder to forecast compared to spring. Future work should seek to understand how other environmental drivers such as temperature will interact with light quality to affect leaf pigments and leaf phenology. It will be important to understand how climate change could produce potential phenological mismatches in cues between the canopy and understorey, and even between different organisms such as plants, herbivores, and pollinators.
  • Laurinmäki, Pasi (Helsingin yliopisto, 2020)
    Technological advances especially in the last two decades have made it feasible to study virus structures to atomic or near-atomic resolution by a combination of cryogenic electron microscopy (cryo-EM) and image reconstruction or by X-ray crystallography. Drawing from these data, intertwined concepts of “viral self” and structure-based viral lineages been put forward and refined during the last decade. In order to address these theories, I used cryo-EM, 3D image reconstruction, homology modelling and de novo atomic modelling of proteins to study five viruses with icosahedrally symmetric protein capsids. The capsid structures of bacteriophage Bam35, two African horse sickness virus serotypes (AHSV-4 and AHSV-7 tVP2), archaeal head-tailed virus HSTV-1, and Nora virus (NORAV) that infects Drosophila melanogaster were solved to subnanometer resolution. I examine the methodological advances in the field of cryoEM and image processing that have occurred over the time span of the original articles included in the thesis, giving an empirical perspective on the important changes required to reach atomic resolution in virus structural determination. These included improved imaging, data recording, automation, and software developments. Notably, grid preparation is the area where the next strides need to be made to improve reproducibility and throughput. The results contribute to the general field of structural virology as well as shedding light into more specific areas, such as biological membrane modulation, archaeal viruses and vaccine development. Moreover, the hosts of the viruses studied span all three domains of life (bacteria, archaea and eukaryotes). This unusually wide sampling of the viral universe, or virosphere, creates an excellent basis for testing the utilisation of capsid structure in structure-based virus classification as well as verification of structure-based viral lineages. The results allowed unambiguous structure-based classification of the five studied viruses into the four previously postulated virus lineages; the picornavirus-like lineage, the HK97-like tailed-phage lineage, the PRD1/adenovirus lineage and the icosahedral dsRNA virus lineage. Furthermore, the NORAV reconstruction at 2.7 Å resolution provided structural evidence suggesting that NORAV is a representative of a new virus family within the order Picornavirales; a result not achievable by genetic evidence alone and a benchmark example of structure-based virus classification.
  • Tverin, Malin (Helsingin yliopisto, 2020)
    Growing populations of the piscivorous top predators the grey seal (Halichoerus grypus) and the great cormorant (Phalacrocorax ssp) in the Baltic Sea have created predator-fishery conflicts. Responsible mitigation measures require that correct and adequate information on the predator foraging habits is available. Adipose tissue fatty acid (FA) profiling is an established method for such dietary and food web studies since the FAs are transferred from the prey to predator tissues in a predictable way. However, sampling procedures and the way of using the FA profiles need to be optimized for each predator phyla to create globally applicable and reliable procedures for the diet assessment. One prerequisite is to build a reference library of the FA compositions of the available prey selection (fishes in the Baltic Sea), and the predator tissue metabolism and other factors affecting the tissue FA profiles need to be studied in order to avoid misinterpretation of results. We analysed tissue FA profiles of fishes, grey seals and great cormorants from the Baltic Sea area by gas chromatography, and used multivariate Principal Component Analysis (PCA) along with Soft Independent Modelling of Class Analogy (SIMCA) to analyse the multivariate data. Pinniped blubber FA profile differs vertically through the blubber column from skin to muscle, which raises a question whether sampling depth causes discrepancy in the results. We found that the vertical FA stratification patterns show constant trends, but also large individual differences. All studied adult grey seal males (n = 30) had enrichment of monounsaturated FAs (MUFAs) in their outer blubber, whereas polyunsaturated FAs (PUFAs) did not show similar strong stratification pattern along the blubber column as the MUFAs did. The degree of vertical FA stratification did not depend on blubber thickness and was slightly age-dependent only for two FAs, 16:0 and 16:1n-7. In the absence of an effect caused by other factors such as sex, blubber thickness, capture area and water temperature, we found that the driver of the vertical stratification of MUFAs was the mismatch between the individually varying FA composition of the inner blubber, regarded as the main incorporation site of dietary FAs, and the outer blubber FA composition, endogenously enriched in MUFAs. This highlights the importance of choosing sampling protocols in which the outer blubber layer is omitted from the analysis, and preferably only the innermost blubber, which carries the most recent dietary information, is included to gain valid information of the diets of the seals. To evaluate the pros and cons of the FA profiling in comparison to other available methods, we analysed the blubber of 108 grey seal individuals collected from the Finnish and Swedish coast of the Baltic sea, and compared dietary information obtained by blubber FA analysis to the information from gut content hard part or DNA analyses, and tissue stable isotope (SI) ratios. We found that in a large data set, PCA of blubber FA profiles did not manage to clearly detect any dietary groups, which was likely due to the large individual variation in the foraging habits of the seal individuals. In smaller scale, however, blubber FA profiles distinguished groups of individuals with accurately known collection site and which likely had used the same type of prey for a longer period of time. This information is valuable since these individuals likely cause the largest economical losses in the form of loss of catch and destroyed fishing gear, which in turn is the culprit to the risen seal-fishery conflict in the Baltic Sea. The population of the avian top predator of the Baltic Sea, i.e. the great cormorant (including two subspecies Phalacrocorax carbo sinensis and Phalacrocorax carbo carbo), has also grown and caused local conflicts with fisheries. In the absence of studies of chemical tissue markers of the great cormorants of the Baltic Sea area, we analysed the subcutaneous knee adipose tissue of 77 cormorant individuals and found that the adipose tissue FA composition of the studied birds differed both spatially and temporally. Thus, the FA profiling is even more sensitive to indicate dietary differences in sea birds than in pinnipeds, presumably due to the high metabolic rate and short turnover time of the adipose tissue lipids and FAs. In addition to the spatial differences observed, which were likely due to locally differing diets, we also observed high relative amounts of ocean FA marker 22:1n-11 in the adipose tissue of the cormorants collected in Sundsvall in June 2017, which likely had got their 22:1n-11 from hatchery-reared salmonids fed ocean fish flour and oils. Since levels of certain FAs or their ratios are characteristic of specific dietary sources and since dietary FAs are assimilated in predator tissues in a predictable way, FA profiling is a useful tool for dietary studies both on individual and population level. To describe the variability of prey sources at population level, we calculated the relative standard deviations (RSD, i.e. SD/Mean) for the FA variables (the mean value of which exceeded 0.2 mol%, and thus excluded methodological variation) in the adult cormorant and adult seal individuals. The FA variables with the highest RSD values were the most useful markers of individual dietary differences in the population. We propose that the RSDs for FA variables could be used to create an “opportunistic feeding index”, providing information on the variability of prey of the studied population. The works that this thesis is based on show, that standardized sampling protocols that take into account tissue metabolism significantly, increase the power of FA profiling in indicating individual diets of aquatic top predators, and allow creating indices to describe population level dietary opportunism. The improved dietary information can be applied for monitoring the diets of aquatic top predators and for ecosystem-based management.
  • Jokinen, Henri (Helsingin yliopisto, 2020)
    Initiated by signs of drastic declines of once abundant flounders in the northern Baltic Sea, this thesis characterised/quantified these declines and studied their potential reasons. Multiple approaches were used to describe population trends, reveal intricacies in stock structure, and assess links to key environmental drivers. The results from the thesis verified clear negative trends in both adult and juvenile flounders in the northern Baltic Sea over the last 2–4 decades. Genetics revealed well-defined genetic structure and evidence for far gone speciation among Baltic Sea flounders, leading to the recognition of two instead of one flounder species, one of which was recently described as the only endemic fish species known to the region. Contrary to previous belief, flounders on the coast of Finland in the northern Baltic Sea were shown to be a mixed assemblage of this cryptic species pair. The thesis further showed that temporal variability in local species composition of the flounder assemblage explained some of the observed stock decline and was related to changing local and regional environmental conditions, of which reproductive volume, salinity, temperature and eutrophication were identified as potentially important factors. Finally, the thesis presented a new method for modelling environmental suitability for long-term population maintenance of the newly described Baltic flounder. The knowledge obtained has great value for how we understand and investigate stock composition and population dynamics of Baltic Sea flounders, and relates to issues of source-sink mechanisms, population connectivity, biological traits, resilience to exploitation and environmental change, among others. The results are likely important for future management and conservation of these fishes in the changing environment of the Baltic Sea.
  • Mavrovic, Martina (Helsingin yliopisto, 2020)
    KCC2 is the main neuronal K-Cl cotransporter, which lowers the intracellular Cl- concentration and thus maintains GABAA receptor-mediated inhibition during network activity in mature neurons. Independently of its function as an ion transporter, KCC2 was shown to be necessary for the formation of excitatory synapses, where it regulates the development of dendritic spines via modulation of the actin cytoskeleton, notably cofilin phosphorylation. KCC2 expression undergoes developmental up-regulation, which parallels the rostro-caudal CNS maturation in rodents, and underlies the robust negative shift in EGABA away from the depolarizing responses characteristic of immature neurons, as well as functionally regulates the intense spino- and synaptogenesis of cortical principal neurons. These findings posit KCC2 as a key molecule for coordinating the maturation and balance of excitatory and inhibitory neurotransmission during the brain growth spurt. While the role of KCC2 has been extensively studied during the first month of postnatal development in rodents, its functions in the perinatal cortex are only beginning to emerge. KCC2 knockout mice have altered hippocampal network activity already at embryonic day 18.5 when KCC2 is considered to be expressed at physiologically non-significant levels. Moreover, KCC2 has been found to regulate the migration of cortical interneurons by acting as a migratory stop signal in an ion transport-dependent way. On the other hand, in pyramidal neurons, KCC2-mediated ion-cotransport seems to be under kinetic suppression until around birth. Finally, the time window of the transition period of GABAergic signaling from depolarizing to hyperpolarizing, and the underlying upregulation of KCC2, coincides with the time when GABAAR modulators, notably general anesthetics, have been shown to induce lasting adverse effects on cognition and behavior in rodents. Thus far, it is not known why there is this defined time window of increased vulnerability to anesthetics, and the underlying molecular and cellular mechanisms warrant investigation. In this Thesis, the primary goal was to study the functional significance of early KCC2 expression in the developing neocortex and hippocampus of mice and rats and to assess the effects of genetic and pharmacological modulation of KCC2 expression on the formation of cortical networks. Particular focus was put on differentiating between the ion transport-dependent and -independent roles of KCC2. This work describes a novel ion transport-independent role of KCC2 in developmental neuroapoptosis, as well as ion transport-dependent functions of KCC2 in the regulation of hippocampal network events and developmental-stage dependent vulnerability to general anesthetics.

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