Faculty of Biological and Environmental Sciences

 

Recent Submissions

  • Rai, Neha (Helsingin yliopisto, 2020)
    Solar UV-B radiation (290–315 nm), UV-A radiation (315–400 nm), and blue light (400–500 nm) regulate multiple aspects of plant growth and development, and these are mediated by different photoreceptors. In plants, UVR8 is described as a UV-B photoreceptor, while cryptochromes (CRYs) are described as UV-A/blue photoreceptors, based on their absorption maxima and action spectra. However, these photoreceptors are also sensitive to other wavelengths outside the wavelengths of maximum absorption. Based on this property, their roles could differ in full-spectrum sunlight than those reported from experiments performed in controlled environments. In sunlight, both UV-B and UV-A/blue photoreceptors are simultaneously activated, and there is a possibility that their signaling pathways interact. However, an interaction between UVR8 and CRYs regulating transcriptome-wide responses remained unexplored. Furthermore, persistent high solar irradiance is often followed by drought in the field, and studies have indicated that UV and drought interact to regulate plant physiological responses. However, an interaction for metabolic and transcript abundance responses has not been well-described. These gaps in knowledge are addressed in my thesis through three main aims: (1) to identify the individual roles of UVR8 and CRYs in the perception of solar UV-B, short-wave UV-A (315–350 nm, UV-Asw), long-wave UV-A (350–400 nm, UV-Alw) radiation, and blue light by plants, (2) to test the interaction between UVR8 and CRYs under solar UV radiation, and (3) to determine if pre-exposure to solar UV radiation could provide acclimation to subsequent drought stress in plants. To achieve the first two aims, I used Arabidopsis thaliana wild type and mutants impaired in UVR8 and CRYs photoreceptors and exposed them to different ranges of wavelengths of solar or simulated solar UV radiation and blue light under optical filters. To achieve the third aim, I used two accessions of Medicago truncatula (Jemalong A17 and F83005-5). I exposed them to solar UV radiation using optical filters and subjected them to drought stress by restricting watering in a factorial experiment. The results indicated that UVR8 mediates the perception of both UV-B and UV-Asw radiation. In contrast, CRYs mediate the perception of UV-Alw radiation and blue light. A further novel finding is that UVR8 and CRYs interact antagonistically to regulate transcriptome-wide responses under UV-B and UV-Asw radiation. My thesis also provides evidence that UV-B+UV-Asw radiation and mild drought can interact positively to trigger acclimation through an increase in epidermal UV screening in the drought-intolerant accession, F83005-5, and through an increase in transcript abundance of CHALCONE SYNTHASE in the moderately drought-tolerant accession, Jemalong A17. Furthermore, all three studies showed a distinct response to solar or simulated solar UV-B+UV-Asw and UV-Alw radiation, suggesting a need to split UV-A into short and long wavelengths for future studies on UV-A radiation.
  • Koskelainen, Susanna (Helsingin yliopisto, 2020)
  • Jerney, Jacqueline (Helsingin yliopisto, 2020)
    Phytoplankton plays a pivotal role for aquatic ecosystem functioning and global biogeochemistry. Climate change has affected phytoplankton community composition and distribution in the last decades, including a higher prevalence for harmful algal blooms in many areas. The globally distributed dinoflagellate Alexandrium ostenfeldii has for example started to form dense toxic blooms in the Baltic Sea and a new bloom location was recently discovered in western Japan. To survive unfavorable conditions this species forms resting stages, which may accumulate in sediments, forming a “seed bank”. The aim of this thesis was to investigate the relevance of the seed bank for the ecology and evolution of A. ostenfeldii and to understand the implications of these findings for persistence and possible expansion under ongoing global change. A combination of field surveys in Finland and Japan, experimental work and genotyping were carried out to address these aims. The results indicate that the seed bank stores a large clonal diversity, underlining its importance for stabilizing local populations against environmental fluctuations. No population structure was detected in temporal parts of a pelagic population, showing that differentiation does not happen during one season. The life cycle of A. ostenfeldii was found to be highly versatile, allowing overwintering of asexual resting stages without a pronounced dormancy period, and sexual reproduction throughout the season. Predicted future temperature and salinity did not affect germination of A. ostenfeldii, but affected growth rates, demonstrating their selective effect on the pelagic part of the population when detached from the seed bank. In addition, the importance of resting stages for colonizing new habitats, was stressed by the close relationship found between a recently discovered bloom population in Japan and geographically distant populations of similar habitats. Low genetic diversity indicated a recent introduction, potentially due to anthropogenic dispersal of resting stages. In conclusion, the seed bank plays a pivotal role for evolution and ecology of A. ostenfeldii. It ensures survival of a genetically diverse population, and slows down evolution, by linking contemporary populations to past populations via frequent re-seeding of resting stages. Although selection is buffered by phenotypic plasticity, future temperature and salinity may affect the pelagic part of the population, in the long run. A generalist life cycle of A. ostenfeldii and the presence of a seed bank support persistence and potential future temporal and spatial expansion under global change.
  • Roslund, Marja (Helsingin yliopisto, 2020)
    The incidence of immune-mediated diseases has increased rapidly in developed societies. According to the biodiversity hypothesis, the core reason is the evident biodiversity loss in urban areas. This biodiversity loss limits exposure to a diverse microbiota, which is associated with the human commensal microbiota and immune regulation. In addition, urban pollutants, such as polycyclic aromatic hydrocarbons (PAHs), may alter microbial communities and interfere with immune regulation. However, studies linking urban biodiversity loss, PAH pollution, environmental and human commensal microbiota and immune regulation are lacking. This study is one of the first to estimate the connections between environmental exposure, the commensal microbiota, and the immune response of urban children using both intervention trials and comparative studies. The aim of this study was also to develop practices to reduce the risk of non-communicable immune-mediated diseases that are globally recognized as emerging public health problems. These diseases comprise over 80 inflammatory disorders including allergies, type 1 diabetes, asthma and inflammatory bowel disease. The research focused on two aspects: the effect of biodiversity and pollution on the commensal microbiota of children and immune regulation. First, I estimated PAH induced bacterial shifts in polluted urban landscaping materials, and whether environmental exposure to PAHs can affect children’s commensal bacterial communities on the skin and in the gut. Secondly, we set up a human intervention trial in which urban environmental biodiversity was manipulated and examined its effects on environmental and commensal microbiota and immune regulation in children. The PAH pollution studies showed that PAHs may induce shifts in environmental and human commensal bacterial communities that are associated with human health and immune regulation. Bacterial shifts in urban landscaping materials depended on soil material type, indicating that in the future it is possible to design gardening and landscaping materials that are more resilient to bacterial shifts induced by PAH pollution. Soil PAH pollution in day-care center yards was associated with altered Actinobacteria, Bacteroidetes and Proteobacteria communities on children’s skin and in day-care yard soils. However, altered genera differed between skin and soil, excluding Mycobacterium, the abundance of which increased on skin and in soil with increasing surface soil PAH levels. Associations were not found between gut microbiota and PAH levels in day-care yard surface soils or ambient air. However, gaseous chrysene levels in the ambient air were associated with the endocrine signaling pathways predicted from the gut bacterial metagenome with the Kyoto Encyclopedia of Genes and Genomes. The peroxisome proliferator-activated receptor (PPAR) is a crucial signaling pathway in the regulation of inflammation, metabolism, and tumorigenesis. The PPAR signaling pathway together with the adipocytokine signaling pathway can regulate immune cells and affect hormonally-mediated diseases, including obesity, insulin sensitivity, puberty, and fertility. The PPAR and adipocytokine signaling pathways both decreased among children, with higher gaseous chrysene levels in the day-care center’s ambient outdoor air. These findings indicate that PAH concentrations that are below the risk assessment safety limits may alter the human commensal microbiota and interfere with endocrine signaling. The imbalance in human microbiota and the decrease in endocrine signaling pathways might contribute to inflammatory disorders. Therefore, optimal risk assessments should take into account the possibility of the disruption of endocrine signaling pathways and the microbiota–health nexus. The 28-day biodiversity intervention trial included 75 children in three different day-care environments (standard urban, biodiversity intervention, and nature-oriented). During this intervention, the environmental and intervention children’s commensal microbiota was diversified, which in turn promoted their immune regulation and eventually may have beneficial health consequences. Surface soil bacterial communities differed between intervention and standard day-care yards and, in particular, differences were seen within alpha-, beta-, and gammaproteobacterial classes. The relative abundance of bacteria typically found in the forests of Finland increased in intervention day-care yards. These environmental changes in day-care yards remained for 2 years. The diversity of proteobacterial communities in soil and on the skin of the day-care children increased during the 28-day intervention. Importantly, an increase in skin gammaproteobacterial diversity was associated with beneficial effects in immune regulation, promotion of the plasma transforming growth factor-β level and proportion of regulatory T cells, and a decline in pro-inflammatory interleukin 17A (IL-17A) levels. In addition, among intervention children the ratio between anti-inflammatory IL-10 and pro-inflammatory IL-17A increased, indicating that the biodiversity intervention promoted children’s immune regulation. In addition, among intervention children, I observed shifts within gut Ruminococcaceae and Lachnospiraceae communities that have earlier been associated with gut health. Interestingly, the microbiota on the skin and in the gut of intervention day-care children shifted toward those in nature-oriented day cares. I followed the environmental and commensal bacterial shifts on the skin, in the saliva, and in the gut for a 2-year period among children in the intervention group. This long-term study showed that the biodiversity intervention shifted the environmental and commensal bacterial communities at the intervention day cares, and these shifts include important primers for the immune system. In particular, environmental shifts were permanent based on the 2-year period. These results are proving valuable since now that we understand the effect of biodiversity in the living environment, we can shape children’s commensal bacteria and thus affect immune regulation. The challenge will be to design novel pathogen-free nature-based solutions for urban people that include a high diversity and richness of anti-inflammatory health-promoting bacteria. Future research should target this challenge. The results of this thesis support the biodiversity hypothesis: environmental biodiversity is associated with the commensal microbiota of humans and immune regulation. Indeed, both biodiversity loss and pollution in the urban environment may lead to an altered environmental microbiome. This in turn can lead to an imbalanced immune system and consequently increase the prevalence of emerging public health problems, including allergies, asthma, type 1 diabetes, and inflammatory bowel disease. Importantly, this study has demonstrated that modifying the living environment of children with microbiologically diverse natural materials might provide a feasible approach for decreasing the risk of immune-mediated diseases in urban populations.
  • Merezhko, Maria (Helsingin yliopisto, 2020)
    Neurodegenerative disorders are progressive, age-dependent, devastating conditions with only symptomatic treatment available. The progressive accumulation and spread of misfolded proteins in the nervous system is the common attribute of multiple neurodegenerative diseases. In Alzheimer’s disease (AD), two types of aggregates accumulate and spread through the brain: extracellular amyloid plaques composed of β-amyloid peptide and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. β-amyloid peptide originates from the pathological processing of amyloid precursor protein (APP). APP processing is susceptible to various stimuli and controlled by multiple proteins and interactions with lipids. The regulation of APP processing, however, is not fully understood. Tau is one of the major microtubule-associated proteins in neurons. In AD and other tauopathies, however, tau becomes hyperphosphorylated, detaches from microtubules, and first forms small soluble oligomers and then larger, insoluble aggregates. Like several other neurodegeneration-related proteins, pathological tau spreads through the brain via cell-to-cell transmission, which involves secretion and internalization stages, and can initiate templated misfolding of normal tau in recipient cells. Unfortunately, the mechanisms of cell-to-cell transfer and templated misfolding of tau are rather elusive. The aim of this thesis is to (1) develop novel assays to advance the understanding of the regulation of processing and trafficking of neurodegenerative-related proteins and (2) investigate the molecular mechanisms of tau secretion. In this thesis, two novel in vitro live-cell assays were developed based on protein-fragment complementation to study APP, tau, and other neurodegeneration-associated proteins. The first assay can generate multiple readouts, reflecting cellular fates of APP: total cellular APP level, total secreted sAPP level in the media, APP-BACE1 interaction in cells, and in culture media. The second assay can monitor protein localization to dynamic nanoscale cholesterol/sphingomyelin-rich microdomains at the plasma membrane, usually called lipid rafts. This assay may be beneficial for neurodegenerative disease research, as many misfolded proteins associate with lipid rafts, including APP and tau. Additionally, this thesis addressed the molecular mechanisms of tau secretion. In N2A cells overexpressing human tau as well as in primary neurons, tau secretion to the extracellular space was shown to occur via an unconventional, vesicle-free mechanism. Imaging studies have revealed that tau clusters at the plasma membrane in the discrete microdomains and does not localize to membranous intracellular organelles. Instead, tau secretion depended on the lipid composition of the plasma membrane, particularly on lipid-organizing lipid rafts, such as cholesterol and sphingolipids. Tau secretion was also shown to depend on its oligomerization state and heparan sulfate proteoglycans at the cell surface. The data collectively suggest that tau secretion happens via translocation through the plasma membrane, which likely occurs in lipid rafts. In summary, the studies included in this thesis provide both methodological and conceptual insights in the field of neurodegeneration.
  • Kielosto, Mari (Helsingin yliopisto, 2020)
    c-Jun, a member of the AP-1 transcription factor family, is involved in numerous cell activities such as proliferation, differentiation, tissue morphogenesis, tumorigenesis, and apoptosis. c-Jun is a basic leucine zipper (bZIP) transcription factor that can form homodimers and heterodimers with other AP-1 family members. As a dimer, it is able to bind to DNA and regulate transcription of different genes. Numerous extracellular stimuli, such as ultraviolet (UV) radiation, and cellular stimuli, such as reactive oxygen species (ROS), induce signaling cascades leading to phosphorylation and activation of c-Jun. The main kinase phosphorylating c-Jun is c-Jun N-terminal kinase (JNK). c-Jun is constitutively activated in many human cancers, including melanoma, breast, pancreatic, and colorectal cancers, and transformed cell lines, like Ha-ras transformed fibroblasts. Thus, better knowledge of the activation of c-Jun and the genes it regulates in cell transformation is needed in the fight against cancer. The aim of this study was to clarify the role of c-Jun in cell transformation by using fibrosarcoma and melanoma cells as models. In the first part of the study, the significance of phosphorylation and activation of c-Jun in S-adenosylmethionine decarboxylase (AdoMetDC)-, ornithine decarboxylase (ODC)-, and Ha-ras oncogene-transformed mouse fibroblasts (Amdc, Odc, and E4 cells, respectively) was examined by exploiting transactivation domain deletion mutant of c-Jun (TAM67) and phosphorylation-deficient c-Jun mutants. Further, the upstream kinases of c-Jun were evaluated by using dominant negative mutants of SEK1 (MKK4) and JNK1 as well as JNK inhibitors. The transformed morphology of the cells was reversed with differing efficacies when transfected with these mutants, most effectively when using TAM67. Due to the highest potency of TAM67, Amdc, Odc, and E4 cells carrying a tetracycline-inducible expression system of TAM67 were then generated (Amdc-, Odc-, and E4- pLRT-TAM67 cell lines). These inducible cell lines provide good, reversibly regulatable models to identify the mechanisms of c-Jun-related transformation. Indeed, expression of TAM67 inhibited cell growth in soft agar and three-dimensional (3D) Matrigel matrix, and, most importantly, tumor formation in nude mice. In the second part of the study, the transformation-relevant genes regulated by c-Jun in Amdc, Odc, and E4 cells were identified by utilizing the above-mentioned cell lines (Amdc-, Odc-, and E4-pLRT-TAM67). After TAM67 induction, the differentially expressed genes in the morphologically normalized cells compared with the transformed cells were identified by Incyte Genomics’ cDNA microarray analysis. Relatively few changes were identified, including those of integrins alpha 6 and beta 7 (ItgA6 and ItgB7), which were upregulated in transformed cells, and lysyl oxidase (Lox), which was downregulated. In addition to Lox, also Lox-like-1 and 3 were found to be downregulated in Odc and E4 cells by Affymetrix’s microarray and RT-PCR analyses. In the third and fourth parts of the study, the functional roles of the up- and downregulated genes were examined. ItgA6 was found to pair mainly with ItgB1 to form integrin A6B1 heterodimer, and function-blocking antibodies against ItgA6 or ItgB1 inhibited the binding of Amdc cells to laminin and cell invasion in 3D Matrigel. Importantly, similar results were seen with human HT-1080 fibrosarcoma cells. Further, downregulated Lox (pro-LOX) was observed to be involved in the invasion of Odc cells. In addition to fibrosarcoma cells, the expressions of LOX family members were also examined in different human melanoma cell lines, where they were variably expressed. LOXL2 and LOXL3 were upregulated in nearly all melanoma cell lines studied. Upregulated LOX family members and their activities were found to be associated with invasion in melanoma cells, especially when co-cultured with fibroblasts in 3D Matrigel. In conclusion, we have demonstrated that the transformed phenotype of ODC-, AdoMetDC-, and Ras-transformed mouse fibroblasts is reversibly regulatable by dominant negative mutants of c-Jun and identified ItgA6 and Lox as transformation-relevant target genes of c-Jun. Inactive pro-LOX is suggested to act as a tumor suppressor in these cells. In human melanoma cells, in turn, active LOX and LOXL2 were identified as molecules promoting invasive growth and could offer potential new targets for therapeutic approaches in melanomas.
  • Korkonen, Sanna (Helsingin yliopisto, 2020)
    The plankton of Finland’s vast amount of lakes are often dominated by diatoms and chrysophytes that, together with periphytic forms, end up in the sediment. All chrysophytes are known to produce siliceous resting stages called cysts that are endurable, considered species-specific, and well preserved in sediments. Thus far, the focus on utilizing these algal remains in palaeolimnology has been on diatoms, due to the ease of their identification compared to chrysophyte cysts. While cyst research in Finland has remained scarce, researchers elsewhere have successfully linked cyst morphotypes with several environmental variables. This has enabled the reconstruction of several environmental variables such as eutrophication, pH and temperature history, for several areas. In this thesis, cysts in lake sediments in Finland are studied for their distribution, seasonality and ecological preferences. The main aim is to create a cyst-based transfer function – a mathematical equation that formalizes the relationship between species and the environment – that could later be used in interpreting past environmental changes to simulate the impacts of future climate change. The reliability of the usage of sedimentary cysts in palaeolimnological environmental research was studied by comparing seasonal cyst assemblages accumulated during two hydrologically and weather-wise different years in sediment traps to the topmost surface sediment of a lake. In addition, previously unencountered cyst morphotypes are officially named and described, along with their ecological preferences. In total, 59 lakes in Finland, ranging from southern lakes to lakes above the treeline in Finnish Lapland, were sampled for their surface sediment for the cyst survey. In addition, sediment traps were employed and the surface sediment collected from Lake Nautajärvi to study cyst seasonality, sensitivity to hydrological and weather changes, and to verify that the surface sediment represents cyst production in the lake. All cyst identification was performed using a scanning electron microscope to enable the detection of minuscule ornamentation details. Altogether, 265 chrysophyte cyst morphotypes or collective categories were found among the 60 Finnish lakes. These cysts represent a variety of different cyst types, most of which are familiar from other cool, oligotrophic locations. However, 18 are new, previously undescribed morphotypes, currently exclusive to Finland. The most common cysts in Finland are unornamented types, most likely produced by several different species; there is also a variety of cysts that can be used in palaeolimnological investigations in the future. Temperature, pH, conductivity and total phosphorus were found to have a significant effect on cyst assemblages in Finland. Therefore, optima and tolerances for the most commonly occurring cyst types for these variables were calculated. Cysts in Finland show seasonality, with the majority of cysts being produced during spring and summer, while winter is essentially a standstill in cyst production. The most common cysts occur throughout the growing season, but there are also cysts that are linked to a specific season. Seasonal variation has a more profound effect on cyst occurrence than short-lived weather events between years. Surface sediment cyst assemblages comprised well with the sediment trap assemblages, supporting the assumption that produced cyst assemblages are well represented in lake sediment, and can hence be used in palaeolimnological investigations. This thesis sets the groundwork for chrysophyte cyst-related research in Finland, and fills a geographical void in cyst biogeography. Cysts in Finland show great potential, specifically in future pH and spring temperature reconstructions.
  • He, Liye (Helsingin yliopisto, 2020)
    Cancer is a dynamic disease driven by complex molecular and environmental interactions. Therefore, the traditional “one gene, one drug, one disease” strategy is insufficient to treat most cancer patients. Drug combination therapy targeting various molecular mechanisms has become increasingly popular in treating cancer and other complex diseases. In comparison to monotherapy, combination therapy has the following advantages: possibility to reduce the dose of each single drug to minimize toxic side effects; achieving at least additive, multi-targeting effects, or even “greater-than-additive” effects, so called synergy; and reducing the likelihood of treatment resistance. However, even with the advanced high-throughput technologies currently used in drug combination screening, it remains infeasible to test systematically all the possible drug combinations across different cancer types, as the number of combination experiments grows exponentially. In addition, it remains difficult to understand the synergy mechanisms of many drug combinations, which poses further challenges for their clinical approval. Therefore, there is a timely need for computational tools that can help in identifying synergistic drug combinations for each individual patient, revealing the mechanisms of action of the drug combinations in the specific cellular context, as well as discovering potential predictive biomarkers for the synergistic responses in a systematic and reproducible way. In this thesis, I implemented a systematic computational framework for identification and validation of synergistic drug combinations for each individual patient. Firstly, I developed machine learning models to predict drug combination effects by utilizing drug-target interaction networks, drug sensitivity profiles as well as genomic profiles of each patient. The models further enable one to identify both synergistic and cancer-selective drug combinations specific for each patient, therefore avoiding broadly toxic combinations. Secondly, to experimentally validate the predicted synergistic drug combinations, I developed software tools to help designing multi-dose drug combination experiments, to facilitate the semi-automated drug screening process, as well as to analyze the high-throughput drug combination screening data. Thirdly, I demonstrated the potential of constructing patient-specific cancer vulnerability networks to investigate the mechanisms of action of the personalized drug combinations, which is of importance to accelerate anticancer therapy discovery for precision oncology. All of the computational tools developed in this thesis are distributed as open-source tools, making it possible for others to reproduce the results and apply to their data, or to extend the tools to new applications, as well as to integrate the tools as part of in-house drug combination analysis pipelines.
  • Ruokolainen, Satu (Helsingin yliopisto, 2020)
    The flowering process of plants is of great importance - both for the sexual reproduction of plants and human nutrition. Floral diversity has fascinated scientists for centuries, but the first important steps towards explaining the molecular puzzle of flowering were taken only thirty years ago when plant MADS box genes were discovered, and the classical ABC model was proposed. The focus of research has shifted from studying single genes to working on whole networks of genes and proteins - genomics and proteomics. The genomics of Arabidopsis (Arabidopsis thaliana), the well-known model plant, is advanced, but phenomena present in Arabidopsis might be lacking in other species, and vice versa. The model plant, Gerbera (Gerbera hybrida), belongs to the large Asteraceae (sunflower) family. While Gerbera flowering process shares several features with Arabidopsis and other model plants, it also shows great specialization. Gerbera bears a complex inflorescence containing hundreds of flowers of three different types, which differ in morphology and sex. The major aim of this thesis was to characterize Gerbera floral developmental genes, with special interest in genes related to A and E functions. The ABC model and the extended ABCDE model are applicable to Gerbera. It has been previously shown that the Gerbera B and C function genes behave as the model predicts. The E function genes affect the development of the whole flower in Arabidopsis, being redundantly active in all four floral whorls. However, the Gerbera E function genes, GRCD1 and GRCD2, are non-redundant and specialized in their tasks. Based on GRCD4 and GRCD5 expression patterns and PPI data presented in this thesis we proposed them to provide general E function in Gerbera. However, later this hypothesis was shown not to be completely accurate by RNAi transgenic lines that showed GRC4 and GCRD5 to be involved in Gerbera petal development. The ABCDE model proposes that A function genes determine the developmental fate of sepals and petals. This function, however, is the most problematic of the model since a true A function seems only to be present in Arabidopsis. Majority of homologous genes from the other model plants execute only partial functions of AP1. Gerbera contains several genes related to AP1 and its homologues CAL and FUL, but none of them supply the A function in the sense of the ABC model. All Gerbera AP1- and FUL-like genes GSQUAs display wide expression patterns, some of them present in all floral organs. Different GSQUA genes were transformed into Gerbera, but only GSQUA2 overexpression lines produced a recurrent phenotype that was an early flowering, dwarf Gerbera. The function of GSQUA2 was shown to be linked to floral transition. Phylogenetic analysis showed Gh-SOC1 to be distantly paralogous to Arabidopsis SOC1. In contrast to Arabidopsis SOC1, Gh-SOC1 was expressed only in the floral parts of Gerbera and it did not promote flowering but altered inflorescence identity towards vegetative characteristics. Temporal expression pattern late in floral development and floral abundancy suggested Gh-SOC1 to have a role in the late stages of Gerbera floral organ development. The results presented in this thesis add to our understanding of inflorescence and floral development of Gerbera. The ABCDE model is applicable to Gerbera for B and C function, A function does not seem exist in Gerbera and E function is differentiated from Arabidopsis general E function. Despite close sequence similarity to Arabidopsis SOC1, Gh-SOC1 function in Gerbera is related to floral development.
  • Ruhanen, Hanna (Helsingin yliopisto, 2020)
    Cardiometabolic diseases such as metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) are risk factors for cardiovascular disease and type 2 diabetes. NAFLD can be seen as the hepatic manifestation of the metabolic syndrome and obesity increases the disease risk, but also a genetic component plays a role in the development of NAFLD. The I148M variant of PNPLA3 (PNPLA3-I148M) and E167K variant of TM6SF2 (TM6SF2-E167K) have been strongly associated with NAFLD. However, these variants cause a fatty liver without systemic metabolic complications, and TM6SF2-E167K has even been shown to protect from myocardial infarction. New treatment possibilities for cardiovascular diseases have risen from studies of loss-of-function (LOF) variants of ANGPTL3. Subjects lacking ANGPTL3 have increased activity of lipoprotein lipase (LPL), low plasma levels of VLDL, LDL and HDL as well as increased insulin sensitivity. In this thesis study we aimed to elucidate the function of PNPLA3 and TM6SF2 in lipid metabolism of human hepatocytes, and to clarify the mechanism underlying the association between the variants of these genes and increased hepatic lipid accumulation. We also investigated the function of ANGPTL3 in human hepatocytes and characterized the plasma lipoprotein lipidomes of subjects homozygous for ANGPTL3 LOF variants. In these studies, we utilized different lipidomics approaches as well as complementary methods such as microscopy and transcriptomics. We found using labelled lipid precursors that overexpression of PNPLA3-I148M in hepatocytes leads to a net accumulation of unlabelled triacylglycerols (TAGs) when compared to PNPLA3 wild type (PNPLA3-WT) overexpressing or control cells, but the level of newly synthesized TAGs did not change. Closer examination of the lipid species profiles and further experiments led us to the conclusion that PNPLA3 is a remodelling protein that transfers fatty acids from TAG to phosphatidylcholine (PC) and that PNPLA3-I148M performs this function less efficiently, which may lead to increased hepatic TAG levels. The noticed lipid accumulation could also be related to a more extensive association of PNPLA3-I148M to lipid droplets compared to PNPLA3-WT, which was also observed in our study. We mimicked the effect of TM6SF2-E167K by knocking down TM6SF2 in hepatocytes. TM6SF2 depletion increased the level of TAGs and cholesterol esters (CEs) and changed the membrane lipid composition of the cells by reducing the amount of polyunsaturated fatty acids (PUFAs) and increasing the levels of saturated and monounsaturated fatty acids in the lipids. The size of the lipoprotein-like particles secreted by the TM6SF2 deficient cells was reduced, as was β-oxidation of fatty acids. Both of these observations could explain the increased lipid accumulation caused by TM6SF2 depletion. In addition, TM6SF2 knock-down increased lipid turnover and the amount of late endosomes/lysosomes in the cells. Depletion of ANGPTL3 in hepatocytes lead to PUFA enrichment in major membrane phospholipids and CEs, and the production of PUFA-derived lipid mediators was also increased. In addition, the total level of CEs as well as their synthesis was reduced in ANGPTL3 depleted cells. An examination of the lipidome of lipoproteins derived from ANGPTL3 deficient or control subjects revealed that, in addition to reducing the total levels of all lipid classes, ANGPTL3 deficiency modifies the species composition of the core and surface lipids of lipoproteins, which likely reflects the increased activity of LPL. These findings increase the knowledge on how genetic NAFDL caused by PNPLA3-I148M or TM6SF2-E167K variant develops and how ANGPTL3 depletion affects the liver and the secreted lipoproteins. This information provides tools for creating future prevention and treatment strategies for cardiometabolic diseases.
  • Koskela, Sanna (Helsingin yliopisto, 2020)
    At the sensitivity limit of vision, the quantal fluctuations of light and neural noise in the retina and the brain limit the detection of light signals. The challenge for vision, as for all senses, lies in separating the weakest signals from the neural noise originating within the sensory system. In this thesis, I studied sparse signal detection in the vertebrate visual system (mouse and frog) at low light levels from single retinal neurons to behavioral performance. First, we determined the sensitivity limit of amphibian color vision at low light levels. Unlike most vertebrates, amphibians are potential dichromats even at night, with two spectrally distinct classes of rod photoreceptors: common vertebrate rods (peak sensitivity at 500 nm) and an additional class called “green rods” (peak sensitivity at 430 nm). We showed that frogs in a phototaxis experiment can distinguish blue from green down to their absolute visual threshold, meaning that they have wavelength discrimination as soon as they start seeing anything. Remarkably, the behavioral blue/green discrimination approached theoretical limits set by photon fluctuations and rod noise, highlighting the sensitivity of the system comparing signals from the two different photoreceptors. Additionally, we show that the amphibian threshold for color discrimination is task- and context-dependent, underlining that sensory discrimination is not universally driven to absolute physical limits, but depends on evolutionary trade-offs and flexible brain states. In the second paper, we studied the impact of the circadian rhythm on the sensitivity limit of mouse vision. The retina has its own intrinsic circadian rhythms, which has led to the hypothesis that the sensitivity limit of vision would be under circadian control. We used a simple photon detection task, which allowed us to link well-defined retinal output signals to visually guided behavior. We found that mice have strikingly better performance in the visual task at night, so that they can reliably detect 10-fold dimmer light in the night than in the day. Interestingly, and contrary to previous hypotheses, this sensitivity difference did not arise in the retina, as assessed by spike recordings from retinal ganglion cells. Instead, mice utilize a more efficient search strategy in the task during the night. They are even able to apply the more efficient strategy at day once they have first performed the task during the night. Measured differences in search strategy explain only part of the day/night difference, however. We hypothesize that in addition there are diurnal changes in the state of brain circuits reading out the retinal input and making decisions. In the third paper, we determined the sensitivity limit of decrement (shadow) detection of mouse vision. Compared with the question of ultimate limit for detecting light, the question of sensitivity limits for detecting light decrements (negative contrast) has been remarkably neglected. We recorded the OFF responses of the most sensitive retinal ganglion cells at dim background light levels and correlated the thresholds to visually guided behavior in tightly matched conditions. We show that compared with an ideal-observer model most of the losses happen in the retina and remarkably, the behavioral performance is very close to an optimal read-out of the retinal ganglion cells. I have shown across visual tasks and in two different species how closely behavior in specific conditions can approach the performance limit set by physical constraints, rejecting noise and making use of every available photon. However, the actual performance strongly depends on the behavioral context and relevance of the task and state of the brain.
  • Kuukka-Anttila, Hanna (Helsingin yliopisto, 2020)
    In addition to their major biological significance, parasites interest scientists due to their importance in agriculture, aquaculture and medicine. In this thesis I look at local adaptation between salmonid fishes and Diplostomatidae parasites as well as genetic differences in rainbow trout resistance and tolerance against Diplostomum sp. parasite infection. I also study phenotypic and genetic correlations between fish resistance and tolerance and other fitness-related traits to find possible trade-offs. The results are combined with the literature on host-parasite coevolution to gain more understanding on how host-parasite dynamics work and how the health, welfare and productivity of the fish could be further enhanced in captive populations. The results indicate local adaptation of parasites and genetic differences in fish resistance against eye flukes in salmonid fishes. The heritability of resistance was estimated to be of moderate degree (h2 = 0.25–0.35) in farmed rainbow trout. There was genetic variation also in fish tolerance against eye flukes, contradicting the hypothesis that tolerance traits should quickly become fixed. Phenotypic and genetic correlations between parasite resistance, tolerance and other fitness-related traits were estimated to find out possible trade-offs, and also for the purposes of the rainbow trout breeding programme. There was no indication of trade-offs between fish resistance and the other studied traits. Fish with lower resistance tended to grow fast during the first months of their life but over the time, parasite-induced cataracts blinded the fish and growth slowed down. Survival was also reduced among the families having lower resistance and more severe cataracts. Yet, a trade-off between fish performance in the absence of parasites and tolerance emerged when parasite load increased. The fish that had the highest weight and the lowest mortality in the absence of parasites tolerated the parasitism the worst. Resistance and tolerance were not genetically correlated, indicating that the two defence mechanisms are independent from each other. In the arms-race of coevolving host and parasite populations, host resistance and tolerance have different consequences. Resistance favours an increase in parasite infectivity and virulence, whereas tolerance does not cause such selection pressure on parasites. The results of my thesis give new information to improve rainbow trout breeding stock. Resistance and tolerance can both be selected for without compromising productivity.
  • Mozumder, Mohammad (Helsingin yliopisto, 2020)
    This doctoral dissertation takes a qualitative case study approach to investigate the social-ecological resilience of a small-scale fishery. The research focuses on small-scale hilsa shad fishery of Bangladesh, intending to contribute valuable information to the global project of sustainable natural resource management. This dissertation's primary goal is to provide state-of-the-art knowledge regarding the status of small-scale hilsa fisheries in Bangladesh, including existing management practices, their constraints, and challenges, as well as highlighting the potential and prospects for co-management as one possible means of enhancing social and ecological resilience. The dissertation is comprised of three original research articles, through which I seek to: (i) understand the social-ecological challenges of a small-scale fishery (article I); (ii) investigate the dynamics of, and needs for enhancing, social resilience of coastal fishing communities, including the implications of over-fishing (article II); and (iii) analyze governance and power dynamics that are considered essential for managing a small-scale fishery sustainably (article III). This dissertation draws on theories of social-ecological systems (SESs), driver-pressure-state-impact-response (DPSIR), social-ecological resilience (particularly social resilience), the power cube, and co-management as analytical frameworks. The dissertation's main findings are presented according to the key issues addressed in each article as follows. (i) Understanding the social-ecological resilience of a small scale fishery: The study shows that the main driving forces causing decreased catches for fishers are the use of illegal fishing gear, overpopulation in the coastal areas, overfishing, the harvesting of juveniles, river water pollution, climate change, upstream dam construction, and cross-border smuggling of hilsa. All these factors compromise sustainable management strategies and have led to disincentives for compliance and increased pressures on hilsa fishery. These pressures include habitat destruction and biodiversity loss, which in turn result in a reduced hilsa catch, poverty, malnutrition, stakeholder conflicts, insecurity, and social tensions. To address these challenges, multilevel responses are recommended for ensuring the sustainability of hilsa fishery, including enhancing the social resilience of the fishing community, increasing incentives for compliance for all fishers and major stakeholders at the ground level to support hilsa conservation, promoting community-supported fisheries, improving financing mechanisms for the fishers, and introducing hilsa-fishing-based eco-tourism. (ii) Enhancing the social resilience of the coastal fishing communities: The findings of the study reveal that while the establishment of hilsa sanctuaries has improved the previously low ecological sustainability of local small-scale fishing, the current hilsa fishery management challenges the social resilience of hilsa fishers by creating new inequalities in the distribution of power and privilege, particularly in terms of how seasonal fishing bans are enforced and compensation distributed for income lost during the fishing ban periods. Based on these findings, specific measures are suggested to strengthen social resilience at the local level, including building community networks, developing community infrastructures, updating existing rules and regulations, providing alternative means of generating income for fishers during low-catch periods (natural disasters or fishing bans), and more active sharing of responsibility between stakeholders and government for management of hilsa fishery. These findings also stress the need to move beyond top-down rules and regulations to a system of co-management to improve social resilience among resource-dependent communities. (iii) Governance and power dynamics in a small-scale fishery: The analysis here displays an imbalance in the present hilsa fishery governance structure, with some stakeholders exercising more power than others, sidelining fishers, and encouraging more non-compliance in terms of illegal fishing that ultimately harms both fishery and its users. To overcome this issue, a co-management system is proposed as essential for dealing with the power asymmetry among hilsa fishery stakeholders to ensure effective hilsa fishery governance. The results of this study suggest that the recognition of analyzed power dynamics has substantial implications for the planning and implementation of co-management regimes and the long-term sustainability of hilsa fishery. Using the DPSIR framework, the findings of this dissertation bring forth the cause–effect interactions between human pressures and environmental components and thus contribute valuable knowledge for fishery managers and policymakers. In so doing, it helps to bridge the gap between research and decision-making for hilsa and other fishery. This work also lays out some necessary foundations for a social resilience framework by identifying key issues underpinning social resilience and moving beyond a reduced focus on environmental management rules to the more holistic perspective of co-management. Finally, the findings help to understand the power dynamics that set the context for and exacerbate the persistent marginalization of resource users, especially in developing countries. Although this study focuses on Bangladesh’s four coastal fishing villages, the results are potentially applicable across a broader perspective with a similar tropical context.
  • Raulinaitis, Vytautas (Helsingin yliopisto, 2020)
    Staphylococcus aureus is a pervasive pathogen, whose infections frequently result in serious medical complications and death. Its encounters are yet more perilous in clinical settings where professional care and financial resources alone do not suffice to ensure successful treatment results. The virulence of the bacteria is enforced by numerous cellular mechanisms that have allowed it to develop resistance to every drug used to this date. The bacterial cell wall (CW) is the primary line of defense, the most common target in treatment strategies, and is likely to remain the prioritized candidate for future therapeutic solutions. The main structural component of bacterial CW is peptidoglycan (PG) that forms protective layers. PG is administered by a large number of enzymes that are involved in its synthesis, maintenance, and cleavage. One family of enzymes, M23 peptidases, cleaves pentaglycine bridges that link chains of PG and are specific to S. aureus. These enzymes can be used by the bacteria to manage its own PG in a controlled manner or, alternatively, by hostile microorganisms and cause cell death. Therefore, M23 peptidases of S. aureus are important as potential targets for drugs as well as pharmacological tools themselves that are already employed by nature. Substrate recognizing SH3b domains enhance the effectiveness of M23 endopeptidases. Previous research had identified a putative M23 peptidase gene, transcription of which is upregulated under S. aureus exposure to compounds harmful to cell wall. We examined and characterized the product of the gene. The protein, which we named LytU, is an M23 family zinc-dependent enzyme that cleaves pentaglycine. It is anchored in plasma membrane and is extracytoplasmic, residing in a periplasm-like space. The physiological role of LytU is not confirmed, but evidence suggest it can recycle PG fragments and participate in daughter cell separation. A distinct feature of the enzyme is its ability to strongly bind a second zinc ion, which incapacitates catalytic residues. We propose that together with pH, the binding of second ion serves a regulatory function in situ. Solution structure of the LytU catalytic domain has been determined. Binding of substrate pentaglycine to catalytic M23 domain is very transient at least in vitro. The binding, nevertheless, is accomplished by SH3b domain of enzymes bearing it. Contrarily to previous beliefs, we found that SH3b domain binding to substrate is primarily driven by interactions with PG branching peptides, rather than by weaker interaction with pentaglycine. The binding of SH3b to substrate is independent of catalytic domain and it targets and binds the PG peptide moieties that are proximal to but different from the pentaglycine cleaved by catalytic domain. In summary, we have introduced and characterized a new M23 family endopeptidase, proposed a regulation mechanism, and changed the paradigm of substrate binding by M23 peptidases. Our results are expected to contribute to a better understanding of S. aureus physiology and provide means for the development of cures.
  • Oja, Sofia (Helsingin yliopisto, 2020)
    Mesenchymal stromal cells (MSC) are a potential tool for cell-based therapies subject to intensive investigation. MSCs display immunomodulatory functions and a broad differentiation capacity, and thus have several potential therapeutic applications, such as treatment of immunological disorders and correction of tissue defects. MSCs have been widely studied and utilized in treatment of Graft-versus-Host disease, a severe complication of stem cell transplantation, and have also been evaluated in treatment of Crohn’s disease, multiple sclerosis, and chronic inflammation. The repair of bone and cartilage defects is another application of significant interest. MSCs are classified as advanced therapy medicinal products (ATMPs), which are manufactured under supervision by regulatory authorities. The aim of regulation is to produce safe and effective medicinal products, and therefore safety risks and sources of alterations or impairment of functionality should be evaluated carefully. MSCs are somatic cells, which can be isolated from various sources, such as from bone marrow, adipose tissue, or umbilical cord. For production of medicinal products, MSCs are usually expanded extensively in cell cultures. Culture conditions are known to affect to the characteristic and functionality of MSCs. Animal-derived components during culture such as bovine serum are to be avoided if possible, due to risks of immune reactions and zoonotic infections. Therefore, animal serum-free culture medium is considered safe option for MSC cultures. This thesis focuses on determining optimal expansion and differentiation conditions for clinical-grade bone marrow-derived MSCs by using platelet-derived culture medium supplements to replace bovine serum. This study also aimed to improve quality of MSCs products by introducing an imaging-based screening method to detect aging-related morphological changes in MSC cultures. Finally, we investigated how additional freezing steps during the manufacturing process affect the basic manufacturing parameters and alter the cellular aging process. We found in this study that platelet lysate with two freeze-thaw cycles effectively supported MSC expansion and maintained their functionality in ambient oxygen concentration. Platelet lysate also promoted osteogenic differentiation at least equally with bovine serum in two-dimensional plate culture and slightly better if a three-dimensional matrix was used. We were able to detect and quantify aging-related morphological changes from MSCs cultures and found that a rapid increase in cell size reflects the expression of aging markers. We found that freezing at early phases of cell cultures did not alter the characteristics or functionality of the MSC. This study has yielded insights into the establishment and scaling up of animal-serum free MSC cultures. In addition, our screening method for aged cells could be implemented into the clinical-grade manufacturing of MSCs to monitor cell quality during processing.
  • Pentinmikko, Nalle (Helsingin yliopisto, 2020)
    Aging is a biological process where tissue functions deteriorate leading to morbidity. Gastrointestinal tract is a key system for organismal health converting external energy sources to a useful form for the body. With age, intestinal function declines in humans as the absorptive capacity decreases and recovery from damage slows down. Absorptive epithelium is constantly renewed by actively proliferating intestinal stem cells (ISC) that are intermingled between specialized secretory cells named Paneth cells. These cells are located at the bottom of intestinal crypts that forms a specialized microenvironment, the stem cell niche, participating to the maintenance of ISCs and their function. What is the role of the niche in age related changes of ISC function remains poorly characterized. In this dissertation age-associated changes in the communication between ISCs and the neighboring Paneth cells are analyzed and the outcome for regenerative capacity and homeostatic maintenance of the epithelium is studied. Under homeostasis, old epithelium is functional, but ISCs are biased to differentiate towards the secretory lineage. Using in vitro organoid culture, we show that reduced regenerative function of old epithelium is due to defects in both ISCs and the supporting Paneth cells. Aged Paneth cells produce excess amount of Notum, a secreted Wnt-inhibitor, that reduces the canonical Wnt-activity in the neighboring ISCs. Moreover, old crypts have enlarged morphology reducing the physical curvature of the niche, and potentially increasing the size of ISCs in vivo. Alterations in size and shape of old ISCs can contribute to decreased capacity to receive signals from the niche. Supplementation of Wnt-ligands, genetic ablation of Notum or culture on bioengineered topologically young scaffolds improved the regenerative growth of old epithelium in vitro. Moreover, inhibition of Notum in vivo with a small molecule ABC99 enhanced recovery of old intestine from 5-Fluorouracil induced chemotherapeutic insult. These findings demonstrate that Notum inhibition could be used as a prophylactic treatment to reduce harmful side-effects in elderly patients undergoing chemotherapy. In addition, cellular shape as a facilitator of cell-cell communication in the ISC niche suggest that modulation of the tissue topology could result in enhanced stem cell function.
  • Kilpi-Koski, Johanna (Helsingin yliopisto, 2020)
    In this thesis we studied the effects of Chromated Copper Arsenate (CCA) compounds in Finnish forest ecosystems. The research site was located in Hartola, southern Finland. Between 1958 and 1966, there was a small wood impregnation plant which used the so-called Lahontuho K-33 impregnating agent. The aim of this thesis is to use a combination of applied bioassays, uptake and elimination kinetics and binary mixture toxicity experiments to gain better understanding of the bioavailability of Cu, Cr and As and their potential eco(toxico)logical risk at the Hartola site. This approach fits the TRIAD method, which uses a combination of chemical analyses, bioassays and field observations to assess the actual risk of contaminated soils. The thesis contains three articles that approached the risk assessment of CCA compounds using different methods. In article I, bioassays were used to assess whether Cr, Cu and As are bioavailable to plants (Lemna minor and Lactuca sativa) and invertebrates (Enchytraeus albidus and Lumbricus rubellus) and whether they pose a risk to the ecosystem. Article II focused on the bioavailability of chromium, copper and arsenic in Hartola soils using an uptake and elimination kinetics approach with the earthworm Eisenia andrei. Article III determined the toxicity of the CCA metals, single and in binary mixtures, to earthworms (Eisenia andrei), in particular using reproduction as the endpoint. Based on the studies in article I, it was concluded that the CCA metals in the Hartola soils cause damage to organisms and pose a risk to the ecosystem. Metals did bioaccumulate, as shown by the metal concentrations in earthworms and plants. The toxic effects obtained in bioassays correlated best with the concentration of arsenic in the Hartola soils. In article II, earthworms were shown to take up and excrete chromium and copper in the same way: fast uptake and fast elimination occurred reaching steady state within 1 to 3 days. Arsenic uptake in the earthworms, however, was slow and did not reach equilibrium. Uptake and elimination rate constants were used to calculate the bioaccumulation factor (BAF), which was highest for As, supporting the conclusion of article I. In article III, at low concentrations copper and chromium had a hormetic effect on earthworm reproduction, which was not seen with As. Arsenic was already toxic to the earthworms at low concentrations. Arsenic-containing mixtures also were highly toxic. The binary mixtures of CCA metals generally acted antagonistic on earthworm reproduction when tested against to the Concentration Addition model. It seems that the CCA metals react with each other, leading to a reduced toxicity in the mixtures. The results of this thesis showed that at the study site the metals were bioavailable as shown from their bioaccumulation in invertebrates and plants, and that the risk of CCA pollution is mainly due to As. Based on our results we suggest that the TRIAD method is a useful approach to determining the potential eco(toxico)logical risk of CCA metals.
  • Tsur, Itamar (Helsingin yliopisto, 2020)
    The evolution of whales into fully aquatic, deep-diving animals, which began over 50 million years ago, entailed a dramatic change in their sensory environment and a corresponding reorganization of their sensory systems. For hearing, this had two major consequences. (1) The physics of sound transmission from water into the inner ear required a complete redesign of the sound-transmitting structures (outer and middle ears) of their terrestrial ancestors, which were exquisitely tuned for hearing in air. (2) In the dark deep-sea environment, hearing overtook vision as the primary sense for object detection and localization at a distance. Toothed whales (Odontoceti) are crucially dependent on hearing as they use active high-frequency sounding (echolocation) to localize prey and predators and to communicate with conspecifics. The present thesis addresses aspects of both sound transmission (1) and sound localization (2) in odontocetes. The first aim was to elucidate the mechanical functioning of the middle ear as part of the transmission chain from sound-receiving head structures to the inner ear. The odontocete tympano-periotic complex (TPC) differs significantly from temporal bone complexes of terrestrial mammals. We studied 32 pairs of formaldehyde-glutaraldehyde-fixed TPCs of the short-finned pilot whale Globicephala macrorhynchus. The distribution of vibration amplitudes on the TPC was measured by Laser Doppler Vibrometry while vibrations at different frequencies were applied at a point near the exit of the acoustic nerve. The results suggest that the tympanic plate acts as a lever amplifying the force driving high-frequency vibrations (> 12 kHz) into the inner ear through the ossicular chain. The second aim was to assess whether there exists asymmetry between the left and right middle ears with respect to critical ossicular parameters, and whether this may help in localizing targets in the vertical direction. The malleus, incus and stapes on both sides were weighed with μg accuracy and their lengths as well as the stapedial footplate area measured with 10 μm accuracy. Further, the incudo-stapedial angle was measured from computer tomography images. Significant differences (left values > right values) were found in the weights of the ossicles and in the incudo-stapedial angle. Modelling the amplitude responses of the two ears shows that these asymmetries result in an elevation-dependent difference signal that may support localization of a sound source in the vertical direction. When relating the asymmetry found in Globicephala to earlier reports of ear asymmetries in mammals and birds (one bat species and several owls hunting in the dark), I hypothesized that asymmetry evolves to support sound localization in species that hunt in darkness in three-dimensional space (whether in air or water). As a pilot test of this working hypothesis, we carefully dissected the middle ears and measured ossicular parameters with high accuracy in two mammals, where left-right asymmetry, if present, would necessarily implicate other factors. (1) The sheep, as a close relative of the immediate terrestrial ancestors of whales, might reveal if whale asymmetry depends on phylogenetic heritage. (2) The cat, as a dark hunter active mainly in two dimensions, might reveal if asymmetry is a more general property of nocturnal predators. We found no signs of asymmetry between the middle ears in either of these species.
  • Pljusnin, Ilja (Helsingin yliopisto, 2020)
    The speed of DNA and RNA sequencing has long ago surpassed the capacity of laboratories to assign function to these sequences by direct experiment. Fortunately, function and other information can be effectively transferred to novel data from previously accumulated knowledge by sequence homology. This has resulted in the development of hundreds of novel homology-based methods. However, the tendency of method developers to be overoptimistic about their own results, biases in the evaluation metrics used to rank methods, inconsistency between different rankings and evaluation metrics, misplaced popularity of methods relative to their performance all indicate that, in many cases, clear knowledge of the comparative performance of different methods is lacking. This has two main consequences. First, researchers use suboptimal tools. Second, method development may go astray because the merits used for guiding method optimization are biased or unclear. To avoid these difficulties, further research is needed into methodology of evaluation and comparative studies. One core approach for transferring function by sequence homology is to create a multiple sequence alignment (MSA) that represents a given group of similar sequences. The resulting alignment can be applied to annotate novel sequences using profile hidden Markov models (HMMs), to create phylogenetic trees or to compare structural features. The application of MSAs and profile HMMs for genome annotation was explored in publication (I). Creating MSA has been addressed by a vast field of research, however there is a lack of independent comparative studies and no comparative studies for alignment strategies. In publication (II) a novel modular MSA aligner was implemented to aid in comparative evaluation of different MSA strategies. Different MSA strategies were then compared to each other and to the state-of-the-art MSA software on three benchmark databases. Another core approach has been to combine homology searches with assignment of annotation terms from a controlled vocabulary such as the Gene Ontology (GO). Hundreds of methods that assign GO terms to novel sequences have been introduced. The research community has also invested into the objective evaluation of these methods via third party competitions. However, the evaluation metrics and merits used in these competitions are still under active debate and need further research and development. In publication (III) a novel framework was introduced for the development of unbiased high-quality evaluation metrics. By testing 37 variations of popular metrics, our approach revealed strong differences between metrics, a list of clearly biased metrics, and a list of high-quality metrics that are well suited for the evaluation of GO annotations. In summary, this thesis presents novel frameworks and implementation platforms for comparative evaluation of two important classes of homology-based methods: MSA aligners and GO sequence classifiers. These results will be instrumental for developing more accurate MSA aligners, for eliminating many forms of bias inherent in contemporary evaluation protocols, for producing informative method rankings for non-specialist users and for guiding method development towards merits that truly reflect the utility of the designed tools.
  • Tiili, Emmi (Helsingin yliopisto, 2020)
    Nicotine dependence is a major cause of tobacco-related illnesses. Although tobacco smoking continues to be the leading cause of preventable death, and although the harmful health effects resulting from it are well known, about 20% of the global population continues to smoke. The addiction-causing substance in tobacco, nicotine, acts through dopamine pathways in the brain to produce several pleasurable experiences as a result of cigarette smoking. There are significant individual differences in how nicotine affects the body, and these differences in both the neurological and metabolic pathways of nicotine may determinate the degree of the subsequent nicotine addiction. Thus, both genetic and epigenetic factors related to nicotine metabolism and nicotine-induced dopamine metabolism can have an important role in influencing individual smoking behaviour. In this thesis, in addition to the potential nicotine metabolism-related gene, CYP2D6, a few selected genes acting in dopamine routes, namely, MAOA, MAOB, and SLC6A3, were genotyped for their several known variants, together with their selected DNA methylation sites. The polymorphisms and variations in DNA methylation levels of the genes included in the study were examined in relation to numerous smoking-related phenotypes in a well characterized study population of 1,230 Caucasians of Russian origin. The aim was to discover novel relations among several smoking-related phenotypes and methylation of the studied genes, as well as to verify some findings of the previous genetic studies on this topic. Our results indicate that the poor metabolic capacity associated-CYP2D6 genotype, defined by the data derived from eight functional polymorphic sites of the gene, is related to cigarette consumption such that the carriers of this genotype have reduced risk of heavy smoking compared to the carriers of the extensive metabolism-associated genotype. We also discovered that the poor metabolizer genotype occurred more frequently with higher methylation values, which was found to be inversely related to heavier smoking. The studies on dopamine related genes revealed that the MAOA 1460 T variant allele, variable number of tandem repeat (VNTR) high-activity alleles (3.5R, 4R, and 5R), and MAOB int13 common G allele were more prevalent in female smokers than non-smokers. Additionally, the 5R allele of 30bp VNTR and the 9R allele of 40bp VNTR in SLC6A3 were associated with negligible smoking history. Moreover, although the carriers of 9R allele had somewhat higher Fagerström Test for Nicotine Dependence (FTND) scores, they were also more likely to successfully cease smoking. When methylation levels were explored, we found that current smokers were 2.5 times more likely to have increased SLC6A3 mean methylation levels compared to ex-smokers, whereas in MAOA and MAOB genes the methylation levels of single CpG sites were independently related to smoking behaviour. In addition, some of the alterations in DNA methylation levels at individual CpG sites showed a high degree of dependence on the genetic polymorphisms studied, while others did not. As expected, age and gender were found to be major contributing factors in determining the DNA methylation levels. Our findings indicate that the CYP2D6-related metabolic capacity may be associated with cigarette consumption both through genetic and epigenetic mechanisms. Additionally, our results suggest that both the genetic and the epigenetic profiles of MAO and SLC6A3 genes modify the availability of dopamine and thereby shape the risk of developing nicotine dependence. Due to the complexity of nicotine addiction and the related phenotypes, more high-quality research is needed to verify these novel findings.

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