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Tiivistelmä – Referat – Abstract Telomeres are cap shaped structures at the very end part of each chromosome that protect DNA from degradation or unwanted chromosome-chromosome attachments. Telomere lengths show considerable heterogeneity in different cells of the same cell population. Reasons for heterogeneitiy and mechanisms inside cells causing them are not fully understood. In this study, we explored the correlation between telomere length and different gene expressions. First, using FACS technique we sorted each single cell into each well of 96-well plate. Second, we used SYBR green based qPCR for telomere length measurement. Third, we used Illumina-seq for sequencing extracted mRNAs. [6] We found a set of genes that were in strong correlation with telomere length, giving opportunity to explore the biological pathways. We compared pathways between different samples and found strong connections between genes involved in viral cycles and immune system with extracted genes that were in high correlation with telomere. We found heterogeneity of telomere lengths and transcriptomes in different cell lines. Telomere related proteins, specifically those involved in shelterin complex, are expressed highly in cancer cell lines and LPS-stimulated monocytes compared to the non-stimulated monocytes. In our study, SLC38A2, PURB, UBR3, SSR1, NCAPH2, AIMP2, PHF21A genes were highly correlated with telomere in mutual way and can therefore be considered as new biomarkers/novel candidates for telomere-related studies. The importance of these genes has been reported in aging/mortality. Concurrent with our findings, a recent report also suggested that NCAPH2 plays role in regulating telomere stability and maintenance through its interaction with TERF. [65] We found new genes in correlation with telomere regulation, and our findings are therefore of high importance in research of cancer, neurodegenerative diseases and aging. Further studies are, however, required as our data is limited by small number of samples and inability to properly validate our technique.

Adolescent ill-being has in recent years become a prominent health concern globally. Ill-being during adolescence can have negative consequences for future health and wellbeing, as important patterns of health are formed during this time. This highlights the importance of early identification of risk factors and overarching patterns of mental and physical ill-being and arguments for early intervention during adolescence. The aim of this study was to identify risk factors and co-occurrence of subjective ill-being symptoms in the form of depressive symptoms and subjective health complaints. This study also examined whether the subjective ill-being of students was reflected in cortisol patterns in a naturalistic setting. This since stress has been identified as a key etiological factor in ill-being, through the damaging effect of prolonged exposure to elevated cortisol levels. By applying a novel measure of school atmosphere, the study also aimed to examine the potential protective role of the social atmosphere in school on subjective ill-being and cortisol levels. A total of 329 students from eleven Finnish-Swedish upper secondary schools participated in the cross-sectional study by answering a questionnaire. The salivary cortisol samples were collected from a subsample of the participants, with of a total of 209 participant that met the salivary sampling criteria applied in the study. The methodological framework for the statistical analysis of the study consisted of independent samples t-test, ANOVA, Pearson’s correlation, and multiple linear regression. The results showed a higher prevalence of ill-being in girls and second year students. A significant co-occurrence was found for the subjective ill-being measures of depressive symptom and subjective health concerns. The subjective ill-being was however not reflected in the daily cortisol patterns of students in a naturalistic setting. Furthermore, a positive school atmosphere was significantly negatively associated with subjective ill-being of student in the form of depressive symptoms and subjective health complaints. When controlling for covariates, the subjective meaning of school experienced by the students was identified as a significant protective factor against symptoms of ill-being. These findings identify students in need of additional support and highlights the need of applying an overarching view on student ill-being in future adolescent research. Since no associations was found between daily cortisol patterns and subjective ill-being this study contributes to the understanding of HPA axis in early disorder onset. This study also highlights the importance of subjective meaning in a school context and posits increasing the subjective meaning as a prominent strategy to decrease ill-being among Finnish-Swedish upper secondary school students. Further studies are however needed to assess the causality and to examine these relationships further.

Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER, which leads to the activation of unfolded protein response (UPR) through three transmembrane protein sensors located in the ER membrane. The sensors correspond to three branches of the UPR, namely protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) branches. Upon ER stress, IRE1 dimerizes and oligomerizes, and its endonuclease domain is activated. It specifically targets X-box-binding protein 1 (XBP1) mRNA, from which a 26 nt intron is spliced. This allows a complete translation of spliced XBP1 mRNA into a functional protein that acts as a transcription factor. Together with the other pathways, the UPR leads to a decrease in the protein folding load by causing a reduction in the general level of protein translation, and by inducing the expression of protein folding machinery. However, if the UPR is activated continuously for a long time, the apoptotic pathway will be triggered, and the cell will die. ER stress and UPR are associated with various disorders, such as some types of cancer, diabetes, chronic inflammatory syndromes, and particularly neurodegeneration. For example, in Parkinson’s disease, it was suggested that prolonged ER stress induces the extensive apoptosis of dopaminergic neurons in substantia nigra pars compacta region of the midbrain. This hinders the normal functioning of the nigrostriatal pathway, and hence results in the progressive development of Parkinson’s motor symptoms. In order to study the regulation or IRE1 branch of the UPR, and to identify the ER-stress-modulating compounds, a human luciferase reporter cell line (XBP1-NLuc) was created in this work. The reporter was expressed when IRE1 splicing was activated, since the XBP1 intron fragment was fused to the Nano luciferase gene. The expression of the reporter was observed with luciferase assay at several time points during treatments. The treatments were done with ER stress inducers thapsigargin and tunicamycin, and with IRE1 inhibitors KIRA6 and 4μ8c, or the combination of those. Quantitative PCR (qPCR) was used to validate the expression of the reporter and to monitor the expression of the other branches of the UPR. Additionally, the oligomerization of IRE1 was observed with IRE1-GFP cell line that was treated identically to the XBP1-NLuc cell line, fixed, stained for nuclei, and imaged with fluorescent microscopy. After imaging, the IRE1-GFP clusters were analysed and quantified with CellProfiller and CellAnalyst softwares. Both cell lines were used to test the effect of neurotrophic factors CDNF, MANF, and MANF mutant isomers on the UPR with and without tunicamycin treatment. Collectively, the experiments confirmed that XBP1-NLuc cell line was created successfully and that it accurately reports IRE1 splicing activity. As expected, ER stress treatment increased the reporter expression, while IRE1 inhibitors decreased the expression of the reporter. qPCR revealed that the other observed UPR markers were activated as well upon thapsigargin treatment, however, they were not decreased with the treatment with IRE1 specific inhibitors. In line with XBP1-NLuc cell line, the IRE1-GFP cell line demonstrated an increased oligomerization of IRE1 upon ER stress induction. The KIRA6 inhibitor of IRE1, which prevents IRE1 oligomerization, decreased the formation of IRE1-GFP clusters. Additionally, the IRE1-endonuclease-activity inhibitor 4μ8c induced the formation of IRE1-GFP clusters. Curiously, the distribution of the intensity of IRE1-GFP clusters was bimodal and could point to two manners of IRE1 clustering and/or activation. Together, the experiments done with cells transfected with CDNF, MANF or MANF mutants, suggested that the tested neurotrophic factors decreased IRE1 oligomerization and its activation. However, there were substantial problems in the quantification of viable cells, which should be considered in the interpretation of these results. No significant difference among the tested neurotrophic factors was observed. In conclusion, the XBP1-NLuc reporter cell line provided a reliable reporter of IRE1 endonuclease activity, whose expression is increased during the ER stress. Together with IRE1-GFP cell line, it revealed the amount of IRE1 oligomerization and activation under various treatments and at different time points relative to treatments. Due to the effectiveness and accuracy, the XBP1-NLuc cell line can be further used in studying the regulation and activation of IRE1, as well as for the identification of ER-stress modulating molecules, which can be used for development of novel treatments for ER stress associated diseases, such as Parkinson’s disease.

Normal sex differentiation depends largely on the healthy development of the bipotential gonad, which is identical in both sexes during early stages of embryonic development. Sex differentiation towards the female phenotype is initiated by the expression of pro-ovarian genes, among which Forkhead Box L2 (FOXL2) is an important regulator. Moreover, FOXL2 was found to be one of the genes most widely implicated in female disorders of sex development (DSD). However, there is a lack of understanding regarding its precise role during ovarian differentiation and development. In order to study the gene during early gonadal development, human embryonic stem cells (hESCs) were used as a model. An inducible FOXL2 activation line was generated in vitro, by applying the CRISPR/Cas9 technique in combination with the tetON and destabilized DHFR systems. The cells were also subjected to gonadal differentiation, based on a previously established protocol. The results showed that the establishment of the activation line was successful, and expression of FOXL2 could only be observed in cells that were treated with trimethoprim and doxycycline. Similar findings were observed in the differentiated activator cells, as again only the induced cells expressed FOXL2. On the other hand, both induced and non-induced differentiated cells showed expression of bipotential gonadal marker genes LHX9, EMX2, GATA4 and WT1. However, in the induced cells a lower relative expression of these markers could be observed. Therefore it seems that relative expression of bipotential gonadal markers was affected by FOXL2 activation. The expression of female gonadal marker genes RSPO1, FSHR, WNT4, AMH and FST was not influenced by FOXL2 activation during gonadal differentiation, as most of the markers showed similar levels of expression in both induced and non-induced cells. Therefore further research needs to be conducted to determine optimal time point of FOXL2 activation during differentiation. Nevertheless, an in vitro model could be generated, which could help in the future to further study the role of FOXL2 in gonadal differentiation, and to better understand pathological mechanisms underlying female DSDs.

Animals regulate their metabolism dynamically as a response to changes in nutritional landscape. Intestine is emerging as a key regulator of systemic metabolism. It possesses secretory enteroendocrine cells (EECs), which have a central role in intestinal nutrient sensing and signaling. However, how the number and function of EECs is regulated in response to nutrients remains poorly understood. Previous work in Hietakangas lab has shown that a transcriptional cofactor, C-terminal binding protein (CtBP), regulates the number of EECs in response to sugar feeding and loss of CtBP function in EECs causes sugar intolerance in Drosophila. CtBP’s transcriptional activity is modulated through homodimerization, which is controlled by redox coenzyme NAD+/NADH, whose levels are dependent on sugar metabolism. Therefore, I hypothesise that CtBP is a sugar- and redox-responsive regulator of EEC function. In this thesis, I aimed to understand how CtBP is regulated and what are its downstream effectors. My results show that the formation of CtBP homodimers is responsive to dietary sugars and cellular redox state. In addition, I observed that CtBP heterodimerizes with EEC fate determining transcription factor Prospero. Functional analysis of CtBP downstream effector genes shows significant overlap with those of Prospero. In conclusion, CtBP is a sugar- and redox-responsive cellular regulator of EEC function, which acts in cooperation with Prospero.

Background: Epithelial ovarian cancer is the most common type of ovarian cancer and is the most lethal gynecologic cancer due to its late diagnosis. Compared to ovarian cancer, endometrial carcinoma, as the most common gynecologic malignancy, is referred to as the “curable cancer”, as it can be detected early. As aberrant promoter methylation patterns are a common change in human cancer, detection of promoter methylation status may help in early diagnosis. In this study, we used a custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay as a rapid and easy method, to simultaneously detect the methylation status of multiple genes in ovarian and endometrial cancer samples. Aims: To design and test an MS-MLPA assay for analyzing promoter methylation of four genes associated with ovarian and endometrial cancers. The selected genes were HNF1 homeobox B (HNF1β), Ten-eleven translocation 1(TET1), L1 cell adhesion molecule (L1CAM), and AT-rich interactive domain 1A (ARID1A). These genes are known to have expression changes by DNA methylation. Methods: The promoter DNA methylation patterns of these four genes were analyzed in 15 cancer cell lines and 5 normal cell lines and DNAs using bisulfite sequencing. Six synthetic probe pairs were designed and optimized by applying them to cancer and normal cell lines and normal DNAs and comparing the results with those of bisulfite sequencing. Finally, the MS-MLPA assay was performed on patient specimens according to the MRC-HOLLAND MS-MLPA general protocol and methylation frequencies were calculated from MS-MLPA data. Results and conclusion: The MS-MLPA assay gave accurate methylation results with the 170 samples assayed. The HNF1B, L1CAM, and TET1 Genes were observed methylated in tumor samples whereas they were not methylated in the normal samples or showed very little methylation, suggested to be favorable diagnostic markers. MS-MLPA robustly and sensitively detects the promoter DNA methylation status.

Aging is the progressive accumulation of cellular dysfunction, stress and inflammation. The mitochondrial network plays a central role in the maintenance of cellular homeostasis, with a growing body of evidence assigning dysfunctional regulation of this network as cause or effect of age-related diseases including metabolic disorders, neuropathies, various forms of cancer and neurodegenerative diseases. Neuronal sensitivity to changes in energy supply and metabolic homeostasis make neurons especially susceptible to alterations in the mitochondrial network. Mitophagy, a specified form of autophagy, is the selective degradation and quality control mechanism of mitochondria by engulfment and fusion with acidic endolysosomal compartments of the cell. Mitophagy has been extensively characterised in cultured cells and short-lived model organisms. However, our understanding of physiological mitophagy during mammalian aging is unknown. This study utilizes mito-QC mitophagy reporter mice that enable in vivo detection and monitoring of mitochondrial turnover due to the distinct physicochemical properties of the tandem GFP-mCherry reporter. Using cohort groups of young and aged reporter mice, age-dependent alterations of mitophagy were quantified in the cerebellum and the outer nuclear layer (ONL) of the retina. Specific autophagy and mitophagy markers were used to assess the longitudinal alterations in the mitophagic landscape. Images of fixed brain tissue sections were attained by high-speed spinning disc confocal microscopy for the quantitative and histological analysis. This study characterises the longitudinal alterations of mitophagy in distinct regions of the central nervous system (CNS) of mitophagy reporter mice, demonstrating tissue-specific alterations in mitochondrial turnover throughout physiological time. Åldrande kan definieras som den successiva ackumuleringen av cellulär dysfunktion, stress och inflammation. I upprätthållandet av cellens funktioner och homeostas har det mitokondriella nätverket en central roll. Omfattande forskning visar att åldersrelaterade sjukdomar såsom neuropati, ämnesomsättningssjukdomar, olika cancerformer samt neurodegenerativa sjukdomar föranleds av mitokondriell dysfunktion. Neuroner är beroende av oavbruten energitillförsel och upprätthållen metabolisk homeostas, vilket gör dem speciellt mottagliga för förändringar i det mitokondriella nätverket. Mitofagi är en selektiv form av autofagi som degenererar och kvalitetskontrollerar mitokondrier genom att leverera dem till lysosomer där de bryts ned av hydrolytiska enzymer. Den aktuella kunskapen inom regleringen av och mekanismerna bakom mitofagi baserar sig på gedigen forskning av kortlivade organismer och cellkulturer. Däremot är vår kunskap inom åldrandets inverkan på mitofagi i däggdjur begränsad. I denna studie används musmodellen mito-QC vars rapportörgen består av ett binärt GFP-mCherry-komplex som besitter olika fysikaliska och kemikaliska egenskaper, vilket möjliggör upptäckt och analys av mitofagi in vivo. En kvantitativ jämförelse av mitofagi i unga och åldrande möss genomfördes i vävnadssnitt av cerebellum och av det yttre nukleära lagret av retinan. Specifika autofagi- och mitofagimarkörer användes för att utvärdera de longitudinella förändringarna i mitokondriell degenerering. Bilder för kvantitativ och histologisk analys erhölls med höghastighets spinning-disk-konfokalmikroskop. Denna forskning karaktäriserar de longitudinella förändringarna av mitofagi i definierade regioner av det centrala nervsystemet i musmodellen mito-QC och presenterar vävnadsspecifika förändringar i degenereringen av mitokondrier under åldrandets framskridande.

As water flow encounters an object on the sea floor, its hydrodynamics change. Accelerated currents and vortices develop around the object with changing intensity as a function of distance from its proximity. This leads to erosion and aggradation of sediment, known as scour. Studies focusing on formation processes of scour often involve locating visible scour sites by sonar scanning the geomorphology of the seafloor. However, the effects of scour on macroinfauna and small-scale sediment characteristics are not visible in sonar images. In this Master’s thesis, scour at a shipwreck of a timber-built historic sailing ship, the Joskär shipwreck, was first identified by scanning the study area with side-scan sonar, and by measuring water depth contours around the shipwreck by scuba diving. Sediment samples were then taken inside the area assumed to be under the most pressure from scour. Samples from three separate distances on two transects drawn outwards from the hull of the shipwreck were collected and analysed for sediment grain size, organic content, and species assemblages of macroinfauna. In addition, macrofauna were analysed for individual lengths, number of individuals, diversity index, and functional groups. All samples were collected with a core tube sampler operated by a scuba diver. The methods used in this Master’s thesis widen the concept of scour past the sole physical processes observable with sonar to a more holistic level that considers the quality of biological, geological, and chemical characteristics of the benthic environment. The results of the present Master’s thesis show that the quality of the sediment near Joskär shipwreck varies within a relatively small scale. Organic content of the sediment was the most potent descriptor of scour at the study site, exhibiting a consistent decreasing trend as distance to the shipwreck increased on both sampled transects. Sediment grain size became finer as distance to the shipwreck increased. However, compared to grain size, based on visual observations of the sediment samples, shell debris content of the sediment could possibly act as a better measure of presence of scour. The variability of characteristics of macroinfaunal communities as a function of distance from Joskär shipwreck was not a viable tool to describe the presence of scour, as no consistent trends of the variables were observed. As no control site was included in the study design, the characteristics of the benthic environment inside the scour around Joskär shipwreck could not be compared to the seafloor unaffected by scour. Further research could reveal possible variation between these distinct habitats, and that way produce valuable indicators of scour. The hypothesis in the present thesis was that macroinfaunal assemblages and sediment characteristics would exhibit variation between the sampling sites as a function of distance from the shipwreck. The observed trends of sediment characteristics validated a part of the hypothesis, showcasing the utility of sediment characteristics in describing scour at Joskär shipwreck. However, a part of the hypothesis was rejected, as no consistent trends of macroinfaunal features were present.

Arctic ecosystems face drastic changes in community structure due to warming, shrubification, permafrost loss, and other environmental changes. Due to the spatial heterogeneity of these ecosystems, understanding such changes on a local scale requires high-resolution data. Earth observation using satellite imagery and aerial photography has become a staple in mapping large areas and general patterns. Advances in sensor technology, the proliferation of unmanned aerial vehicles (UAVs), and increases in processing capacity enable the use of higher spatial and spectral resolutions. As a result, more detailed ecological observations can be made using remote sensing methods. In this thesis, I assess how increased spectral resolution affects the remote-sensing based modelling of plant communities in low-growth oroarctic tundra heaths. Based on a large field observation dataset, I estimate biomass, leaf area index, species richness, Shannon's biodiversity index, and fuzzy community clusters. I then build random forest models of these with image data of varying spectral, spatial, and temporal specifications and topographical data. Finally, I create maps of the vegetation. Leaf area index and biomass are best estimated of the response variables, with R2 values of 0.64 and 0.59, respectively, with multispectral data proving the most important explanatory dataset. Biodiversity metrics are best estimated with R2 values of 0.40–0.50 with the most important explanatory variables being topographical and hyperspectral, and community cluster with R2 values of 0.27–0.53, with the importance of various explanatory variables depending on the cluster being estimated. These results can help choose a suitable high-resolution remote sensing approach for modelling plant communities in similar conditions.

In the past few years, there has been an increased consideration on the stem cell niche as a key factor to regulate stem cell maintenance and differentiation. Research on characterization of the stem cell microenvironment boosted after the determination of long-term three-dimensional (3D) tissue cultures, or so-called organoids. Organoids are derived from stem cells which self-organize in 3D multicellular structures upon embedding in an extracellular matrix mimic, such as Matrigel®. Their main advantage is these structures resemble the architectural distribution of the tissue of origin in vivo. Likewise, the cellular components of organoids vary depending on multiple variables as the tissue of origin and the growth factors they have access to. As a result of advances in this technique, some stem cell niches have been well characterized, as in the case of intestinal stem cells (ISCs), while others remain elusive as in case of the human gastric stem cells (hGSCs). Along with the remarkable development of 3D cultures, the interest of ECM proteins in stem cell regulation increased. Matrigel® is a rich matrix composed of several adhesive proteins such as laminins and collagens. Aside from providing structural support, the extracellular matrix (ECM) proteins forming this matrix contribute to cell adhesion and signalling. However, Matrigel® composition cannot be modified or even well-characterized due to its origin from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Additionally, it has been demonstrated that contains a high batch-to-batch variability. Other techniques to study the effects of individual ECM proteins have been used such as coating of tissue culture plates with ECM proteins. However, the biomechanical properties in this model are far from being physiological. Therefore, although preliminary results can be obtained using this technique, results extrapolation to an in vivo model can be questioned. To date, there is a lack of a reproducible, high-throughput and reliable technique to test the effect of ECM proteins on human gastric stem cells behavior. This Master’s thesis presents a novel transwell device containing a polyethylene glycol (PEG)-based hydrogel enriched with human ECM proteins to test their effect on human gastric stem cell regulation. Preliminary results showed that gastric organoid-derived epithelial cells (GODE) grown on hydrogels with ECM proteins that are localized at base of the gastric glands, such as Laminin-211, had a higher stem cell marker expression than the control grown on ECM proteins that are uniformly localized in vivo. Additionally, when GODE were grown on hydrogels containing ECM proteins that are localized at the surface of the native gastric epithelium, expression of surface gastric mucins markers was enhanced. These preliminary results highlight the utility of the optimized transwell device to further shed light on how the human gastric stem cells are regulated and what is the effect of the ECM proteins surrounding them.