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Light is crucial for aquatic ecosystems, as photosynthesis supports the higher trophic levels. Light attenuates in water due to absorption and scattering by optically active substances (OAS), the main ones in coastal environments being chlorophyll-a (chl-a), particulate matter and coloured dissolved organic matter (CDOM). Light attenuation measurements are also important indicators of many environmental changes. Traditionally, Secchi depth has been used to monitor changes in light attenuation. However, Secchi depth (ZS) is a not an accurate estimate of light attenuation, and conversion to light attenuation coefficient (Kd) is needed to study light conditions as aquatic organisms observe them. Therefore, calibration between methods is important. In this study, light attenuation was estimated with Secchi disc, a light meter and light loggers to scrutinize the possibilities of autonomous measurements in this context. The aims of the study were 1) to compare three methods for estimating light attenuation and 2) to identify the environmental drivers affecting the relationships and accuracy of these methods, and 3) to assess the possibility of using conductivity as a proxy for light attenuation. The main hypotheses were that the differences in conversion between ZS and the two sensor-based methods in different concentrations of optically active substances can be explained by contribution of scattering to light attenuation, and that variation in different Kd-estimates is due to the different measurement spectra. The effect of three OASs (chl-a, particulate matter, CDOM) on differences among light attenuation estimates were quantified in Pohjanpitäjänlahti bay. Light attenuation was mainly controlled by CDOM, followed by particulate matter and chl-a. Conductivity was found to be a good proxy for light attenuation. However, there was a mixed signal if the differences among methods could be explained by OAS concentrations or conductivity, as the hypotheses were supported with one device pair but not with the other. Therefore, the differences among light measurement methods might stem from the characteristics of the devices or measurement errors. However, all the methods were found suitable for tracking changes in light attenuation and a summary table of the advantages and disadvantages of each method is presented to help choose a suitable method to estimate light attenuation e.g., in future studies or environmental monitoring.

Ympäristökriisi on yksi aikamme suurimmista globaaleista ongelmista. Voimakkaana tekijänä kriisissä ovat ihmisten valinnat niin arjen kuluttamisessa kuin yleisessä käyttäytymisessä luontoa kohtaan. Ympäristömyönteinen käyttäytyminen kumpuaa luontoyhteydestä, jonka nähdään rappeutuneen nyky-yhteiskunnassamme. Tästä johtuen lisäämällä ihmisten luontoyhteyttä voisimme edistää heidän ympäristömyönteistä käyttäytymistään. Tämä tutkielma käsittelee luontoyhteyden ja ympäristömyönteisen käyttäytymisen välistä yhteyttä. Tavoitteena on hahmottaa, millä tavoin tämä yhteys muodostuu, jotta voimme lisätä ihmisten ympäristömyönteistä käyttäytymistä syvällä ja perusteellisella tasolla. Tutkielmassa pyritään vastaamaan tähän systemaattisen kirjallisuuskatsauksen keinoin perehtymällä aiempien tutkimusten aihepiireihin ja niiden teoreettiseen pohjaan koskien luontoyhteyden ja ympäristömyönteisen käyttäytymisen suhdetta. Katsauksessa löydettiin 38 kappaletta soveltuvia artikkeleita, joita analysoitiin luokittelemalla niitä seuraavin perustein: 1) millaisia yhteyksiä aiempi tutkimus on luonut luontoyhteyden ja ympäristömyönteisen käyttäytymisen välille, ja 2) mitä välittäviä muuttujia luontoyhteyden ja ympäristömyönteisen käyttäytymisen välillä on havaittu aiemmissa tutkimuksissa. Tutkimuksen mukaan aineistosta voidaan muodostaa kolme kategoriaa, jotka ovat: 1) CTN-PEB, 2) x-CTN-PEB, and 3) CTN-x-PEB. Tutkimuksessa syvennyttiin eniten kategoriaan numero kolme, jossa luontoyhteyden ja ympäristömyönteisen käyttäytymisen suhdetta selvitettiin erilaisten välittävien muuttujien avulla. Jatkotutkimusta kausaalisen yhteyden selvittämiseksi tarvitaan.

Reptiles have long been studied in search of the mechanisms behind neuronal regeneration. This thesis delves into the regenerative areas of two emerging model species to the field of regenerative research: Pogona vitticeps (bearded dragon) and Pantherophis guttatus (corn snake). This fluorescent immunohistochemical study maps out and compares the constitutive proliferative zones in these two species to better define the focus of future comparative neurodegenerative experiments. A BrdU pulse chase experiment in conjunction with PCNA reveals proliferative zones in the lateral ventricular ependyma of both species. Stem cell niches were found in the ependymal lining adjacent to the medial cortex and dorsal ventricular ridge in both species, however, the nucleus sphericus ependyma was an active proliferative zone only in Pantherophis. Imaging of further markers in this study support the findings of the pulse chase experiment. High levels of the stem cell marker Sox2 was found in lateral ventricular ependymal cells in both species. The glial marker GFAP reveals a highly ordered array of radial glia in the cortical areas of Pogona, which is significantly reduced or absent in Pantherophis. And lastly the neuronal marker HU was found in the same cells that were BrdU positive and had migrated a short distance from the proliferative zones, which shows that the proliferative areas in the lateral ventricular lining do indeed produce neurons. The BrdU and PCNA marked cells were quantified in both species, and a brief comparison between the species showed that Pogona had a significantly higher number and concentration of proliferative cells in the proliferative zones than Pantherophis. Scattered BrdU positive cells that were neither neuronal nor positive for any other marker were also found scattered throughout the parenchyma of Pogona, and these cells remain uncharacterized. Differences between these two species are not surprising, as lizards are known to have better regenerative capabilities than snakes, however, more comparative research between these species is needed to gain further insight into the mechanisms behind their contrasting regenerative capabilities.

Knowledge co-production has become more widespread in sustainability research as it possesses the potential as a tool to provide policymakers with usable knowledge in collaboration with researchers to increase the impact of scientific knowledge. This thesis complements the current literature by providing empirical insights of the perception of Finnish national-level policymakers who have participated in Strategic Research Councils (SRC) projects. SRC is a funding instrument with the aim to produce high-quality research with societal impact in collaboration with relevant stakeholders. The perception of Finnish researchers is already somewhat covered in literature and therefore the material for this thesis was collected by interviewing non-researcher participants in SRC projects. The analysis of the material was conducted as a theory-bound qualitative content analysis. The first coding was conducted with a material-based approach to allow the perception of the interviewees to prevail. The final analysis is conducted with three different modes of interaction obtained from the literature: science-push, policy demand, and iterative modes used as main categories. The analysis provides a standpoint on the research question: How do policymakers perceive the interaction in the co-production process? Methods to include policymakers and their participation in national-level co-production processes are versatile in Finland. The knowledge co-production between researchers and national-level policymakers results in usable knowledge and furthers policy uptake especially when collaboration and participation are genuine and profound. This study concludes that the inclusion of policymakers in the early stage of research, two-way communication, iterative interaction, and trust building are factors related to successful interaction, as the policymakers perceive success. The success factors could also help to overcome barriers of co-production presented in earlier literature.

Spinal muscular atrophy of Jokela type (SMAJ) is an autosomal dominant motor-neuron disease caused by a missense mutation c.197G>T, p.G66V in the gene CHCHD10. Coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) is a nuclear-encoded mitochondrial protein located in the intermembrane space (IMS) of mitochondria with an unknown exact function and disease-causing mechanism. In this project, the overarching aim was to correct a heterozygous SMAJ-causing mutation in patient myoblast cells with CRISPR-Cas9 genome editing. The goal was to create a genetically identical, isogenic, cell line to study only the effects of the mutation on cellular phenotype in vitro. Human myoblast cells isolated from patient biopsies provide the most pertinent experimental model to study neuromuscular atrophy-associated mutations in their natural genomic environment. More specific aims included genome editing optimization with myoblast cells, since it is not as widely conducted as with some other cell types, such as iPSCs. CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology-directed repair (HDR) in the genome of patient-derived myoblast cells and correct the mutation. After optimization of electroporation conditions for myoblast cells, guide RNAs were designed and transfected into patient myoblasts. Clonal cell lines were made by utilizing techniques such as fluorescence adjusted cell sorting (FACS) and manual colony picking. The success and precision of genome editing were analyzed by Sanger sequencing, comparing the performance of the different guide RNAs with restriction enzyme analysis and Synthego ICE CRISPR web tool, and screening regions of potential off-target genome editing. A genome-edited myoblast cell line with the CHCHD10 c.197G>T mutation corrected, was successfully generated to provide an isogenic control for the patient myoblast cell line. Optimization of myoblast electroporation was successful and conditions used proved to be effective. Clonal cell line creation proved to be challenging with myoblast cells and work is still needed to improve the viability of single-cell clones after FACS. Nevertheless, the advances taken here regarding myoblast genome editing with CRISPR-Cas9 offer a fertile avenue for future research of myoblasts genome manipulation, myogenic disorders, and the role of CHCHD10 in skeletal muscle and SMAJ. Comparing the CHCHD10 protein level and mRNA expression between patient cells, corrected myoblasts, and differentiated myotubes is an area of future research. Future work also includes measuring the mitochondrial integrated stress response in both cell lines and co-culturing myotubes and iPSC derived motor neurons to study the effects of p.G66V on neuromuscular junction (NMJ) formation.

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.