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Epileptic patients experience spontaneous recurrent seizures and interictal epileptiform discharges that lead to brain injuries, triggering neuroinflammation and waste product accumulation. Due to the detrimental effect of waste products on brain homeostasis, their removal from the central nervous system is (CNS) is crucial. Meningeal lymphatic vessels (mLVs) located in dura matter contribute to CNS clearance by the drainage of metabolites, waste products, and immune cells from subarachnoid space into cervical lymph nodes. Therefore, because of its role in brain homeostasis, the study of mLVs in different neurological conditions and diseases, including TLE, has gotten increased attention in the last decade. In this study, we sought to understand mLVs role in neuroinflammation and changes in rapid eye movement (REM) sleep stage during epilepsy. For this purpose, we induced mLVs ablation followed by kainic acid (KA) epilepsy model in mice. Shortly, animals were inoculated with AAV-VEGFR3-1-4 to induce mLVs ablation and subsequently challenged with KA to induce status epilepticus. Simultaneously, a control group of animals were injected with a sham AAV and later injection of KA. Afterward, spontaneous EEG activity was registered continuously, and data analysed to compare durations of REM sleep. Also, immunohistochemistry of brain samples was performed to investigate neuroinflammatory changes between experimental groups. Ex-vivo analyses of Iba1 and GFAP expression in brain tissue did not show statistically significant changes in neuroinflammation between experimental groups. However, we observed a trend towards lower expression of inflammatory markers in mLVs ablated animals. The analysis of REM sleep duration shows a progressive reduction of this sleep stage in both groups during the first recording period with a subsequent stabilization during the second one. Our data also indicate that mLVs ablated animals present prolonged REM sleep duration compared to the control group. Although this data contradicts our initial hypothesis it is consistent with the well-established negative correlation between neuroinflammation and REM sleep duration. Future studies should consider a deeper analysis of the glial cell profile for a better understanding of the effect of mLVs dysfunction on epileptic pathology. Moreover, the impact of mLVs ablation on REM sleep duration should be characterized in healthy animals.

Colorectal cancer (CRC), which refers to the cancer of the colon and the rectum currently ranks as the second leading cause of cancer related death worldwide and as the third most common form of cancer in both males and females. The latest reports show that approximately 10% of all new cancer cases globally are diagnosed as CRC annually. Initiation of sporadic CRC is commonly caused by somatic mutations causing the loss of function of the tumor suppressor gene APC. This leads to aberrant activation of the canonical Wnt signalling pathway. The ApcMin/+ mice model the progression of CRC as they carry a constitutive heterozygous nonsense mutation in Apc allele and develop intestinal adenomas. TCF/LEF transcription factor family members are best known as the main downstream effectors of canonical Wnt signalling. In the presence of nuclear β-catenin, TCF/LEF proteins bind to it through their β-catenin-binding domain and activate the transcription of Wnt target genes. The TCF7 gene encodes several isoforms of TCF1 protein, which are traditionally divided into long and short isoforms, transcribed from different promoters. Previously, it has been shown that Tcf7 deletion (Tcf7-/-) in ApcMin/+ mice increases the formation of adenomas. The aim of my study is to better understand the role of Tcf7 and its isoforms in CRC tumorigenesis. To study the Tcf7 deletion in intestinal adenoma development, ApcMin/+; Tcf7mut/mut; Villin CreERT2 mouse strain was established. The expression of the full-length isoforms (p45) is constitutively prevented in the Tcf7mut/mut mice. Moreover, tamoxifen administration to these mice led to the deletion of all isoforms in the intestinal epithelium. The number of intestinal tumors, their sizes and the survival of the Tcf7 deleted ApcMin/+ mice were analyzed and compared to ApcMin/+ mice. Intestinal tissues of the mice were collected after euthanasia. The tissue samples were preserved in paraffin, and later cut into sections for IHC, stained and imaged. The deletion of Tcf7 was confirmed at the RNA level by qPCR, and at the protein level by immunohistochemistry (IHC). IHC and single-cell RNA sequencing was used to further analyze the effect of Tcf7 deletion in mouse intestinal adenomas. The deletion of all Tcf7 isoforms or only the p45 isoforms in ApcMin/+ mice increased robustly the numbers of intestinal tumors. IHC analysis of the intestinal adenomas showed that the deletion of p45 isoforms was sufficient to cause a dramatic decrease in total Tcf1 expression in the adenoma cells. These results were supported by the qPCR results. In summary, our results lead us to believe that the deletion of p45 isoforms causes an acceleration of tumorigenesis in the adenoma model. Without the Apc mutation, the mice did not develop intestinal adenomas. Interestingly, the expression of the Wnt-target gene Prox1 in intestinal adenomas was decreased when Tcf7 was deleted. We next aim to optimize our protocol for single cell dissociation of adenomas and re-run the single-cell RNA sequencing analysis for further analysis of the mechanisms behind the increased tumorigenesis.

Climate change poses an ever-increasing threat on biodiversity as the global mean temperature rises causing changes in weather patterns. Species will have to adapt to the circumstances or follow their climatic niches across space to avoid decline and extinction. Many species are already threatened by extinction due to climate change. Understanding how species are reacting to rising temperatures can help us preserve biodiversity. Genetic adaptation is a long process and takes several generations to occur. A more immediate means to cope with variation is adjusting through phenotypic plasticity, which can help species cope with environmental changes in the short-term. Plasticity can help individuals maintain fitness in different environments and with fluctuating environmental conditions. Flowering phenology is a plastic trait which can have a large impact on reproductive success. Flowering is an important part of a plant’s life cycle as it can produce offspring with new combinations of genes. In this thesis I examine how temperature affects the flowering phenology of Hypericum species and how this thermal plasticity affects fitness. Populations of Hypericum perforatum, H. maculatum and H. montanum from different parts of their distribution across Europe were studied in greenhouse experiments. The plants were grown in four different temperature treatments (16/6°C, 20/10°C, 24/14°C, 28/20°C) and the timing of first flowering was monitored. Seed mass and flower count were recorded and used as measures of fitness. In general, the plants flowered later in the colder temperature treatments. The results differed between species: in H. maculatum the leading-edge populations were less plastic while in H. perforatum differences between areas were negligible. More plastic accessions produced more flowers due to earlier flowering. There was no effect on seed mass. The possible effects of plasticity on overall fitness highlight the need for detailed information on plasticity for predicting species response to climate change.

In this study, a greenhouse experiment was used to assess if temperature sensitivity, specifically, thermoregulatory plasticity, has a functional role in floral reflectance and pigmentation in a population of P. lanceolata grown in three different temperature treatments, reflecting past, present, and future summer temperatures. Spectrophotometry, surface temperature readings, and near-infrared (NIR) region image analysis were used to identify how the spectral absorbance properties and biochemical makeup of P. lanceolata flowers differed between treatments. Reflectance and phenolic absorbance were both found to be influenced by ambient temperature. However, surface temperature of flower spikes was not affected by growing temperature, reflectance, or phenolic absorbance. The results suggest that Plantago lanceolata may utilize thermoregulatory plasticity in reflectance and phenolic absorbance to adjust to rising temperatures. These findings have important implications in species reactions to climate change and denotes that increased selection on thermal function traits may occur under a future climate scenario of continued warming in temperate and boreal biomes.

Peatlands are ecosystems of global importance for biodiversity conservation. Peatlands are wetland ecosystems which provide a critical habitat for many rare and specialized species. Biodiversity maintains the provision of ecosystem services, which are the benefits people obtain from ecosystems. The loss of biodiversity and peatland degradation constitute global challenges. One of the main reasons why biodiversity loss and peatland degradation remain challenges is nested in the failure to account for the range of economic benefits of ecosystem services and biodiversity in relevant policy making. The role of peatlands in maintaining global biodiversity has often been underestimated in global, regional and local land use planning and conservation measures. This thesis undertakes a systematic literature review on the empirical economic valuation literature on peatland ecosystem services and biodiversity. The two main aims of this literature review are to synthesize the current state of knowledge on this phenomenon and analyse the role of biodiversity in the economic valuation of peatland ecosystem services by answering specific research questions. This systematic literature review employs the data of 23 peer-reviewed English language papers published between 2006 and June 2021. The studies were chosen for analysis based on a selected search strategy and screening process. The data analysis was undertaken using the qualitative data analysis software Atlas.ti which is a tool used to help organize analysed material with the help of descriptive codes. Based on the findings of this thesis, the inclusion of biodiversity in the economic valuation of peatland ecosystem services has become a standard procedure. This is showcased by the number of studies applying stated preference approaches. The sample included studies applying different valuation methods in order to value many ecosystem services. The studies including biodiversity in economic valuation often find that biodiversity conservation policies can be cost-effective, and that people are generally willing to pay for biodiversity conservation and would derive economic benefits from this. Biodiversity is included in the studies as different elements, mainly as a specific species or as a reference to wildlife. Biodiversity is also found to be closely related to cultural ecosystem services and their benefits. Many studies find that people value familiar peatland landscapes, and biodiversity plays an important part in defining that value. Some studies find that human activity plays an important role in maintaining biodiversity in semi-natural peatland landscapes. Hence, biodiversity conservation needs to be in some accordance with local interests. Biodiversity provided founding principles for policy making, but previously implemented practices, such as existing conservation measures and the extractive use of peatlands exerted much influence on final economic values. Moreover, biodiversity plays different roles in determining the objectives of the studies. Eight studies use biodiversity as the justification for conducting the economic analysis. Most studies include biodiversity as a study component among others under valuation. The portrayal of biodiversity influences the focus of the studies and how biodiversity contributes to the findings of the literature. The body of literature on the economic valuation of peatland ecosystem services remains small. The geographical distribution of the sample is skewed towards Europe and Southeast Asia. There is a notable upward trend in the number of studies which have been published in the last five years. The literature demonstrates that the economic valuation of peatland ecosystem services and the need to adopt sustainable peatland management with biodiversity conservation have become relevant and topical issues in policy making. There is a significant need to address the issue of peatland degradation and biodiversity loss by increasing awareness. Further research is needed to establish a more comprehensive understanding of the links between peatlands, ecosystem services and their values. In addition, future research could study how the provision of information and the contribution of biodiversity awareness and knowledge influence economic valuation.

Cities are responsible for many of the current environmental changes in the world. Even though the need for urban sustainability transitions is apparent, city governments have been, so far, uncapable of governing the change. In this thesis, the possible urban transition and its multi-level governance is examined through the case example of Eko-Viikki neighbourhood and the urban planning changes around it. The framework of multi-level perspective on socio-technical transitions (MLP) is used for the analysis. This theory on transitions is widely used, while criticized for its inadequate conceptualization of the regime (the dominant system) and its spatial application, especially in cities. Traditionally, the framework has not been used to study transitions in cities, due to which, there is a lack of case examples of urban sustainability transitions and their governance. This thesis addresses the deficiency in question and aims to find out which types of policy instruments could be essential in bringing about urban sustainability transitions and whether the dominant regime, in this case the urban planning of Helsinki, can actively influence the steering of the transition. The materials of this thesis consist of two different sources. The document analysis was used to gather the materials for the policy instrument analysis. In addition, six semi-structured expert interviews were conducted to provide supporting material for it, as well as to examine the change of the urban planning context in Finland. The materials were analysed by qualitative content analysis using the MLP framework and a commonly used environmental policy instrument classification as a frame. The results indicate that regime actors can have endogenous power to somewhat steer the urban sustainability transitions. The regime actors of urban planning of Helsinki were active in the Eko-Viikki project, and sustainability issues have become more integrated part of the everyday urban planning in Helsinki. As for the niche level of Eko-Viikki, regulation and collaboration related policy instruments were the most successful in advancing the sustainability issues. In fact, the site transfer conditions containing sustainability demands as well as the area working group method have been scaled up to the use of the regime. The city of Helsinki has, moreover, committed in advancing sustainable city development through international agreements and its own city strategies. As regards the landscape level, the land use and construction legislation changes have remarkably tightened the requirements for sustainability of urban planning, especially for the energy efficiency. On the contrary, the lack of certain types of instruments seem to remarkably hinder the urban sustainability transitions. According to the results, information related instruments need to be developed to make the most of the technical solutions available and, ultimately, to make urban planning more like continuous learning processes rather than individual projects. Also, the issue of short-term profit seeking should be addressed by economic instruments that involve long-term investments and set sustainability issues as a first priority. All in all, it can be concluded, that much has done for promoting sustainability in urban planning of Helsinki even though a profound urban sustainability transition cannot be said to have happened. Particularly, systemic and process focused policy instruments are needed to take into account the multiple different stakeholders involved, and the governance levels where urban sustainability transitions take place. The urban sustainability transitions can be enabled only together with other governmental bodies of the city, state, construction companies, maintenance companies and, ultimately, residents.

Host factors play crucial roles in virus infections. Viruses exploit various cellular processes and are counteracted by an arsenal of host antiviral defenses. Characterization of these interactions is crucial for understanding the viral life cycle and developing novel antiviral treatments. Semliki Forest virus (SFV) is a positive-strand RNA alphavirus that has been used as a model virus for multiple clinically significant diseases such as lethal encephalitis. The aim of this thesis was to identify host factors that affect SFV infection to better understand the biology of SFV, and to provide candidate targets for therapies against more serious alphavirus infections. Here I have conducted follow up studies on a previously performed genome-wide siRNA screen that hinted that a number of genes have novel functions in SFV infection. I used an automated high-throughput imaging-based approach to confirm the roles of these host factors in SFV infection. For comparison, I also used a similar strategy to test if these genes affect negative-strand RNA virus infections, using vesicular stomatitis virus (VSV). Additionally, I studied whether the host factors affecting SFV infections perform their roles in the entry and penetration, or post-penetration steps using a previously developed endocytic bypass assay. I identified the γ-aminobutyric acid (GABA) transporter, SLC6A13, as a potential receptor for SFV. I also describe other novel genes that have roles in SFV or VSV infections. In addition, I show that TNP01, RPL18, ETF1, DMN2, and GNDPA1 promote, and HDAC6 counteracts SFV infection in the entry and membrane penetration steps. Furthermore, I report that in the later stages of the infection DDX54 boosts and EIF2B3, EIF4G1, PHB2, EDF1, DDX47, and DHX57 hinder SFV.

Abstract Faculty: Faculty of Biological and Environmental Sciences Degree programme: Master’s programme in Neuroscience Study track: Neuroscience Author: Enija Stoka Title: The Role of Meningeal Lymphatic Vessels in the CNS clearance Level: Master’s thesis Month and year: April 2022 Number of pages: 28 Keywords: meningeal lymphatic vessels (mLVs), brain clearance, glymphatic system, perivascular spaces Supervisor or supervisors: Anaϊs Virenque, Francesco Mattia Noe Where deposited: the Helsinki University Library Additional information: - Abstract: The lymphatic system is a drainage pathway for metabolic waste products, soluble proteins and cerebro-spinal (CSF) as well as interstitial (ISF) fluids. Classically, the lymphatic system has been described all over the body, except the central nervous system (CNS) and the retina. This fact created the question of how the brain is being cleared from harmful solutes. The first system described to being responsible for the clearance of the brain was the glymphatic system, and only recently the existence of lymphatic vessels in the meninges (the meningeal lymphatic vessels, mLVs), has been recognized. However, it is still unknown how these two systems interact in removing solutes from the brain. Here, we analyse if the absence of mLVs affects diffusion and clearance of two tracers with low and high molecular weight (3 kDa and 70 kDa), which have been injected intraparenchymally in wild type (WT) and transgenic (TG) mice lacking functional mLVs. Diffusion of 3 kDa dextran tracer in the surrounding tissue was noticeably increased in WT compared to TG mice, associated with an overall decreased accumulation of the tracer in the parenchyma of the mice lacking mLVs. At the same time, we did not observe a genotype difference in the diffusion or clearance of the 70 kDa dextran tracer. Overall, these results indicate that mLVs dysfunction affects the intraparenchymal diffusion and clearance of low molecular weight molecules.

In this master’s thesis project, I studied the association of lipid molecules phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 3-phosphate (PI3P) with autophagy in neurons. One of the aims of the study is to determine the level of basal autophagy in primary hippocampal neurons and to come up with a protocol for autophagosome observation without forcing radical changes in cell culture conditions. Other mammalian cells have extremely weak basal autophagy, but they increase it significantly in response to starvation, for example. However, neurons are extremely sensitive to any changes in their surroundings. They change their morphology, behaviour and biochemical properties, and often they simply do not survive. Therefore, the goal is a protocol for successful autophagy observation with minimal external influence. Despite the debate around basal autophagy in neurons, I observed high levels of basal autophagy in neuronal cells incubated in media without supplements. Also, my observations revealed that the inhibition of the last step of autophagosome processing with Bafilomycin A1, was enough to cause the massive accumulation of large autophagosomes. Results demonstrated that primary hippocampal neurons exhibit high levels of basal autophagy, suggesting that on the contrary to other mammalian cells neurons might not have enough potential to increase autophagy when it is induced pharmacologically or by stressful conditions. This would explain why autophagy induction is often claimed to be ineffective for neuronal cultures. The main goal is to observe and compare PI4P presence on autophagosomes in normal conditions and when autophagosome:lysosome fusion is inhibited with Bafilomycin A1. The side goal is to observe PI3P presence on autophagosomes as well. I transfected primary hippocampal neurons with fluorescent probes for PI4P or PI3P as well as for autophagosome-related protein LC3. Localization data was collected with live-cell imaging on a confocal microscope. As expected, PI3P was not detected on autophagosomes located in soma. It is involved in the initial vesicle biogenesis in distal axons but not in later events taking place closer to the cell body. PI4P showed high degree of colocalization with LC3, indicating PI4P presence on autophagosomes, but only when the fusion was presumably inhibited by Bafilomycin A1. These results suggest that PI4P appears on autophagosomes either as a result of compensatory pathway, where autophagosomes fuse with late endosomes instead of lysosomes; or as a molecule normally involved in autophagosome:lysosome fusion. Literature supports the latter explanation, but it cannot be confirmed without further research. These results give an insight into PI4P role in neuronal autophagy and might be relevant for the future research of autophagy disruption and aggregate accumulation in neuronal diseases as a consequence of abnormal lipid signalling, lipid metabolism and transport.

Rare mutations in the primate specific ZNF808 gene are a novel cause of pancreatic agenesis, a congenital developmental disorder that leads to neonatal diabetes. ZNF808 loss-of-function has been shown to lead to aberrant activation of regulatory MER11 elements, followed by upregulation of genes in proximity to these elements and increased expression of hepatic lineage markers. These findings suggest ZNF808 to play a key role in balancing the differentiation of endoderm progenitor cells between pancreatic and liver lineages during early human development. This thesis work aimed to study the gene regulatory mechanisms of ZNF808 in the differentiating endoderm progenitor cells to understand its function in controlling pancreatic lineage specification. This was achieved by comparing the lineage specification processes in wild-type (H1) and ZNF808 knockout (H1-ZNF808-KO) human embryonic stem cells (hESCs) during pancreatic differentiation. Further characterization of cellular heterogeneity and gene expression profiles upon ZNF808 loss was done using single-cell RNA sequencing (scRNA-seq). To validate the role of ZNF808 as the mediator of the observed lineage specification bias, the phenotype rescue was examined in a ZNF808 knockout overexpression cell line (H1-ZNF808-KO-OX). The results of this study demonstrate a clear lineage specification bias in the ZNF808 knockout, seen as divergence of the multipotent endoderm progenitors towards alternate hepatic and biliary fates at the posterior foregut stage. By modifying the pancreatic differentiation protocol, we were able to observe phenotype manifestation and cellular heterogeneity suppressed in the standard differentiation conditions. The scRNA-seq data analysis revealed the emergence of a biliary cell population showing upregulation of several hepatic markers, suggesting an alternative lineage specification process governed by ZNF808. Additionally, preliminary results from ZNF808 overexpression showed rescue of the ZNF808 knockout phenotype, further supporting its critical role in the normal pancreatic lineage development. In conclusion, these findings demonstrate the important role of ZNF808 in early human pancreatic development and warrant further studies on the detailed gene regulatory network guiding pancreatic lineage specification.