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Adeno-Associated Viruses (AAVs) are quickly becoming one of the most applied vectors for gene therapy applications. In the recent years three new AAV-based gene therapies have been approved by U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The regulatory bodies require accurate and reliable characterisation of the clinical grade viral vectors during and after production. Analytic methods measuring the purity, potency and safety of the product support the up-stream and down-stream processes during the production and are used for final-drug substance characterisation. Median Tissue Culture Infectious Dose (TCID50) is a well-established method for measuring the infectious titer of a virus. Here, an assay for determining the infectious titer of AAVs, which has previously been used to characterise the existing AAV2 Reference Standard Material (AAV2RSM) was set up and optimised for research use at Kuopio Center for Gene and Cell Therapy (KCT). The assay utilizes the HeRC32-cell line, a HeLa clone, that stably expresses AAV Rep and Cap -proteins and in presence of adenovirus, enables the replication of recombinant AAV-vectors. The cells were grown in 96-well plates and infected with 10-fold dilution series of AAV vectors (AAV2 and AAV6) using human adenovirus type 5 as the co-infector. 72 hours post infection the vector genome replication of AAV was detected with quantitative PCR (qPCR). Thresholds for qPCR determined copy number and cycle threshold (Ct) were set and used for the determination of infection-positive wells. The 50-percent endpoint was observed and used to calculate the infectious titer according to the Spearman-Kärber method. The assay was set up and optimised with the AAV2 Reference standard material (AAV2RSM) using two different primer-probe sets (targeted sequences were; SV40 polyadenylation signal and AAV inverted terminal repeats (ITRs)). Plates infected with AAV2RSM were analysed separately with both primers resulting in mean infectious titers of 8.07 ± 3.13 x 108 TCID50 Infectious Units (IU) / mL (n = 9) and 1.27 ± 0.464 x 109 TCID50 IU/mL (n = 9) for SV40 and ITR, respectively. After the assay was set up with the AAV2RSM, an in-house AAV6 product was analysed with the ITR primers yielding 6.09 ± 3.94 x 109 TCID50 (IU) / mL (n = 5). The assay protocol was successfully set up for research use at the KCT laboratory. Improvements were added to the original protocol to increase assay robustness, accuracy and precision as well as to minimize the possibility of over-estimation of the infectious titer. The assay can be further optimised for a particular therapeutic AAV product in the research laboratory or technology transferred to a production facility for optimisation and validation for the analytics needs of a production pipeline.

The focus of this thesis is on environmental agency in two different video games, The Sims 4 Eco Lifestyle and Final Fantasy VII Remake. The research questions aim to answer how the player can act as an environmental agent in these two games and what are the key differences between these games. The study is executed in a form of qualitative two-case case study with the help of close reading. With close reading eight different types of agencies that form the typology of this study, will be analysed in order to answer the research questions. The data for this thesis was collected by playing both games and taking notes by following close reading. The notes were then analysed with the different types of agencies. The results showed clear overlapping of the types of agencies, and it could be said that environmental agency can be used better in the game when the overlapping is happening. The agencies complemented each other and made the possible learning process in the game more fulfilling. The main difference between the game seems to be that The Sims 4 Eco Lifestyle as a life simulation game offers more diverse possibilities for environmental player agency. The studies about environmental games are mainly focused on serious games and not so much on commercial games. Video games hold great potential to engage people in environmental things especially with the help of player agency. It offers the player the ability to make meaningful choices and if they are structure well, the player can see the consequences of their agency which serves as an effective feedback which could lead to positive learning. In this case, the environmental agency in the game could be transformed into real-life environmental agency. As video games have become more immersive and their environments more realistic, it could be worth considering that separating virtual environment from the real-life one might not be necessary anymore. Therefore, games like The Sims 4 Eco Lifestyle and Final Fantasy VII Remake could serve as an example of how environmental agency within them could be harnessed into wider use.

The climate crisis is such major environmental threat that it has negative impacts on both nature and people. Recently, the effects of climate change on people’s mental health have also been noticed. Children and adolescents are especially vulnerable group in terms of climate anxiety. The amount of climate anxiety among youth has been found to be increasing, yet the studies on it are still scarce. It is important to find out how much climate anxiety there is among youth and does it influence their behavior. The objective of this master’s thesis is to find out how much climate anxiety there is and is climate anxiety interconnected with the pro-environmental behavior among youth. Also, its aim is to find out whether attending the climate course affects the amount of climate anxiety. In addition, the study focuses on whether gender, home country and parental education level are interconnected with the amount of climate anxiety. This study was conducted as part of the D.Game project which includes a climate course for youth aged 12–18 years. Data for this research was collected through a survey during the climate course. The schools and students participating in the D.Game climate course and the climate anxiety survey were selected through an open recruitment during autumn of 2021. The data consists of 48 students who responded to the pre survey and of 13 students who responded to the post survey. The participants were 15–17 years old. On the contrary to other research, there is not much climate anxiety among youth. There is also a strong positive correlation between climate anxiety and pro-environmental behavior, which differs from other research. On the other hand, there were no significant differences in climate anxiety between the groups divided by divided by gender, home country and parental education level, which was expected result. Based on the data, the climate course did not have statistically significant effect on the amount of climate anxiety among youth. Also, there is need for further research. Larger sample could provide more valid results. The causality of the correlation between climate anxiety and pro-environmental behavior would be interesting to resolve. Whether attending to a climate course affects the amount of climate anxiety could be found out by development of climate courses and comparing different courses.

The production and consumption of foodstuffs has a strong impact on climate change, and vice versa. Agriculture and the food industry are responsible for over 25% of man-made greenhouse gas emissions. Thus, food choices are a significant way in which individuals can influence greenhouse gas emissions. By comprehensively changing one’s food consuming habits to align with the principles of sustainability, an individual can decrease the climate impact of their total consumption by approximately ten percent. Sustainable habits, such as food consumption, are part of wider social and cultural processes. Therefore, in research relating to food consumption, it is important to consider both the background of the research participants and the community and society in which they live. Through food choices, an individual expresses their identity, status, and belonging in the community. Eating is a social event that is affected by the values and attitudes of the surrounding community and society. These things strongly influence an individual’s food choices, but on the other hand, individuals can also reshape the attitudes and values of their community through their choices. In order to advance sustainable food decisions on a societal level, it is important to examine what factors influence people’s consuming and eating habits. There has been a considerable amount of research done on sustainable foods, but the research focus has not often been on aware consumers. Studying aware consumers provides information about which factors hinder the making of sustainable decisions when the obstacle is not a lack of awareness. As more is known about the reasons behind people’s food choices, it becomes possible to consider new methods for getting people to make more sustainable choices. The students of the Environmental Change and Global Sustainability program at the University of Helsinki are interesting subjects of research, because they presumably are aware of and interested in the impacts of their food choices. Therefore, in studying them it is possible to focus on other factors determining their food choices, rather than obstacles related to a lack of awareness. In addition, Helsinki as a study location offers good possibilities for making sustainable choices, because there is a diverse supply of sustainable foods in the urban centre. In this master’s thesis, I aim to answer the question: What kinds of perceptions of a sustainable diet do the students of the University of Helsinki program of Environmental Change and Global Sustainability have and what kinds of obstacles do they face when making sustainable food choices? My study is a qualitative case study. I gathered the research material by conducting semi-structured theme interviews with eight students. I analyzed the material by employing thematic analysis methods. My study indicates that the students found it most difficult to follow a totally plant-based diet, even though they consider it to be a sustainable choice. Choosing plant-based food was challenging for them especially in certain social situations. These included situations in which the students felt pressured into eating animal-based food, or situations in which they wanted to please another person by eating the animal-based food they were offering. In such situations, the students were inclined to make choices that differ from those they make in their everyday lives. In social situations that involve food and eating, people have a human need to indicate a sense of community and respect towards people important to them. The students I studied have a lot of knowledge and awareness of the sustainability impacts of their food choices. However, my study indicates that in some situations the need for social cohesion is more important than the need to make a sustainable food choice. The social meaning of eating is important to take into account when considering how to get people to make more sustainable food choices.

Attention deficit hyperactivity disorder (ADHD) is a neuropsychiatric disorder characterized by inattention, hyperactivity and impulsivity. The symptoms appear in childhood and, if left untreated, can continue into adulthood affecting the quality of life. Currently, diagnosing a child's ADHD relies on subjective questionnaires filled out by a parent and an interview. The detection of changes in brain activity especially during everyday activities could bring important information that could help inform the diagnostics of ADHD. The changes in brain activity in persons diagnosed with ADHD during familiar, everyday events have been studied very little. However, ADHD brain imaging studies done during resting state and simple tasks have found changes in large-scale brain networks. These networks can be studied using functional connectivity approach, where the degree of synchronous activity in different brain regions is used to determine the connection strength between these regions. In this thesis, using functional magnetic resonance imaging, the differences in the functional connectivity of the whole brain between children diagnosed with ADHD (n=17) and controls (n=19) were investigated during a virtual reality task that simulates everyday life (EPELI), watching natural-like videos, and resting state. Connectivity matrices were generated with the NiLearn Toolbox program using Seitzman and colleagues' (2018) 300-area parcellation. The connectivity of the whole brain was examined using Network-Based Statistics. During movie watching the ADHD group showed increased connectivity compared to the control group in a network that included several areas of the motor cortex. This may indicate a role for these regions in the hyperactivity symptoms of ADHD. The same network also included the right superior temporal gyrus, which has previously been linked to impulsivity symptoms in individuals diagnosed with ADHD. During the virtual reality task and resting state, no differences in connectivity were observed between the groups. However, differences between the experimental situations were revealed in several networks when the connectivity was compared within the groups. Many of these networks were very extensive and included several subcortical and cerebellar structures in addition to cortical areas. Both the control and ADHD groups showed increased connectivity in the resting state compared to EPELI. This could possibly be due to the differences in the participants' actions during the task performance. To the best of the author's knowledge, this study is the first to examine the functional connectivity of brains diagnosed with ADHD during naturalistic stimuli. The clear differences between the controls and the ADHD group during movie watching are promising for future naturalistic brain studies. Based on the results, network models are effective in studying the functional connectivity of ADHD under different conditions. However, consideration for similarity of activities during the virtual task could have led to the detection of larger differences.

Aims of this study. Previous studies have shown cyanobacterial dominance and harmful cyanobacterial blooms to increase due to recent climate warming. The increase of aggressively blooming species and toxin-producing strains of cyanobacteria has been predicted to further increase in the future. However, information on the response of cyanobacteria communities to environmental forcing in the Arctic region – which is experiencing warming at over twice the rate compared to the global average – has been insufficient. Thus, it is crucial to study how algal and cyanobacterial communities have developed after industrialization to better understand and predict future trends of subarctic algal communities as well as changes within cyanobacteria communities experiencing environmental forcing. This study aims to provide information on the effect of recent climate warming and lake browning on algal communities in subarctic lakes, with a special focus on cyanobacteria and cyanotoxins. Materials and methods. Modern and historical primary producer group abundances of 23 subarctic lakes located on an ideal temperature and vegetation gradient were studied using sedimentary algal pigments as a proxy. The top-bottom method was used to study both changes within algal communities during the last ca. 150 years and the broader trends in algal communities of subarctic lakes. Pigment data was analyzed together with environmental data using ordination analyses (principal component analysis (PCA) and redundancy analysis (RDA)) as well as other statistical analyses in order to determine possible trends of change and to reveal the environmental variables that have the strongest impact on cyanobacterial abundance. Results and conclusions. Algal communities have changed during the last ca. 150 years and show a general trend of increased primary production as well as lake browning in the spruce, pine and birch (SPB) vegetation zone. Siliceous algae generally dominate modern algal communities, and relative abundances of cyanobacteria have declined throughout the vegetation gradient. Within the Barren (Ba)- and mountain birch woodland (MBW) vegetation zones, cyanobacteria communities show a marked decline in the abundance of assumed benthic species based on pigment data, and low abundances of planktic picocyanobacteria. However, due to climate warming and lake browning, abundances of cyanobacteria have increased in several sites within the SPB vegetation zone and are suspected to indicate an increase of harmful planktic species. The most significant environmental variables controlling the abundance of cyanobacteria were total phosphorus, temperature and the amount of organic matter. The results highlight the urgent need to mitigate climate warming in order to preserve the unique biota and characteristics of Arctic and subarctic lake ecosystems, and to prevent the possible harmful increase of cyanotoxins in these sensitive ecosystems.

Pulmonary arterial hypertension (PAH) is a progressive and devastating disease with poorly understood pathogenesis. It is characterized by abnormal remodelling of pulmonary vasculature due to uncontrolled apoptosis and proliferation of endothelial (ECs) and smooth muscle cells (SMCs) in vascular wall. In severe PAH pulmonary ECs exhibit hyperproliferative and apoptosis resistant phenotype contributing to the formation of neointima and development of plexiformic lesions. Structural changes promote occlusion of vascular lumen, and thus, increase in pulmonary vascular resistance. To date we lack efficient therapy to prevent vascular remodelling and restore normal vascular function in PAH. Purinergic signalling is potential modulator of pulmonary vascular homeostasis. It comprises of extracellular nucleotides, such as ATP, which signal through their receptors on cell membrane. Ectoenzymes with nucleotide hydrolyzing activity have an essential part in controlling homeostasis and physiologic concentration of extracellular nucleotides. Ectoenzyme CD39 plays a crucial role in dephosphorylating ATP, which is a known mediator of inflammation, angiogenesis, thrombosis and vasoconstriction according to previous research. Aims of this project were to study the role of extracellular ATP in pulmonary endothelial dysfunction during PAH pathogenesis. The goal was to evaluate the significance of ATPases, such as CD39, in the disease process and to identify significant ATP receptors on pulmonary ECs. We utilized a previously unused strategy to monitor ATPase activity in vivo in pulmonary endothelium of rats with PAH. With this strategy we could identify changes in a time-line manner. Our results indicate that ATPase activity is significantly attenuated in ECs during disease process. Similar finding was also observed in human pulmonary EC isolated from PAH patients suggesting that loss of ATPase activity mediated increase of extracellular ATP could play a role in disease pathogenesis. Our in vitro experiments reveal that loss-of CD39 in human pulmonary ECs leads to an apoptosis resistant and hyperproliferative phenotype. We also identify that purinergic receptor P2Y11 is a critical mediator of ATP responses in these ECs. Suppression of ATP mediated P2Y11 response in apoptosis resistant PAH patient ECs restores normal EC phenotype and thus, suggests a novel therapeutic strategy for pulmonary occlusive vasculopathy.

Typpi on perustuotantoa rajoittava ravinne lähes koko Itämerellä. Typen kierto perustuu mikrobien välittämiin hapetus-pelkistysreaktioihin, joissa typpeä muunnetaan erilaisiin muotoihin. Ammonifikaatiossa mikrobit mineralisoivat liuennutta tai partikulaarista orgaanista typpeä ammoniumiksi ja DNRA- prosessissa (dissimilatorinen nitraatin pelkistys ammoniumiksi)mikrobit pelkistävät nitraattia ammoniumiksi. Ammonium on biologisesti kaikkein käyttökelpoisin typen muoto ja siksi sen pitoisuudet ovat vesipatsaassa yleensä hyvin pieniä. Mikrobiprosessien mittaamiseksi myös pienten ammoniumpitoisuuksien määrittäminen on tärkeää. Pro gradu-tutkielmassa määritettiin DNRA:n ja ammonifikaation prosessinopeuksia Varsinaisen Itämeren vesipatsaassa hyödyntämällä stabiili-isotooppitekniikkaa. Tutkimuksen edellytyksenä oli selvittää kokeellisesti alin luotettava määritysraja ammoniumin 15N-pitoisuudelle. Menetelmäkehitystä varten valmistettiin koesarjoja ultrapuhtaasta vedestä ja tunnetun 15N:14N -suhteen omaavista ammoniumkloridijauheista. Minimimääritysrajakokeessa etsittiin eri ammoniumin ainemääriä sisältävän koesarjan avulla massaspektrometrisen analyysin tarvittava alin ammoniumin ainemäärä. Minimimääritysrajan alapuolelle pyrittiin pääsemään tekemällä kantoliuoskoe. Kantoliuoskokeessa valmistettiin eri ammoniumin ainemääriä (0,5 atom. %) sisältävä koesarja, joiden ammoniumin ainemäärää kasvatettiin lisäämällä näyteliuoksiin kantoliuosta, jonka määrä ja isotooppisuhteet tunnettiin. Kantoliuoslisäysten jälkeen näytteet analysoitiin massaspektrometrillä ja tuloksista seurattiin 15N:14N – suhteen muutosta eri laimennoksissa. Pienin luotettavasti määritetty ammoniumin ainemäärä oli 0,5 µmol. Kantoliuoskokeilla ei onnistuttu optimoimaan määritysrajaa pienemmäksi, eikä menetelmää siten hyödynnetty vesinäytteiden DNRA- ja ammonifikaatiomittauksissa. DNRA- ja ammonifikaatiomittauksia varten kerättiin vesinäytteitä Varsinaiselta Itämereltä neljältä eri näytteenottoasemalta (Läntisen Gotlannin, Itäisen Gotlannin , Fårön ja Landsortin syvänteet) toukokuussa 2011. Lisäksi Itäiseltä Gotlannin syvänteeltä otettiin vesinäytteet DNRA- ja ammonifikaatiomittauksia varten myös heinäkuussa 2011. Vesinäytteitä rikastettiin 15N- ammoniumilla siten, että näytteiden 15N- pitoisuudeksi saatiin 5 µM. Näytteitä inkuboitiin 24 tuntia in situ- olosuhteissa. Inkuboinnin jälkeen näytteistä mitattiin 15N- leima ammonium diffuusio- menetelmällä. Tuloksista laskettiin DNRA- potentiaalit sekä ammonifikaation in situ- prosessinopeudet. Keväällä mitatut ammonifikaation in situ- prosessinopeudet olivat matalia (0,36- 8,35 nM d-1). Tuloksia saattoi selittää se, ettei kesäisen perustuotannon tuottama partikulaarinen orgaaninen typpi ollut vielä ammonifioijien käytettävissä. Eri asemilta mitatuissa prosessinopeuksissa ei ollut tilastollisesti merkitsevää eroa. Tutkittujen ympäristömuuttujien ja ammonifikaation prosessinopeuksien välillä ei havaittu korrelaatiota. DNRA- potentiaalituloksia saatiin vain toukokuussa Läntiseltä Gotlannin syvänteeltä ja heinäkuussa Itäiseltä Gotlannin syvänteeltä kerätyistä näytteistä. Läntiseltä Gotlannin syvänteeltä mitattu DNRA- potentiaalinopeus (1,14 nM d-1) oli lähes nelinkertainen heinäkuussa Itäiseltä Gotlannin syvänteeltä mitattuun DNRA- potentiaaliin (0,38 nM d-1) nähden. Ammonifikaation merkitys Itämerellä on todennäköisesti pienempi ulapalla kuin estuaareissa, joissa suuri orgaanisen aineksen kuormitus tuo jatkuvasti substraatteja ammonifikaatiolle. Keväisen piileväkukinnan nopeasta sedimentoitumisesta johtuen saattavat ammonifikaation prosessinopeudet Itämerellä keväisin olla suurempia sedimenteissä kuin vesipatsaassa. Itämeren erityispiirteet, kuten alhainen suolapitoisuus todennäköisesti inhiboi vesipatsaan DNRA:n esiintymistä, jonka seurauksena mitatut DNRA- nopeudet jäivät mataliksi. DNRA: n kanssa nitraatista kilpailevan kemolitoautotrofisen denitrifikaation on sen sijaan havaittu olevan pääasiallinen typpeä pelkistävä prosessi Varsinaisella Itämerellä.

Glutamine, the conditionally essential amino acid, is a major carbon and nitrogen carrier required for a range of cell functions, such as protein synthesis and maintaining redox balance. While healthy cells adjust their activities in response to glutamine availability, tumor cells display deregulated glutamine uptake and metabolism allowing quick proliferation and survival in cellular stress conditions. Hence, further knowledge of the glutamine sensing network is of interest. Utilizing Drosophila melanogaster, the roles of formerly identified glutamine sensing regulator candidates, Forkhead box O (FoxO), Super sex combs (Sxc), Spalt major (Salm) and Spalt-related (Salr), were explored. Drosophila is an efficient model organism for analyzing gene regulatory mechanisms, with its simple genome but conserved genes and metabolic pathways. Loss-of function and gain-of-function mutants of the candidates were cultured with/without glutamine, and their physiological response and gene expression changes were analyzed. The results show the glutamine intolerant phenotype of FoxO and Sxc deficiency, not dependent on altered food intake levels of larvae. However, glutamine intolerance of Salr and Salm deficiency was not observed. Moreover, we aimed to gain further insight to the roles of FoxO and Sxc in glutamine metabolism. Since amino acid catabolism produces ammonia, and glutamine metabolism plays a vital role in ammonia detoxification, we performed a pH-based measurement of foxo and sxc mutant larvae hemolymph on food with/without glutamine. However, we could not associate FoxO or Sxc with regulation of glutamine-derived ammonia clearance. In addition, we explored FoxO downstream regulator candidates. Putative promoter areas of Paics, Uro, Sesn, salr, Prat2 and Gdh were cloned into reporter vectors and the luciferase activity was analyzed under the expression of foxo. The results indicate that FoxO is a regulator of all of the 6 genes. Next we could utilize the here constructed plasmids to see whether the FoxO-mediated regulation is affected by altered glutamine levels in cell culture.

Uterine leiomyoma (also known as myoma) is the most common neoplasia in women during reproductive age and it represents a burden for public health care. Approximately 70% of Caucasian women develop myomas, although only 25% of cases are symptomatic. The genetic background of myomas varies significantly and the most common genetic causes are mutations in genes Mediator complex subunit 12 (MED12), Fumarate hydratase (FH) or YEATS Domain containing 4 (YEATS4) , rearrangements affecting the High Mobility Group AT-hook 2 (HMGA2), and deletions in COL4A5/6 locus. MED12 mutations represent the most common genetic alteration in myomas, being present in approximately 70% of cases. Genome organization comprises different levels of complexity, spanning from regulation of individual genes to changes in the architecture of large portions of chromosomes. Literature offers massive evidence of changes in genome organization among different cell types and between several tumor and related healthy cells, but information about these changes in myoma is lacking. The aim of this study is to determine the main features of genome organization in myomas belonging to the aforementioned five genetic subclasses, in order to identify which are the underlying common pathways that are dysregulated in the neoplasia. This is achieved by mapping regions of open chromatin in myomas and related my-ometrium samples with ATAC-seq. Sample’s clustering seems not to be individual-dependent, while tumors belonging to FH, YEATS4 and COL4A5/6 subclasses form distinct clusters, unlike MED12 and HMGA2 subclasses. Six open chromatin regions located within genes were identified in 19/25 tumors and not in myometrium. Seven myometrium-specific open chromatin regions were identified in 21/25 myometria and in less than 10 tumors. As expected, Gene Ontology enrichment analysis revealed that myomas belonging to FH subclass are characterized by deregulation of metabolic pathways. Many of the identified genes in the open chromatin regions have been linked to other tumors in previous studies. Tumor-specific open chromatin regions locate within oncogenes, while myometrium-specific ones are found in proximity of tumor suppressor genes, suggesting a biological role in myomagenesis for these genes. Further investigation on the identified genes (e.g. transcriptional regulation, gene expression and protein level) and addi-tional studies on chromatin architecture are needed to fully unravel the mechanism of myomagenesis.