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  • Larkiala, Taru (2020)
    Calsyntenin-3 is a type I transmembrane protein, that is mainly expressed on the post-synaptic cell membranes. It belongs to the calsyntenin family that is part of the cadherin superfamily. Calsyntenin-3 consists of a cytosolic C-terminal region, a transmembrane domain and an extracellular N-terminal part, that consists of a laminin G-like domain (LNS) and two cadherin domains (CAD). Calsyntenin-3 is mainly expressed in the brain, but it can also be found in the heart, liver, pancreas, lung, skeletal muscle and placenta. Calsyntenin-3 has an effect on neurogenesis by affecting the development of excitatory and inhibitory synapses. It might also play a role in Alzheimer’s disease, as it has been found to be able to bind β-amyloid peptide, that is known to play a key role in the development of Alzheimer’s disease. Calsyntenin-3 acts as a synaptic adhesion protein, that binds to the post-synaptic neurexins with its extracellular region. However, the previous studies have contradicting results regarding the calsyntenin-3 domains that mediate the interaction between the calsyntenin-3 and neurexins. There is also disagreement whether calsyntenin-3 binds neurexin-α, neurexin-β or both. Because of these discrepancies, the aim of this master’s thesis study was to produce the calsyntenin-3 ectodomain constructs that contained either the two CAD domains, the LNS domain or all three domains, using baculovirus mediated protein production in insect cell cultures. These purified protein constructs were meant to be used for the determination of the binding domains. Unfortunately, only the purification of the calsyntenin-3 LNS domain was successful and the purification of the constructs, containing the CAD domains, was unsuccessful. A SEC-MALLS experiment, that was performed for the calsyntenin-3 LNS domain, revealed that it forms dimers in a solution, which is consistent with experiments performed with the LNS domain of human sex hormone‐binding globulin. The second aim of this master’s thesis study was to express the calsyntenin-3 ectodomain constructs on the surface of HEK293T cells and to test the binding between calsyntenin-3 and neurexins in a cell surface binding assay. The results of the cell surface binding assay indicated that the binding is mediated by the calsyntenin-3 CAD domains and that calsyntenin-3 binds to neurexin-α, but the binding to neurexin-β was not detected. However, the results from the cell surface binding assay were conflicting: the binding between the calsyntenin-3 full ectodomain construct and neurexin-α was not detected, but the binding was detected between calsyntenin-3 CAD ectodomain construct and neurexin-α. Therefore, the cell surface binding assay cannot be considered entirely reliable and should be repeated before making further conclusions.
  • Schwenk, Cindy Emilia (2022)
    Breeding-dispersal and philopatry are important life-history traits when it comes to the ecology of animals. A number of factors such as sex, age, habitat stability, population density, predation and various environmental factors influence the movement patterns of species. Philopatry, in general, can be used by organisms as a predator avoidance strategy and to improve feeding efficiency. Dispersal on the other hand has been shown to help avoid inbreeding and competition within groups or between kin. Among the different types of dispersal and philopatry, breeding dispersal and site fidelity to breeding sites have been studied in many vertebrates. Many birds and some ungulate species have been the focus of these studies and have shown that breeding success influences site fidelity. Among ungulates, the reindeer genus (Rangifer tarandus spp.) however, has received little attention for this topic. A species of special conservational value is the wild forest reindeer (Rangifer tarandus fennicus), which is a rare, and near-threatened subspecies of reindeer living in Finland. Knowledge of factors influencing the dispersal and site fidelity for this species is lacking but has important implications for the conservation and management of this and other vulnerable species. In my thesis I investigated the philopatry of wild forest reindeer females from eastern Finland and tested if the calving site fidelity was influenced by breeding success, predator pressure and the female’s age. Given the high densities of predators in the study area (Kainuu) and thus high predator pressure on reindeer, I expected (i) the philopatry of the wild forest reindeer female’s to be stronger in areas with higher predator pressure but (ii) higher dispersal movement when wild forest reindeer females had no breeding success the previous year. Additionally, I expected (iii) the dispersal distances of wild forest reindeer females to be negatively correlated with their age. For this study I used long-term GPS monitoring data of 53 collared wild forest reindeer females, which were followed for varying periods (2-6 years) between 2010-2021 from Eastern Finland. I found that the inter-year distances between calving sites per female had a median distance of 2.84 km, confirming that female wild forest reindeer show philopatry to calving sites, as do other reindeer subspecies. Even though the movement ranged from 4.8 m to 36.99 km, showing substantially longer dispersal for some individuals. The model outcomes indicate some association between predation pressure and site fidelity, and reduced philopatry following calf mortality, although none of these associations were significant. The age of the mother did also not show any significant influence on a female’s site fidelity. Nonetheless, these results would suggest that a mother's choice to leave or return to the same calving site might be influenced by the previous year's calf mortality but more complex variables like anthropogenic disturbances, environmental factors, as well as intrinsic factors and the physical condition of the mother most likely also play a role in this.
  • Fredrikson, Linda (2019)
    The consumption of omega (n-) 3 polyunsaturated fatty acids (PUFA) from fish has been associated with lower rates of cardiovascular diseases with one mechanism being lowering LDL cholesterol levels in blood. When incorporated into LDL particle n-3 PUFAs can modify the lipid composition and reduce atherogenicity of the particle, e.g. by influencing inflammatory processes. The effects of n-3 PUFA of plant origin are less studied. This study investigated the effects of Camelina sativa oil (CSO), a rich source of alpha-linolenic acid (ALA), on lipid species of human LDL including phosphatidylcholines (PC), lysophosphatidylcholines (LPC), sphingomyelin (SM), triacylglycerols (TAG) and cholesterol esters (CE). A total of 38 subjects with a history of impaired fasting glucose, were randomly divided into two groups; CSO (ALA 10 g/day) and the control group (limited fish and ALA intake) for 12 weeks. Blood samples were collected from the subjects at the beginning and at the end of the experiment after 12 weeks. LDL particles were isolated from blood and the lipids were analyzed by mass spectrometry. The CSO affected more the LDL core lipids (TAG and CE) than lipid species of the shell (PC, LPC, SM). CSO is high in ALA and linoleic acid (LA). Thus, the diet reduced mole fractions of lipid species containing saturated acyl chains while acyl chains in the core lipids with ALA, LA and EPA, that is formed in the body from ALA, were increased. Based on the results, having CSO in the diet changed the LDL particle lipid composition in a favorable direction for cardiovascular health.
  • Mikkola, Reija (2012)
    Carbon capture and storage may become an inevitable means in mitigating climate change. However, it is a new technology involving a great deal of uncertainties. It is of utmost importance to understand on one hand, the risks caused by the technology and on the other, what is holding it back. This way unforeseen setbacks and environmental or other damage could be avoided. This thesis is a part of a wider research project on the risk governance of carbon dioxide capture and storage (RICCS). The present study gives additional insights to CCS risk analysis by diving into the stories that the media tells about the risks. I analyze the media coverage on the risks of CCS in the most wide spread newspapers of Norway and Finland with the aim of identifying what kind of risk framings are portrayed by the media; how strong is the presence of uncertainties and what kind of uncertainties are brought up. The media is seen as a mirror of public perception, but also one of the players influencing it. The possible effects that the analyzed articles could have on public perception of risks are discussed. The theoretical framework consists of theories of systemic risks, narrative policy analysis and framing of environmental risks in the media. I describe the nature of systemic risks. Then I move on to framing, more specifically how environmental risks are framed in the media and how it can effect public perception. After this I explain how narrative analysis can be used as a tool for identifying framings. Then I describe Klinke and Renn's Prometheus theory that I will use for analyzing the level of uncertainty in the framing of the articles and for discussing the implications of my findings. The results show that the risks caused by CCS are mainly the lock-in in fossil fuels, it's possible negative effect on developing renewable energy and environmental and health risks in general. The risks towards successful CCS seem to be mainly connected to funding, which connects to emissions' prices, the climate agreement and viability of investments. The differences between the two countries are quite related to the situation in which each country is in terms of CCS development. Norway is very active and pushing CCS forward. Consequently, the Norwegian articles are generally not very critical of the technology itself, but discuss what is holding it back. Generally, the Finnish articles bring out more aspects on the issue, both positive and negative, leaving quite an ambiguous image to the reader. The implications of my findings for future policy practices are quite extensive and therefore not very useful, since most policy recommendations seem more or less relevant. What is interesting though, is that based on my findings I could identify the turning points in which public perception is most relevant. These are: What kind of energy production is supported? Is CCS an acceptable mitigation means? Is the risk of leakage taken as severe? These issues represent turning points for the future of CCS technology and deliberative processes can be crucial when discussing them.
  • Zhang, Huizhong (2014)
    Lakes play an important role in both global and regional carbon cycling, especially, the role of lakes is pronounced in the boreal zone, where lakes cover up to 20% of the land area. Allochthonous carbon is discharged mainly from terrestrial processes, but also through anthropogenic eutrophication. The role of allochthonous carbon load in lacustrine ecosystems is important, which can have far-reaching effects on lacustrine biogeochemistry in general and especially on carbon cycling. Part of dissolved organic carbon is transformed to carbon dioxide (CO2) and methane (CH4) in biological processes that result in carbon gas concentrations in water that increase above atmospheric equilibrium, thus making boreal lakes serve as sources of the important greenhouse gases. Lake Vesijärvi is renowned for its clear-water, but it has suffered eutrophication for a long history. The severely affected Enonslkä lake basin is the most eutrophic part since it surrounded mainly by urban area (28%) and forests (31%). To improve the water quality, large scale aeration in the autumn 2009 was started with the Mixox-oxygenerators. Aeration is used to weaken thermal stratification and recharge oxygen by increasing vertical flow circulation within the water columns. This leads to most chemical cocnetrations become more homogenous with depth and concentrations of reduced forms decrease in the hypolimnion. The aeration units were operated on campaign basis in summer 2013. One aim here was to investigate carbon gas concentrations and fluxes from the urban boreal lake basin, and examine the impacts of artificial aeration on greenhouse gases during the open water period in 2013. Besides CO2 and CH4 concentrations and fluxes, I measured also the water temperature and dissolved oxygen concentration of the water columns for monitoring the efficiency of artificial aeration. All gas samples were analyzed at the Lammi Biological Station of University of Helsinki with gas chromatography using the head space technique. As background data I used the information on temperature and oxygen profiles collected from the measuring platform on Lake Vesijärvi, and a reference study on CO2 and CH4 concentrations and fluxes dating back to 2005 when there was no aeration going on. In 2013, the studied lake basin was a source of CO2 to the atmosphere during the open water period, although the lake basin showed uptake of CO2 from the atmosphere for short times. The lake acted as a steady source of CH4 to the atmosphere throughout the measuring period. The mean CO2 flux was 34.1 mmol m-2 d-1, which was over 2.5 times higher than in the reference year 2005 when there was no the aeration yet. During the campaigns, the CO2 fluxes were higher from aerated than non-aerated water column and from the longer aerations as well. Therefore, aeration mixed stratified water columns and thus enhanced release of gases such as CO2 from surface water to the atmosphere. The mean CH4 flux was 0.2 mmol m-2 d-1. The CH4 fluxes from the Enonselkä basin were slightly decreased in 2013. These decreased CH4 fluxes in Enonselkä basin can be attributed to the limited CH4 production and high rate of CH4 oxidation in the oxygenated hypolimnion and surface of sediment. The daily CH4 fluxes fluxes during the long time aerated operations decreased. The significance of aeration to carbon cycling processes dpended on the residual CH4 concentrations in the water column.
  • Lehtonen, Ilmari (2020)
    In this paper, I examine the discussions around the concept of carbon sinks. From those discussion of Finnish forestry, I identify frames based on a media material of 108 news articles combining the methodologies of frame analysis and content analysis. I aim to contextualize the carbon sink discussions of the latter half of 2010s and examine how the natural science-based term is used to support varying policy agendas. Building from background literature on the media as a societal actor and a context around Finnish forest discussions and mismatches between science and forest policy, I reflect on the ways that Finnish media frames and contextualizes carbon sink-related forest discussions. Eventually, I identify three dominant and eight secondary frames that describe the ways of using and the transforming of carbon sink as a term in detail. The dominant frames divide the discussion into two clashing ways to communicate carbon sink issues and a third middle ground way of understanding and using the term. The middle ground frame identifies the conflict between the clashing frames and suggests reaching to an understanding as a priority goal in terms of optimal climate change policy. I discuss the results in terms of the frames' policy implications. In addition, I ask how they signal potential developments in forest and climate policy and discourse. The analysis shows that the clearest disagreements in the carbon sink conflicts raise from how forestry restricting policies are seen to affect carbon sink levels and how prominent a role should forest industry have in meeting national and international climate policy targets. The study confirms that carbon sink as a term transforms into altering forms to support distinct, even controversial policy goals because of both definitional and calculative uncertainties.
  • Jalanko, Petri (2021)
    Physical fitness has declined during the last decades in adolescents. Furthermore, several studies have found a positive association between physical fitness and brain volume in adolescents, which is noteworthy since the adolescent brain undergoes substantial changes during growth and maturation. However, despite the importance of the cerebellum on adolescents' cognition and coordination, there remains a paucity of evidence on the associations between physical fitness and cerebellum characteristics. Thus, a cross-sectional approach was used to explore the relationship of cardiorespiratory fitness (CRF), power, speed-agility, coordination and overall neuromuscular performance index (NPI) with total gray matter (GM) volume of the cerebellum as well as lobules VI & VIIb, and crus I volume in 40 (22 girls; 18 boys) adolescents. Peak oxygen uptake (V̇O2peak) was measured by the maximal ramp test on a cycle ergometer, lower limb power was determined with standing long jump (SLJ), speed-agility was assessed with the 10 x 5-m shuttle-run test, upper limb coordination was determined with the Box and Block Test (BBT) and NPI was calculated as the sum of SLJ, BBT and shuttle-run z-values. Lean mass (LM) and body fat percentage (BF%) were measured using a bioelectrical impedance analysis. Cerebellum GM volume, lobules VI & VIIb, and crus I volumes were measured by magnetic resonance imaging (MRI). Results demonstrated that V̇O2peak/LM was negatively associated (β = -.045 P= .014) with cerebellum GM volume. No statistically significant associations were found between SLJ, shuttle-run, BBT scores or NPI and cerebellum characteristics in all participants. However, a poorer shuttle-run time was associated (β = -.363 P = .024) with smaller crus I volume in girls and V̇O2peak/LM was negatively associated (β = -.501 P = .031) with lobule VIIb volume in boys. These findings suggest that, in general, CRF and speed agility are associated with cerebellum characteristics in adolescents and there may be sex differences. The results extend our knowledge of the associations between physical fitness and brain volume, but more studies should be conducted to understand the relationship further.
  • Jha, Sawan (2014)
    Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.
  • Tsui, Ka Man (2015)
    Forest certification has been used as a tool to promote forestry responsibility towards sustainable forest management. Forest Stewardship Council (FSC) is one of the certification systems that is well recognised in Europe. Nevertheless, compared to other European countries, the number of FSC chain of custody (CoC) certifications in Finland is relatively low. Semi-structured interviews were conducted with six FSC CoC certified companies to explore their experiences towards implementing and maintaining the system in their companies. The sample group was comprised of wood and paper product industries in manufacturing and trading sectors. Thematic analysis of the interviews revealed the challenges companies encountered. The results indicated that there were eight types of challenges, including three internal and five external ones, hindering the development of FSC CoC certification in Finland. Internal challenges included competence, financial resources, and a lack of motivation to change. External challenges included insufficient marketing and demand, uncertain cost benefit, keen competitor programmes, limited supply, and long trademark approval time. Meanwhile, the relevant solutions these companies adopted to deal with the challenges were discussed. Since external challenges out-numbered internal ones, it seemed that certified companies are not able to tackle the existing challenges alone. Joint-effort among other actors in the forestry sector, for instance, the national authority, FSC national office, certification bodies are essential to influence the rate of certification uptake. Furthermore, participants discussed about the future development of FSC CoC certification system in Finland. Interviewees believed that the enactment of the EU Timber Regulation (EUTR) and the introduction of forest certification into national public procurement policy could positively impact the development of the system. The results of this study could be used as a reference for potential certificate users to prepare themselves for implementing FSC CoC certification system. In addition, the study could shed a light on the development of FSC in Finland in the future.
  • Courroux, Maxime (2021)
    The changes in lake diatom assemblages as a response to climate warming over the past three decades were examined in 26 lakes across Northwestern Finnish Lapland using multivariate statistical techniques. The lakes are distributed along a steep climatic and vegetational gradient, covering three distinct vegetation zones spanning boreal coniferous forest, mountain birch woodland, and treeless tundra. Lakes were selected following a study realised by Weckström and Korhola in 2001, who had sampled the same lakes for surface-sediment diatom assemblages, physical, and chemical limnological variables. Climate data from the past 30 years was retrieved, showing a slow and steady yearly increase in temperature, with strong seasonal fluctuation and fall months experiencing the strongest warming. Surface sediment samples were taken from the lakes and their diatom communities analysed. A total of 185 diatom taxa representing 27 genera were recorded. Ordination techniques (DCA, CCA) at the genus and species level were performed to identify the main patterns of variation between diatom data from the original data set and the current study, and their relationship to environmental variables. Strong changes were recorded in four of the lakes with major shifts in dominant diatom species. Moderate changes were recorded in eight lakes, where dominance changes were recorded for a few species while the majority remained unchanged. The remaining 14 lakes did not show noticeable changes over the 30-year period. Changes observed in the studied lakes did not follow a widely observed pattern in northern Hemisphere lakes. The results indicate that while climate change is a driving factor behind changing lake dynamics with increasing temperatures and decreasing lake ice cover duration, it cannot be the only force responsible.
  • Sarasma, Juho Johannes (2021)
    Mobility, the somewhat regular and recurring physical movement of people from place to place, is a very important part of a broader transition to sustainability. In Finland the transport sector accounts for 20 % of total greenhouse gas emissions and while emissions have been steadily declining, the pace is not sufficient to meet current emission cut targets. When looking at household generated greenhouse gas emissions, mobility is the single largest contributor. Previous research has focused a lot on technological advancements and individuals’ choices as causes and solutions to sustainable mobility. These approaches have been criticized for underemphasizing the importance of social conditions. Practice theories have been presented as an alternative way of understanding mobility behaviors, challenging the mainstream individualistic explanations. Practices are routinized human behaviors that are made of several elements of materials, meanings, and competences. This thesis adopts a practice theoretical view in analyzing people’s mobility before and during the COVID-19 pandemic. The aim is to learn what practice theory can teach us about sustainable mobility, and how the pandemic has affected people’s mobility in Finland. Nine semi-structured interviews were conducted, asking the participants about their mobility practices before and during the COVID-19 pandemic, forming a comprehensive picture of their daily lives from a mobility point of view. The results were analyzed using qualitative theory-based content analysis. The results indicated that people’s mobility is a complex system which was largely affected by the COVID-19 pandemic. Various elements either enabling or hindering the use of different transport modes were identified, as were important connections between different mobility practices. Practice theory has been often used to research one mobility practice at a time and the broader look of this study, focusing on multiple mobility practices, is potentially the most important contribution this thesis makes to previous mobility research. While not providing direct answers to how people’s mobility could be made more sustainable, this thesis makes an important contribution to practice theoretical mobility research which in a Finnish context is very scarce.
  • Olgac, Ezgi (2022)
    Background– The BCL-2 protein family members are major regulators of apoptosis, and the anti-apoptotic (pro-survival) members of the family is commonly targeted with BH3 mimetic drugs in haematological cancers. However, these treatments have not been very impactful when administered as single agents and they have long been investigated for combination therapy with other agents. Acute myeloid leukaemia (AML) is one of the difficult-to-cure haematological malignancies. A recently approved therapy for AML consists of the combinatorial administration of venetoclax (a selective BCL-2 inhibitor) and a DNA methyltransferase (DNMT) inhibitor such as azacitidine or decitabine. Although this novel therapy has shown promising clinical results, the majority of the patients still relapse under this treatment. These relapsed patients typically become highly resistant to treatment and have poor prognosis, emphasising the need for new effective drug combinations. Apart from BCL-2, other family members like BCL-xL and MCL1 are also common targets of BH3-mimetic drugs. This project thus aims to understand and characterise the resistance against BH3-mimetics and investigate new therapeutic approaches to overcome the challenges of resistance. Aims– This study aims (i) to characterise BH3-resistant AML cell lines for uncovering the mechanisms of drug resistance, and (ii) to identify possible combination treatment options for overcoming drug-resistance. Methods– Viability assays with Cell Titer Glo® (CTG) and Drug Sensitivity and Resistance Testing (DSRT). The long-term effectiveness of venetoclax, azacitidine and talazoparib (a PARP inhibitor) as single agents, double combinations and triple combination were investigated with Time-to-Progression (TTP) assay. For the resistant cell line models, underlying resistance mechanisms were assessed by checking protein expression of pro- and/or anti-apoptotic members of the BCL-2 family members with western blot (WB). Real-time quantitative PCR (RT-qPCR) and WB were carried out for transcriptional and translational expression analyses of certain DNA damage-associated genes in PARP inhibitor-resistant cell lines. Results– Drug screening with DSRT has revealed promising results for two combination treatments of a BCL-xL inhibitor (A-1331852) (i) with an Aurora kinase A inhibitor (alisertib) and (ii) with an MCL1 inhibitor (S63845) for BCL-xL inhibitor-resistant cells. WB analyses of BCL-2 family members showed translational upregulation of un-inhibited members of the anti-apoptotic proteins in BH3-mimetic-resistant cell lines. A venetoclax-resistant AML cell line showed increased levels of the DNA damage marker P-γ-H2Ax upon treatments containing venetoclax, as well as increased levels of cleaved-PARP1, indicating induction of apoptosis. RT-qPCR analyses revealed increased mRNA expression of PARP1 in two resistant cell lines, whereas no significant expression changes in other DNA repair mechanism genes on the transcriptional level. Conclusions– In BH3-mimetic-resistant AML cell lines, apoptosis is avoided through translational upregulation of un-inhibited anti-apoptotic members of the BCL-2 family, and this resistance can be countered by combination treatment for additional inhibition of the compensatory anti-apoptotic proteins. Venetoclax is still effective on cells resistant to it, by inducing DNA damage and sensitising these cells against inhibitors of the members of DNA repair pathway. The transcriptional upregulation of PARP1 and the increase in its auto-catalytic activity suggests the DNA damage-inducing effects of the triple combination treatment [Ven + Aza + Tal].
  • Reinikka, Siiri (2020)
    Endometrial polyps are one of the most common benign uterine lesions, affecting approximately 10% of all adult women. While endometrial polyps have a high prevalence, their molecular pathogenesis and genetic background are largely undefined. Accordingly, the aim of this thesis was to characterize the somatic mutational landscape of endometrial polyps – to identify mutations in cancer-associated genes, and to identify mutational signatures contributing towards the somatic mutational spectrum. The present study was conducted using whole exome sequencing of 23 endometrial polyps and 18 matching normal blood samples. Mutational signature analysis was conducted using MutationalPatterns and SigProfiler. Endometrial polyps were found to carry varying number of somatic mutations in their exomes, most of them present at a low allelic fraction. Moreover, 43% (10/23) of the polyps were identified to carry one to four cancer-associated mutations, including mutations in genes such as PIK3CA 17% (4/23), KRAS 13% (3/23) and ERBB1 9% (2/23), which are well-established cancer driver genes. Cancer-associated mutational signatures do not have a notable contribution towards the somatic mutational spectrum of endometrial polyps. However, a novel signature, ‘signature B’, characterized by T>G mutations, was found to affect a subset of polyp samples. To conclude, the whole exome sequencing of endometrial polyps revealed several mutations in cancer-associated genes and a novel mutational signature, which may contribute to the development of these benign tumours. However, further research is required to confirm and validate the novel signature, and to define the genetic alterations leading to the polyp pathogenesis.
  • Lamichane, Nicole (2019)
    Over the past years sugar consumption has seen great increases worldwide, together with a rise in the prevalence of metabolic diseases. There is a growing need for a comprehensive characterisation of the genes involved in sugar metabolism, yet the mechanisms by which cells sense and respond to sugars in vivo have remained incompletely understood. Here, I analyse members of a protein family best known for their regulation of differentiation during development with regards to their role in sugar metabolism. The Hairy and Enhancer of Split (HES) protein family are a group of basic helix-loop-helix (bHLH) transcription factors that function as major downstream effectors of the Notch signalling pathway. In mammals, the HES proteins have mostly been studied for their role in cell differentiation, but HES1 has been implicated in metabolic control. Drosophila has several transcription factors belonging to the HES family, including Hairy and seven bHLH transcription factors located in the Enhancer of split complex (E(spl)-C). The E(spl)-C bHLH transcription factors display high homology and are considered to be genetically redundant, and therefore little is known about their individual functions. The other HES family members in Drosophila have not previously been linked to metabolic regulation, but Hairy has been shown to repress the tricarboxylic acid cycle. In light of the findings implicating HES1 and Hairy in the regulation of metabolism, I systematically investigated the role of the HES transcription factors in sugar metabolism. By using the GAL4/UAS system in Drosophila melanogaster, I knocked down gene expression of each of the family members, and raised the flies on diets varying in sugar content to identify possible sugar intolerance phenotypes. Here, I show that knockdown of one of the E(spl)-C bHLH genes led to severe sugar intolerance that affected both survival and organismal growth, but did not alter the levels of circulating carbohydrates and storage lipids as measured with colorimetric assays and lipid staining. Furthermore, I identify the tissues in which this transcription factor functions to provide sugar tolerance. Using analysis of publically available chromatin-immunoprecipitation sequencing data coupled with quantitative RT-PCR, I uncover mTOR target Thor/4E-BP as a putative target gene. Additionally, I show that Hairy is similarly required for complete sugar tolerance, but that the mechanism differs from the E(spl)-C bHLH transcription factor. Hairy binds to and positively regulates expression of genes involved in glycolysis and the pentose phosphate pathway, suggestive of a cooperation with earlier known regulators of sugar sensing. In conclusion, I have shown that only two HES family members are involved in the regulation of sugar metabolism and that their regulatory mechanisms are distinct, implying that the HES family members have more diverse roles than previously assumed.
  • Puutio, Johanna (2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Rönkkö, Julius (2020)
    Charcot-Marie-Tooth disease (CMT) is a collective name for inherited neuropathies affecting the peripheral nerves. CMT affects 1:2500 children and adults worldwide. The disease is genetically highly heterogeneous, and the pathogenic mechanisms are largely unknown. Thus far, there is no cure known for the Charcot-Marie-Tooth disease. Therefore, the study of the genetic factors involved in the disease and the understanding of the underlying molecular mechanisms will benefit the development of strategies to prevent or treat these diseases. In this thesis, a new candidate gene for CMT was investigated in patient fibroblasts. The novel gene variant was originally found at University of Helsinki in a pair of Finnish brothers with CMT; and in later examinations, in their affected family members. The gene encodes an ER calcium channel receptor that is responsible for Ca2+ release from the endoplasmic reticulum (ER) and plays an important role in the regulation of various cellular processes. In this thesis, I studied the effect of the variant in patient fibroblasts by Western blotting, quantitative reverse transcriptase PCR (RT-qPCR) and calcium imaging. I also knocked down the gene using siRNA in healthy fibroblasts to investigate if the loss of the receptor has a similar effect on calcium signaling as the patient variant. My results showed that siRNA treatment significantly decreased the targeted protein levels and delayed the ATP-evoked Ca2+ release from ER without profound effect on the amplitude of the release. Similar effects of the studied mutation were observed in one patient cell line, but not in the other. Patient cell line, which did not have alterations in the levels of the protein and Ca2+ release, had elevated levels of mRNA of the affected gene. The results suggest that the gene variant does not impair the total volume of the ATP-evoked Ca2+ release from ER. The possible effect of the studied mutation may be related to the decreased levels of the mutated protein, which at the functional level may affect the timing of total Ca2+ release from ER. However, the functional effect of the variant could not be confirmed with the fibroblast cells; further experiments are needed to clearly confirm the variant’s effect on calcium signaling.
  • Kemppinen, Jasmin (2020)
    Reactive oxygen species (ROS) are one of the prominent groups of signal compounds that are produced in stress conditions such as excess light. Nuclear protein RADICAL-INDUCED CELL DEAT (RCD1) is sensitive to ROS and controls the expression of organelle components, e.g. mitochondrial alternative oxidases (AOX), thus balancing the redox-status of a plant cell. Plants have fast responses to fluctuating light conditions that happen even before gene expression: i.e. readjusting the capability to receive light energy between the two photosystems by state transitions and increasing the capacity to remove excess energy by non-photochemical quenching (NPQ). Various small auxiliary proteins function in these fast acclimation events. However, many of them are identified on gene level only. The goal of this master’s thesis is to describe the role of a hypothetical protein, PPD8 in Arabidopsis thaliana. We evaluate how PPD8 is associated with RCD1 and a chloroplast thiol-regulator enzyme NTRC. We created double (rcd1 ppd8) and triple mutant plant lines (rcd1 ppd8 ntrc) by crossing single knockout lines ppd8, rcd1 and ntrc. Photosynthetic performance, NPQ and sensitivity to ROS were observed in each line by using two different chlorophyll fluorescence measurement methods: pulse-amplitude-modulation (PAM) and novel OJIP imaging fluorometry. The leaves were exposed to methyl viologen (MV), which accelerates the chloroplastic ROS production in light, and also to hypoxic conditions in order to study how the effect of MV is altered in low concentrations of oxygen. Additionally, we examined the amount of photosynthetic proteins and stoichiometry of photosystems in ppd8, rcd1 and rcd1 ppd8 by immunological methods. Finally, PPD8 gene with attached hemagglutinin encoding tags was generated by cloning and reintroduced back to the ppd8 knockout lines. Plants lacking RCD1 are very tolerant against MV and ROS, but when rcd1 was crossed with ppd8 the resistance was suppressed. Both rcd1 ppd8 and ppd8 exhibited elevated chlorophyll fluorescence and NPQ values. The removal of PPD8 gene had an impact on the abundance and the stoichiometry of photosynthetic proteins reducing the plants’ performance. When RCD1, PPD8 and NTRC were simultaneously absent the plants had major defects: their NPQ and fluorescence values were drastically increased. Furthermore, several results hinted towards possible issues in the function of ATP synthase in ppd8 background plants. It is also known that NTRC regulates ATP synthase: taken together, the results suggest that PPD8 is necessary for a fully operative ATP synthase and photosynthetic machinery. By reintroducing PPD8 to knockout line ppd8, the phenotype could be reverted back to wild type -like, thus confirming the significance of the PPD8 gene product in plant.
  • Zaki, Urfa (2019)
    Cerebral dopamine neurotrophic factor (CDNF) belongs to the the family of neurotrophic factors that are evolutionary conserved, having a unique structure, with two domains: C-terminal domain and the N-terminal domain, and a cysteine bridge. It is known to be involved in the repair of the dopaminergic neurons when studied in the animal models of PD, which shows their different mode of action as compared to other neurotrophic factors, highlighting their therapeutic potential. Analysis of the crystal structure shows that CDNF and MANF consist of two domains: the saposin-like N-terminal domain with five α-helices stabilized by three disulphide bridges, and presumably unstructured C-terminal domain with a disulphide bridge. Characteristic feature of saposin-like proteins is their ability to interact with membranes or lipids. The lipid interaction may be crucial for the activity of CDNF and MANF proteins. In the first part of this project, the binding of CDNF was tested with several oxidized lipids, using two methods; Co-sedementation assay and lipid fluorescence assay;with two different types of probes. According to the results, CDNF seemed to show binding with POVPC. The second part of the project involved testing the binding and internalization of CDNF to mouse myoblast cells in the presence of oxidized lipid; POVPC. It was observed that CDNF seemed to show binding to the cell surface of the mouse myoblast cells (C2C12) and is also observed to be internalized to the cells as well. However, as these are the preliminary results, so we need to further test the binding between the protein and other lipids and devise more precise protocols for the testing the internalization to the cells.
  • Aksentjeff, Katri (2020)
    The progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a neurodegenerative disease caused by loss-of-function mutations in the cystatin B gene (CSTB) with juvenile onset, stimulus sensitive action-activated myoclonus, generalized tonic-clonic seizures and ataxia. The cystatin B (CSTB) protein inhibits cysteine proteases, such as cathepsin L, which has been reported to cleave histone H3 N-terminal tails in mouse embryonic stem cell differentiation. We have shown previously that histone H3 cleavage is an irreversible epigenetic chromatin modification, which occurs in cystatin B-deficient (Cstb-/-) mice derived neural progenitor cells during differentiation. In this study, first, we used the wild-type E13.5 mice brain derived neural cells in culture to determine the effect of extrinsic signaling factors to our earlier developed ex vivo neurosphere cell model. We also confirmed that the histone H3 cleavage positive progenitor cells are primarily neuronal cells. Then, we used phenotype rescue of Cstb-/- neural progenitor cells and showed that CSTB is a negative regulator of histone H3 cleavage. In wt mouse neurosphere cryosections, we showed that cathepsin B and L are not expressed in the nucleus of neural cells before differentiation.
  • Pennonen, Jana (2017)
    Puberty is a process of physiological changes, through which an immature individual becomes sexually mature. In humans, timing of puberty is highly variable within and between sexes and populations. Timing of puberty represents a complex trait, which is controlled both genetically and environmentally. Precocious pubertal timing is associated with development of metabolic diseases later in life, such as obesity and diabetes, and other disorders as ovarian and testicular cancer. Despite the estimated high heritability (50-80%) of pubertal timing, its genetic background is still poorly understood. Recently, the genome-wide association studies (GWASs) revealed many novel pubertal timing associated loci. Nevertheless, molecular mechanisms behind these associations remain elusive. This thesis focused on gene vestigial-like family member 3 (VGLL3), which is associated with pubertal timing in humans and maturation in Atlantic salmon (Salmo salar). Since the main physical structures, such as the hypothalamus and the pituitary gland, needed in reaching puberty are evolutionary conserved and start to develop in vertebrates during embryogenesis, the aim was to study the expression pat-terns and role of vgll3 in zebrafish (Danio rerio) during this period. In order to localize expression patterns of the vgll3 gene in zebrafish embryos, a whole-mount in situ RNA hybridization (ISH) was performed. mRNA overexpression and morpholino oligonucleotide (MO) knockdown techniques were used to alter the vgll3 gene expression levels in 0-5 dpf zebrafish. The combined injections of both mRNA and MO were performed to validate MO specificity. The ISH experiment showed the expression patterns in 0-1 dpf embryos. The expression was ubiquitous up to 6 hours post fertilization becoming more localized to specific regions in the head and trunk of the embryos during the later stages. Altering vgll3 expression with high concentrations of synthetic mRNA or MO lead to phenotypical abnormalities such as shortened and curved body axis, pericardial and yolk sack edemas, deformed heads and eyes. However, it remained unclear if these malformations appear only due to the alteration of vgll3 expression levels. The results suggest that vgll3 may play an important role in the embryonic development. However, the study does not show that vgll3 has impacts on the pubertal timing in vertebrates by affecting the development of the structures required for sexual maturation.