Browsing by Title

Protein kinases are signaling molecules that regulate vital cellular and biological processes by phosphorylating cellular proteins. Kinases are linked to variety of diseases such as cancer, immune deficiencies and degenerative diseases. This thesis work aimed to identify direct substrates for protein kinases in the CMGC family, which consists of the cyclin-depended kinases (CDK), mitogen activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and CDC-like kinases (CLK). CMGC kinases have been identified as cancer hubs in interactome studies, but large-scale identification of direct substrates has been difficult due to the lack of efficient methods. Here, we present a heavy-labeled 18O-ATP-based kinase assay combined with LC-MS/MS analysis for direct substrate identification. In the assay, HEK and HeLa cell lysates are treated with a pan-kinase inhibitor FSBA which irreversibly blocks endogenous kinases. After the removal of FSBA, cell lysates are incubated with the kinase of interest and a heavy-labeled ATP, which contains 18O isotope at the γ-phosphate position. Resulting phosphopeptides are enriched with Ti4+- IMAC before the LC-MS/MS analysis, which distinguishes the desired phosphorylation events based on a mass shift caused by the heavy 18O. With this pipeline of methods, we managed to quantify and identify direct substrates for 26 members of CMGC kinase family. A total of 1345 substrates and 3841 interacting kinase-substrate pairs were identified in cytosolic cell lysates, from which 165 were annotated in the PhosphoSitePlus® database. To identify substrates for kinases with nuclear localization, ten kinases were tested with nuclear HEK cell lysate. We identified 194 kinase-substrate pairs, 141 of which were unique to the nuclear fraction and 27 annotated in the PhosphoSitePlus® database. Finally, kinases with outstandingly high amounts of novel substrates were subjected to gene ontology analysis. We were able to link the gene ontology classifications of novel substrates to the biological processes regulated by the kinase of interest. These results indicate that heavy-labeled 18O-ATP-based kinase assay linked LC-MS/MS is a useful tool for large-scale direct kinase substrate identification.

Plants control the exchange of gases through the stomatal pores. Stomata are formed by guard cells and the closure of stomata are regulated via a complex signaling network in response to various biotic and abiotic stimuli, such as pathogens, elevated levels of CO2 and darkness. The leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) is part of the network regulating stomatal closure. GHR1 is an inaktive pseudokinase that can activate SLOW ANION CHANNEL-ASSOCIATED1 (SLAC1), an anion channel that is crucial for stomatal closure, via interacting proteins. The exact role of GHR1 is still partly unknown, however, it has been suggested that GHR1 could function as a scaffold or as an allosteric regulator of additional components required for stomatal closure. The aim of this study was to identify novel interactors of GHR1. First stable plant lines expressing fusion proteins GHR1-YFP, GHR1W799*-YFP and plain YFP as a negative control were generated and from these lines fusion protein expression levels and the subcellular localization were studied. Next the plant lines were used for purifying GHR1 interacting proteins with the use of co-immunoprecipitation and identification of the proteins with mass spectrometry. The unlikely GHR1 interactor candidates were then filtered from the mass spectrometry data. The subcellular localization and the protein expression of the interacting proteins were studied with the use of internet databases. Literature of the GHR1 interacting proteins were studied in order to make possible connections with GHR1 and stomatal closure. In this study 38 GHR1 interactors were identified. Literature search revealed that many of the identified interactors had a known role in stomatal movements. These included proteins such as PLASMA MEMBRANE INTRINSIC PROTEIN2-1 (PIP2-1) and BETA CARBONIC ANHYDRASE 4 (BCA4), that are known to have a role in stomatal closure. Future work includes confirming the interactions with independent methods and studying the molecular mechanisms related to stomatal movements. The GHR1 interactome identified here for the first time reveals novel parts of the network regulating stomatal movements and thus increases our understanding of molecular mechanisms behind stomatal functions.

This study aimed to identify conservation landscapes with potential to be mutually beneficial for people and African savanna elephants (Loxodonta africana) in South Africa through spatial conservation planning analyses that integrate ecological and socioeconomic data. The research questions were: (i) what are the most ecologically suitable areas for the reintroduction of elephants, and (ii) which of these areas provide the best opportunities for also sustaining socioeconomic development of local people. The first question was answered with an ecological model that predicts habitat suitability for elephants, developed by a combination of literature review, expert opinion, and GIS-based methods. The second question was answered by combining the ecological model with socioeconomic criteria in Zonation spatial conservation planning software. The results show that the central part of South Africa holds most potential for elephant conservation as it has the largest uniform area of high-quality habitat, while the area also meets the socioeconomic criteria. The priority areas for the conservation of elephants were classified into top priority classes of 1%, 2%, 5%, 10% and 20%. The identified areas hold an unrealized opportunity in the wildlife and ecotourism sectors, and the reintroduction of elephants to those areas could provide the foundation for long-term economic activity of local communities e.g. in the form of elephant-based ecotourism, while contributing to the conservation of elephants. Conserving just the top 5% priority areas would grow South African protected area estate by approximately three million hectares and increase the current elephant range by approximately 75%. Ideally, the results of this study could be used to inform the on-going decision-making process on where to allocate resources for elephant conservation in South Africa.

Education research has for decades acknowledged that prior knowledge is a strong predictor of academic success. This idea is largely based on constructivist theory of learning which postulates that all learning occurs by actively building on existing knowledge. When this prior knowledge conflicts with the normative scientific understanding, students are dealing with incompatible knowledge structures, or misconceptions. Misconceptions need to be revised and sometimes even replaced through a learning process called conceptual change. Research shows that the level of prior knowledge can determine students’ academic success and performance. Undergraduate biology students enrol to university with diverse levels of prior knowledge and concepts regarding topics such as photosynthesis, cellular respiration, primary production in ecosystems, and Darwinian evolution. These topics present challenges for learning because of their complexity. At the same time, a robust understanding of them is essential. These topics are at the heart of mitigating and resolving the climate crisis and other global natural threats. This study explored the level of prior knowledge and the nature of misconceptions held by undergraduate biology students at the beginning of their academic degree in fall of 2019, and further sought to describe how their conceptual understanding developed during the first academic year. Students (N = 41) completed a questionnaire consisting of eight open-ended questions that were designed to assess declarative knowledge of facts and meaning, and procedural integration and application of knowledge. This pre-test measurement was conducted in September 2019. In the post-test measurement, the same questionnaire was repeated a year later. The data were analysed with a mixed methods approach where the answers were quantitatively scored as well as qualitatively analysed for misconceptions. The qualitative content analysis of the answers relied both on existing literature and on the content of the answers themselves. Results showed that the students’ prior knowledge was relatively poor in the beginning of their studies. Most students performed well in tasks measuring knowledge of facts and meaning but struggled in tasks measuring integration and application of knowledge. During the first academic year, the students’ understanding generally improved as demonstrated by the improvement in mean scores of the tasks. Misconceptions were robust and pervasive. The most pervasive misconceptions reflected difficulties in understanding emergent properties and processes. Misconceptions related to the process of Darwinian evolution became more prominent in the post-test. Persistent misconceptions became integrated with the new conceptual frameworks that the students acquired during the first academic year. If students held no misconceptions in the post-test, they performed significantly better in both tests than those with misconceptions. During this first academic year learning seemed to be mainly additive as conceptual change turned out to be rare. The need for more encompassing biology teaching at least in the University of Helsinki became evident. Introductory courses should acknowledge the large degree of variation in students’ prior knowledge and assess the most common and serious misconceptions even over course theme disciplines to ensure more equal learning outcomes.

Distal myopathies are a group of rare progressive genetic muscle disorders that are extremely varied both genetically and clinically. Typical symptoms include weakness and atrophy limited to the skeletal muscles of distal extremities in hands and legs. The age of onset ranges from early childhood to late adulthood depending on the disease. Currently around 30 genes have been associated with distal myopathies, most of them causing a dominant disease. The objective of the thesis was to identify the disease-causing variant in a family affected by autosomal dominant distal myopathy with early adulthood onset. Affected family members expressed weakness and atrophy in muscles of both hands and legs. To narrow down the chromosomal location of the disease-causing variant, linkage analysis was conducted with genome-wide single nucleotide polymorphism data of family members. Because of the progressive nature of the disease and uncertain disease status of one family member, linkage analysis had to be repeated a few different times with different settings. Both disease statuses and pedigree size were altered to account for the possibility of presymptomatic carriers or incomplete penetrance. Analyses with different parameters led to discovery of multiple possible co-segregating regions. Rare co-segregating small-scale and structural variants as well as repeat expansions in these regions were examined from next-generation sequencing data with multiple bioinformatic detection tools. The segregation of possible candidate variants was validated with Sanger sequencing and PCR. Ultimately, no likely rare co-segregating variant of any type of genetic variation with a likelihood to cause a disease such as distal myopathy was identified by any detection method used. Lack of potential disease-causing variant could be due to incomplete penetrance of the variant or if it was in non-coding regions, such as a deep intronic splicing variant in a gene currently not known to be connected to muscles.

Tässä tutkimuksessa selvitettiin, millaisia kehyksiä ilmastoahdistus saa kahdeksassa, vuonna 2021 julkaistussa suomalaisessa podcast-jaksossa. Näin pyritään lisäämään ymmärrystä ilmastoahdistuksen ilmiöstä, sekä siitä, millaisia kehystyksiä aihe mediassa saa. Tutkimus on tehty laadullisella kehysanalyysilla, joka perustuu Erwin Goffmanin (1974) kehittämään kehysteoriaan ja sen myöhempiin sovellutuksiin. Lyhyesti sanottuna, tutkimus pohjaa siihen olettamukseen, jossa median tavat kehystää ja esittää jokin aihe tai asia ohjaavat sitä, millä tavalla asiat meille esitetään ja millaisina me asiat näemme. Tutkimuksen aineisto kerättiin suoratoistopalvelu Spotifysta, Yle Areenasta ja Suplasta. Kehyksien muodostamisessa hyödynnettiin Robert Entmanin (1993) median kehysanalyysin periaatetta, jossa kehyksillä on neljä tarkoitusta: Ongelmien määritteleminen, ongelman aiheuttavien tekijöiden tunnistaminen, moraalisten arvioiden tekeminen sekä ratkaisujen ehdottaminen. Tutkimuksen tuloksena syntyi kolme kehystä, jotka nimettiin luonnollisen ilmastoahdistuksen, ilmastoahdistus sukupolvikysymyksenä sekä ilmastoahdistus tarvitsee vertaistukea, käsittelyä ja toimintaa kehyksiksi. Jokainen kehys kuvaa omalla tavallaan sitä, millaisena ilmastoahdistus nähdään viime aikaisissa podcast-keskusteluissa. Luonnollisen ilmastoahdistuksen kehyksessä korostuvat näkemykset siitä, kuinka ilmastoahdistus voidaan nähdä luonnollisena ja normaalina, sekä hyvänä reaktiona motivoivan vaikutuksensa kautta. Kaiken kaikkiaan kehys kuvaa ilmasto- ja ympäristöahdistuksen ilmiön monitahoisuutta ja yksilöllisyyttä, sekä pyrkii vahvistamaan näkemyksiä siitä, mitä ilmastoahdistus on ja mitä se ei ole. Lisäksi ilmastoahdistuksen sisältämä ahdistus sana kyseenalaistetaan. Ilmastoahdistus sukupolvikysymyksenä - kehys avaa puolestaan näkemyksiä nuorten ilmastoahdistuksen syihin ja korostumiseen, sekä ottaa kantaa aikaisempaan ilmastoahdistuskeskusteluun, joka joidenkin keskustelijoiden mielestä näyttäytyi ilmiötä vähättelevänä. Kokonaisuudessaan kehyksessä kuitenkin korostuu näkemys siitä, että ilmastoahdistus ei ole vain nuoria koskettava asia, vaikka se tällä hetkellä korostuu ja ilmenee eniten nuoremmissa sukupolvissa. Kehys ilmastoahdistus tarvitsee vertaistukea, käsittelyä ja toimintaa, tuo esiin keskustelijoiden näkemyksiä ja kokemuksia liittyen ilmastoahdistuksen käsittelyyn ja lievittämiseen, sekä osallistuu keskusteluun koskien ilmastotekojen merkityksellisyyttä. Tutkimuksen tulosten perusteella voidaan sanoa, että ilmastoahdistus saa vuonna 2021 julkaistuissa, suomalaisissa podcastkeskusteluissa monia eri sävyjä ja ilmastoahdistusta käsitellään niissä laajasti. Ja vaikka keskustelijoiden näkemykset ovat pitkälti linjassa ilmastoahdistusta käsittelevien julkaisujen kanssa, muutamia kiinnostavia ja tutkimattomiakin näkökulmia ja kehystyksiä ilmenee. Osaltaan tutkimuksen tulokset täydentävät kuvaa siitä, miten valtavirtamediaa hitaammat lajityypit – kuten podcastit – mukautuvat muualla esitettyihin määritelmiin ja kehystyksiin, mutta myös täydentävät niitä. Tällöin podcast-keskustelujen voidaan nähdä tarjoavan vaihtoehtoisia kehyksiä, jotka osittain haastavat ja täydentävät julkisuudessa hallitsevia kehyksiä.

The impacts of climate change are going to be significant in Finland, thus the need to adapt is inevitable. Municipalities are the key to adaptation because the impacts of climate change are met locally. Several cities have developed their measures to climate impacts. However, multiple barriers may hinder the planning and implementation of adaptation measures in the cities. The purpose of this research is to identify and overcome the barriers in urban climate change adaptation in the City of Helsinki. The main data of this research was collected in a workshop and it consists of the blank form replies collected with 6-3-5 method and focus group discussions. 11 civil servants from the City of Helsinki who deal with adaptation issues in their daily work participated in the workshop. The participants identified the barriers and evaluated the most important ones in the workshop: lack of cost-benefit analyses, rivalry of the resources with other interests, lack of urgency regarding adaptation, lack of information, fragmentation of the organization and unclear roles and responsibilities. Identifying the barriers does not solely promote the resilience of the cities but it is an important step in the development of adaptation work. It is essential to seek possible solutions to overcome the identified barriers. Six solutions that can tackle simultaneously several barriers emerged from the data gathered in the workshop: costbenefit analyses, increasing training and information, concrete examples, increasing co-operation, clear modes of action and responsibilities and the support and commitment of the management. With these solutions, the City of Helsinki has the possibility to simultaneously overcome several barriers that were identified in this research. To conclude, the responsibility of climate change adaptation should be clarified in the new city organization and silos between different sectors should be addressed, if possible. It would be useful to utilise the multi-criteria decision analysis in prioritising and argumenting of the adaptation measures in the city. As additional conclusions, it seems that improved co-operation with the universities and research institutions, and legislation indicating clear roles and responsibilities in terms of adaptation might benefit the adaptation work in the City of Helsinki. As for the need for further research, the analytical framework developed and utilized in this research needs to be tested in other case studies also.

Tämä pro gradu -tutkielma tarkastelee vuosina 2021-2022 Suomessa esitettyjä ilmastoyksiköiden käyttöön liittyviä mainos- ja markkinointiväittämiä ja niiden laatua. Tällaisia väittämiä ovat esimerkiksi yleisesti käytetyt hiilineutraalius- ja kompensaatioväittämät. Työ esittää katsauksen siihen, kuinka tarkkaa ja asianmukaista tietoa vuosina 2021-2022 mainos- ja markkinointiväittämät ovat esittäneet. Työssä väittämiä tarkastellaan tulevien EU-sääntelyn edellytysten sekä helmikuussa 2023 julkaistujen kansallisten suositusten valossa (Laine ym., 2023). Sääntelyn ja kansallisen toimintakentän muutoksien kautta työ tutkii väittämien mahdollista tulevaisuutta ja muutoksia väittämien tulevassa käytössä. Merkittävin yksittäinen ilmastoyksiköihin perustuvien väittämien tulevaisuuden käyttöön vaikuttava tekijä on väittämien tekemiseen käytettyjen ilmastoyksiköiden kaksoislaskennan välttäminen. Kapea-alaisen pilottitutkimuksen avulla työssä testataan kaksoislaskennan riskin arviointiin esitettyä menetelmää (Laininen ym., 2022) sekä arvioidaan väittämiin liittyvää riskiä. Työssä hyödynnetään kahta erillistä aineistoa, joiden avulla tutkitaan mainonnassa ja markkinoinnissa esitettyjen väittämien eroja. Väittämien laatua tutkitaan analysoimalla niiden syvyyttä eli sitä, kuinka tarkkaa tietoa väittämä tarjoaa sekä asianmukaisuutta eli sitä, onko väittämässä annettu tieto selkeää ja yksiselitteistä vai mahdollisesti harhaanjohtavaa. Työssä toteutetun analyysin perusteella suurin osa vuosina 2021-2022 mainonnassa ja markkinoinnissa esitetyistä ilmastoyksiköihin perustuvista väittämistä oli harhaanjohtavia. Mainonnassa ja markkinoinnissa esitettyjen väittämien välillä esiintyi kuitenkin merkittäviä eroja. Analyysin perusteella mainonnassa esitetyt väittämät olivat markkinointiväittämiä heikompilaatuisia sekä syvyydeltään että asianmukaisuudeltaan. Arvioinnin perusteella väittämiin liittyvät nykyiset markkinointikäytännöt eivät yleisesti vastaa niille esitettyjä kansallisia suosituksia eivätkä tulevia EU-sääntelyn edellytyksiä. Väittämien tarkkuuteen ja yksityiskohtaisuuteen liittyvien markkinointikäytäntöjen lisäksi tulee tulevaisuudessa ottaa huomioon väittämiin liittyvä kaksoislaskennan riski, joka pilottitutkimuksen perusteella koskee oletettavasti merkittävää osaa nykyisistä markkinoilla esitetyistä väittämistä.

Tumors contain variable number of different immune cells that infiltrate the tumor microenvironment, such as tumor infiltrating lymphocytes (TILs). More research is needed to understand the functional and clinical importance of various TIL subgroups in cancer. Understanding the differences between individual cancer patients will help development of new treatment methods and discovering why only some patients respond to immunological treatments. Renal cell carcinoma (RCC) is the most common kidney cancer type with good overall survival prognosis when the tumor is surgically removed before it has metastasized. However, the prognosis of RCC is significantly decreased when the cancer has spread. The aim of this master’s thesis project was to characterize the tumor infiltrating lymphocyte populations in patient derived RCC samples. Characterization was done with flow cytometry and a custom antibody panel designed to detect various lymphocyte subpopulations. We also wanted to further study the TILs by expanding the lymphocytes from the tumor samples and test their function in an impendence-based assay against matched autologous tumor cells. Based on the flow cytometry results, the different RCC subtypes in the cohort showed some variation in TILs. Still, more research is needed to investigate these differences. We were able to culture the TILs from the RCC tumor samples, and most of them were CD4+ T cells expressing memory markers CD45RO and CCR7. Some expanded TILs expressed markers related to T cell activity and terminal differentiation. In conclusion, this thesis provided material and insights for future RCC TIL experiments as well as considerations for optimization needed in further studies.

The vagus nerve is the longest nerve of the autonomic nervous system. It innervates, among other organs, the stomach, the lungs and the heart, and it reaches several areas of the brain, including the locus coeruleus and the amygdala. The invasive stimulation of this nerve (vagus nerve stimulation, or VNS) is a currently used method for the treatment of refractory epilepsy and pharmaco-resistant depression (Englot et al. 2011; O’Reardon et al., 2006), but the impact that this technique might have on the brain physiology and functions is still under investigation. Various studies (Frangos et al., 2015; Yakunina et al., 2016; Hansen, 2019) have shown that VNS increases noradrenaline production in the brain, a neurotransmitter that is involved in several cognitive processes, such as sleep and mood control. Furthermore, in a study on patients with epilepsy, by Sun et al. in 2017, VNS appeared to have a clear effect on working memory and emotion-attention interaction. Nevertheless, VNS presents all the risks and potential complications that characterize invasive procedures requiring surgery. Therefore, research is now focusing on safer, non-invasive alternatives, such as transcutaneous vagus nerve stimulation (tVNS). This technique allows to stimulate the nerve through its sensory fibres, located in the cymba and tragus of the ear. The scope of the present study was to see whether tVNS would have the same effects on cognitive and affective functions as VNS. The sample for this single blind placebo-controlled study was composed of 30 healthy subjects between 18 and 45 years old. Exclusion criteria included a history of psychiatric, neurological or cardiovascular diseases. All subjects were asked to complete a computer-based task, the Executive Reaction Times-Test. Throughout the test the subjects alternately received an active or a placebo stimulation, and their brain activity was recorded for the whole duration of the test using a 64-channel EEG cap. The Executive-Reaction Times-Test was chosen for this study because it allows to test multiple executive functions simultaneously. The subjects were presented with a series of stimuli on a screen and were asked to react as fast and accurately as possible to “Go” signals, and to refrain from responding when “NoGo” signals appeared. The test started with a triangle pointing either up- or downwards, followed by a brief pause and a traffic light image. The traffic light showed either a red or a green light and included an emotional distractor in the form of a spider or a flower. The red and green lights were alternately used as “Go” or “NoGo” signals, and the rule changed at each test block. In order to complete the task, subjects needed to keep the image of the triangle in their working memory, stay focused on the stimuli and be ready to react or be able to inhibit any responses, thus several main executive functions are being tested: inhibitory control, working memory, attention and emotion-attention interaction. Active stimulation was delivered through clip electrodes that were attached to the tragus of the left ear, whereas placebo stimulation was delivered through clip electrodes that were attached to the left ear lobe. The subjects were not aware of the difference between the two locations. Only the data of 18 subjects was used for the results analysis, because of technical difficulties with the EEG data (some recordings were too noisy, some presented flat channels). The behavioural data was divided into reaction times and errors, which were separately analysed. The EEG data was used to extract the amplitudes of the ERP peaks N2 and P3. The former is a negative peak visible at 200-350ms; the latter is a positive peak visible at 300-500ms. Previous studies have shown the peaks to be associated with response conflict and inhibition (Falkenstein et al., 1999; Donkers et al., 2004; Smith et al., 2013). The behavioural data analysis did not show any significant effect of stimulation on reaction times or error amounts. The ERP analysis, instead, returned interesting results. We observed a main effect of stimulation (p=0.04) in “NoGo” conditions. There was a significant reduction in the N2P3 amplitude and the N2 amplitude in “NoGo” conditions, with active stimulation compared to placebo. These results seem to suggest that with tVNS, fewer cognitive resources are allocated to resolve the inhibitory task, without worsening the subjects’ performance. The lack of significance in the behavioural results might have been due to a ceiling effect, with the Executive Reaction Times-test being too easy for our sample. Overall, the number of errors was too low to conduct a reliable statistical analysis. Nevertheless, the effects we observed on brain physiology would suggest that further research is needed to explore the actual impact of tVNS on cognitive and affective functions.