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This project was about the molecular mechanisms involved in the generation of eicosanoids in human mast cells with particular emphasis on lipid bodies as a source and/or site of lipid mediator biogenesis. The cells to be used are isolated from human peripheral blood provided by Finnish Red Cross Blood Transfusion Service and collected from healthy donors. Human mast cells are found in connective tissue. They contain granules filled with histamine, heparine and proteases. Human mast cells are potent effector cells in host-defense mechanisms of innate immunity, including inflammatory diseases such as atherosclerosis. Activation of mast cells by different stimuli triggers the release of a huge range of mediators, including de-novo synthesized eicosanoids, which are highly biologically active lipid mediators. The major eicosanoid released by activated mast cells is prostanoid prostaglandin D2 (PGD2). The aim of this project was to find out whether mast cell lipid bodies are the cellular compartments of PGD2 synthesis, what are the enzymes involved in AA liberation from TGs, and whether TG-derived AA is a source for PGD2 production. The enzymes of special interest were hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). We were also interested about hematopoietic prostaglandin D synthase (HPGDS), the key enzyme in the production of D and J series of prostanoids. Methods used in this pro gradu work include siRNA transfections, RNA isolation, cDNA synthesis, qPCR, immunoblotting, ELISA and conventional fluorescence microscopy. Immediate increase in the amount of PGD2 released from mast cells sensitized with human IgE (1 µg/ml) and activated by polyclonal rabbit anti-human IgE (1 µg/ml) was observed. The increase was most prominent after one hour of activation, and slowly decreased to basal levels at 48 h post-activation. siRNA transfection affected the amount of enzyme DNA in mast cells and the amount of PGD2 released. HSL, ATGL and HSL+ATGL double knockdowns all reduced the amount of PGD2 released in acute (5 to 30 minutes) term activation compared to control cells. However, no significant changes were observed in the mRNA expression levels of ATGL, HSL, CGI-58, HPGDS or COX-1 under mast cell activation. The only significant changes in mRNA expression levels were observed with COX-2. However, the relative expression of HPGDS increased in IgE treated mast cells compared to control treated cells and the expression was even greater in mast cells treated with αIgE also. Both ATGL and HPGDS were recognized throughout the cytosolic area in the non-activated Ctrl cells. Although HPGDS located also in the circumference of mast cells, no clear localization of HPGDS was observed in the circumference of mast cell lipid droplets. The experiments carried out at the Wihuri Research Institute, including those presented here, have established that, in addition to phospholipids, the triglycerides present in mast cell lipid droplet core are also an important source of eicosanoids, and that also ATGL and HSL, not just cPLA, can release arachidonic acid for eicosanoid production. The ramifications of this study include the possibility that arachidonic acid release from triglycerides for the formation of eicosanoids could take an indirect or a direct route to supply precursors for cellular eicosanoid biosynthesis. The key is the pathway of AA release. In the direct pathway, AA is released from LD TGs by ATGL or HSL and this free AA is used for the generation of PGs by either COX-1 or COX-2, depending on the status of the cell. In the indirect pathway, AA is liberated from LD TGs by ATGL or HSL and then further re-esterified into phospholipids from where AA is then finally released by cPLA2 for the generation of eicosanoids.

Ilmastonmuutos on yksi aikamme viheliäistä ongelmista, jonka yhtenä ratkaisukeinona pidetään kiertotaloutta. Kaupungit ovat ilmastonmuutoksen torjunnassa avainasemassa muun muassa suuren asukaslukunsa ja hiilijalanjälkensä takia. Kiertotalouskaupunkeja syntyy enenevissä määrin, ja myös Helsingin kaupunki on julistautunut kiertotalouskaupungiksi. Matka lineaarisesta kaupungista kohti kiertotalouskaupunkia on kuitenkin pitkä, ja siinä matkalla tarvitaan myös muita toimijoita kaupungin lisäksi. Tutkimuskirjallisuuden mukaan yhteistyö on avainasemassa niin itse kiertotaloudessa kuin kiertotalouskaupungeissakin. Myös Helsingin kaupunki on tunnistanut tarpeen toimia yhteistyössä muiden toimijoiden kanssa kiertotalouskaupungin rakentamisessa. Avainasemassa olevia yhteistyön osapuolia ovat niin yritykset kuin kaupunkilaisetkin. Helsingin kaupunki on lanseerannut Kiertotalousvahti- palvelun, jossa on 31 kiertotalouteen liittyvää toimenpidettä. Näitä toimenpiteitä voidaan pitää Helsingin kärkihankkeina kiertotalouden saralla. Näissä hankkeissa uskoisi siis myös yhteistyön näkyvän kaupunkilaisten ja yritysten kanssa. Tutkimuksen tavoitteena oli analysoida teorialähtöisen sisällönanalyysin avulla Kiertotalousvahdin 31 toimenpidettä ja etsiä toimenpiteistä yhteistyötä. Tarkempaan syynäykseen otettiin yhteistyö kaupunkilaisten ja yritysten kanssa. Tutkimuksessa oli tarkoitus selvittää myös kaupungin, kaupunkilaisten ja yritysten rooleja yhteistyön saralla. Tutkimuksessa selvisi, että Helsingin kiertotalouden kärkihankkeissa yhteistyötä näkyy yllättävän vähän. Yhteistyö kaupunkilaisten kanssa on lähes olematonta, ja yritystenkin kanssa vähänlaista. Yhteistyön avulla kiertotalous saataisiin leviämään laajemmalle ja sen vaikutukset olisivat laajempia. Kaupunki jättää paljon potentiaalia käyttämättä kiertotalouden suhteen.

Abstract Faculty: Faculty of Biological and Environmental Sciences Degree programme: Genetics and Molecular Biosciences Study track: Molecular and Analytical Health Biosciences Author: Annie Stendahl Title: Measurement repeatability of flow cytometry and nanoparticle tracking analysis for optimization of extracellular vesicle measurements Level: Master’s thesis Month and year: 11/2022 Number of pages: 92 Keywords: extracellular vesicles, repeatability measurements, metrology, traceability, flow cytometry, nanoparticle tracking analysis, reference material, METVES Supervisor or supervisors: Virpi Korpelainen, Katariina Maaninka and Pia Siljander Collaborative partner: VTT Technical Research Centre of Finland Ltd. Where deposited: E-thesis Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles secreted by all cells, containing variable cargo from nucleic acids and proteins to carbohydrates, metabolites, and lipids. EVs are considered to be involved in many physiological and pathological cell functions. Due to their presence in biofluids hence enabling semi-invasive liquid biopsies, EVs have indicated great promise for utilization as biomarkers in clinical settings. The innate properties of EVs and their cargo could also be harnessed into therapeutic use. However, the current methods and reference materials for determining EV concentration and size have not yet achieved the metrological level of repeatability and traceability, which is needed for EV measurements to be utilized in clinical settings. The aim of this thesis project was to evaluate repeatability of the methods typically used for EV quantification and size determination, flow cytometry (FCM) and nanoparticle tracking analysis (NTA). The repeatability was analyzed with reference material made of hollow organosilica beads and biological EV test samples, both developed in an ongoing EU metrology-project METVES II for EVs. A similar biological EV test sample was also prepared as part of the thesis project. Finally, the repeatability measurements were conducted with calibration beads recommended by the instrument manufacturers. The calibration beads gave repeatable results with FCM and one of the two NTA instruments tested, but neither the reference beads nor the biological EV test samples produced repeatable results to enable determination of repeatability. However, valuable understanding was gained on what can be optimized during the measurements and operation of the instruments to generate more repeatable results with FCM and NTA in EV analysis. Prior knowledge of both the sample type and method used for measuring would enable optimization of the measurement and instrument operation. Whether the aim is EV quantification or size determination, instrument errors and bias could then be minimized by adjusting the settings according to sample type. Furthermore, EV quantification and size determination would benefit from combining different methods to ensure more reliable and repeatable results. It is clear that more research needs to be done, for i.e., the tested reference beads need to be further developed to be established as EV reference material and enabling standardization of EV measurements. Standardizing EV quantification and size determination is required to achieve metrological repeatability and ultimately, traceability, and thus for EVs to be utilized in clinical settings as biomarkers or therapeutic use.

Growth differentiation factor 15 (GDF15), a member of TGF-β super family is a soluble cytokine that is associated with different pathological conditions including cancer, cardiac and renal failure and obesity. Its high serum levels are linked with symptoms like cachexia/anorexia in cancer patients and can be used as a marker for these diseases. Its crucial role in weight regulation and energy homeostasis has been demonstrated by treating obese mice with GDF15, which results in weight lose along with improved glucose metabolism and increased insulin tolerance. It is now known that GDF15 exerts its metabolic effect by binding to a GDNF receptor -α-Like (GFRAL) receptor along with co-receptor RET. Interestingly, these two receptors co-localize only in the brain stem area of mice and humans indicating involvement of a neuronal circuit in GDF15 mediated effects. Despite its implications in major health disorders, little is known about the interaction of GDF15 with its receptors and how this interaction in turn modulates different cellular signalling and functions. The aim of the thesis was to study the mechanism and factors involved in endocytosis of GDF15. I employed high content imaging and flow cytometry techniques to visualize and analyse the internalization of ligand-receptor complex and investigate the role of actin, dynamin and phosphoinositide 3 kinase in the process. The results suggest that similar to the internalization of other cellular growth factors, the uptake of GDF15 is affected by disruption of the actin cytoskeleton. The role of dynamin is still unclear. I also discovered that the internalization of GDF15 was inefficient even in cells that expressed the receptor GFRAL, with large cell-to-cell variation. By following the intracellular localization of the receptor GFRAL, my results revealed that the receptor GFRAL is not efficiently exported to the plasma membrane and most of the protein is retained in the Golgi compartment of cells. This phenomenon was stronger in murine fibroblast cells, where the receptor was almost exclusively trapped in the secretory compartment, explaining why the uptake of the ligand GDF15 is so inefficient in these cells. The system developed during this project will now be used to analyse different factors involved in the uptake of GDF15 and eventually uncover the possible endocytic pathway. Moreover, the Golgi retention of the receptor opens up new questions to investigate like whether the physiological function of GDF15 is regulated by receptor export signals. This will help deciphering the complex and mysterious interaction of GDF15 with its receptor GFRAL.

Evolving societies force universities to transform from the producers of new information sat in their ivory towers towards the role of entrepreneurial universities. The theme of entrepreneurial universities is widely studied internationally, but studies concerning the University of Helsinki (UH) are scarce. The aim of this thesis is to map the current UH bioentrepreneurship ecosystem and the services it provides. The services were mapped and assessed based on how they match the needs of academic bioentrepreneurs. Measures are also suggested on how to develop the ecosystem. This thesis links strongly to the previous literature on entrepreneurial universities and academic entrepreneurship. Entrepreneurial university as a term encompasses an organisation, which strongly supports and encompasses entrepreneurial action in its different functions. Strong technology transfer and commercialization of research via licensing and spinout company formation, is usually linked to entrepreneurial universities. University spinouts are strongly linked to academic entrepreneurship. In spinouts research results and academic tacit knowledge are transformed into enterprises to produce value. The thesis was conducted as a qualitative case study. For the study UH affiliated entities offering entrepreneurship services and bioentrepreneurs originating from within the university were interviewed. The data was analysed with content analysis methods. The results show that UH bioentrepreneurship ecosystem is just in the beginning with multiple useful services but also with some significant flaws. The most significant obstacles preventing the growth of the ecosystem are the university’s negative culture towards entrepreneurship, non-existent communication about the subject and the absence of relevant supportive networks for academic entrepreneurs. Via changing these the critical mass to enable sustainable ecosystem can be achieved.

The objective of this study is twofold: Firstly, to investigate how men and women living in Tanala villages in the Ranomafana National Park buffer zone differ in their natural resource use and livelihood. Secondly, based on this information, the intention is to find out how the establishment of the park has influenced people living in the buffer zone from the gender point of view. The data have been gathered mainly by using semi-structured interviews. Group interviews and individual interviews were carried out in three buffer zone villages. In addition, members of the park personnel were interviewed, observations were made during the visits to the villages and documents related to the planning and the administration of the park were investigated. The data were analysed using qualitative content analysis. It seems that Tanala women and men relate to their environment in a rather similar way and that they have quite equal rights considering the use and the control of natural resources. Probably this is the reason why the management of the Ranomafana National Park has practically not taken gender issues into account in the planning of the park or in administering activities related to it. Both Tanala men and women can own and inherit land, and in most cases they also exert full control over it. Similarly, women and men seem to control their own labour as well as the produce of their work. There is a strong gendered division of labour in Tanala villages, based on the conception of men doing the hard and difficult work and women s tasks being easy. This idea does not appear to correspond with reality, but it rather seems to be a social construction highlighting the importance of men s work at the expense of women s tasks. At the household level, men and women have equal say considering environmental resources management as well as other kinds of issues, but at the community level women are significantly less active than men in decision making. Since the park management has not paid any special attention to this, women s voices and their interests have not been heard as much as those of men in park related meetings. In consequence, there have been no development projects focused on women s cultivation activities, for example. The establishment of the park has also had another important gendered impact. Since the creation of the park local people have been forbidden to enter the park forest. In practise this ban has mostly affected women, and the forest has become a men s space considering both the access to it and it s resources as well as the feeling of authority. As a conclusion it can be stated that for achieving socially just nature conservation, it is important to take gender issues into consideration even if gender relations seem to be quite equal. In Ranomafana area the situation could be improved, for example, by listening more carefully to women s interests and needs.

The Great Cormorant (Phalacrocorax carbo) is among the most recent additions to the Finnish avifauna: the current breeding population dates back to 1996. Historical and archeological data clearly demonstrate that the 20th century breeding range expansion of the species in the Baltic region is a re-colonisation event. The two subspecies of Cormorant in Europe are the nominate subspecies P. carbo carbo and the continental subspecies P. c. sinensis. The Cormorants currently breeding in Finland belong to the subspecies sinensis. P. c. carbo lives on the coasts of the North Sea and North Atlantic and visit Finland only during migration. However, osteological evidence suggests that carbo was prevalent subspecies in the Baltic area during prehistoric times, only to disappear in the Late Middle Age (by circa 1500 AD). The subspecies of the Cormorant differ in average size and weight, the details of the plumage and the angle of the gular pouch. P. c. carbo is on average larger than sinensis. However, males are larger than females in both subspecies. The subspecies differ in their ecological characteristics, e.g. preferred prey, breeding sites and migratory behaviour. However, only bones remain from the prehistoric Baltic Cormorants and therefore, soft tissues or behavioural characteristics cannot be used for their identification. The known osteological measurements of the subspecies overlap, and it has previously been possible to distinguish only the two extremes, large P. c. carbo males and small P. c. sinensis females. Here, I looked into features that would enable distinguishing of the skeletons of all the specimens of both subspecies. The primary material of my Master’s thesis consists of 65 specimens representing birds of both subspecies and sexes. The material has been collected from the Baltic basin and the western Gulf of Bothnia in Sweden and the Åland islands in Finland. In addition, I examined 11 specimens of the subspecies carbo from the collections of the Swedish Museum of Natural History (Naturhistoriska riksmuseet). I focused on the most diagnostic elements of the skeleton: skull, sternum and the large bones of the limbs. Moreover, I examined bones that may be associated with ecological differences between the subspecies, such as the sclerotic ring, the os nuchale, and the length of the distalmost phalanx bone of the opposable hallux toe. I wanted to find out if there are osteological differences between the males and females or the two subspecies. The statistical method used for testing the hypotheses and examining subspecific averages was the analysis of variance (ANOVA). The measurements were also analysed with Principal Component Analysis to investigate the proportional differences. The bones of the two subspecies differed in particular in the length of the forearm bones. Reliable discriminating features also included the depth of the bill and the measurements of leg bones. These are subspecific adaptations to their preferred habitats, prey, nesting sites and migratory behaviour. The relatively short wings of P. c. carbo make it an excellent diver, but a less efficient flyer. This subspecies migrates shorter distances, and is able to overwinter relatively far up in the north. In contrast, P. c. sinensis is able to fly with greater speed and efficiency during migration to avoid adverse conditions. The depth of the bill is an important factor determining the snapping power of the bill, which makes P. c. carbo better suited for handling big or powerful prey. The contemporary Baltic Sea is warmer and less marine than during earlier periods. The current brackish environment seems to favor the subspecies sinensis over carbo, although human persecution may also have had an effect on the previous demise of subspecies carbo. My results support the view that the subspecies of the Great Cormorant are clearly distinct morphologically. My result also suggest that skeletal research is an underutilised method in bird taxonomy, and studying the proportions of bones helps to perceive ecological and biological differences between bird subspecies.

Insects (Insecta) are the most species-rich class of organisms on earth. Due to their vast biodiversity, insects are found in almost every environment. However, approximately 90 % of the over 1 000 000 insect species described are terrestrial. The ocean covers over 70 % of the earths surface, but less than 2 000 marine insect species are known. The purpose of this thesis is to give the reader a comprehensive understanding of marine insect biodiversity and what has caused its sparceness in the seas. Such a multi-faceted review of the topic has not previously been written. The first aim of this review is to catalogue all insect taxons containing marine species, and to examine the habitat choices and ecology of these species. For this purpose, marine insect species have been researched using the World Register of Marine Species (WoRMS) and reference literature, as well as reviews and other articles. Based mostly on WoRMS, the number of marine insect species is determined to be 1 318, divided between 10 insect orders. In addition, seven insect orders are noted to include an unknown number of marine species. As well as examining marine insect biodiversity order-by-order, this work considers the littoral and pelagic habitats in which marine insects live, and the adaptations that are required of insects living in these environments. The second aim of this review is to investigate the lack of insects in marine environments as a phenomenon. The groundwork is laid at the beginning of the review with an examination of why and how insects have diversified so successfully. Later on, possible factors to prevent a similarily successful diversification in marine environments are presented, such as issues with osmoregulation and breathing systems, the typical features of insect reproduction and life cycles, and the fact that transitions between physically contrasting habitats are rare. Pertaining to this the evolutionary history of insects, which has produced the aforementioned features impeding radiation to the seas is also explored. Ultimately insect species in the oceans are few, because insects are a terrestrial evolutionary branch of Pancrustacea. Even though marine habits are uncommon among insects, they are not unheard of. Marine species have emerged in several insect clades and these species have adapted to their environment in numerous ways. For the purposes of further analyses and research on the radiation and biodiversity of marine insects, further surveying of the species and their habits and adaptations is recommended. This work will hopefully offer a point of reference for more detailed future studies and reviews of marine insect biodiversity and the factors that have led to the near-exclusion of insects from the seas.

Breast cancer remains as the leading cause of cancer deaths among women. Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer subtypes and lacks targetable receptors, consequently, cannot be treated with current hormone of anti-HER2 targeting therapies. Thus, there is a need for discovering novel and well-tolerated therapies. MYC is a proto-oncogene and a transcription factor, that is frequently amplified and overexpressed in breast cancers. MYC is involved in many cellular processes promoting cell proliferation, however, overexpression of MYC can also sensitize cells to replicative stress and apoptotic cell death. In our previous studies we have shown that pharmacological activation of AMPK, a cellular energy sensor, synergises with Bcl-2 family inhibitors, such as navitoclax and venetoclax, and activates MYC-dependent apoptosis in breast cancer cell lines, transgenic mouse models of MYC-dependent mammary tumorigenesis and in MYC-high patient-derived explant cultures (PDECs). In subsequent study we observed, that indirect AMPK activator metformin alone inhibited tumor growth in vivo, but did not induce apoptosis in mouse tumors or in PDECs. Metformin, a type II diabetes mellitus drug, has shown anti-cancer effects in some population studies and is under investigation for a cancer therapies, however the whole mechanism of action in cancer is still not well-known. To elucidate metformin’s effects on MYC overexpressing triple-negative breast cancer cells, I will present, that metformin has anti-proliferative effects and show that long term metformin treatment induces senescence biomarkers in MYC-high TNBC breast cancer cell lines. To study metformin's short and long-term anti-proliferative activity, cell proliferation during and after drug treatment was investigated, which showed, that metformin’s effects do not seem to persist long after drug withdrawal. In conclusion, the key observation of this thesis was, that metformin does inhibit the proliferation of MYC overexpressing cancer cells and presents a senescence phenotype that possibly can be exploited to find new targeted therapies for triple-negative breast cancer patients.

Animal coloration is as striking as it is diverse; however, the transcriptional basis of coloration is not deeply understood. Cichlid fishes are a tractable system for studying coloration as they exhibit a wide range of phenotypic diversity while remaining genetically similar. This facilitates the study of genotype-phenotype correlations and the identification of causative genes. RNA sequencing is a powerful approach to investigate the genes which characterize chromatophores. However, RNA-seq results can be plagued by the high abundance of rRNA in cells. This thesis aims to investigate differential gene expression between differently pigmented regions as well as explore the effects of tissue treatments and rRNA depletion on gene expression. Gene sets acquired with polyA selection, riboPOOL probes optimized for zebrafish, and zebrafish probes complemented with newly designed riboPOOL cichlid probes were compared to assess the functionality of these different rRNA depletion strategies. The use of zebrafish probes complemented with newly designed cichlid probes captured the greatest diversity of genes, many transcripts of which were missing from the other gene sets. Furthermore, as experiments such as scRNA-seq rely on a dissociation step, the effect of dissociation on gene expression was examined and found to promote the expression of stress response genes. The results of this upstream optimization were applied in the analysis of differential gene expression between the vertical stripes of the cichlid Pseudotropheus demasoni to better understand the molecular basis of vertical striping in fish. The dark stripes exhibited upregulation of melanic marker genes and the light, iridescent stripes showed an increase in iridophore marker gene expression. These findings were corroborated with cell count data from FACS to link transcriptional profiles and cell type quantifications. Overall, the study provides insight into the transcriptional basis of coloration in cichlid fishes and underscores the importance of optimizing methods drawing meaningful conclusions.