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Puberty initiation is a crucial physiological process in human development. A group of hypothalamic neurons secreting the gonadotropin-releasing hormone (GnRH) and expressing the kisspeptin receptor (KISS1R) plays a key role in launching puberty. Furthermore, cellular KISS1R signaling has been shown to regulate GnRH expression and secretion. Although the in vitro differentiation of human pluripotent stem cells into GnRH-secreting neurons has been successful, it is of high interest to generate KISS1R expressing GnRH neurons. By utilizing the CRISPR activation technology, this study aimed to establish a conditional KISS1R-activation cell line using H9 human embryonic stem cells. Through controlling dCas9VP192 abundance using the Tet-On system combined with the dihydrofolate reductase destabilizing domain, the transcriptional activation of KISS1R was temporally regulated by the addition of two antibiotic drugs - doxycycline and trimethoprim. KISS1R expression was primarily assessed by qPCR and verified by immunocytochemistry and the use of a KISS1R-GFP reporter cell line. The main finding of this study is the achievement of a 6217 ± 2286 fold change in KISS1R transcription by introducing two guide RNAs (N = 3). Nevertheless, leaky gene activation was observed without drug treatment (fold change of 63 ± 51). Concludingly, this study successfully led to the generation of a KISS1R-activation cell line. After further characterization and refinement of the activation protocol, the established cell line will enable to investigate whether KISS1R upregulation modulates in vitro GnRH neuron differentiation, electrophysiology, hormone expression, and secretion in the future. Respective outcomes may lead to advances in understanding and treating pubertal disorders.

Faculty: Faculty of Biological and Environmental Sciences Degree programme: Master’s Programme in Neuroscience Study track: Cell and Systems Physiology Author: Mélina GUILLON Title: Inflammatory activation of Macrophages by Triglyceride-Rich Lipoproteins in Atherosclerosis Level: Master’s thesis Month and year: August 2023 Number of pages: 38 Keywords: Atherosclerosis, Inflammation, Triglycerides-Rich Lipoproteins, Emulsion Particles Supervisor or supervisors: Dr. Katariina Öörni Where deposited: Helsinki University Library Additional information: Background: Inflammation is a key factor in atherosclerotic cardiovascular disease (ASCVD) and is present at all phases. It has been shown that reducing inflammation by blocking cytokine pathways diminishes the risk of stroke and myocardial infarction. Despite the well-established linked between lipoproteins and atherosclerosis, little is known on the specific effect of lipids on inflammation. In this study, we investigated the impact of triglycerides-rich lipoproteins’ (TRLs) lipids on inflammation in the context of atherosclerosis. Methods: TRLs were isolated and purified from pooled plasma of healthy volunteers, and emulsion particles (EPs) generated by sonication using lipids extracted from TRLs. TRLs and EPs were characterized in size, triglycerides, and cholesterol content. THP-1 cells were treated with EPs, TRLs, and modified EPs (oxidation, vortexed, and lipolysis with PLA2), and the release of pro-inflammatory cytokines (IL-1β and TNF-α) was detected with ELISA. Results: EPs were successfully synthesized by sonication using an ultrasonic probe. EPs induced cytokine secretion from THP-1 cells (N=4). Modified EPs (Oxidized EPs, vortexed EPs, and PLA2-treated EPs) did not increase cytokine secretion (N=4). Conclusion: Our findings suggest that TRLs’ lipids contribute to inflammation and that TRLs may play a crucial role in the pathogenesis and pathophysiology of ASCVD. Inflammatory properties of TRLs should be extensively investigated in the future for the development of preventive and curative strategies.

Plastics are important materials in construction due to their longevity, durability and lightness. However, the use of plastics should be reduced as they are made mainly from fossil fuels and unnecessarily end up in the environment where they cause damage to various organisms. The purpose of this Master's thesis was to investigate whether the plastics currently used in infrastructure construction can be replaced by better choices in terms of recyclability and carbon footprint. The subject of the thesis was two infrastructure construction sites in the city of Helsinki, where it was studied how much and what types of plastics were purchased for the site, excluding packaging plastics. Thereafter, circular economy experts and waste management companies as well as pipe manufacturers were interviewed to clarify how well polyvinylchloride (PVC) and high density polyethylene (HDPE) plastic materials can currently be recycled. In addition, it was investigated whether nowadays used cable protection pipes could be replaced by recycled plastic pipes or biobased plastic pipes. Plastics that were used at the construction sites were PVC, HDPE, polystyrene (XPS), polypropylene (PP), cross-linked polyethylene (XLPE) and ethylene-propylene rubber (EPDM). There is no further processing of recycled PVC into the recycled plastic granulates in Finland, which is why PVC plastic is sold abroad. In addition, due to the chlorine content of PVC, incineration is not recommended for PVC products. For this reason, it would be good to replace PVC plastic in cable protection pipes with HDPE plastic. HDPE plastic can be recycled in Finland, and further processed into recycled plastic granulates, which can be used for pipe production instead of virgin material. There are no bio-based pipes yet in Finland, which is why waste management companies do not have experience in recycling bio-based pipes. Based on the literature review, no clear differences were found in the carbon dioxide emissions of HDPE and PVC plastic manufacturing. However, HDPE and PVC pipes made from recycled plastics were clearly lower in emissions than similar virgin plastic pipes. At the moment, within the framework of for recyclability and carbon footprint, the best option would be an HDPE cable protection pipes made of recycled plastic, as the use of recycled plastic itself reduces the carbon footprint of the pipe.

Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.

The ends of eukaryotic chromosomes are formed by a special heterochromatic structure, the telomere, which is essential to guarantee chromosome stability. Telomeres protect chromosomic ends from DNA degradation, repair, and recombination events. However, they are difficult to replicate due to their repetitive and heterochromatic nature, which hinder DNA replication fork progression. In yeast, Mph1 helicase promotes replication fork regression, cross-over suppression during homologous recombination (HR), and telomere maintenance. Moreover, Mte1 is a D-loop binding protein involved in response to DNA damage and maintenance of telomere length, which interacts with Mph1, thereby stimulating its regression capacity as a helicase and fork. Thus, the Mte1-Mph1 complex is recruited to stressed telomeres. Mte1 also shares a domain of unknown function, DUF2439, with Rad51 and Rdh54. Additionally, Esc2 protein is involved in the regulation of DNA damage through template switch (TS) recombination, preventing HR events caused by Mph1. This thesis aimed to uncover the potential roles and interactions of proteins involved in telomere maintenance, such as Mph1, Mte1, Esc2 and Rdh54, for which two main assays were conducted: (1) Telomere Stability assay, consisting of Tus/Ter barrier based on the high-affinity binding of the E. coli protein, Tus, to specific DNA sequence called Ter; (2) Template Switching assay, focused on the capability of the proteins in reconstructing a functional LYS2 gene by TS. The obtained results demonstrated that (1) the absence of Rdh54 enhances replication fork regression, (2) Mte1 and Esc2 show opposite roles in telomere maintenance, (3) the interaction between Mte1 and Rad51 plays a crucial role in ensuring telomere stability and nuclear foci formation, (4) Mph1 and Mte1 promote cell survival through the break-induced replication (BIR) pathway. Further studies should assess the plausible interaction between Mph1 and Rdh54 proteins and characterize the function and interplay of the proteins involved in TS.

The intersections of climate change and sexual and reproductive health and rights (SRHR) have increasingly received attention from international organisations but also from academia. For some, establishing these intersections is about reducing human pressure on the Earth systems, while for others it is about the human rights of vulnerable individuals and communities. Many have lauded these connections for providing a win-win solution for both. While these benefits are championed, there has been little reflection on the underlying motives and justifications for establishing these connections in the first place. Given the problematic past of population control policies, understanding these justifications is necessary to break away from the neo-colonial practices of the past. This thesis investigates the motives and justifications for establishing such intersections between SRHR and climate change. Specifically, the thesis addresses two questions, namely: 1) In what ways are the interconnections between SRHR and climate change justified in academic literature? 2) What are the implications of the ways in which these interconnections are justified? By drawing on a postcolonial feminist theoretical framework rooted in understanding this nexus critically, and carefully reflecting on the implications of these discourses, the thesis answers these questions by systematically drawing on a sustained body of research. The data consist of 88 academic publications that are systematised through discourse analysis. The findings identify six distinctive intersectional discourses which reflect the ways in which SRHR, and climate change are justified, namely: public health, population dynamics, reproductive rights, critical, sustainable development and environment discourses. Largely reflecting adherence to liberal feminist and populationist frameworks, these findings imply that the discourses, justifications, and motives do not sufficiently address the neo-colonial practices and structural inequalities that shape intersections between SRHR and climate change. Analytically, therefore, this thesis suggests that postcolonial feminism offers a more effective way for understanding intersectional discourses because it recognises how power inequalities manifest in the discourses, while contributing towards more justice-based approaches to sustainability.

Climate change is applying pressures to plant populations, which must adapt or move to retain fitness. A changing climate highlights the need for us to understand the potential that species possess to evolve in addition to any plastic responses. Approaches that allow the study of contemporary evolution, such as resurrection studies, have the capacity to provide insights into the responses of populations to these changes. In this resurrection experiment, seeds from seven populations of Hypericum perforatum collected from the UK and France, and their historic counterparts, were grown and subjected to four temperature treatments. Three traits were measured and compared between historic and contemporary populations: date of flowering, average seed weight and flower abundance. I found that temperature influenced date of flowering and flower abundance, leading to an overall earlier flowering time and an overall decrease in flower abundance with increase in temperature. The only significant difference between historic and contemporary populations was found in flower abundance - where, whilst flower abundance declined with increasing temperature, contemporary populations produced proportionally more flowers than historic populations per degree of temperature increase. These results suggest that plasticity allows this species to adjust its flowering phenology to retain fitness in warmer conditions but that evolution during the past decades may have selected for a decreased flower abundance at higher temperatures. These findings contribute to our overall understanding of how species have and will react under climate change, as we try to disentangle the roles that plasticity and evolution play in enabling populations to retain fitness under changing conditions.

Establishing and maintaining cell polarity is critical to all multicellular organisms. Apicobasal polarity is a type of cell polarity specific to epithelial cells, which is established and maintained by three distinct protein complexes. Among them, the Scribble (Scrib) complex plays a role as a basolateral determinant. Scrib is a scaffold protein with multiple functions, including maintenance of the basolateral polarity of epithelial cells and a tumor suppressor, acting as a regulator of the Hippo signalling, an evolutionarily conserved pathway which controls organ size through regulation of cell proliferation and apoptosis by inhibiting the transcriptional co-activator protein Yorkie (Yki). A recent experiment proposed that Scrib is involved in maintaining tissue homeostasis through relaying apicobasal polarity regulation across the tissue. This mechanism can be used both by normal cells to rescue hypomorphic scrib cells and by loss of scrib cells to spread loss of polarity. The signal is likely related by cell-cell contact and the junctions present in epithelial cells may be involved in this communication. This project aims to identify the genes involved in tissue homeostasis through intercellular alignment of apicobasal polarity together with Scrib. First, a screening protocol was established by studying genetic interactions and tissue structure. Second, a systematic screening was carried out by using deficiency lines of left arm of the third chromosome in Drosophila. Fly stock expressing spatially and temporally controlled scrib RNAi was established and crossed with deficiency lines to identify genes that have synergy with Scrib. The wing discs of the offspring were dissected, imaged, and the phenotypes were sorted into categories according to the degree of overgrowth. Five strong candidates and four candidates with milder phenotype were identified. The results show the screening method is robust and suitable to carry out a finer, single gene level screen of the candidates, as well as screening for additional candidates in the rest of the Drosophila genome. The identified candidates provide new leads to develop the theorical model of intercellular alignment of apicobasal polarity. Understanding how apicobasal polarity is maintained in the dynamic environment of a living organism is important for physiological and pathological conditions. This study provides an important insight into further understanding tissue development and homeostasis.

Loisten vaikutus isäntäpopulaatioihin ja isäntäyksilöiden elinkiertoon vaihtelee isäntä–lois-suhteittain. Loisten leviämistä ja vaikutuksia isäntäpopulaatioissa on kuvattu erilaisilla matemaattisilla malleilla, joissa on keskitytty joihinkin loisen yleistymisen kannalta olennaisiksi arvioituihin tekijöihin, kuten loisen transmissioon ja isäntien kuolleisuuteen, syntyvyyteen ja vastustuskykyyn. Mallien taustaoletukset ja painotukset vaihtelevat. Isäntä–lois-suhteiden muodostumisen taustalla onkin monia vaihtoehtoja tai syitä, joita on hyvä selvittää. Isäntä–lois-suhteiden muodostumista ja loisen leviämistä voidaan tarkastella myös ohjelmallisesti esimerkiksi simulaatioilla, joilla voidaan selvittää eri tekijöiden tai parametrien arvojen vaikutusta loisintaan. Simulaatio voi mallintaa loisintaa isäntäpopulaation tasolla ja kuvata tartunnan leviämistä isäntäpopulaatiossa eri hetkinä. Se voi mallintaa loisintaa myös yksilötasolla, jolloin siinä voidaan huomioida eri tavoin yksilöön liittyviä ominaisuuksia, kuten yksilön liikkumista tai parinvalintaa. Tällaisten isäntäpopulaation ja loispopulaation yksilöiden suhteita, sijainteja ja tilanteita eri hetkillä mallintavien ohjelmien suunnittelun tueksi on hyödyllistä luoda käsitteellinen malli, joka kuvaa loisintaa yksilötasolta lähtien samalla selventäen isäntien ja loisten ominaisuuksia ja loisintaan liittyviä vuorovaikutussuhteita. Tutkimuksen tavoitteena olikin luoda loisintasuhteita yleisesti kuvaava käsitteellinen malli. Tutkimuksen aineisto muodostui loisinnan biologiasta ja siihen kytkeytyvistä ilmiöistä. UML-mallinnuskieltä (Unified Modeling Language) käytetään yleisesti oliopohjaisten ohjelmistojen suunnittelussa ja järjestelmien kuvauksessa. Loisintaakin voi tarkastella isäntien ja loisten välisenä järjestelmänä, jossa loisinnan osapuolet ovat olioita. Menetelmänä käytinkin UML-luokkakaavioita. Tutkimus selvittää isäntä–lois-suhteiden olennaisia piirteitä ja muodostaa niiden pohjalta loisintaa yksilötasolla yleisesti kuvaavan UML-luokkakaavion. Osoittautui, että ’tavanomaisen’ loisinnan, jossa loinen elää kiinni isännässään hyödyntäen sitä ravintolähteenään, sekä kleptoparasitismin tai sosiaalisen loisinnan yhdistäminen samaan UML-luokkakaaviomalliin saattaa johtaa tulkinnallisiin epäselvyyksiin. Osoittautui myös, että isäntä–lois-suhteisiin liittyy erilaisia vuorovaikutuksia ja ilmiöitä esimerkiksi loishyökkäysten torjunnan osalta. UML-luokkakaaviomallista jäi pois muun muassa isännän loisintaa ehkäisevä käyttäytyminen, jotta mallin rakenne säilyi selkeänä. Isäntä–lois-suhteisiin liittyvät myös eliöiden liikkuminen ja lisääntyminen, jotka vaihtelevat lajeittain ja kehitysvaiheittain. Niitä voi tarkastella omina malleinaan, jotka kytkeytyvät loisinnan osapuoliin tai niiden kehitysvaiheisiin. Loisinnan osapuolten suhteisiin liittyy koevoluutiokin. Nopeasti ja runsaasti lisääntyvillä ja muuntuvilla mikroloisilla evoluutio voi olla nopeaa. Kuitenkin evoluutiota tarkasteltaessa pitäisi tietää, mitä mallinnetaan ja mitä rajoituksia evoluution mallinnukseen sisältyy. UML-luokkakaaviossa ei mallinnettukaan evoluutiota, vaan se vaatisi tarkempaa analyysia.

Panssarisiimalevät ovat suuri, morfologisesti ja ekologisesti monimuotoinen kasviplanktonryhmä.Panssarisiimalevien tuntemus on painottunut suurikokoisiin, yhteyttäviin ja laboratoriossa kasvatettaviin lajeihin, jaerityisesti pienten hetero- ja miksotrofisten panssarisiimalevien monimuotoisuus tunnetaan puutteellisesti. Itämerion maantieteellisesti ja ekologisesti eristynyt merialue. Panssarisiimalevien suhteellinen osuus Itämerenkasviplanktonista on kasvanut viime vuosikymmeninä. Tunnistamisen vaikeuden vuoksi Itämerenkasviplanktonseurannoissa pienikokoiset panssarisiimalevät kuitenkin usein sivuutetaan tai niitä käsitellään yhtenä,vain lahkotasolle määriteltynä ryhmänä. Pfiesteriaceae-heimon lajit ja Karlodinium veneficum ovat pienikokoisiahetero- ja miksotrofisia, mahdollisesti toksisia, haitallisia leväesiintymiä muodostavia panssarisiimaleviä. Näistäpanssarisiimalevistä ei ole vahvistettuja havaintoja Itämerestä. Lajeja pidetään kosmopoliittisina ja niidenasettuminen Itämeren murtoveteen on mahdollista. Tutkimukseni tavoite on selvittää pienikokoisten (alle 20 μm)panssarisiimalevien monimuotoisuutta Itämeressä ja arvioida näiden panssarisiimaleväkantojen suhdetta kantojenmuilta merialueilta eristettyihin lajitovereihin. Aineisto kerättiin Hankoniemen itäpuolelta Tvärminnen eläintieteellisen aseman läheisyydestä ja AhvenanmaanFöglöstä matalista ja suojaisista lahdelmista. Vesinäytteistä eristettiin kuoppalevylle valomikroskoopin avullapieniä (alle 20 μm) panssarisiimaleviä puhdasviljelmiä varten. Menestyksellisesti eristettyjäpanssarisiimaleväkantoja kasvatettiin Itämeren murtoveteen valmistetussa f/2-Si-kasvatusliuoksessa. Tiheiksikasvaneista soluviljelmistä eristin DNAn ja selvitin kantojen suku- ja lajitason identiteetin sekvensoimallaribosomaalisen DNAn SSU-, LSU- ja ITS-alueet. Tutkimuksessa luotujen sekvenssien ja GenBank-tietokannastaladattujen sekvenssien avulla laskin aineistosta neljä fylogeneettistä puuta. Kuvasin havaitut lajit morfologisestipyyhkäisyelektronimikroskoopilla. Toksisuusanalyysejä varten suodatin panssarisiimaleväkannoista näytteetlasikuitusuodattimille, jotka lähetettiin tutkittaviksi Yhdysvaltoihin. Tutkimuksessa eristetyistä 512 panssarisiimalevästä 100 kantaa jakautui kuoppalevyillä. Lopullisiin analyyseihinvalikoitui 12 kantaa. Fylogeneettisten ja morfologisten analyysien perusteella Itämerestä eristetyt kannat kuuluvatCryptoperidiniopsis brodyi-, K. veneficum- ja Pfiesteria piscicida-lajeihin. Itämeren K. veneficum -kantojen eitässä tutkimuksessa havaittu tuottavan karlotoksiineja. Itämerestä eristetyt C.brodyi-, K.veneficum- ja P.piscicida-kannat eivät eroa geneettisesti tai morfologisestimuilta merialueilta eristetyistä lajitovereistaan. Taustalla voi olla jatkuva geenivirta lajien eri populaatioidenvälillä. On myös mahdollista, että ribosomaalinen DNA on liian konservatiivinen alue kuvastamaan lajinsisäistävaihtelua. K. veneficum ja P. piscicida voivat tuottaa toksiineja ja muodostaa haitallisia leväesiintymiä. Vaikkatässä tutkimuksessa toksiinituotantoa ei havaittu, luonnonpopulaatiot voivat sisältää toksisuusfenotyypiltäänerilaisia kantoja, ja haitallisten leväesiintymien seurannan ja ennakoinnin kannalta tulisikin selvittää mahdollisestitoksiineja tuottavien kantojen esiintymistä Itämeressä. Suurin osa tutkimuksessa eristetyistä panssarisiimalevistä eimenestynyt laboratorio-olosuhteissa. Tutkimuksessa havaitut lajit eivät siis todennäköisesti edusta Itämerenpienikokoisten panssarisiimalevien koko monimuotoisuutta. Panssarisiimaleviin kohdistettu ympäristönäytteidensekvensointi eristyneessä ja vesimassoiltaan kerrostuneessa Itämeressä voisikin tuottaa lisätietoa niinpanssarisiimalevien monimuotoisuudesta yleensä kuin niiden erityispiirteistä Itämeressä.