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  • Pursiainen, Annie (2020)
    Introduction: High anthropogenic pressure exercised on the Baltic Sea causes a decline in valuable reproduction areas of coastal fish, such as the northern pike (Esox lucius L.). Hence, the conservation and restoration of the reproduction areas of pike are increasingly of importance. The Kvarken Archipelago, located in the Gulf of Bothnia, presents valuable reproduction habitats for fish. The pre-flads, flads, glo-flads and glo-lakes provide warm and sheltered areas that are preferred by spring-spawning fish, such as pike. However, the precise environmental factors influencing pike reproduction in the region have not yet been identified. In this study, different environmental factors influencing pike reproduction in the Kvarken Archipelago were assessed. Furthermore, as technology advances, there is a need to evaluate the use of new techniques alongside the generally time consuming and expensive traditional research methods applied for surveying the pike reproductive areas. Henceforth, the present study assessed the suitability of the high-resolution aerial photography for identifying potential reproduction areas of northern pike. Methods: The data of pike fry density, the vegetation, the abiotic and biotic environmental factors, as well as the aerial images of the 45 sampling sites were collected by Natural Resources Institute Finland and Metsähallitus Parks and Wildlife Finland as part of the Kvarken Flada -project. Pike fry were sampled with a flat dipnet amongst vegetation along a 100 m sampling line during spring. Concurrently, the vegetation, the occurrence of sticklebacks (Gasterosteidae) and the abiotic factors affecting water quality, including salinity, pH, oxygen content and turbidity were determined. The temperature was recorded with temperature loggers from early spring until late summer. The temperature sum for June showed the greatest effect on pike fry densities and was thus used in the analyses. The archipelago zones, which are based on previously modelled temperature sums, were defined for the sampling sites in the geographic information system (QGIS). Additionally, factors describing the morphology, the vegetation and bottom substrate coverage of the sampling sites were utilized in the analyses. The presence of pike fry was analysed with binary logistic regression. The pike fry densities were studied using non-parametric analyses due to the high number of zero observations in the data. The aerial images were taken in late summer of 2017 and 2018, when the vegetation was fully developed. The vegetation types, i.e. reed, fallen reed, wetland grasses, submerged vegetation and filamentous algae, identified in the images were compared to the vegetation data gathered during field survey in QGIS. The performance of the aerial images was assessed by calculating the accuracy, sensitivity and specificity for the different vegetation classes, first for the whole dataset and then for the sampling points with pike fry observations. Results and conclusions: This study determines two key factors defining suitable reproduction areas of pike, the temperature sum for June and the morphology of the sampling site. The results indicate that pike prefer warm and sheltered areas with suitable vegetation, such as reed and wetland grasses. In particular, glo-lakes provide favorable conditions for pike and the temperature sum for June was significantly higher in the glo-lakes compared to the pre-flads. The temperatures of the glo-lakes were constant throughout the archipelago, providing suitable conditions for pike reproduction also in the outermost archipelago. In all of the glo-lakes pike fry were observed and generally the highest pike fry densities were found in glo-lakes. However, the year class strength of pike can be affected by the possibility of the juvenile pike to emigrate from the enclosed glo-lakes back to the sea. Salinity reflects the connectivity of the site to the surrounding sea and had a significant negative effect on pike fry density. Additionally, the presence of sticklebacks affected negatively the presence of pike fry, which might be traced back to the different use of the archipelago zones and site morphologies of these species. Increasing pH negatively influenced the pike fry density. However, pike fry were observed also in the highest pH conditions and therefore pH alone is not sufficient to explain the presence and density of pike fry. Due to the small sample size of the dataset, it can be assumed that not all of the factors influencing pike reproduction were identified and thus the use of a larger dataset is recommendable in future studies. This study provides new methodological information on reproduction area surveying of pike with high-resolution aerial photography. The different vegetation classes were successfully identified from the aerial images. The seasonal differences were noticeable in the results, as in the aerial photographs taken in late summer, the vegetation was fully developed compared to the field study in early spring. Nevertheless, aerial photography proved to be an effective method for identifying reproduction areas of pike based on the vegetation. Furthermore, reproduction areas in need of restoration could be assessed with aerial photography.
  • Hyppölä, Ronja (2019)
    Peatlands are roughly divided into bogs, which are dry and ombotrophic, and fens, which are wet and minerotrophic. Bog vegetation typically consists of dwarf shrubs, lichens, and especially of peat forming white mosses Sphagna spp. White mosses, yet different taxa, also occur on fens and are accompanied by various sedges Carex spp. and Eriophorum spp. Human activities are changing the climate. These activities increase greenhouse gas concentrations in the atmosphere, which in turn causes global climate warming. It is not yet resolved how the warming is affecting the ecosystems and what are the response mechanisms. Peatlands cover a considerable area, i.e. 4 million square kilometres of the Northern hemisphere, including continuous, intermittent or sporadic permafrost, the latter especially vulnerable to thawing. Since their formation following the deglaciation ca. 11 700 years ago, the Northern hemisphere peatlands have acted as carbon sinks storing great amount of atmospheric carbon as accumulated peat. This is due to the slow decomposition rate under cold climate. Simultaneously peatlands release carbon back to the atmosphere via microbial activity, which is largely regulated by hydrological conditions and associated plant composition. Carbon dioxide (CO2) release in aerobic conditions happens mainly through respiration of the living organisms, while in wet anaerobic conditions methane (CH4) is released due to methanogenic processes. The rate of peat, i.e. carbon, accumulation and release has varied over time due to for instance prevailing climate conditions. Mire surface is a mosaic of hydrological and thus vegetational patterns. Different taxa thrive in different growing conditions and furthermore, respectively, can affect the carbon dynamics. While long-term autogenic succession changes surface vegetation and microtopography, also allogenic forcing, such as climate and warming alter habitat conditions and for instance lead to permafrost thawing. Currently, especially thawing of permafrost is a concern worth noticing. Thawing ice might cause water saturated conditions, which in turn may lead to increase in methane emissions. Past changes in prevailing vegetation can be studied through stratigraphical examinations of peat. The accumulated peat layers reflect drier and wetter conditions, and these can be identified by distinguishing the remaining plant macrofossils. The reconstructed changes in past vegetation can be dated with radiometric methods and then compared with paleoecological climate data or meteorological measurements to evaluate if the changes in vegetation and carbon accumulation correspond to changes in climate. In this study I investigate the relationship between past changes and peatland vegetation, carbon accumulation and climate. The focus time period covers the last centuries. My thesis consists of two case studies from northern Sweden permafrost peatland areas, Abisko and Tavvavuoma. The two main taxa, which dominated the plant assemblage were Sphagnum fuscum and Dicranum elongatum. In Tavvavuoma the plant composition varied more than composition in Abisko. Also, the oldest peat section was from Tavvavuoma, where bottom age of the peat section was ca. 8000 years old. The peat and carbon accumulation data indicate similar patterns for both study sites. No clear signs of the generally warmer Medieval Climate Anomaly or colder Little Ice Age were found in my data, but at both study sites the warming after Little Ice Age has shifted vegetation towards a drier assemblage and this has affected the carbon accumulation rates positively especially since the turn to 20th century. According to my data, it can be suggested that vegetation composition and carbon accumulation follow the climatological and thus hydrological conditions, thus my results are in accordance with earlier studies.
  • Suvanto, Maija Tellervo (2019)
    Sindbis virus belongs to the Alphavirus genus and it has spread around the globe. Sindbis virus is transmitted by mosquitoes and spread birds. Mosquitoes from Culex and Culiseta genera are able to spread Sindbis virus. In Finland game birds from Tetrao genus have been observed to act as host for Sindbis virus thus enabling viral amplification. In Finland Sindbis virus causes annual disease called Pogosta disease. Typically, Pogosta disease occurs from August to September. Symptoms of Pogosta disease include for example joint pain, arthritis, rash and fever. Pogosta disease is endemic in Finland and known endemic regions are e.g. Ilomantsi region. Even though Sindbis virus causes annual disease in Finland there is currently only six Sindbis virus strains isolated from Finland. These isolations were done in 2002 and in 2005. There is a need for new data. In this master thesis the aims were to describe how two new Finnish Sindbis virus strains were isolated and to compare them to each other and to other Sindbis virus strains isolated from Finland and around the globe. Mosquito samples were collected from Ilomantsi region in 2018 summer. Serum samples from serologically diagnosed patients with Sindbis virus infection were obtained from HUSLAB and included in this study. The mosquito and patient serum samples were cultivated on cells and later virus isolates were plaque purified. Conventional nested reverse transcriptase polymerase chain reactions (RT-PCR) for Alphavirus and Bunyavirus genera were used in order to screen mosquito and patient serum samples for viruses. Later a Sindbis virus specific real time reverse transcriptase PCR was used to test mosquito and patient serum samples. Cell cultures were also tested with immunofluorescence assays. Isolated Sindbis viruses were sent for next generation sequencing and based on that a phylogenetical tree was constructed. The phylogenetical tree represents the new Sindbis virus strains relation to Finnish and to other Sindbis virus strains around the globe. Mosquito samples were morphologically identified and subjected to genetic species identification analysis via COI sequences. These COI PCR products were sent for Sanger sequencing and compared to sequences in NCBI database in order to determine the mosquito species. In this master thesis two new Finnish Sindbis virus strains were isolated from mosquito and from human serum sample. This is the first time in Finland when Sindbis virus has been isolated from human serum sample. These two new Finnish Sindbis virus strains differ from each other. The strain isolated from the patient sample is more closely related to German strains than to Finnish Sindbis virus strains. The strain isolated from mosquitoes is closely related to pre-existing Finnish Sindbis virus strain Ilomantsi region-2005M which is isolated from mosquitoes. These finding suggest that currently there is two different Sindbis virus strains circulating in Finland. This master thesis provides valuable information of Finnish Sindbis virus strains which can be used in future studies e.g. when studying Sindbis virus pathogenesis, distribution, relation to other Sindbis virus strains or mapping Sindbis virus prevalence in Finland.
  • Mäkelä, Tiina-Kaisa (2020)
    Tiivistelmä – Referat – Abstract Celiac disease (CD) is a serious lifelong condition, in which the immune system attacks an individual’s own tissue when eating gluten. This leads to inflammation and damage to the small intestine. Celiac disease often goes undiagnosed because many of its symptoms are nonspecific. The prevalence of combined undiagnosed and diagnosed CD is estimated to affect 1 in 100 people throughout Europe and USA. CD is a polygenic disease, it is known that the human leukocyte antigen (HLA) system plays a crucial role. HLA-DQ2/DQ8 risk allele genotyping screening test from a whole blood sample (B -HLAKeli) is routinely used to estimate the genetic risk of a patient having CD. HLA genotyping test result is routinely used to rule out celiac disease rather than confirming it; if an individual does not have celiac disease related risk alleles, it is very unlikely that he or she has celiac disease. The Celiac disease diagnosis decision making process is based on the classic triple combination of serological antibody tests, the HLA-DQ2/DQ8 genotyping test and duodenal biopsies. The aim of this master’s thesis was to study evaluate how the two different risk classification praxis for HLA-DQx.5 allele used for celiac disease diagnostics in SYNLAB Finland and Estonia central laboratory and in SYNLAB Suomi central laboratory might influence the clinical process and final diagnosis. In SYNLAB Suomi central laboratory HLA-DQx.5 is classified and interpreted as a risk allele predisposing to celiac disease. In SYNLAB Finland and Estonia central laboratory this allele is classified as CD-non-risk-allele based on recommendations in international guideline. In addition, the aim was to get a general understanding of celiac disease prevalence and risk allele distributions among the study population. From the study population of 196 celiac disease suspect patients, 9% had a celiac disease positive laboratory result and the HLA risk genotype distribution among positive cases was well aligned with the expected values described in the literature. Study results indicated that there’s no additional clinical value if HLA-DQx.5 is classified as a celiac disease predisposing risk allele; the study data implies that it is very unlikely to find celiac disease positive cases from laboratory test perspective among HLA-DQx.5 carriers. Based on the study, approximately 7% of the celiac disease suspects carry the allele HLA-DQx.5 and therefore probably go through additional celiac disease related laboratory testing if this allele is interpreted as a risk allele. According to the study findings and general recommendations based on international guideline of celiac disease diagnosis, it seems that there is no clear clinical benefit if HLA-DQx.5 is classified as a CD risk allele.
  • Lehtonen, Anna (2017)
    Yersinia pseudotuberculosis voi aiheuttaa ihmiselle suolistoinfektion, yersinioosin. Se tarttuu kontaminoituneiden elintarvikkeiden välityksellä, ja Suomessa on todettu useita epidemioita viimeisen 20 vuoden aikana. Yersinia-suku kuuluu Enterobacteriaceae-heimoon, ja siihen kuuluu tällä hetkellä 18 lajia. Y. pseudotuberculosis-lajin lisäksi sukuun kuuluu kaksi ihmispatogeenia, Yersinia enterocolitica ja Yersinia pestis. Y. enterocolitica on myös elintarvikevälitteinen patogeeni, Y. pestis puolestaan tunnetaan ruton aiheuttajana. Y. pseudotuberculosis kasvaa hyvin jääkaappilämpötiloissa, jolloin nykyaikaiseen elintarvikehygieniaan oleellisena osana kuuluva kylmäsäilytys ei estä sen lisääntymistä elintarvikkeissa. Sen vuoksi onkin tärkeä tutkia tekijöitä, joilla on merkitystä Y. pseudotuberculosis-bakteerin kylmänsiedossa ja muissa stressiolosuhteissa. RNA-helikaasit ovat proteiineja, jotka avaavat kaksijuosteista RNA:ta. Niihin kuuluu useita proteiiniperheitä, mutta suurin niistä on DEAD-box-helikaasiperhe. DEAD-box-helikaaseja löytyy kaikista eliöryhmistä, ja ne osallistuvat kaikkiin RNA-metabolian vaiheisiin. Ne ovat ATP-riippuvaisia helikaaseja ja ne kykenevät avaamaan vain lyhyitä kaksijuosteisia alueita. Y. pseudotuberculosis IP32953- genomi sisältää viisi DEAD-box-helikaasia koodaavaa geeniä, csdA, dbpA, rhlB, rhlE ja srmB. RhlB osallistuu mRNA-molekyylien hajottamiseen osana RNA-degradosomia. Muut proteiinit osallistuvat pääasiassa ribosomin rakentumiseen. Escherichia coli -bakteerilla aiemmin tehdyn tutkimuksen perusteella tiedetään, että CsdA:n ja SrmB:n puuttuminen soluista saa aikaan kylmäherkän fenotyypin. CsdA:n roolia on tutkittu myös Y. pseudotuberculosis-lajilla, jossa toimimaton CsdA heikentää kasvua kylmässä. Tämän tutkielman tarkoituksena oli tutkia RNA-helikaasien vaikutusta Y. pseudotuberculosis IP32953-kannan kasvuun erilaisissa stressiolosuhteissa. RhlE-, DbpA- ja SrmB-deleetiomutanttikantoja kasvatettiin optimilämpötilan (28 °C) lisäksi kylmässä (3 °C), korkeassa suolapitoisuudessa sekä alhaisessa ja korkeassa pH:ssa. Kasvun aikana mitattiin bakteerien kasvua OD600nm-mittausten avulla, ja aineiston perusteella kannoille piirrettiin kasvukäyrät kaikissa olosuhteissa. Lisäksi aineistosta laskettiin käyrän alle jäävä pinta-ala, jonka perusteella tutkittiin kasvuerojen tilastollista merkitsevyyttä Studentin t-testillä. Kannoille määritettiin myös kasvunopeudet eri olosuhteissa. Deleetiomutanttien kasvussa huomattiin eroa alhaisessa lämpötilassa, jossa kaikki deleetiomutanttikannat tilastollisesti merkitsevästi kasvoivat villityypin kantaa huonommin. Lisäksi ΔsrmB -kanta kasvoi tilastollisesti merkitsevästi heikommin optimilämpötilassa ja korkeassa pH:ssa. Näyttääkin siis siltä, että DEAD-box-helikaasit ovat Y. pseudotuberculosis-bakteerilla suuremmassa roolissa kuin E. coli-bakteerilla, ja niitä tarvitaan erityisesti alhaisissa lämpötiloissa.
  • Raineva, Iona (2022)
    High-grade serous carcinoma (HGSC) is the most common and lethal subtype of ovarian cancer. To date, HGSC has typically been diagnosed late, and the survival rate is poor. Relapses are common despite standardised treatment options, and platinum-based chemotherapy resistance remains frequent. The tumours are generally heterogeneous, which makes HGSC complex. Molecular mechanisms of tumour initiation, progression and chemoresistance are insufficiently understood. Thus, efficacious treatment is challenging, and current options do not help some patients. In addition, a population of cancer cells having stem cell-like properties are suggested to play a role in tumour initiation, progression and chemoresistance. There is an urgent need to better understand prognostic biomarkers and treatment responses in HGSC. With diverse analytical methods, the treatment-unresponsive patients and their outcomes could be identified by predictive biomarkers. This thesis aimed to validate potential tissue biomarkers associated with cancer stem cells and poor prognosis in HGSC, evaluate the usability of the RNAscope technique and briefly review the hypotheses on cancer stem cells. Four putative biomarkers were studied: ALDH1A1, BMI1, MYC and SOX2. The RNAscope technique was used to detect and quantify the biomarker expression. Using diagnostic tumour tissue specimens from 95 patients allowed capturing the expression in situ. With comprehensive clinical information, we could test whether the biomarkers distinguished patients with similar background information but different outcomes. This thesis shows that BMI1 could be a potential prognostic biomarker in high-grade serous carcinoma. The results provide information about the expression patterns of previously identified potential HGSC stemness markers. Proteomics studies such as immunohistochemistry could provide complementary information. When the molecular mechanisms and prognostic markers are better understood, they will provide a promising opportunity to develop novel diagnostic methods for predicting the outcomes and deciding treatments for this complex disease.
  • Aung, July (2021)
    Epithelial cells line the surfaces of organs and tissues in a continuous and tightly packed manner, thereby functioning as a protective barrier between the tissue and the external environment known as the epithelium. During development, the epithelium undergoes a series of morphogenetic events which alters the shape and size of epithelial cells, enabling them to perform tissue specific functions in mature tissue. During morphogenesis, cells sense the mechanical forces and establish polarity through cell proliferation and rearrangement according to morphogenetic signalling pathways. This manoeuvre is achieved by the underlying actin cytoskeleton network which enables cells to resist the tension and stresses of morphogenesis via alteration of filament dynamics and network architecture. In vivo, numerous actin-regulatory proteins generate various polymerized forms of straight, branched, or contractile actin-myosin filaments, regulating dynamic actin filament turnover. The robust actin cytoskeleton provides the cell with protrusive and contractile forces that enable cells to migrate, maintain, and change its shape and form during morphogenetic events. Actin filament depolymerization is accomplished by ADF/cofilin (Drosophila homolog twinstar) binding to actin monomers (G-actin) and actin filaments. However, ADF/cofilin alone is not very efficient in promoting disassembly of actin monomers, especially in subcellular regions where ADF/cofilin is highly concentrated. AIP1 (Drosophila homolog flare) then enhances actin depolymerization via preferential binding to ADF/Cofilin rich regions in vitro. The aim of my thesis was to study the localization and roles of AIP1 and cofilin in follicular epithelium during Drosophila oogenesis. My results showed that Actin-Interacting-Protein-1 (AIP1) was expressed throughout oogenesis. AIP1 expression was increased in cell type-specific manner and AIP1 showed spatiotemporal localization in follicular epithelium during oogenesis. Silencing of AIP1 led to accumulation of ectopic F-actin aggregates, localization of which may reflect the cellular sites of dynamic actin reorganization in the follicular epithelium. My results also indicate that AIP1 may be indirectly responsible for maintaining epithelial integrity as its silencing resulted in formation of epithelial gaps throughout follicular epithelium. Also delays in border cell migration were observed. Considering the above, understanding how AIP1 functions in Drosophila morphogenetic events would therefore pave the way for a greater understanding of how this protein works in other organisms. The knowledge gained may also be used to extend the current understanding of the role of actin binding proteins in diseased states.
  • Gelman, Valeria (2014)
    The increased rates of population growth and urbanization worldwide raises the question of food security and self-reliance in cities. In view of this situation, in recent years there has been a re-emergence of urban agriculture in its traditional form and in new variations, such as on urban rooftops. A number of rooftop urban farms exist in the world; however, very few studies have been done to establish the quality of crops they produce, specifically concerning the concentrations of contaminants. The main purpose of this study was to investigate levels of contamination in edible plants grown on urban rooftops. I determined concentrations of polycyclic aromatic hydrocarbons (PAH) and trace metals in the biomass of three types of horticultural crops grown in the city of Helsinki, Finland. Lettuce, radish and peas were planted on five rooftops in various areas of Helsinki and control samples were acquired from local food stores and markets. Both groups of crops were analyzed for concentrations of 11 trace elements using the Elan 6000 ICP-MS and 16 PAHs using Shimadzu GC-MS-QP2010 Ultra system with the AOC-20i /AOC-20s autosampler. Additionally, lettuce and pea samples from the roofs were analyzed washed and unwashed to establish levels of particulate contamination on the surface of plants that can be mechanically removed through washing. Results obtained suggest that concentrations of PAHs and trace metals in rooftop vegetables in Helsinki are very low and the differences in their concentrations compared to control (store) samples are insignificant. This demonstrates that the consumption of vegetables produced in uncontaminated soil on urban roofs in Helsinki is safe. All samples showed concentrations well below the safety limits for heavy metals and PAHs established in the European Union (EC, 2006). Finally, there was a difference in concentration of PAHs and trace metals between washed and unwashed samples, however most of the results did not show statistical significance.
  • Kiviluoma, Juha (2003)
    Työn tavoitteena oli muodostaa kokonaiskuva sähköjärjestelmän sisäisistä ja ulkoisista vuorovaikutussuhteista. Sähköntuotantoa tarkasteltiin ympäristöönsä kytkeytyvänä systeeminä tieteidenvälisestä näkökulmasta. Tätä tarkoitusta varten rakennettiin menetelmä laajan kokonaisuuden hahmottamiseksi ja sovellettiin sitä sähköntuotannon alalle. Menetelmäperustuu grounded theoryyn ja systeemiajatteluun: Aineisto kerättiin ja luokiteltiin grounded theorya soveltaen. Systeemiajattelua käytettiin aineiston analyysissä ja aihealuetta kuvaavien mallien luomisessa. Aineisto kerättiin haastattelemalla sähköntuotannon ja -kulutuksen asiantuntijoita. Sen pohjalta rakennettiin useita alamalleja kuvaamaan sähköntuotantokompleksin osajärjestelmiä sekä näitä yhdistävä päämalli. Alamallien aihealueiksi muodostuivat tekninen sähköjärjestelmä, sähköjärjestelmän talous, sähkönsäästö, yhteiskunnallinen vaikuttaminen, ympäristö sekä tuotantorakenteen uusiminen. Lisäksi työssä esitettiin aineistosta nousseita kriteerejä hyvälle sähköjärjestelmälle ja sähköjärjestelmään liittyviä toimijoita. Mallien yhteydessä käsiteltiin myöseri tuotantomuotojen ominaisuuksia. Työn tuloksia tarkasteltiin kriittisesti kirjallisuuden valossa ja arvioitiin luotujen mallien sovellettavuutta. Malleja on mahdollista hyödyntää tieteenalarajoja ylittävän energiatutkimuksen jäsentäjänä sekä arvioitaessa kestävämmän energiapolitiikan edellytyksiä järjestelmätasolla.
  • Mäkilouko, Miia (2019)
    Antibioottien ylenmääräinen käyttö ja uusien antibioottien puute ovat johtaneet antibiooteille vastustuskykyisten bakteerien aiheuttamien sairauksien yleistymiseen. Lisääntynyt antibioottiresistenssi on maailmanlaajuinen ongelma, joka uhkaa globaalia terveyttä ja ruoan turvallisuutta. Staphylococcus aureus ja Pseudomonas aeruginosa ovat sairaaloissa yleisiä infektioita aiheuttavia mikrobeja. Metisilliiniresistentti S. aureus eli MRSA pystyy aiheuttamaan infektioita lähes missä tahansa kudoksessa. P. aeruginosa on akvaattisissa ympäristöissä yleinen mikrobi, joka on usein luonnollisesti vastustuskykyinen useille antibiooteille. Lisäksi molemmat bakteerit kykenevät biofilmin muodostamiseen, joka heikentää entisestään antibioottien tehoa. Jätevesien puhdistamoilla on yleisesti havaittu esiintyvän S. aureus- ja P. aeruginosa -bakteereita, mutta suurin osa tutkimuksista on keskittynyt tulevan ja käsitellyn jäteveden mikrobimääriiin ja/tai aktiivilietteeseen. Jätevesillä on ehdotettu olevan merkittävä rooli antibioottiresistenssin kehittymisessä ja leviämisesessä. Jätevesien puhdistamot keräävät yhteen kotitalouksien, teollisuuden ja sairaaloiden jätevesiä ja luovat niiden mukana tulleille mikrobeille tilaisuuden sekoittua ja vaihtaa geneettistä materiaalia, kuten antibioottiresistenssigeenejä. Toisaalta ne ovat myös paikkoja, joissa antibioottiresistenssejä bakteereita vastaan voi kehittyä uusia antimikrobiaalisia aineita tuottavia mikrobeja. Pro Gradu tutkielmani on osa TWIN-A konsortion hanketta ”uusia antibiootteja jätteistä”, jonka päämääränä on uusien antimikrobiaalisten aineiden löytäminen jätevesistä ja teollisista komposteista. Pro Gradu tutkielmassani kartoitan S. aureus ja P. aeruginosa bakteerien esiintymistä jätevedenpuhdistamoiden eri prosesseissa reaaliaika-PCR:n perusteella. Tutkimukseni tuloksia voidaan käyttää hankkeen jatkotutkimuksissa sekä jätevedenpuhdistamoiden riskinarvioinnissa. S. aureus -bakteeria kartoitettiin metisilliiniresistenttiä koodaavan mecA-geenin avulla ja S. aureus -bakteerille spesifistä nukleaasia koodaavan nucA-geenien avulla. P. aeruginosa -bakteeria kartoitettiin gyrB- ja ecfX-geenien avulla. Lisäksi näiden geenien kartoituksessa oli apuna koettimet.GyrB- ja ecfX-geeneissä olevilla muutoksilla on havaittu olevan vaikutusta bakteerin virulenssikykyyn. Kartoitettuja geenejä havaittiin esiintyvän yleisesti jätevedenpuhdistamojen prosesseissa, mutta pitoisuudet olivat alle määritysrajan. MecA-geenin esiintymisfrekvenssi oli nucA-geeniä suurempi, joka voi johtua siitä, että mecA-geeniä esiintyy myös muilla stafylokokki-lajeilla, kun nucA-geeni on spesifinen S. aureus-lajille. Myös gyrB-geenin esiintymisfrekvenssi oli korkeampi kuin ecfX-geenin, joka selittynee gyrB-geenin heikommalla lajispesifisyydellä. Kaikkien kartoitettujen geenien esiintyminen oli painottunut välppeeseen, aktiivilietteisiin, raakalietteeseen, palautuslietteeseen ja tiivistämölietteisiin. Välppeen läheinen kontakti ihmisen kanssa ja suuri orgaanisen aineen määrä selittävät korkeita esiintymisfrekvenssejä tässä prosessissa. Mikrobeille otolliset olot ja mikrobien sorptio aktiivilietteeseen selittävät kartoitettujen bakteerien yleisyyden aktiivilietteissä ja sen jälkeisissä prosesseissa mädättämölle asti. Mädättämöllä anaerobinen mädätys johtaa kartoitettujen geenien vähenemiseen. Mädättämöliete käsitellään Suomessa pääosin kompostoimalla, jossa lämpötilan nousu tappaa suurimman osan patogeeneistä. Kartoitettujen geenien poistuminen jätevedenpuhdistusprosesseista aktiivilietteen mukana, selittää myös geenien matalamman esiintymisfrekvenssin käsitellyssä jätevedessä verrattuna tulevaan jäteveteen. Tulosten perusteella kartoitetut bakteerit ja niiden antibioottiresistenssigeenit eivät aiheuta riskiä ympäristölle. Vaikka havaitut pitoisuudet olivat alle määritysrajan on kuitenkin hyvä pitää mielessä, että ympäristötekijöistä riippuen antibioottiresistenssigeenit ja bakteerit voivat kertyä ympäristöön ja sopivissa olosuhteissa lisääntyä ekspotentiaalisesti. Multiresistenssien bakteerien on myös havaittu selviävän paremmin jätevedenpuhdistusprosesseista, jonka vuoksi tilannetta olisi hyvä seurata tulevaisuudessa.
  • Kiuru, Jukka (2020)
    Tiivistelmä – Referat – Abstract Ikääntymiseen liittyvien biologisten ilmiöiden, kuten rappeutumisen ja sairauksien tutkiminen on ensiarvoisen tärkeää, koska näistä ilmiöistä on haittaa niin yksilölle kuin koko yhteiskunnalle. Erityisen kiinnostavaa on, jos sairaudet ja rappeutuminen pystytäisiin parantamaan tai niiden haitallisia vaikutuksia voitaisiin vähentää. Ikääntymiseen voidaan yhdistää monia vakavia sairauksia kuten hermostorappeumasairaudet, syöpä ja diabetes. Hermostorappeumasairauksille on tunnusomaista proteiiniaggregaattien muodostuminen. SBC003-yhdiste on luonnon yrtistä eristetty molekyyli, joka on potentiaalinen lääke hermostorappeumasairauksiin. Aikaisemmin tehdyt tutkimukset hermostorappeuma sairausmalleilla osoittavat yhdisteellä olevan voimakas konsentraatiosta riippuvainen hermosoluja suojaava vaikutus. Malliorganismit ovat tärkeitä, koska niillä pystytään testaamaan potentiaalisten molekyylien vaikutusta vahingoittamatta kuitenkaan ihmistä. Hiivasta on tullut mielenkiintoinen malliorganismi ikääntymisen ja ikääntymiseen liittyvien sairauksien tutkimuksessa. Monet biologiset prosessit ja aineenvaihduntareitit ovat konservoituneet hiivasta aina ihmiseen asti. Lisäksi geneettiset muutokset on helppo jäljittää. Ikääntymisen tutkimisessa leivinhiiva (Saccharomyces cerevisiae) on osoittautunut erinomaiseksi malliorganismiksi. Hiivan kronologinen ikääntyminen on osoittautunut myös hyväksi menetelmäksi tutkittaessa solujen post-mitoottista ikääntymistä. Hiivasoluissa pystytään havaitsemaan nopeasti, edullisesti ja helposti proteiiniaggregaattien aiheuttamat haitalliset vaikutukset. Käytettäessä hiivaa malliorganismina tutkimuksessa pystytään luomaan suora linkki geenien ja kemiallisten yhdisteiden välille. Hiivalla tehdyt solujen sairausmekanismien tutkimukset ovat vertailukelpoisia ihmisten sairauksien kanssa. Nämä seikat tekevät hiivasta kiinnostavan malliorganismin myös hermostorappeumasairauksien lääkeainetutkimuksessa. Työn tavoitteena oli selvittää, voidaanko hiivasairausmalleja käyttää SBC003-yhdisteen vaikutuksien arvioimiseen hermostorappeumasairauksissa, ja onko tutkittavalla yhdisteellä pelastava vaikutus haitallisia aggregaatteja muodostavia proteiineja vastaan hiivasairausmalleissa. Lisäksi tarkoitus oli tutkia, onko SBC003-yhdisteellä vaikutusta ikääntymisestä aiheutuvaan elinvoimaisuuden heikkenemiseen hiivasoluissa. Tutkimuksissa käytettiin seuraavia menetelmiä ja koejärjestelyjä 1. spottitestejä ja 2. elinkiertotestejä ravistelukasvatuksina, joiden elinvoimaisuutta mitattiin näytteillä, joita puolestaan analysoitiin edelleen Bioscreen-kasvatuksia apuna käyttäen. Spottitestien ja elinkiertotestien tuloksien mukaan SBC003-yhdisteellä saattaa olla pelastava vaikutus haitallisia FUS-aggregaatteja vastaan hiivan ihmisen sairausmallin soluissa. Tutkimustulosten mukaan SBC003-yhdisteellä on solun elinvoimaisuutta parantava vaikutus soluihin, jotka rappeutuvat ikääntymisen seurauksena. Saatujen tulosten varmentamiseksi pitäisi elinkierotestit ja spottitestit toistaa ja lisäksi tehdä sekä geneettisiä että epigeneettisiä tutkimuksia.
  • Coles, Eric Anthony (2019)
    Tiivistelmä–Referat–Abstract Background: Cancer is one of the leading causes of death around the world and in Finland. Ambitious research projects have been carried out for decades investigating cancer and how it spreads. Over 35 years ago, the systems that regulate vascular formation were discovered; the vascular endothelial growth factor (VEGF)-VEGF receptor VEGFR system and the more recent Angiopoietin-TIE system. These are the main endothelial growth factor receptor pathways involved in regulation of vessel quiescence and angiogenesis. The VEGF-VEGFR system is the first discovered endothelial cell (EC) specific receptor tyrosine kinase (RTK) signaling system. VEGF is a major growth factor involved in proangiogenic activity and vascular permeability when bound to its receptor, VEGFR2. Tumor cells take advantage of the VEGF-VEGFR2 system by secreting VEGF to stimulate angiogenesis in surrounding tissue to create new blood vessels allowing for greater access to nutrients and oxygen for tumor growth. The Angiopoietin-TIE system is the second EC specific RTK signaling system that was discovered. Angiopoietin-1 (ANG1) is the ligand for the TIE2 RTK. ANG1 is an obligatory TIE2 agonist and its effects on intracellular signaling, cell cytoskeleton, and junction-related molecules allows ANG1 to restrict the amount and size of gaps that are formed at EC junctions in inflamed vessels, increasing barrier function and decreasing vascular permeability. Angiopoietin-2 (ANG2) is an autocrine context-dependent TIE2 agonist/antagonist which is implicated in stimulating pathological angiogenesis, inflammation and vascular permeability. Integrins are important cell surface receptors that all cells use to communicate with their environment. Recently, it has been discovered that ANG2 is capable of inducing pathological angiogenesis, and can destabilize ECs when bound to integrin, specifically β1-integrin, via ANG2 N-terminal region. Objectives: The general aim of this study was to discover which part of angiopoiten-2’s N-terminus region was responsible for integrin activation. Materials and Methods: Fibronectin fragment containing type III 7-10 domains was produced and fluorescently labeled with Alexa 647. Integrin activation was measured using the fluorescently labeled Fibronectin III 7-10 and angiopoietins. Fluorescence activated cell sorting (FACS) was used to collect the results, which were analyzed using excel. Chimeric angiopoietins were produced using retroviral vectors and used for FACS experiments. A cell internalization assay was performed in Hela cells using CellTracker™ Orange CMRA and angiopoietin proteins, stained with secondary antibody anti-human Alexa 488 and Texas Red Phallodin. Results: Optimization of the FACS assay defined the minimum number of cells required to reliably measure integrin activation and showed that BD Accuri FACS machine was better suited than Guarva FACS machine for the assay and that the amount of integrin varied between cell passages used for the assay. In addition, it was essential to ensure a homogenous mix of cells and recombinant proteins during the assay and the quality of the produced FN III 7-10 was critical for the success of the assay. Results from the FACS assay confirmed that ANG2 is capable of activating integrin. In addition, chimeric angiopoietins that were expressed and secreted from CHO cells, were capable of activating integrins to a variable degree. The results confirmed the importance of ANG2 N-terminus in integrin activation. Cell internalization assay visually demonstrated angiopoietin binding to Hela cells. ANG2 was internalized by the cell and resistant to the acid wash, while the majority of ANG1 bound to the cell surface was washed away by acid wash. Conclusions: In this thesis work, integrin activation assays were optimized and carried out, along with cell internalization assays, to determine which specific part of ANG2 is responsible for inducing integrin activation. The findings from this work confirmed that ANG2 is capable of activating integrin. Several chimeric constructs were successfully expressed in CHO cells, and the cell supernatants were used to activate integrins. However, more studies are needed to determine which specific region of ANG2 is responsible for integrin activation. Investigating angiopoietin induced integrin activation would allow for a better understanding of the angiopoietin signaling pathway with potential translational significance.
  • Rajala, Salla (2022)
    Tiivistelmä * Referat * Abstract Brownification is a serious environmental problem, which means a substantial increase in water color. Suggested reasons for brownification are soil recovery from acidification, land-use change, especially ditching, climate change. Brownification changes the light conditions in the lakes, increases thermal stratification, and sedimentation. Despite the considerable effects of brownification on lake ecosystem, currently in the EU Water Framework Directive, there is no sufficient biological indicator for lake browning. Biological indicators of the Water Framework Directive are mainly targeted to eutrophication, and thus are not sufficient for measuring the effects of brownification. Macrophyte chlorophyll a:b ratio has been shown to decrease with decreasing light conditions, thus making it a potential indicator for browning. The seasonal variation of macrophyte chlorophyll content is not well studied. This master’s thesis aims to find out, how the chlorophyll content of yellow water lily (Nuphar lutea) changes during the growing season in lakes with variable water quality. N. lutea was chosen as a study species, as it is very common in different lakes in Finland. It is important to study the seasonal variation of the macrophyte chlorophyll content, because if the chlorophyll content reacts rapidly for example changes in the weather, it might be too sensitive to be used as a long-term indicator for lake browning. It is also needed to study, if the seasonal variation is different in lakes with variable water quality, so that in the future, the possible sampling can be timed right in different lakes. The study lakes were clear water lakes, humic and eutrophic lake, and their water quality and the chlorophyll content of N. lutea were examined with two weeks intervals from June to September. The concentration of chlorophyll a and chlorophyll b were measured from the floating leaf and from the petiole at 10 cm intervals. Based on these, the total chlorophyll concentration (CHL a+b) and CHL a:b ratio was calculated. In addition to macrophyte samples, water samples were taken from each lake from both epilimnion and hypolimnion. From the water samples, dissolved organic carbon, water color, iron concentration and specific ultraviolet absorbance at 254 nm were measured. In addition, water temperature, light intensity, oxygen concentration and water turbidity were measured at each sampling time. The results of this thesis showed, that the CHL a:b ratio, and total CHL concentration (a+b) were significantly lower in the humic than in the clear water lake. In the eutrophic lake, CHL a:b ratio and CHL a+b concentration was higher than in the humic lake but lower than in the clear water lake. In each lake, the CHL a:b ratio increased during the growing season, together with the increasing water clarity. The change in the CHL a:b ratio was detected best in the 10-30 cm petiole depth. In the floating leaf, CHL a:b ratio and CHL a+b concentration varied a lot, meaning that the floating leaf is not a sufficient indicator for brownification. The change in CHL a:b ratio took approximately 5 to 7 weeks, and the seasonal variation was similar in each lake. Based on the results of this thesis, the CHL a:b ratio of the N. lutea petiole seems to be a very promising long-term indicator for lake browning. In the future, it would be, however, important to study, how the carbon sequestration changes as a result of brownification, if the macrophyte chlorophyll concentration decreases, since this might have considerable effects on lake carbon sequestration. Also, the eutrophic study lake of this thesis had a water color over 100 mg Pt l-1, so it would be important to study, what is the macrophyte chlorophyll content in clear, but eutrophic lakes.
  • Liao, Wenfei (2017)
    Urbanisation has caused many environmental problems, such as air pollution and the loss of biodiversity. One way to mitigate these problems is to expand green spaces. Roofs, as the last frontier, could be made full use of. Green roofs have become a hot topic in recent years. In this study, I investigated the ability of green roofs to support urban biodiversity by conducting a literature review, and then I sought the criteria for biodiversity roofs under Finnish conditions by interviewing ecologists. My research questions in this study were 1) What kinds of habitats could be 'ideal ecosystems' to be mimicked on biodiversity roofs in Finland; 2) which plant species could exist on roofs and whether they contribute to biodiversity; 3) what kinds of substrates support the biodiversity on roofs; 4) whether green roofs support faunal diversity and what faunal taxa could exist on roofs; 5) if and how roof structural characteristics influence roof biodiversity; 6) what kinds of management are practiced on biodiversity roofs; 7) what are people s attitudes towards or perceptions of biodiversity roofs in general. In this study, I conduct that 1) Sunny dry habitats, such as meadows and tundra, can be regarded as 'model ecosystems' for biodiversity roofs in the Finnish context. 2) Substrate heterogeneity is a key to biodiversity on green roofs. Different materials and different combinations of materials could be applied on the same roof to mimic diverse types of soil types in the most biodiverse Finnish ecosystems. 3) Native species from the model ecosystems are ideal plants for biodiversity roofs. Combining multiple greening methods on the same roof can be a solution to achieve 'instant greening effects' with only native species. 4) An ideal biodiversity roof in the Finnish context could support birds, bats, and invertebrates, such as spiders. To attract and support fauna, a roof needs a diverse plant community, as well as extra elements, such as deadwood. 5) Roof structural characteristics (i.e. roof height, size, slope, direction, location, and age) impact biodiversity by determining the accessibility to and the dispersal of flora and fauna, as well as microclimates on roofs. 6) Management, such as irrigation, might help biodiversity at least for newly established biodiversity roofs, but biodiversity roofs aim at being self-sustaining eventually. 7) People have generally positive attitudes towards green roofs, but their willingness to actually install a biodiversity roof is influenced by other issues, such as the financial cost and roof safety.
  • Razumov, Vitali (2022)
    Climatic cycles lead to changes in habitat suitability, which in turn can lead to allopatry, i.e. isolation, between populations. Lack of gene flow between allopatric populations causes them to diverge through accumulation of genetic differences that can create incompatibilities between lineages upon secondary contact in the form of lowered survivability or reproduction rate in hybrids. Incompatible genes act as reproductive barriers and keep lineages isolated by selection against hybrids, while gene flow and recombination work as a counterforce to selection promoting admixture. Reproductive barriers like these are most often found inside hybrid zones and are well demonstrated in nature, but the isolating effect of individual genetic incompatibilities on genome-wide gene flow is still an open question. Here we test if selection counteracting gene flow maintains a narrow hybrid zone between two subspecies of the meadow grasshopper Pseudochorthippus parallelus. We targeted 0,01 % of the 13 GB genome, recovering a 29,1 mean coverage per locus per individual in targeted regions, when mapping against a transcriptome. We find that, for the nuclear markers, the hybrid zone is narrower than expected under a neutral scenario of no selection, suggesting that it is maintained by selection against hybrids. We also find significant isolation by distance, suggesting gene flow across the hybrid zone despite selection against hybrids. Different parts of the genome show significant excess or deficit of heterozygotes, suggesting that selection and gene flow are heterogeneous throughout the genome. Combined, our results show that reproductive isolation between recently diverged lineages can evolve quickly despite gene flow in neutral and positively selected sites.
  • Hiltunen, Antti Olavi (2022)
    Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases and has the worst clinical outcome. Characterizing features of TNBC are high recurrence and mortality rates, and the absence of three commonly targetable breast cancer biomarkers estrogen receptor, progesterone receptor, and HER2, limiting the number of targetable therapy options. Cytotoxic CD8 positive T cells play a crucial role in the anticancer immune response and act as a major component of successful cancer immunotherapies. However, cancer cells can evade T cell-mediated killing by overexpressing programmed death-ligand 1 (PD-L1) resulting in T cell exhaustion and limited immune response via the interaction with programmed death protein 1 (PD-1). Systemic anti-PD-L1/PD-1 therapies aim to prevent this immunosuppressive mechanism, but they are burdened with potentially life-threatening autoimmunity-type adverse effects. Therefore, cancer cell-specific targets to downregulate PD-L1 could offer efficacious and less harmful ways to overcome PD_L1/PD-1 mediated immunosuppression. Serine protease hepsin is commonly overexpressed in many solid tumors where it is responsible for the activation of HGF/MET signaling pathway as well as degradation of desmosomes and hemidesmosomes leading to the loss of epithelial integrity, invasion, and metastasis. Earlier studies have linked hyperactive HGF/MET pathway to the upregulation of immune checkpoint molecule PD-L1. In this thesis, I show how pharmacological inhibition of hepsin leads to decreased MET activity and downregulation of PD-L1 in a panel of TNBC cell lines. My results demonstrate the potential of hepsin-mediated regulation of PD-L1 in tumor immunosuppression, and hint at the potential of hepsin as a therapeutic avenue towards safe and efficacious immunotherapy in the future. These results are part of a larger study addressing the role of hepsin as a regulator of PD-L1 breast cancer.
  • Koskinen, Netta (2022)
    SerpinE2 is a serine protease inhibitor (serpin) family protein that inhibits several extracellular proteases, such as thrombin, urokinase-type plasminogen activator and trypsin. Proteases and their inhibitors are often involved in cancer. SerpinE2 transcripts are upregulated in several cancers and found to predict poor prognosis of cancer patients. However, such studies regarding protein levels of serpinE2 are scarce. In this study, serpinE2 protein was analysed in three urological cancers, with patient groups that address the greatest needs for clinical biomarkers. The major aim of this study was to examine the association of serpinE2 staining with patient survival and clinicopathological features in prostate, urinary bladder and kidney cancers, and to evaluate its usability as an immunohistochemical biomarker. Tissue microarray slides from cancer patient tissues were stained immunohistochemically for serpinE2. The staining intensity was scored with four-point scale from 0 (no staining) to 3 (very intensive staining). Prostate and kidney cancer patients had been treated surgically and some of the cancers had relapsed after the surgery. In bladder cancer, association of serpinE2 with treatment response to neoadjuvant chemotherapy was evaluated. SerpinE2 expression was also measured in two prostate cancer cell lines with quantitative PCR and Western blotting. The serpinE2 staining was observed both in cancer cells and epithelial structures of benign tissues. The results showed that cancer tissue serpinE2 is not associated with relapse, treatment response or survival in prostate and bladder cancer patients. However, serpinE2 staining was more pronounced in prostate cancer tissues compared with benign tissues adjacent to cancer, and, surprisingly, the staining in such benign tissues was stronger in tissues from patients who developed metastases after surgery as compared to those without detectable metastases during 10.3-year (median) follow-up (p = 0.017). In addition, higher serpinE2 staining intensity was observed in higher grade bladder cancers (p = 0.034). In kidney cancer, on the other hand, serpinE2 staining intensity was significantly lower in patients whose cancer relapsed (p = 0.048), and high intensity predicted favourable disease-specific survival (p = 0.013). To conclude, serpinE2 is worth of further investigation in urological cancers. In prostate cancer, the possible field effect of cancer on serpinE2 in adjacent benign tissues could be examined more closely. In kidney cancer, the impact of serpinE2 on patient survival was inverse compared to transcript data in the Cancer Genome Atlas/the Human Protein Atlas database, and most other cancers. Thus, further validation studies need to be performed, and if the results hold true, serpinE2 staining could be used as part of a prognostic model predicting kidney cancer-specific survival.
  • Suomalainen, Milla (2021)
    Climate change is expected to increase the frequency and intensity of heatwaves in Finland. Heatwaves increase morbidity and mortality, especially among the elderly and chronically ill. Home care services are in an important role supporting the functioning ability and quality of life among populations vulnerable to heat. This study presents a qualitative empirical case study to understand heatwave adaptation in municipal home care services in Helsinki. The purpose of this study is to explore, describe and analyse the effects of the 2018 heatwave in the context of home care services, and the services’ capacity to adapt to heat. The data was collected by conducting semi-structured interviews with home care workers both on the operational and strategical levels. Theoretical frameworks regarding extreme heat vulnerability and local adaptive practices were used to support the analysis. The results suggest that heatwaves have caused challenges for both workers and customers. While the working ability of home care staff may be reduced due to thermal stress, the customers’ reduced functioning ability may simultaneously increase the need for care. Some workers had experienced fatigue, and customers were described having typical heat exhaustion symptoms. Workers adaptive practices were mostly reactive responses aimed at reducing the vulnerability of customers to heat. Interviewees described having taken preventive measures to limit customers’ exposure to heat and prevent adverse heat-related health outcomes by monitoring customers’ hydration more carefully. No long-term preparedness measures were identified on the strategical level. The city had supported workers by offering mineral drink bottles and providing instructions for coping in hot weather. In conclusion, workers have an active role in adaptation, but it might be limited by their own thermal stress, customers’ different health conditions and resources, as well as service-level stressors such as lack of staff. Because heatwaves are expected to increase in the future, home care services’ preparedness to heatwaves might need to be improved. It would be important to consider adaptation needs of both workers and customers as they can be interrelated in the sense, that care is dependent on workers ability to function. In a wider societal framework, the adaptive capacity of home care should be secured through providing sufficient resources for these services. The latter is even more important in the light of future trends regarding the aging of population, increase in need for home-based services and decrease in workforce availability. More research is required on the effects of heatwaves on the most vulnerable populations, what adaptation needs there might be as well as how this relates to service provision.
  • Warren, Andrew (2019)
    A novel method of somatic cell reprogramming employing CRISPR/Cas mediated gene activation (CRISPRa) may lead to improvements in the quality and efficiency of induced pluripotent stem cell (iPSC) generation by directly activating the endogenous factors of the cell. However, this method is yet to be optimized and is inefficient in its current form. Thorough characterization of the molecular events that occur during CRISPRa-mediated reprogramming could permit the fine-tuning of this method to improve iPSC production. B-lymphoblastoid cell lines (LCLs) adhere to culture plates during reprogramming, offering a form of selection for reprogramming cell populations. This thesis aimed to establish a system using LCLs for the characterisation of CRISPRa-mediated pluripotent reprogramming at the single-cell transcriptomic level. In this thesis LCL reprogramming conditions were characterized using alkaline phosphatase staining, immunocytochemistry, embryoid body formation, and live cell imaging. CRISPRa-mediated reprogramming efficiency was greatly increased by the targeting of the miR-302/367 cluster, a group of microRNAs known to improve the efficiency of transgenic reprogramming. Samples were collected for single-cell RNA sequencing (scRNA-seq) at multiple stages of reprogramming, the pluripotency of the iPSC samples was assessed, and a subset of the samples was sequenced. Clustering analysis of the sequencing data showed that the samples clustered apart from one another distinctly based on gene expression. The expression of notable genetic markers of LCLs, pluripotency, and developmental stages was consistent with the loss of somatic cell identity and rise of subpopulations characteristic of reprogramming. These results show that this is a functional system for scRNA-seq sample preparation that can be used to investigate reprogramming kinetics, and the samples collected will be part of a larger study of CRISPRa reprogramming.