Browsing by Subject "differentiation"

This study aims to find out if differentiation of the features of the 4P of the product milk can create value to customers in a particular customer segment. Creating value is in the core in every company´s actions. Customers buy products and services that create value to them and are willing to pay from the value they get. The more the product creates value, the more the customers are willing to pay. This study focused on the customer segment of young women with academic background who live in the capital region. Milk is a bulk product consumed widely in Finland. The product also has significant role in the nutritional history of the country and has a big role in Finnish nutritional recommendations. However the consumption of milk has decreased over the years. The theoretical background of this study is based on the following theories: marketing strategy and the 4P, customer value and differentiation. These theories formed the theoretical framework which gave the focus to the empirical experiment. The approach of the study was qualitative because qualitative research aims to understand the phenomenon it studies and the approach is more suitable for analysing verbal data. Qualitative research wants to get a deeper understanding and it gives room for individual´s thoughts and experience. The study was executed and a group interview using theme interview methods. For the study chosen features of the 4P of the product milk were differentiated and group interview focused on them. The differentiated features were the following. For product organic milk and milk with added protein, for price milk with a lower price, for place online shopping and for promotion advertising milk on social media. The findings of this study were that in some cases differentiation of the features if the 4P of the product milk can create value for customers but in most cases it does not. The focus group felt that the most valuable differentiated feature of the 4P was organically produced milk and other factors that supported the well-being of the production animals and the environment. If a company selling or producing milk tries to create value to its customers through differentiation of the features of the 4P, it needs to consider carefully which features to focus on.

In the past few years, there has been an increased consideration on the stem cell niche as a key factor to regulate stem cell maintenance and differentiation. Research on characterization of the stem cell microenvironment boosted after the determination of long-term three-dimensional (3D) tissue cultures, or so-called organoids. Organoids are derived from stem cells which self-organize in 3D multicellular structures upon embedding in an extracellular matrix mimic, such as Matrigel®. Their main advantage is these structures resemble the architectural distribution of the tissue of origin in vivo. Likewise, the cellular components of organoids vary depending on multiple variables as the tissue of origin and the growth factors they have access to. As a result of advances in this technique, some stem cell niches have been well characterized, as in the case of intestinal stem cells (ISCs), while others remain elusive as in case of the human gastric stem cells (hGSCs). Along with the remarkable development of 3D cultures, the interest of ECM proteins in stem cell regulation increased. Matrigel® is a rich matrix composed of several adhesive proteins such as laminins and collagens. Aside from providing structural support, the extracellular matrix (ECM) proteins forming this matrix contribute to cell adhesion and signalling. However, Matrigel® composition cannot be modified or even well-characterized due to its origin from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Additionally, it has been demonstrated that contains a high batch-to-batch variability. Other techniques to study the effects of individual ECM proteins have been used such as coating of tissue culture plates with ECM proteins. However, the biomechanical properties in this model are far from being physiological. Therefore, although preliminary results can be obtained using this technique, results extrapolation to an in vivo model can be questioned. To date, there is a lack of a reproducible, high-throughput and reliable technique to test the effect of ECM proteins on human gastric stem cells behavior. This Master’s thesis presents a novel transwell device containing a polyethylene glycol (PEG)-based hydrogel enriched with human ECM proteins to test their effect on human gastric stem cell regulation. Preliminary results showed that gastric organoid-derived epithelial cells (GODE) grown on hydrogels with ECM proteins that are localized at base of the gastric glands, such as Laminin-211, had a higher stem cell marker expression than the control grown on ECM proteins that are uniformly localized in vivo. Additionally, when GODE were grown on hydrogels containing ECM proteins that are localized at the surface of the native gastric epithelium, expression of surface gastric mucins markers was enhanced. These preliminary results highlight the utility of the optimized transwell device to further shed light on how the human gastric stem cells are regulated and what is the effect of the ECM proteins surrounding them.

Lukivaikeus on verrattain yleinen oppimisvaikeus, johon jokainen opettaja luultavasti törmää uransa aikana. Koska lukivaikeuden taustalla on kielitietoisuuteen ja erityisesti äännetietoisuuteen liittyviä ongelmia, se aiheuttaa erityisiä hankaluuksia lukemisen ja kirjoittamisen lisäksi myös vieraiden kielten oppimisessa. Näin ollen olisi tärkeää, että vieraiden kielten opettajat ymmärtäisivät lukivaikeuden taustoja sekä eriyttämiskeinoja, jotka hyödyttävät erityisesti lukivaikeudesta kärsiviä vieraiden kielten oppijoita. Siksi tämän tutkimuksen tarkoituksena oli kartoittaa suomalaisten englanninopettajien kokemuksia lukivaikeuteen liittyen. Tutkimuksen tavoitteena oli selvittää, (1) mitä englanninopettajat tietävät lukivaikeudesta ja mistä he ovat tietonsa saaneet, (2) millaisia asenteita heillä on lukivaikeudesta kärsiviä oppilaita ja opiskelijoita kohtaan, (3) millaisina he näkevät lukivaikeuden vaikutukset englannin oppimiseen ja (4) miten he ovat ottaneet lukivaikeuden huomioon opetuksessaan. Kysymyksiin etsittiin vastausta kyselytutkimuksen keinoin. Kyselyä jaettiin sosiaalisessa mediassa ja sen täyttäminen tapahtui internetissä. Kaikki osallistujat (n = 72) olivat muodollisesti päteviä opettajia ja opettivat englantia suomenkielisissä alakouluissa, yläkouluissa, lukioissa ja/tai ammattikouluissa. Kyselyllä kerätty aineisto sisältää sekä määrällisiä että laadullisia tietoja. Määrällinen aineisto analysoitiin deskriptiivisin tilastollisin menetelmin ja laadullinen aineisto sisällönanalyysin keinoin. Tutkimukseen osallistuneilla opettajilla oli verrattain hyvä tietämys lukivaikeudesta, vaikkakaan he eivät kokeneet omaavansa tarvittavaa tietotaitoa lukivaikeudesta kärsivien oppijoiden tukemiseen. Lukivaikeutta ei joko ollut käsitelty ollenkaan tai ei riittävästi osallistujien opettajaopinnoissa. Sen sijaan osallistujat olivat saaneet tietonsa lukivaikeudesta muista lähteistä, kuten erityisopettajalta tai kirjallisuudesta oman aktiivisuutensa turvin. Osallistujien asenne lukivaikeutta ja siitä kärsiviä oppijoita kohtaan oli laajasti ottaen positiivinen. Opettajan näkökulmasta lukivaikeus vaikeuttaa englannin oppimista sanaston, kieliopin, kirjoittamisen, lukemisen, kuuntelun, ääntämisen ja äänne-erittelyn osalta. Oppijoilla saattaa myös olla erilaisia negatiivisia tunteita itseään, kieliä tai oppimista kohtaan. Lisäksi lukivaikeudesta kärsivät tarvitsevat enemmän aikaa tehtävien tekemiseen kuin luokkatoverinsa. Lähes kaikki osallistujat ovat ottaneet lukivaikeuden huomioon opetuksessaan esimerkiksi eriyttämällä arviointia, materiaaleja ja opetustaan. Osallistujille tutuimpia eriyttämisen keinoja olivat arvioinnin eriyttämiseen liittyvät keinot sekä sellaiset keinot, joita yleisesti käytetään kaikkien oppimisvaikeuksien huomioinnissa ja kaikkien oppiaineiden opetuksessa. Osallistujille vähemmän tuttuja olivat opetuksen eriyttämiseen liittyvät keinot, joita suositellaan nimenomaan lukivaikeudesta kärsiville vieraiden kielten oppijoille. Tutkielmassa pohditaan, miten hyvin opettajakoulutus valmistaa opettajia käytännön työhön, ja kritisoidaan sitä lähtökohtaa, että lukivaikeudesta kärsiviä kielten oppijoita tuetaan kouluissa ensisijaisesti arvioinnin keinoin oppimisen edesauttamisen sijaan.

Kv7.1 is a potassium ion channel comprised of the KCNQ1 protein, which can coassemble with distinct β-subunits modulating the channel functions in different tissues. In 2017, Raivio’s group (from the University of Helsinki) found two missense mutations in the KCNQ1 gene, p.(Arg116Leu) and p.(Pro369Leu), responsible for causing pituitary hormone deficiency and maternally inherited gingival fibromatosis. The facial features and bone structure pointed to a cranial neural crest (CNC)-derived phenotype caused by an alteration in the potassium channel balance, given that these cells form the bone and cartilage of the cranial zone. To understand the implication of the CNC in the KCNQ1 syndrome, I attempted to replicate the CNC differentiation protocol of Suga and Furue (2019) with the aim of obtaining cranial neural crest cells (CNCCs). This would enable future generation of a KCNQ1-related disease model. The differentiation process was carried out thrice, and two BMP4 concentrations (10 and 100 ng/ml) were assayed. The differentiated cells exhibited a CNC-like morphology as well as upregulation of the marker genes (TFAP2A, SOX10, DLX1, MSX1, and DLX2) associated to this cell lineage. However, the gene expression was low according to the qRT-PCR Ct values, which were in most cases higher than 30. Additionally, no differences were found between the two BMP4 treatments. Furthermore, the cells did not express KCNQ1, and thus the impact of the two KCNQ1 mutations was not investigated under this protocol. In conclusion, the protocol had a low efficiency in the generation of CNCCs that was not improved by increasing the BMP4 concentration. Further optimization of the protocol, such as the BMP4 concentration or the cell density of the culture, will be needed to improve its efficiency and obtain an adequate disease model.

Pharmaceutical companies are currently facing increasing developmental costs, and at the same time, less new compounds are being brought to the market. In vitro -metabolism studies and toxicity assessment of new drug candidates are crucial, as early as possible, to prevent their withdrawal in later development phases. Used study systems are, however, limited and new improved technologies are being investigated. Notable, drug induced liver toxicity and alterations in the liver function are frequent reasons for the drug removals from the development. Human embryonic stem cell (hESC) is one of the most powerful cell types known. hESCs have not only the possibility to divide indefinitely but these cells have also the ability to differentiate to all mature cell types of the human body, such as hepatocytes. This makes them potentially very valuable for pharmaceutical development, in order to create a functional in vitro -model, mimicking the liver tissue. In the literature part, the three dimensional (3D) -hepatic differentiation of mouse and human ESCs in vitro, are discussed. Traditional 2D-culture systems do not adequately mimic the microenvironment of three dimensionally organized native tissue. In 2D-cultures cells grow as a monolayer, when the cell morphology is flattened leading to poor cell-cell and cell-matrix contacts and preventing from the tissue formation. In 3D-culture systems, cells are able to form tissue-like cell integrations, spheroids, and thus, remain their functionality and viability significantly longer. Hydrogels are commonly used biomaterials in 3D-cell cultivation and well known in various areas of tissue engineering for their nano scale porosity and ability to surround cells in 3D-polymer network. In addition, they are capable to absorb large volumes of water and functionalized, in various ways, to improve the required biological or mechanical properties. In the experimental part, the main purpose was to differentiate human hepatic progenitor cells to mature hepatocyte-like cells in three dimensional (3D) -biomaterials. Overall, four different hydrogels (cellulose nanofiber (CNF) hydrogel, HydroMatrixTM, ExtracelTM and PuraMatrixTM) were used as 3D-cell culture scaffolds. Several hepatic cell functions (albumin and urea production and cytochrome P450 (CYP) 3A4 activity) were measured in 2D- and 3D-cultures and compared with the human hepatic carcinoma cells, HepG2, which are often used in drug development. Differentiated hepatocyte-like cells did not show CYP3A4 activity and they produced less albumin and urea compared with HepG2 cells. However, working with hESCs is very demanding and the research in this area is only in the beginning. Therefore, the poor cell functionality results did not come up as a surprise.

Bipotential gonads are precursor structures for testes and ovaries. Steroidogenic factor-1 (SF1) is one of the most important transcription factors in an embryo needed for the development and maintenance of bipotential gonads. If SF1 is not expressed, bipotential gonads fail to develop, and genitalia and kidneys are not formed. Later, SF1 expression persists high in testes, where it supports Sertoli and Leydig cell formation and development. If SF1 is not expressed enough in males, the bipotential gonads differentiate into ovaries. The factors activating and regulating SF1 are not currently fully known. By getting more knowledge of how SF1 is controlled, regulatory mechanisms behind normal fetal development of gonads and disorders of sex development (DSD) can be understood better. The aims of this thesis were to study whether growth factors, that naturally regulate differentiation of developing gonads, promote differentiation of human induced pluripotent stem cells (hiPSC) into Sertoli-like cells (SLCs) and whether SF1 expression is induced by the addition of these growth factors. For conducting the study, we used hiPSCs, which have an SF1 activation domain cassette previously introduced to the cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) method. SF1 could be activated by adding doxycycline (DOX) and trimethoprim (TMP). These hiPSCs were differentiated into intermediate mesoderm (IM) on the first four days according to the protocol published earlier by the group. After this, the differentiation to SLCs was guided by adding growth factors to the culture medium. Basic fibroblast growth factor (bFGF), fibroblast growth factor 9 (FGF9) and prostaglandin-2 (PGD2) were tested separately and in a combined cocktail also including follicle stimulating (FSH) and glial cell-derived neurotrophic factor (GDNF). In a control condition, cells were differentiated without additional growth factors. In all tested conditions, cells first differentiated into IM were further differentiated either in the presence or absence of DOX and TMP for 8 days. The differentiation medias were changed to the cells every day and lysis samples for quantitative PCR (qRT-PCR) were taken every other day. The relative gene expression levels of bipotential gonad, testis and steroidogenic gene markers from each condition were monitored with qRT-PCR and compared to the levels of the undifferentiated hiPSCs. Immunocytochemistry was performed to see the changes in protein production. Against our hypothesis and the previous studies by others, none of the tested growth factors induced the cells to differentiate into SLCs. However, SF1 expression was triggered by chemical induction with DOX and TMP. Also, the expression levels of bipotential gonadal and testicular gene markers increased in control conditions with/without chemical induction. PGD2 conditions were the only ones to resemble the gene expression and morphology of control conditions while the others differed. These results indicated that the addition of bFGF, FGF9, FSH and GDNF did not improve the differentiation of iPSCs into SLCs and in fact, bFGF and FGF9 hindered their differentiation into SLCs. As a future perspective the optimal concentrations for each growth factor and the duration of growth factor supplementation ought to be tested to refine the protocol.

In my Master’s thesis, I offer a novel interpretation of Gilles Deleuze’s (1925-1995) conception of transcendent thinking. As a first approximation, transcendent thinking is an unconscious disruption of quotidian thinking (i.e. empirical thinking). Deleuze’s conception is an important attempt at explaining the emergence of thought from material reality. Additionally, it offers insights into the conditions of creating something new in thinking. In Deleuze’s account, these two are closely connected. My interpretation is mainly based on Deleuze’s Difference and Repetition (1968), but I also draw from Deleuze’s other works and philosophers he discusses. Deleuze’s reading of Immanuel Kant (1724–1804) is important for my interpretation. I proceed by close readings of Deleuze and compare my interpretations to others from secondary literature. My thesis is divided into five chapters and I begin by introducing my reading of the relevant features of Deleuze’s overall project in Difference and Repetition. In chapter one, I introduce Deleuze’s novel philosophy of difference. According to Deleuze, all continuity we experience is constituted by the interplay of internal difference and hidden repetition. In chapter two, I introduce the relevant features of Deleuze’s ontological scheme in Difference and Repetition. According to it, actual objects are constituted through the process of different/ciation; two figures of internal difference, the differential relations of virtual Ideas and intensive differences, produce the actual objects we perceive in our experience. Situating Deleuze’s transcendent thinking into his overall project is necessary to interpret it correctly and to grasp its significance. Next, I interpret what Deleuze means by thinking. In chapter three, I read Immanuel Kant’s (1724–1804) determining judgment (e.g. “This is a dog”) as providing a case of Deleuze’s empirical thinking. This kind of thinking is what human subjects experience in the quotidian. However, transcendent thinking goes beyond empirical thinking. In chapter four, I show how transcendent thinking is comprised of a series of encounters where the different faculties (i.e. cognitive capabilities) of the thinker are elevated to their transcendent exercise. This series starts as sensibility encounters sensible intensity and it continues as subsequent faculties are traversed by a virtual Idea. In these encounters, the faculties confront their internal differences, which reveal their limits and what is most singular to them. However, intermediary encounters do not correspond to any conscious empirical experiences, nor does the whole of transcendent thinking either. In the final chapter of my Master’s thesis, I begin by arguing that my interpretation ameliorates on previous readings. First, it reveals that transcendent thinking is a case of different/ciation unravelling through the faculties of a psychic system. Second, my reading distinguishes between empirical thinking and transcendent thinking—both being kinds of thinking, for Deleuze. Third, it clarifies that learning is an instance of transcendent thinking (not vaguely thinking in general). Next, I discuss how transcendent thinking reveals the possibility of creation in thinking. Empirical thinking is incapable of change because in it, the faculties function according to the model of recognition: the thinker only recognizes what is already known using pregiven concepts. Transcendent thinking, as a case of different/ciation progressing through the faculties, changes the faculties and, in doing so, transforms the composition of the psychic system. This process is carried out on the level of being and results in something new emerging in thinking. However, transcendent thinking is involuntary and unconscious, leaving the conception of creative agency in Difference and Repetition restricted.

Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.