Investigating the Regulation of Distinct Epigenetic States in Human Embryonic Stem Cells

Accumulating evidence suggest that pluripotency – the ability to generate all somatic cell types – is not a fixed state. Pluripotent cell populations encompass a heterogeneous group of cells with different phenotypic and functional properties in equilibrium. At least two phases of pluripotency, immature and primed for differentiation, have been identified during early mouse development, and these are typified by the two distinct stem cell populations – mouse embryonic (mES) and epiblast-­‐derived (mEpiS) stem cells – isolated from epiblast layers of pre-­‐ and post-­‐implantation embryo, respectively. Many lines of human ES (hES) cells have also been established in vitro from the inner cell mass of pre-­‐ implantation blastocysts yet the precise in vivo lineage affiliation of these cells remains largely unresolved. Profiling chromatin in a particular cell line has proven to be a valuable signature for cell identity and developmental stage. One approach has been to assay the timing of DNA replication during S-­‐phase of the cell cycle across a panel of loci, as an indicator of chromatin accessibility. This replication timing profiling was notably capable of discriminating pluripotent mES cells from cells with a more restricted differentiation capacity. This study sought to address whether distinct pluripotent states could be reliably discriminated at the chromatin level. In particular, the replication timing profiles of a number of hES cell lines were characterised and compared to those of mES and mEpiS cell lines derived from different genetic backgrounds. Profiles of undifferentiated H1, H7 and H9 hES cell lines typically harboured an increased proportion of late-­‐replicating loci during S-­‐ phase when compared to mES cells, which were confirmed to have a steady and mostly early-­‐replicating profile regardless of their genetic background. Moreover, hES cell replication profile greatly varied between cultures and cell lines; a level of replication timing variability also observed among mEpiS cells, as opposed to mES cells. These results highlight that hES and mEpiS cells most likely share a common unstable epigenetic state or transitional state primed on the verge of differentiation. The epigenetic state of hES cell lines was further interrogated by analysing cells grown under two different culture conditions, both however similarly relying on TGF-­‐β/Activin signalling. Results demonstrated that hES cells could adopt distinct yet reversible epigenetic signatures while retaining their pluripotency. In particular, extensive and dynamic shifts of replication timing, from late-­‐to-­‐early, were consistently observed at many target loci in hES cells as well as in human induced pluripotent cells (iPS) cells, upon increased SMAD2/3-­‐associated P300/CBP histone acetyltransferase (HAT) activity. This was accompanied by fluctuations in the expression of NANOG and REX1 (also known as ZFP42), and a change in hES cell’s functional properties, as judged by their responsiveness to differentiation-­‐inducing signals. Interestingly, inhibiting P300/CBP HAT activity by curcumin treatment in undifferentiated hES cells was sufficient to revert back to a late-­‐replicating profile associated with a histone hypoacetylated state. Stable knockdown of P300 in hES cell cultures, however, resulted in a gradual loss of the pluripotent identity through differentiation or apoptosis, preventing further analysis of effects on replication timing. Collectively, these data strongly support the view that different but interchangeable pluripotent states exist within hES cell cultures and suggest a role for P300/CBP HAT activity in determining distinct epigenetic states in hES cells. As mentioned above, REX1 was also significantly upregulated in H1 hES cells upon culture condition change. REX1 is a developmental stage-­‐specific marker that is expressed in the ICM of both mouse and human embryos, as well as in pluripotent mES, hES and iPS cells. However, its function in hES cells remains largely unclear. Human ES cells overexpressing REX1 were here generated to investigate the role of REX1 in regulating hES cell pluripotent identity. These cells expressed similar levels of key pluripotency markers than their normal counterparts and remained capable of self-­‐renewing and differentiating into the three germ layers in vitro. Interestingly, however, upon withdrawal of exogenous Activin A, REX1 overexpressing hES cells in contrast to control cells retained a comparatively high level of OCT4 and stained positive for alkaline phosphatase, a known marker of undifferentiated hES cells. Taken together, these results point to a possible role for REX1 in sustaining hES cell self-­‐renewal ability

Bannerituotannon työnkulku

Bannerimainonta on edelleen laajasti käytetty digitaalisen mainonnan muoto huolimatta sosiaalisen median valtavasta noususta viimeisen kymmenen vuoden aikana. Eri laitealustat ja ohjelmistoympäristöt luovat tilanteen, jonka vaatimukset poikkeavat merkittävästi bannerimainonnan alkuaikojen kuvabannereiden yksinkertaisuudesta. Insinöörityössä oli tavoitteena tehdä optimoitu työnkulku internetin bannerimainosten tuotantoon. Työ toteutettiin digitaalista markkinointia tekevälle yritykselle. Tavoitteena oli löytää työprosessia helpottavia ja tehostavia työtapoja ja kartoittaa, millaisia mahdollisuuksia HTML5-pohjaisten bannereiden luontiin on olemassa. Osana insinöörityötä tehtiin myös dynaamista sisältöä käyttävää bannerimainontaa asiakasyritykselle. Insinöörityössä kartoitettiin erilaisia työkaluja bannerien toteuttamiseen ja animointiin ja listattiin kunkin toteutustavan hyviä ja huonoja puolia. Myös bannereissa käytettävien kuvaformaattien käyttötarkoituksia ja laadun optimointiin käytettäviä työkaluja vertailtiin. Työnkulkua pohdittiin myös arkistoinnin ja luotujen materiaalien jatkokäyttömahdollisuuksien pohjalta. Tutkimuskirjallisuutta läpikäymällä pyrittiin löytämään tietoa siitä, minkälaiset bannerit toimivat parhaiten internetissä ja perehdyttiin bannerimainonnan markkinatilanteeseen vertailemalla sitä sosiaalisen median mainoskanaviin. Työn tuloksena syntyi ehdotus siitä, millaisia optimointeja työnkulkuun voi tehdä päivittäisessä työssä, ja toisaalta, miten työnkulun hallintaa parannetaan pidemmällä aikajänteellä esimerkiksi banneripohjien ja -arkiston avulla. Bannerisokeutta voisi torjua muun muassa vaihtelemalla mainosten sijoittelua verkkosivulla ja animaation keinoin. Bannereiden animointiin ja toteutukseen ehdotettiin eri tekniikoiden tai ohjelmistojen käyttämistä käyttötapauksesta riippuen, eikä yhtä, kaikkiin projekteihin soveltuvaa menetelmää löydetty. Työnkulun automatisointiin löydettiin ratkaisuja, joilla usein samanlaisena toistuvaa työtä saataisiin vähennettyä. Insinöörityössä kuvatut työnkulun menetelmät mietittiin tilaajayrityksen näkökulmasta ja sen ympäristöön sopiviksi, mutta niitä voidaan soveltaa yleisemminkin verkkokäyttöön tarkoitetun grafiikan ja animaation tuottamiseen. Työ tarjoaa pohjan siinä kuvattujen menetelmien käyttöönottoon tuotantoympäristössä.Banner advertising is still a widely-used method in digital advertising despite the immense rise of social media during the last ten years. Creating advertisements for different types of devices and environments is a process, which reaches far beyond in complexity the relatively simple era of still image banners. The goal of this thesis is to create an optimized workflow for producing modern HTML5 banner advertisements for the web and seek out the possibilities and technologies currently available. As part of the thesis banner advertisements using dynamic content were created for a client. Different tools for creating and animating banners were evaluated and their pros and cons listed. The usage cases and quality optimization tools for image formats commonly used in banners were compared to find out the best quality / file size ratio for images. Workflow was considered also for archival and future usage of created materials. Existing research literature and statistics were used as a reference for assessing the effectiveness of banner advertising: what works and what does not. As a result, suggestions for improving the workflow were created. Some of the suggestions are usable in day-to-day work while others lean more towards improving the overall functionality of the banner production with regards to archiving and re-usage of the produced materials. For banner creation, there was not one method or program that would fit all possible cases, but it was proposed that different tools would be used based on the individual requirements of the project. Banner workflow was found to include many repetitive tasks, which could be automated with solutions such as JavaScript build tools. The methods described in the thesis for improving the workflow in banner creation were considered from the client company's point of view, and requirements may vary for other companies, but they are still applicable as general guidelines on how to create graphics and animation for use on the internet. This thesis acts as a basis for applying the described methods for actual production environment

Clinical Impact of Tumor-Associated Macrophage and T-Cell Contents in Diffuse Large B-Cell Lymphoma

Peer reviewe

Laitteita vai kokonaisuuksia:eheyttävä mediakasvatus varhaiskasvatuksessa

Tiivistelmä. Tutkielma perehtyy varhaiskasvatuksessa toteutettavaan mediakasvatukseen. Sen tavoitteena on selvittää, millaisena eheyttäminen näyttäytyy varhaiskasvatuksen mediakasvatuksessa, kuvailevan kirjallisuuskatsauksen kautta. Aihe on varhaiskasvatuksen kentällä ajankohtainen, sillä sekä eheyttäminen että mediakasvatus ovat osa varhaiskasvatusta ohjaavaa Varhaiskasvatussuunnitelman perusteita. Tutkielma on toteutettu kuvailevana kirjallisuuskatsauksena, jonka tutkimusmateriaalina on käytetty ajankohtaisia koti- ja ulkomaisia lähteitä sekä Varhaiskasvatussuunnitelman perusteita. Opetuksen eheyttämisen pitkät juuret huomioon ottaen, lähdekirjallisuus ulottuu eheyttämisen määrittelyn osalta pitkälle historiaan. Mediakasvatuksen osalta tutkimuskirjallisuus painottuu mahdollisimman tuoreisiin lähteisiin. Käytettäväksi tutkimusaineistoksi on valittu tieteellisiä artikkeleita, väitöskirjoja ja tutkimusaiheeseen sopivia perusteoksia. Tutkimustulosten mukaan eheyttäminen näyttäytyi varhaiskasvatuksen mediakasvatuksessa kokonaisuuksien, lapsilähtöisyyden ja mielekkään työskentelyn kautta. Tulosten mukaan kokonaisuudet sitoutuivat vahvasti kasvattajan omiin näkemyksiin, tietoihin ja taitoihin mediakasvatuksesta. Kasvattajan teknologisen identiteetin nähtiin vaikuttavan siihen, millaista mediakasvatusta kasvattaja toteuttaa. Kasvattajilla havaittiin olevan eheyttävää ja kokonaisvaltaista pedagogiikkaa, mutta sen ei nähty ulottuvan mediakasvatukseen saakka. Eheyttävyyden lapsilähtöisyys näkyi mediakulttuurin asemassa varhaiskasvatuksessa. Tutkimustulosten perusteella lasten omia mielenkiinnon kohteita — kuten medialeikkejä — rajoitettiin monin tavoin, eikä varhaiskasvatusta nähty mediakulttuurisena paikkana. Eheyttävyyden mielekäs työskentely näyttäytyi laitekeskeisyyden kautta. Tulosten perusteella kasvattajat näkivät mediakasvatuksen usein rajoittuneesti, lähinnä laitteiden kautta

Clinical Impact of Immune Cells and Their Spatial Interactions in Diffuse Large B-Cell Lymphoma Microenvironment

Purpose: Tumor-infiltrating immune cells have prognostic sig-nificance and are attractive therapeutic targets. Yet, the clinical significance of their spatial organization and phenotype in diffuse large B-cell lymphoma (DLBCL) is unclear. Experimental Design: We characterized T cells, macrophages, and their spatial interactions by multiplex IHC (mIHC) in 178 patients with DLBCL and correlated the data with patient demo-graphics and survival. We validated the findings on gene expression data from two external DLBCL cohorts comprising 633 patients. Results: Macrophage and T-cell contents divided the samples into T cell-inflamed (60%) and noninflamed (40%) subgroups. The T cell-inflamed lymphoma microenvironment (LME) was also rich in other immune cells, defining immune hot phenotype, which did not as such correlate with outcome. However, when we divided the patients according to T-cell and macrophage contents, LME char-acterized by high T-cell/low macrophage content or a correspond-ing gene signature was associated with superior survival [5-year overall survival (OS): 92.3% vs. 74.4%, P = 0.036; 5-year progres-sion-free survival (PFS): 92.6% vs. 69.8%, P = 0.012]. High pro-portion of PD -L1-and TIM3-expressing CD163- macrophages in the T cell-inflamed LME defined a group of patients with poor outcome [OS: HR = 3.22, 95% confidence interval (CI), 1.63-6.37, P-adj = 0.011; PFS: HR = 2.76, 95% CI, 1.44-5.28, P-adj = 0.016]. Furthermore, PD-L1 and PD-1 were enriched on macrophages interacting with T cells. Conclusions: Our data demonstrate that the interplay between macrophages and T cells in the DLBCL LME is immune checkpoint dependent and clinically meaningful.Peer reviewe

T-cell inflamed tumor microenvironment predicts favorable prognosis in primary testicular lymphoma

Primary testicular lymphoma is a rare lymphoid malignancy, most often, histologically, representing diffuse large B-cell lymphoma. The tumor rnicroenvi.ro.nrnent and limited immune surveillance have a major impact on diffuse large B-cell lymphoma pathogenesis and. survival, but the impact on primary testicular lymphoma is unknown. Here, the purpose of the study was to characterize the tumor microenvironme.nt in primary testicular lymphoma, and associate the findings with outcome. We profiled the expression of 730 immune response enes in 60 primary testicular lymphomas utilizing the Nanostring platorm, and used multiplex imrnunohistochemistry to characterize the immune cell phenotypes in the tumor tissue. We identified a gene signature enriched for T-lymphocyte markers differentially expressed. between the patients. Low expression of the signature predicted poor outcome independently of the International Prognostic Index (progression -free survival: HR=2.810, 95%CI: 1.228-6.431, P=0.014; overall survival: HR=3.267, 95`)/0' CI: 1.406-7.590, P=0.006). The T-lymphocyte signature was associated with outcome also in an independent diffuse large B-cell lymphoma cohort (n=96). Multiplex immunohistochemistry revealed that poor survival of primary testicular lymphoma patients correlated with low percentage of CD3'CD4' and CD3+CD8' tumor-infiltrating lymphocytes (PPeer reviewe

Deltex-1 mutations predict poor survival in diffuse large B-cell lymphoma

Non peer reviewe

What conditions favor the influence of seasonally frozen ground on hydrological partitioning? : a systematic review

The influence of seasonally frozen ground (SFG) on water, energy, and solute fluxes is important in cold climate regions. The hydrological role of permafrost is now being actively researched, but the influence of SFG has received less attention. Intuitively, SFG restricts (snowmelt) infiltration, thereby enhancing surface runoff and decreasing soil water replenishment and groundwater recharge. However, the reported hydrological effects of SFG remain contradictory and appear to be highly site- and event-specific. There is a clear knowledge gap concerning under what physiographical and climate conditions SFG is more likely to influence hydrological fluxes. We addressed this knowledge gap by systematically reviewing published work examining the role of SFG in hydrological partitioning. We collected data on environmental variables influencing the SFG regime across different climates, land covers, and measurement scales, along with the main conclusion about the SFG influence on the studied hydrological flux. The compiled dataset allowed us to draw conclusions that extended beyond individual site investigations. Our key findings were: (a) an obvious hydrological influence of SFG at small-scale, but a more variable hydrological response with increasing scale of measurement, and (b) indication that cold climate with deep snow and forest land cover may be related to reduced importance of SFG in hydrological partitioning. It is thus increasingly important to understand the hydrological repercussions of SFG in a warming climate, where permafrost is transitioning to seasonally frozen conditions

Immune cell constitution in the tumor microenvironment predicts the outcome in diffuse large B-cell lymphoma

Tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here, we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL) and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of DLBCL patients (n=81) utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T-cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes - the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T-cells and macrophages, together entitled as a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T-cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy