Keratins regulate colonic epithelial cell differentiation through the Notch1 signalling pathway

Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8(-/-)) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine. Proximity ligation and immunoprecipitation assays demonstrate that K8 and Notch1 co-localize and interact in cell cultures, and in vivo in the colonic epithelial cells. K8 with its heteropolymeric partner K18 enhance Notch1 protein levels and activity in a dose dependent manner. The levels of the full-length Notch1 receptor (FLN), the Notch1 intracellular domain (NICD) and expression of Notch1 downstream target genes are reduced in the absence of K8, and the K8-dependent loss of Notch1 activity can be rescued with re-expression of K8/K18 in K8-knockout CRISPR/Cas9 Caco-2 cells protein levels. In vivo, K8 deletion with subsequent Notch1 downregulation leads to a shift in differentiation towards a goblet cell and enteroendocrine phenotype from an enterocyte cell fate. Furthermore, the K8(-/-) colonic hyperproliferation results from an increased number of transit amplifying progenitor cells in these mice. K8/K18 thus interact with Notch1 and regulate Notch1 signalling activity during differentiation of the colonic epithelium

Cellular regulation of the Notch signaling pathway by Notch-interacting proteins

The Notch signaling mechanism is a crucial regulator of stem cells during development and tissue regeneration. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD), NICD migration into the nucleus, and assembly of a transcriptional complex. This thesis focuses on the stress-induced interaction of Notch with the small ubiquitin-like modifier (SUMO) and the effect of this interaction on the differentiation of neuronal stem cells in the central nervous system (Study I), and the interaction of Notch with the cytoskeletal intermediate filament (IF) keratin proteins in the differentiation of intestinal epithelial cells (Study II). Study (III) is a review and focuses on discussing different aspects of various post-translational modifications (PTM) of Notch. The Notch receptor interacts with several post-translational modifiers, but the exact mechanisms and consequences of these interactions remain elusive. PTMs of Notch regulate its trafficking, turnover, and transcriptional activity. Study (I) presents a direct interaction between Notch and SUMOduring cell stress in the form of heat shock or the presence of the protea some inhibitor termed bortezomib. The sumoylation took place in the nucleus where SUMO was conjugated to the transcription factor Suppressor of Hairless (RBPJ/CSL)-associated domain(RAM) of NICD. In canonical Notch signaling, the interaction between RAM and the transcription factor Recombination signal binding protein for immunoglobulin kappa J region (RBPJ)/CSL is a prerequisite for activation of Notch target genes. However, the sumoylation site in the RAM domain was easily accessible for sumoylation without affecting the RAM-CSL interaction or any other component within the DNA-bound transcriptional complex consisting of NICD, CSL and the coactivator Mastermind-like (MAML). Neitherdid the SUMO-conjugating protein, termed ubiquitin conjugating enzyme 9 (Ubc9) interfere in the NICD-CSL-DNA interactions. Therefore, NICD sumoylation occurred in the transcriptional complex without disrupting the complex. Cellular stress-induced sumoylation of NICD enhanced the protein level and nuclear localization of Notch, but nevertheless still dampened its transcriptional activity. On a mechanistic level, NICD sumoylation promoted the recruitment of histone deacetylase 4 (HDAC4) to the transcriptional complex to suppress Notch target gene expression. The NICD sumoylation-mediated repression of Notch target genes was abolished by expression of a sumoylation-deficient mutant Notch construct in cells in vitro. Furthermore, the stress-inducible sumoylation provided a regulatory mechanism for dynamic Notch target gene expression also in vivo. In the developing central nervous system of chicken embryos, the expression of Notch target genes was repressed upon cellular stress by wildtype Notch, whereas the target gene activation capacity was retained by a sumoylation-deficient Notch mutant. In addition, consequent differences between the wildtype and the sumoylation-deficient Notch regarding tissue differentiation were displayed. The findings ofthe stress-inducible sumoylation of NICD in study (I), revealed a novel context-dependent regulatory mechanism of Notch target gene expression.Study (II) of this thesis presents a direct interaction between Notch and keratins and the outcomes that this specific interaction mediates on the proliferation and differentiation of colonic epithelial cells. Keratins are cytoskeletal intermediate filament (IF) proteins and have essential roles in cellular stress protection and mechanical support of epithelial tissues. 8 K8, K18 and K19 constitute the largest group of the keratin isoforms that are expressed in the colon.K8 is along with K18 the main component, not only of all keratin isoforms, but also of the entire IF cytoskeleton and is therefore also the main IF protein of simple layered epithelia. K8 is localized preferentially in the cytoplasm and is the most common type II keratin, pairs predominantly with the type I K18, and is mainly expressed in the epithelia of the intestine, liver and exocrine pancreas. Earlier it has been known that mice lacking K8(K8-/-mice) develop colitis, decreased apoptosis and significant hyperproliferation of colonic crypt epithelial cells, but the effect of the loss of K8 on intestinal cell differentiation had so far been unknown. Notch in turn has been known to be a critical regulator of colonic epithelial cell differentiation. In study (II), we showed that K8 together with its polymeric partner K18 regulated Notch signaling activity and cell differentiation in the epithelium of the large intestine.K8 bound directly to Notch1 in colonic epithelial cells, both in vitro in cell culture, and in vivo, displayed in immunoprecipitations and proximity ligation assays. In vitro, the interaction resulted in enhanced Notch levels and activity in a dose-dependent manner, but unlike the Notch-SUMO interaction discovered in study (I), which resulted in repressed Notch target gene expression, the Notch-K8/K18 interaction in study (II) led to increased expression of specific Notch target genes. Accordingly, in vivo in the colon epithelium, K8-/-mice had decreased levels of both the complete form of Notch, called full length Notch(FLN), and NICD, and also repressed expression of the Notch target genes Hey1 and Hey2. The effect of the absence of K8 on Notch activity was observed in cultured colorectal cancer cells by deleting K8 with CRISPR/Cas9. This knockout of K8 led to the downregulation of FLN and NICD levels and reduced Notch downstream target gene Hey1 expression, which was rescued with re-expression of K8/K18. In vivo, the differentiation phenotype in K8-/-colonic epithelium was shifted from an enterocyte cell fate towards increased goblet cells and enteroendocrine cells. Moreover, in the colonic crypts, K8 depleted mice developed increased amount of transit amplifying (TA) cells from which the terminally differentiated cells originate. In study (II), Notchinteracted with K8/K18 and regulated Notch signaling during differentiation in the colonic epithelium

Tilannepaikanjohtaminen pelastuslaitoksissa

Tämän opinnäytetyön tavoitteena oli selvittää, miten pelastuslaitoksilla käytetään tilannepaikanjohtajuutta tällä hetkellä. Tilannepaikanjohtajuus on ollut käytössä noin viisi vuotta, ja tilannepaikanjohtajuuden käyttö vaihtelee pelastuslaitoksilla merkittävästi. Tavoitteena oli myös tutkia, miten pelastuslaitoksilla koulutetaan tilannepaikanjohtajuutta. Tutkimusmenetelmä oli pääosin kvantitatiivinen tutkimusmenetelmä. Tutkimuskysely oli standardoitu survey-kysely, joka lähetettiin kaikkiin 22 pelastuslaitokseen ja sopimuspalokunnille. Tutkimuskyselyssä oli 19 monivalintakysymystä ja kolme avointa kysymystä. Tutkimustulokset osoittivat osittaista puutetta koulutuksessa. Koulutusta tehostamalla voidaan kehittää tilannepaikanjohtajuuden käyttöä ja yhtenäistää toimintamallia pelastuslaitoksilla.The objective of this final project was to study the current state of the use of the on-site commander (OSC) system at the fire departments in Finland. An on-site commander provides important information from the fire scene to the officer in charge. The OSC system was taken in use about five years ago. At the moment, the level of use of the OSC system varies significantly at different fire departments. The second objective of this final project was to study the level of education of OSC arranged by the fire departments in Finland. The research method was mainly quantitative. Information was gathered with a questionnaire which was sent to all 22 fire departments and volunteer fire departments via email. The questionnaire consisted of multiple-choice questions and few open-ended questions. The open-ended questions answered by the officers in charge provided qualitative information about the experiences of using the OSC system during rescue missions. The results shows that there were some shortcomings in providing OSC training. It is recommended that fire departments should increase the amount of OSC training as it would develop the use of the OSC system and unify the working methods in the whole country

Adoption Barrier for Radio Frequency Identification Technology

Radio frequency identification technology (RFID) has during the past decade seen an upwards trend among logistics companies in the supply chain. With increased e-commerce, demand for new and more efficient solutions are ever-increasing. RFID is a technology which Schmidt, Thoroe & Schumann (2013) call the next ‘evolutionary step for auto-identification technology’. The purpose of this research is to investigate and describe how previously found barriers for RFID adoption are perceived by Swedish logistic companies. The significance of barriers often accompanies the choice of investing or not. This thesis was conducted with mixed qualitative research design and used multiple case studies as a pilot study as well as a survey based on the results from the qualitative study to look at different logistic companies in Sweden. The authors collected data using semistructured interviews with a purposive sampling technique. Through thematic analysis, predetermined barrier themes emerged and that were strengthened by coding and categorising the transcripts. The survey data has been collected and analysed in IBM SPSS. The findings in this thesis conclude that RFID has matured over the last decade, and that cost is no longer the most significant barrier when it comes to implementing RFID technology into logistic processes. The most significant barrier now is lack of understanding and the popularity of barcodes. Lack of business case and an extensive knowledge gap surrounding RFID impedes Swedish logistic companies to adopt the technology. Another significant barrier was the popularity of barcodes. It has long been the industry standard, and companies are heavily invested in the infrastructure developed for barcodes. The target group for this research is both companies thinking of investing in RFID technology and RFID solution providers who want to know what challenges logistic companies face regarding RFID. For future studies, researchers should try to investigate how postal services could feasibly adopt this technology or analyse and compare barriers between different logistical segments within the supply chain. 

Factors affecting different payment methods online : A comparative study between invoice, card and multiple alternatives when shopping online.

E-handels snabba utveckling det senaste årtiondet har öppnat upp nya möjligheter för både företag och konsumenter och det har gjort att betalsätten förändrats. I takt med den tekniska utveckling som skett har även konsumenters intentioner och uppfattningar förändrats och faktorer som användbarhet, användarvänlighet, tillit och risk är starkt kopplat till köpbeteende online. De två vanligaste betalsätten för svenska e-handels konsumenter idag är bankkort/kreditkort och faktura. Studiens syfte var att undersöka svenska konsumenters intention och uppfattning av betalningssätten kort och faktura samt situationer med fler betalningsalternativ vid handel online. Till vår hjälp har den teknologiska acceptansmodellen (TAM) används tillsammans Pavlous (2003) e-handels modell för att se om faktorerna de fann också påverkade svenska konsumenter. En enkät togs fram med fokus på tre delar, 1) bankkort/kreditkort, 2) faktura och 3) flera betalsätt. Den insamlade data analyserades med hjälp av SPSS och resultatet visade att faktorn som hade störst påverkan var användbarhet. För betalsätten bankkort/kreditkort och när flera alternativ listades visade analysen att faktorn risk inte påverkade intentionen till att använda betalsätten. Faktura var det betalsätt som upplevdes som minst riskfyllt bland respondenterna. Resultatet visade också att trots bankkort/kreditkort har en högre risk, är de flesta benägna att använda det betalsättet vid nästa köp kontra faktura, som hade en lägre risk vilket kan förklaras att bankkort/kreditkort är det betalsätt i Sverige som används mest.The rapid development of E-commerce over the past decade has opened up new opportunities for both businesses and consumers, and this has changed the payment methods. In line with the technological development that has taken place, consumers' intentions and perceptions have also changed and factors such as usability, ease of use, trust and risk are strongly linked to online purchasing behavior. The two most common payment methods for Swedish e-commerce consumers today are bank cards / credit cards and invoices. The purpose of this study was to examine the intention and perception of payment methods, cards and invoice of Swedish consumers, as well as situations with more payment options when shopping online. To our aid, the Technological Acceptance Model (TAM) has been used together with Pavlous (2003) e-commerce model to see if the factors they found also affected Swedish consumers. A questionnaire was developed focusing on three parts, 1) bank card / credit card, 2) invoice and 3) several payment methods. The data collected were analyzed using SPSS and the results showed that the factor that had the greatest impact was usability. For the payment methods bank card / credit card and when several options were listed, the analysis showed that the risk factor did not affect the intention to use the payment methods. Invoice was the payment method that was perceived as least risky among the respondents. The result also showed that although bank cards / credit cards have a higher risk, most people are inclined to use the payment method in the next purchase, as opposed to invoice, which had a lower risk which can be explained by bank card / credit card being the most used payment method in Sweden

Adoption Barrier for Radio Frequency Identification Technology

Radio frequency identification technology (RFID) has during the past decade seen an upwards trend among logistics companies in the supply chain. With increased e-commerce, demand for new and more efficient solutions are ever-increasing. RFID is a technology which Schmidt, Thoroe & Schumann (2013) call the next ‘evolutionary step for auto-identification technology’. The purpose of this research is to investigate and describe how previously found barriers for RFID adoption are perceived by Swedish logistic companies. The significance of barriers often accompanies the choice of investing or not. This thesis was conducted with mixed qualitative research design and used multiple case studies as a pilot study as well as a survey based on the results from the qualitative study to look at different logistic companies in Sweden. The authors collected data using semistructured interviews with a purposive sampling technique. Through thematic analysis, predetermined barrier themes emerged and that were strengthened by coding and categorising the transcripts. The survey data has been collected and analysed in IBM SPSS. The findings in this thesis conclude that RFID has matured over the last decade, and that cost is no longer the most significant barrier when it comes to implementing RFID technology into logistic processes. The most significant barrier now is lack of understanding and the popularity of barcodes. Lack of business case and an extensive knowledge gap surrounding RFID impedes Swedish logistic companies to adopt the technology. Another significant barrier was the popularity of barcodes. It has long been the industry standard, and companies are heavily invested in the infrastructure developed for barcodes. The target group for this research is both companies thinking of investing in RFID technology and RFID solution providers who want to know what challenges logistic companies face regarding RFID. For future studies, researchers should try to investigate how postal services could feasibly adopt this technology or analyse and compare barriers between different logistical segments within the supply chain. 

Decoding the PTM-switchboard of Notch

The developmentally indispensable Notch pathway exhibits a high grade of pleiotropism in its biological output. Emerging evidence supports the notion of post-translational modifications (PTMs) as a modus operandi controlling dynamic fine-tuning of Notch activity. Although, the intricacy of Notch post-translational regulation, as well as how these modifications lead to multiples of divergent Notch phenotypes is still largely unknown, numerous studies show a correlation between the site of modification and the output. These include glycosylation of the extracellular domain of Notch modulating ligand binding, and phosphorylation of the PEST domain controlling half-life of the intracellular domain of Notch. Furthermore, several reports show that multiple PTMs can act in concert, or compete for the same sites to drive opposite outputs. However, further investigation of the complex PTM crosstalk is required for a complete understanding of the PTM-mediated Notch switchboard. In this review, we aim to provide a consistent and up-to-date summary of the currently known PTMs acting on the Notch signaling pathway, their functions in different contexts, as well as explore their implications in physiology and disease. Furthermore, we give an overview of the present state of PTM research methodology, and allude to a future with PTM-targeted Notch therapeutics

Decoding the PTM-switchboard of Notch

\u3cp\u3eThe developmentally indispensable Notch pathway exhibits a high grade of pleiotropism in its biological output. Emerging evidence supports the notion of post-translational modifications (PTMs) as a modus operandi controlling dynamic fine-tuning of Notch activity. Although, the intricacy of Notch post-translational regulation, as well as how these modifications lead to multiples of divergent Notch phenotypes is still largely unknown, numerous studies show a correlation between the site of modification and the output. These include glycosylation of the extracellular domain of Notch modulating ligand binding, and phosphorylation of the PEST domain controlling half-life of the intracellular domain of Notch. Furthermore, several reports show that multiple PTMs can act in concert, or compete for the same sites to drive opposite outputs. However, further investigation of the complex PTM crosstalk is required for a complete understanding of the PTM-mediated Notch switchboard. In this review, we aim to provide a consistent and up-to-date summary of the currently known PTMs acting on the Notch signaling pathway, their functions in different contexts, as well as explore their implications in physiology and disease. Furthermore, we give an overview of the present state of PTM research methodology, and allude to a future with PTM-targeted Notch therapeutics.\u3c/p\u3

Dupilumab efficacy in subgroups of type 2 asthma with high-dose inhaled corticosteroids at baseline

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