Studies of granulocyte colony stimulating factor signaling to develop tools for clinical assessments of severe congenital neutropenia

Neutropenia is condition characterized by low number of neutrophils in circulation. This leads to an increased risk of infections and is often diagnosed early in life. Extreme cases are known as severe neutropenia and are often associated with inactivating mutations in common neutrophil genes. Granulocyte colony stimulating factor receptor (G-CSFR), encoded by CSF3R, is a growth factor receptor known to induce stem cell release from bone marrow (BM), but also stimulate granulopoiesis. Mutations in this gene leading to low neutrophil counts are associated with the disease severe congenital neutropenia 7 (SNC7). Classification of genetic variants or mutations based solely on sequencing data can be subjective and often lead to misclassification. Pathogenic mutations have the potential to be categorized as variants of uncertain significance (VUS). This also includes mutations in CSF3R, where missense mutations can have a large impact on neutrophile production. A functional test to assess the effects of novel missense mutations in the CSF3R gene would be beneficial for the diagnostic work up of SCN7 patients. This project aims to establish cellular assays for the investigation of G-CSF signaling and for functional characterization of CSF3R mutations. First, an assay was created using phospho-flow cytometry to study changes in protein signaling after G-CSF stimulation of primary human blood cells. Secondly, a reporter assay was developed for assessing the impact of CSF3R-mutations on STAT3 signaling. A specific mutation previously classified as a VUS, p.(Gly27Arg) was also studied to characterize its impacts on STAT3 signaling following receptor stimulation. Stimulation assays on neutrophils showed a lack of signaling downstream from the G-CSF receptor. We did, however, observe significant signal transduction in the phospho-flow assay for two out of the four signaling proteins (STAT3 and STAT5) when studying monocytes. The reporter-assay was successful in quantifying STAT3 signal after G-CSF stimulation but showed some difficulties with activating mutations. The mutation p.(Gly27Arg) was found to be likely pathogenic with STAT3 signaling barely detectable compared to WT. Both assays created show promising results for clinically validating the significance of CSF3R variants.Masteroppgave i molekylærbiologiMOL399MAMN-MO

Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary

<p>Abstract</p> <p>Background</p> <p>Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation <it>in vivo </it>is unclear.</p> <p>Results</p> <p>We have studied glutathionylation of α- and β-actin <it>in vitro </it>using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. α- and β-actin were both glutathionylated when incubated with reduced glutathione (GSH) combined with diamide as a thiol oxidant. However, β-actin was also glutathionylated by both glutathione disulfide (GSSG) and GSH in the absence of diamide whereas α-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of β-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. β-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in β-actin.</p> <p>Conclusion</p> <p>We conclude that glutathionylation of β-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.</p

Goals à la carte : selective translation of the Sustainable Development Goals in strategic municipal planning in Norway

publishedVersio

Mye styr, lite styring? Implementering av FNs bærekraftsmål i samfunns- og arealplanlegging

Regjeringen har vedtatt at FNS bærekraftsmål skal legges til grunn for lokal og regional planlegging. Planleggerne får dermed en sentral rolle i å oversette bærekraftsmålene til en lokal kontekst. På bakgrunn av intervjuer med planleggere i Nordland og Vestland, og analyser av statlige implementeringsdokumenter, utforsker artikkelen hvilke styringslogikker som kjennetegner implementeringen av bærekraftsmålene.publishedVersio

Kommunen på miljødugnad: FNs bærekraftsmål som rammeverk for regional og lokal planlegging

Author's accepted version (postprint).This is an Accepted Manuscript of a book chapter published by Universitetsforlaget in “Introduksjon til miljøplanlegging” on 11/02/2022.Available online: https://www.universitetsforlaget.no/introduksjon-til-miljoplanlegging-1acceptedVersio

Many obesity-associated SNPs strongly associate with DNA methylation changes at proximal promoters and enhancers

Background: The mechanisms by which genetic variants, such as single nucleotide polymorphisms (SNPs), identified in genome-wide association studies act to influence body mass remain unknown for most of these SNPs, which continue to puzzle the scientific community. Recent evidence points to the epigenetic and chromatin states of the genome as having important roles. Methods: We genotyped 355 healthy young individuals for 52 known obesity-associated SNPs and obtained DNA methylation levels in their blood using the Illumina 450 K BeadChip. Associations between alleles and methylation at proximal cytosine residues were tested using a linear model adjusted for age, sex, weight category, and a proxy for blood cell type counts. For replication in other tissues, we used two open-access datasets (skin fibroblasts, n = 62; four brain regions, n = 121-133) and an additional dataset in subcutaneous and visceral fat (n = 149). Results: We found that alleles at 28 of these obesity-associated SNPs associate with methylation levels at 107 proximal CpG sites. Out of 107 CpG sites, 38 are located in gene promoters, including genes strongly implicated in obesity (MIR148A, BDNF, PTPMT1, NR1H3, MGAT1, SCGB3A1, HOXC12, PMAIP1, PSIP1, RPS10-NUDT3, RPS10, SKOR1, MAP2K5, SIX5, AGRN, IMMP1L, ELP4, ITIH4, SEMA3G, POMC, ADCY3, SSPN, LGR4, TUFM, MIR4721, SULT1A1, SULT1A2, APOBR, CLN3, SPNS1, SH2B1, ATXN2L, and IL27). Interestingly, the associated SNPs are in known eQTLs for some of these genes. We also found that the 107 CpGs are enriched in enhancers in peripheral blood mononuclear cells. Finally, our results indicate that some of these associations are not blood-specific as we successfully replicated four associations in skin fibroblasts. Conclusions: Our results strongly suggest that many obesity-associated SNPs are associated with proximal gene regulation, which was reflected by association of obesity risk allele genotypes with differential DNA methylation. This study highlights the importance of DNA methylation and other chromatin marks as a way to understand the molecular basis of genetic variants associated with human diseases and traits

Analysis of transcript and protein overlap in a human osteosarcoma cell line

<p>Abstract</p> <p>Background</p> <p>An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with <it>in situ </it>protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF).</p> <p>Results</p> <p>A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies.</p> <p>Conclusion</p> <p>This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.</p

A global view of protein expression in human cells, tissues, and organs

Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry-based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced

Interrelation of inflammation and APP in sIBM: IL-1β induces accumulation of β-amyloid in skeletal muscle

Distinct interrelationships between inflammation and β-amyloid-associated degeneration, the two major hallmarks of the skeletal muscle pathology in sporadic inclusion body myositis (sIBM), have remained elusive. Expression of markers relevant for these pathomechanisms were analysed in biopsies of sIBM, polymyositis (PM), dermatomyositis (DM), dystrophic and non-myopathic muscle as controls, and cultured human myotubes. By quantitative PCR, a higher upregulation was noted for the mRNA-expression of CXCL-9, CCL-3, CCL-4, IFN-γ, TNF-α and IL-1β in sIBM muscle compared to PM, DM and controls. All inflammatory myopathies displayed overexpression of degeneration-associated markers, yet only in sIBM, expression of the mRNA of amyloid precursor protein (APP) significantly and consistently correlated with inflammation in the muscle and mRNA-levels of chemokines and IFN-γ. Only in sIBM, immunohistochemical analysis revealed that inflammatory mediators including IL-1β co-localized to β-amyloid depositions within myofibres. In human myotubes, exposure to IL-1β caused upregulation of APP with subsequent intracellular aggregation of β-amyloid. Our data suggest that, in sIBM muscle, production of high amounts of pro-inflammatory mediators specifically induces β-amyloid-associated degeneration. The observations may help to design targeted treatment strategies for chronic inflammatory disorders of the skeletal muscle