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  • 1.
    Abreu-Vieira, Gustavo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fischer, Alexander W.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Hamburg, Germany.
    Mattsson, Charlotte
    de Jong, Jasper M. A.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Shabalina, Irina G.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ryden, Mikael
    Laurencikiene, Jurga
    Arner, Peter
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Petrovic, Natasa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cidea improves the metabolic profile through expansion of adipose tissue2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7433Article in journal (Refereed)
    Abstract [en]

    In humans, Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A) is highly but variably expressed in white fat, and expression correlates with metabolic health. Here we generate transgenic mice expressing human Cidea in adipose tissues (aP2-hCidea mice) and show that Cidea is mechanistically associated with a robust increase in adipose tissue expandability. Under humanized conditions (thermoneutrality, mature age and prolonged exposure to high-fat diet), aP2-hCidea mice develop a much more pronounced obesity than their wild-type littermates. Remarkably, the malfunctioning of visceral fat normally caused by massive obesity is fully overcome-perilipin 1 and Akt expression are preserved, tissue degradation is prevented, macrophage accumulation is decreased and adiponectin expression remains high. Importantly, the aP2-hCidea mice display enhanced insulin sensitivity. Our data establish a functional role for Cidea and suggest that, in humans, the association between Cidea levels in white fat and metabolic health is not only correlative but also causative.

  • 2.
    Adler, Jeremy
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Plasma membrane topology and membrane models2009Conference paper (Other academic)
  • 3.
    Agurell, Eva
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Comparative studies of alkylating and N-heterocyclic compounds on different genetic endpoints with special emphasis on amplification of minisatellite sequences1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Several mutational events are involved in the multistage process of cancer. Genetic instability of tumor DNA is shown in a variety of genetic changes such as point mutations, chromosomal aberrations, aneuploidy, DNA amplification and somatic recombination. Highly repetitive sequences, minisatellite DNA, have been changed in tumors, most probably by DNA amplification.

    To better predict carcinogens all these genetic changes should be considered. The present investigation has focused on the study of genetic changes especially DNA amplification with the use of genetic instability of minisatellites. DNA alterations in human bladder tumors were examined. A new in vitro test system was developed to study DNA amplification.

    In this study DNA fingerprint analysis, with the use of minisatellite DNA, of 22 bladder tumor patients is performed. DNA alterations were determined in approximately 50% of the examined bladder tumor patients. In eight of these ten patients a loss of bands was demonstrated.

    To study DNA amplifications an in vitro test system was developed. A haploid yeast strain was constructed, the TR(MS1)-1, which carries a chromosomal integration of the human minisatellite sequence MSI. The spontaneous frequency of new MSI length alleles was approximately 30%. Both amplifications and deamplifications can be detected in this system without selection. A plasmid pop-out frequency can also be meausured.

    A number of model compounds was used during this study, namely three alkylating agents, a nongenotoxic carcinogen and several nitrogen heterocyclic compounds. To compare genotoxicity and the mechanism of action, these compounds are tested in several in vitro test systems. Ethylene oxide (EO) was demonstrated to be more potent than propylene oxide (PO) in several of the measured endpoints in yeast. EO induced changes in the amplification spectrum of new MSI length alleles in TR(MS1)-1 while PO increased the plasmid pop-out frequency. 2,3,7,8-Tetrachlorodibenzo(p)dioxin (TCDD) was also seen to induce changes in the amplification spectrum of new MSI length alleles, thus demonstrating an effect of TCDD at the DNA level. Among the N-heterocyclic compounds, camptothecin increased the plasmid pop-out frequency in TR(MS1)-1. Two tryptophan photoproducts, "284" and "312", were shown to bind to the A A-receptor. They were demonstrated to be nongenotoxic and antimutagenic in bacteria and slightly genotoxic in yeast. These two compounds are antimutagenic by inhibiting the cytochrome P450IA1. "284" increased cell survival or cell division. These two tryptophan photoproducts, ”284" and "312", are postulated to be biological signal substances by being the endogenous ligand to the Ah -receptor.

  • 4.
    Alarcón-Riquelme, Marta E.
    Stockholm University, Faculty of Science.
    Murine Systemic Lupus erythematosus: cellular regulation and molecular mechanisms behind the formation of autoantibodies in autoimmune disease1994Doctoral thesis, comprehensive summary (Other academic)
  • 5.
    Andréasson, Claes
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Rampelt, Heike
    Fiaux, Jocelyne
    Druffel-Augustin, Silke
    Bukau, Bernd
    The endoplasmic reticulum Grp170 acts as a nucleotide exchange factor of Hsp70 via a mechanism similar to that of the cytosolic Hsp112010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 16, p. 12445-53Article in journal (Refereed)
    Abstract [en]

    Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.

  • 6. Aufschnaiter, Andreas
    et al.
    Kohler, Verena
    Diessl, Jutta
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Peselj, Carlotta
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Carmona-Gutierrez, Didac
    Keller, Walter
    Büttner, Sabrina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Graz, Austria.
    Mitochondrial lipids in neurodegeneration2017In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 367, no 1, p. 125-140Article, review/survey (Refereed)
    Abstract [en]

    Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.

  • 7. Bach, Dominik R.
    et al.
    Guitart-Masip, Marc
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Packard, Pau A.
    Miro, Julia
    Falip, Merce
    Fuentemilla, Lluis
    Dolan, Raymond J.
    Human Hippocampus Arbitrates Approach-Avoidance Conflict2014In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 24, no 5, p. 541-547Article in journal (Refereed)
    Abstract [en]

    Animal models of human anxiety often invoke a conflict between approach and avoidance [1, 2]. In these, a key behavioral assay comprises passive avoidance of potential threat and inhibition, both thought to be controlled by ventral hippocampus [2-6]. Efforts to translate these approaches to clinical contexts [7, 8] are hampered by the fact that it is not known whether humans manifest analogous approach-avoidance dispositions and, if so, whether they share a homologous neurobiological substrate [9]. Here, we developed a paradigm to investigate the role of human hippocampus in arbitrating an approach-avoidance conflict under varying levels of potential threat. Across four experiments, subjects showed analogous behavior by adapting both passive avoidance behavior and behavioral inhibition to threat level. Using functional magnetic resonance imaging (fMRI), we observe that threat level engages the anterior hippocampus, the human homolog of rodent ventral hippocampus [10]. Testing patients with selective hippocampal lesions, we demonstrate a causal role for the hippocampus with patients showing reduced passive avoidance behavior and inhibition across all threat levels. Our data provide the first human assay for approach-avoidance conflict akin to that of animal anxiety models. The findings bridge rodent and human research on passive avoidance and behavioral inhibition and furnish a framework for addressing the neuronal underpinnings of human anxiety disorders, where our data indicate a major role for the hippocampus.

  • 8.
    Bajinskis, Ainars
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Studies of DNA repair strategies in response to complex DNA damages2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The main aim of this thesis was to study the role of the indirect actions of γ-rays and α-particles on the complexity of primary DNA damages and the repair fidelity of major DNA repair pathways: non-homologous end joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER). The complexity of radiation-induced damages increases and the proximity between damages decreases with increasing LET due to formation of ionization clusters along the particle track. The complexity of damages formed can be modified by the free radical scavenger dimethyl sulfoxide (DMSO). In addition, the effects of low doses of low dose rate γ-radiation on cellular response in terms of differentiation were investigated.

    Paper I investigates the role of the indirect effect of radiation on repair fidelity of HRR, NHEJ and BER when damages of different complexity were induced by radiation or by potassium bromate. We found that potassium bromate induces complex DNA damages through processing of base modifications and that the indirect effect of radiation has a high impact on the NHEJ pathway. Results in paper II confirmed our conclusions in paper I that the indirect effect from both γ-rays and α-particles has an impact on all three repair pathways studied and NHEJ benefits the most when the indirect effect of radiation is removed.

    In paper III we investigated the effects of low dose/dose rate γ-radiation on the developmental process of neural cells by using cell models for neurons and astrocytes. Our results suggest that low dose/dose rate γ-radiation attenuates differentiation and down-regulates proteins involved in the differentiation process of neural cells by an epigenetic rather than cytotoxic mechanism.

  • 9.
    Bajinskis, Ainars
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Natarajan, Adayapalam T.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The response of HRR-deficient Chinese hamster ovary cell line reveals significant contribution of the indirect effect from both γ-rays and α-particles on NHEJ pathwayManuscript (preprint) (Other academic)
    Abstract [en]

    In order to investigate the relative involvement of the different DNA repair pathways NHEJ, HRR and BER in repair of DNA lesions of different complexity, we have compared clonogenic survival and induction of micronuclei in a panel of repair-deficient CHO cell lines after exposure to γ-rays and α-particle radiation. The complexity of the DNA lesions formed was also modified by exposures to 2 M DMSO, a potent radical scavenger, which is known to interact with the lesions produced by direct hits on DNA.

    The NHEJ pathway gained the most from scavenging of the free radicals after irradiation to γ-rays or α-particles as evaluated by cell survival and the yields of MN. Results presented here also implicate that clustered base damages were induced by α-radiation and contributed to the yield of DNA double-strand breaks.

  • 10.
    Bajinskis, Ainars
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Olsson, Gunilla
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate2012In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 731, p. 125-132Article in journal (Refereed)
    Abstract [en]

    The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by  potassium bromate (KBrO3).

    CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulphoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay.

    The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. 

    The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

  • 11.
    Bartish, Galyna
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Synthesis of proteins is performed by the ribosome, a large ribonucleoprotein complex. Apart from the ribosome, numerous protein factors participate in this process. Elongation factor 2 (eEF2) is one of these factors. eEF2 is an essential protein with a mol. mass of about 100 kDa. The amino acid sequence of eEF2 is highly conserved in different organisms. eEF2 from S. cerevisiae contains 842 amino acids. The role of eEF2 in protein synthesis is to participate in the translocation of tRNAs from the A- and P-sites on the ribosome to the P- and E-sites. This movement of tRNAs is accompanied by a simultaneous movement of mRNA by one codon. eEF2 consists of six domains referred to as domains G, G′ and II-V, belongs to the G-protein super-family and possesses all structural motifs characterizing proteins in this family. eEF2 binds to the ribosome in complex with GTP. After GTP hydrolysis and translocation, it leaves the ribosome bound to GDP. The rate of protein synthesis in the cell can be regulated by phosphorylation of eEF2. Phosphorylation occurs on two threonine residues, situated in the G domain of the factor. Phosphorylation of eEF2 is catalysed by Rck2-kinase in yeast which is activated in response to osmotic stress. Despite the high degree of conservation of the threonine residues, they are not essential for yeast cell under normal growth conditions. However, under mild osmotic stress the growth rate of the cells lacking threonine residues was decreased. Region where threonine residues are located, called Switch I. Cryo-EM reconstruction shows that this region adopts ordered conformation when the eEF2•GTP complex is bound to the ribosome but became structurally disordered upon GTP hydrolysis. Mutagenesis of individual amino acids in Switch I resulted in both functional and non-functional eEF2 depending on the site of mutation and the substituting amino acid. Both functional and non-functional Switch I mutants were able to bind to the ribosome, indicating that mutations did not abolish the capacity of the factor to bind GTP. Yeast eEF2 with Switch I region from E. coli was able to substitute the wild type protein in vivo, though the growth rate of these cells was severely impaired. The eEF2-dependent GTP hydrolysis can be activated by ribosome from heterologous sources as seen in vitro. However, eEF2 from A. thaliana, D. melanogaster and S. solfataricus could not substi-tute yeast eEF2 in vivo. This may indicate additional roles of eEF2 in the yeast cell, apart from translocation itself.

  • 12.
    Bartish, Galyna
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Moradi, Hossein
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Nygård, Odd
    Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 20, p. 5285-5297Article in journal (Refereed)
    Abstract [en]

    Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

  • 13. Bauerschmitt, Heike
    et al.
    Mick, David
    Deckers, Markus
    Vollmer, C
    Funes, S
    Kehrein, Kirsten
    University of Kaiserslautern, Germany.
    Ott, Martin
    University of Kaiserslautern, Germany.
    Rehling, Peter
    Herrmann, Johannes
    Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation2010In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 21, no 12, p. 1937-1944Article in journal (Other academic)
    Abstract [en]

    Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.

  • 14.
    Bayat, Narges
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristobal, Susana
    The effects of ultra-small TiO2 nanoparticle and single walled carbon nanotubes on endothelial cells: next generation sequencing and transcriptome sequencing (RNA-seq) analysisManuscript (preprint) (Other academic)
    Abstract [en]

    The cardiovascular system is a key route of exposure to nanoparticles (NPs). The exposure could costendothelial cell dysfunction and impairment in blood circulation that could lead to cardiovascular diseasessuch as atherosclerosis. Currently, ultra-small nanoparticles (USNPs) at 1-3 nm, are receiving growingattention due to their unique properties. Emerging application for rutile TiO2-USNPs in medicine areexploring due to their insoluble nature, lack of oxidative activity and strong luminescence not observed inlarger NPs. On organic nanoparticle side, single walled carbon nanotubes (SWCNTs) are candidatemolecules for drug delivery from the chemical perspective. However their potential applications arehindered by their high oxidative activity and potential toxicity. Here we used transcriptome sequencing(RNA-seq) to evaluate the effects of exposure to sub-lethal concentration of TiO2-USNPs, TiO2-NPs andSWCNTs on human dermal microvascular endothelial cells. Specific toxicological effects were inferredfrom the functions of genes whose transcripts either increased or decreased. Our results show that TiO2-USNPs mostly induced the up-regulation of transcripts involved in lipid and cholesterol metabolism.TiO2-NPs induced the highest number of differentially expressed transcripts involved in cellularsenescence, endoplasmic reticulum (ER) stress, and heat shock responses as well lipid metabolism.Finally, SWCNTs affected to those genes involved in early stress and inflammatory responses.

  • 15.
    Bayat, Narges
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lopes, Viviana
    Sanchez-Dominguez, Maria
    Lakshmanan, Ramnath
    Rajarao, Gunaratna
    Cristobal, Susana
    Assessment of the safety of functionalized iron oxide nanoparticles in vitro: introduction to integrated nanoimpact indexManuscript (preprint) (Other academic)
    Abstract [en]

    Functionalization of super paramagnetic iron oxide NPs (SPIONs) with different coatings renders them with unique physicochemicalproperties that allow them to be used in a broad range of applications such as drug targeting and water purification. However, it is required tofill the gap between the promises of any new functionalized SPIONs and the effects of these coatings on the NPs safety. Nanotoxicology isoffering diverse strategies to assess the effect of exposure to SPIONs in a case-by-case manner but an integrated nanoimpact scale has notbeen developed yet. We have implemented the classical integrated biological response (IBR) into an integrated nanoimpact index (INI) as anearly warning scale of nano-impact based on a combination of toxicological end points such as cell proliferation, oxidative stress, apoptosisand genotoxicity. Here, the effect of SPIONs functionalized with tri-sodium citrate (TSC), polyethylenimine (PEI), aminopropyltriethoxysilane(APTES) and Chitosan (chitosan) were assessed on human keratinocytes and endothelial cells. Our results show thatendothelial cells were more sensitive to exposure than keratinocytes and the initial cell culture density modulated the toxicity. PEI-SPIONshad the strongest effects in both cell types while TSC-SPIONS were the most biocompatible. This study emphasizes not only the importanceof surface coatings but also the cell type and the initial cell density on the selection of toxicity assays. The INI developed here could offer aninitial rationale to choose either modifying SPIONs properties to reduce its nanoimpact or performing a complete risk assessment to definethe risk boundaries.

  • 16.
    Beckman, Marie
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kihlmark, Madeleine
    Södertörn University, Stockholm, Sweden.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Nucleus and Nuclear Envelope: Methods for Preparation2010Book (Other academic)
    Abstract [en]

    The cell nucleus of eukaryotic organisms contains the genome surrounded by a nuclear envelope consisting of a double-lipid membrane with embedded nuclear pores and an underlying nuclear lamina. The uniformity in size and density makes it possible to isolate pure intact nuclei at high yields from tissue homogenates by centrifugation through a sucrose cushion. Nuclear envelopes can be prepared from isolated nuclei by enzymatic degradation of their nucleic acid content. The resulting nuclear envelope preparations contain structurally well-conserved inner and outer nuclear membranes with attached ribosomes, nuclear pore complexes and nuclear lamina. Reliable methods for preparation of nuclei and nuclear envelopes play an important role in the successful identification of components that are located in nuclei and in nuclear subcompartments.

  • 17. Bembenek, Joshua N.
    et al.
    Meshik, Xenia
    Tsarouhas, Vasilios
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Meeting report - Cellular dynamics: membrane-cytoskeleton interface2017In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, no 17, p. 2775-2779Article in journal (Refereed)
    Abstract [en]

    The first ever 'Cellular Dynamics' meeting on the membrane-cytoskeleton interface took place in Southbridge, MA on May 21-24, 2017 and was co-organized by Michael Way, Elizabeth Chen, Margaret Gardel and Jennifer Lippincott-Schwarz. Investigators from around the world studying a broad range of related topics shared their insights into the function and regulation of the cytoskeleton and membrane compartments. This provided great opportunities to learn about key questions in various cellular processes, from the basic organization and operation of the cell to higher-order interactions in adhesion, migration, metastasis, division and immune cell interactions in different model organisms. This unique and diverse mix of research interests created a stimulating and educational meeting that will hopefully continue to be a successful meeting for years to come.

  • 18. Bergholm, Fredrik
    et al.
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Analysis of Bias in the Apparent Correlation Coefficient Between Image Pairs Corrupted by Severe Noise2010In: Journal of Mathematical Imaging and Vision, ISSN 0924-9907, E-ISSN 1573-7683, Vol. 37, no 3, p. 204-219Article in journal (Refereed)
    Abstract [en]

    The correlation coefficient r is a measure of similarity used to compare regions of interest in image pairs. In fluorescence microscopy there is a basic tradeoff between the degree of image noise and the frequency with which images can be acquired and therefore the ability to follow dynamic events. The correlation coefficient r is commonly used in fluorescence microscopy for colocalization measurements, when the relative distributions of two fluorophores are of interest. Unfortunately, r is known to be biased understating the true correlation when noise is present. A better measure of correlation is needed. This article analyses the expected value of r and comes up with a procedure for evaluating the bias of r, expected value formulas. A Taylor series of so-called invariant factors is analyzed in detail. These formulas indicate ways to correct r and thereby obtain a corrected value free from the influence of noise that is on average accurate (unbiased). One possible correction is the attenuated corrected correlation coefficient R, introduced heuristically by Spearman (in Am. J. Psychol. 15:72-101, 1904). An ideal correction formula in terms of expected values is derived. For large samples R tends towards the ideal correction formula and the true noise-free correlation. Correlation measurements using simulation based on the types of noise found in fluorescence microscopy images illustrate both the power of the method and the variance of R. We conclude that the correction formula is valid and is particularly useful for making correct analyses from very noisy datasets.

  • 19.
    Bergqvist, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The role of nuclear membrane proteins in differentiation and chromatin organization2016Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The nuclear envelope, consisting of an outer and an inner nuclear membrane, surrounds the genomic material. The genomic material (chromatin) is highly structured with (transcriptionally inactive) heterochromatin mostly found in the nuclear periphery and (transcriptionally active) euchromatin mostly found in the nuclear interior. Underlying the nuclear envelope is the nuclear lamina that consists of lamin proteins and nuclear envelope transmembrane proteins (NETs), which organize chromatin in the nuclear periphery. There are several hundred uncharacterized tissue-specific NETs, with only a few linked to cellular differentiation. Induced pluripotent stem cells (iPSCs) enable studies of early differentiation and are a promising tool for cell replacement therapies.

    In this licentiate thesis, we have focused on investigating the role of the inner nuclear membrane protein Samp1 in chromatin organization and cell differentiation. Overexpression of Samp1 induced a fast differentiation of iPSCs, suggesting that Samp1 may be involved in the differentiation process. We have also developed a novel image analysis method to be able to monitor chromatin organization in live cells. Depletion of Samp1 affected chromatin distribution and resulted in increased formation of peripheral heterochromatin, contradictory to what is expected of other characterized NETs. It is possible that Samp1 might have a role in both differentiation and chromatin organization and that future studies might link these two processes together.

  • 20.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Beckman, Marie
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Maxell, Danuta
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sousa, Paulo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Monitoring of the epigenetic state in live cellsManuscript (preprint) (Other academic)
  • 21.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gudise, Santhosh
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells2017In: Stem Cell Research, ISSN 1873-5061, E-ISSN 1876-7753, Vol. 23, p. 33-38Article in journal (Refereed)
    Abstract [en]

    The ability of iPSCs (induced pluripotent stem cells) to generate any cell type in the body makes them valuable tools for cell replacement therapies. However, differentiation of iPSCs can be demanding, slowand variable. During differentiation chromatin is re-organized and silent dense heterochromatin becomes tethered to the nuclear periphery by processes involving the nuclear lamina and proteins of the INM(inner nuclearmembrane). The INM protein, Samp1 (Spindle AssociatedMembrane Protein 1) interacts with Lamin A/C and the INMprotein Emerin, which has a chromatin binding LEM(Lap2-Emerin-Man1)-domain. In this paperweinvestigate if Samp1 can play a role in the differentiation of iPSCs. Samp1 levels increased as differentiating iPSCs started to express Lamin A/C. Interestingly, even under pluripotent culturing conditions, ectopic expression of Samp1 induced a rapid differentiation of iPSCs, ofwhich some expressed the neuronal marker beta III-tubulin already after 6 days. This suggests that Samp1 is involved in early differentiation of iPSCs and could potentially be explored as a tool to promote progression of the differentiation process.

  • 22.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Santosh, Gudise
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cellsManuscript (preprint) (Other academic)
  • 23.
    Björck, Pia
    Stockholm University, Faculty of Science.
    B lymphocyte adhesion and activation1994Doctoral thesis, comprehensive summary (Other academic)
  • 24.
    Björkegren Sjögren, Camilla
    Stockholm University.
    Profilin in cell motility and signal transduction studies using site-specific mutagenesis1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    When a cell is stimulated by signalling molecules such as growth factors, one immediate response is an increased motile activity of the cell. Membrane lamella and spikes appear at the cell surface and perform undulating movements. This is caused by the activation and reorganisation of the highly dynamic microfilament system, present beneath the cell membrane. The major component of the microfilament system is actin, a protein that can polymerise into filaments -microfilaments-, and several actin-binding proteins which act in concert to organise actin into different supramolecular arrangements. To understand how a signal is transmitted form the exterior of the cell to the microfilament system, it is important to reveal how the activities of different actin-binding proteins are regulated.

    This thesis focus on the actin-binding protein profilin, which binds actin monomers and regulates actin polymerisation. Profilin also interacts with polyphosphoinositides, which are important signalling molecules, and this association appears to regulate both the function of profilin and the turnover of polyphosphoinositides. Furthermore, profilin binds poly(L-proline) and proline-rich proteins. The latter interaction seems to be involved in the positioning of profilin within a cell, and may also be important for signal transduction-related processes. In the current investigation site-directed mutagenesis was combined with biochemical methods to further investigate the function of profilin.

    The replacement of two amino acid residues located in a hydrophobic patch on the surface of the profilin molecule was shown to abolish the interaction with poly(L-proline), and enabled the localisation of the poly(L-proline) binding-site to this part of profilin. A new purification method was developed for these profilin mutants and further biochemical characterisation showed that the two mutations also decreased the affinity of profilin for actin. As the poly(L-proline)-binding site is separated from the actin-binding surface of profilin this showed that these amino acid replacements in the poly(L-proline)-binding site also introduced long-range alterations in the profilin molecule.

    Profilin was shown to be phosphorylated on a serine and a tyrosine residue by an epidermal growth factor receptor complex isolated from stimulated cells. The phosphorylated residues were located to the poly(L-proline) binding site of profilin, and the phosphorylation interfered with the binding of profilin to poly(L-proline). This suggests that this phosphorylation regulate interactions between profilin and proline-rich proteins.

    The simultaneous deletion of two amino acid residues adjacent to the actin-binding site of profilin was shown to reduce the affinity of profilin for actin, without affecting the interaction of profilin with other ligands. The effect of this mutant profilin on the organisation of the microfilament system in living cells was compared to that of wild type profilin in microinjection experiments. While wild type profilin caused disruption of actin bundles within the cell, the profilin mutant had no apparent effect, showing that the derangement of the actin bundles by profilin is dependent on the stability of the profilin:actin complex.

  • 25. Boban, Mirta
    et al.
    Pantazopoulou, Marina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Schick, Anna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Foisner, Roland
    A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation2014In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 127, no 16, p. 3603-3613Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of similar to 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.

  • 26.
    Bromstrup, Torben
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Trudell, James R.
    Harris, R. Adron
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Inhibition versus Potentiation of Ligand-Gated Ion Channels Can Be Altered by a Single Mutation that Moves Ligands between Intra- and Intersubunit Sites2013In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, no 8, p. 1307-1316Article in journal (Refereed)
    Abstract [en]

    Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), 1240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.

  • 27. Brunnström, Åsa
    et al.
    Tryselius, Ylva
    Feltenmark, Stina
    Andersson, Erik
    Leksell, Helene
    James, Anna
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Dahlén, Barbro
    Claesson, Hans-Erik
    On the biosynthesis of 15-HETE and eoxin C-4 by human airway epithelial cells2015In: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 121, p. 83-90Article in journal (Refereed)
    Abstract [en]

    Several lines of evidence indicate that 15-lipoxygenase type 1 (15-LO-1) plays a pathophysiological role in asthma. The aim for this study was to investigate the 15-LO-1 expression and activity in primary human airway epithelial cells cultivated on micro-porous filters at air liquid interface. Incubation of human airway epithelial cells with arachidonic acid led to the formation of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and exposing the cells to bacteria or physical injury markedly increased their production of 15-HETE. The cells were also found to convert arachidonic acid to eoxin C-4 (EXC4). Subcellular fractionation revealed that the conversion of EXA(4) to EXC4 was catalyzed by a soluble glutathione transferase (GST). The GST P1-1 enzyme was found to possess the highest activity of the investigated soluble GSTs. Following IL-4 treatment of airway epithelial cells, microarray analysis confirmed high expression of 15-LO-1 and GST P1-1, and immunohistochemical staining of bronchial biopsies revealed co-localization of 15-LO-1 and GST P1-1 in airway epithelial cells. These results indicate that respiratory infection and cell injury may activate the 15-LO pathway in airway epithelial cells. Furthermore, we also demonstrate that airway epithelial cells have the capacity to produce EXC4.

  • 28.
    Böhm, Stefanie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Non-protein-coding RNA: Transcription and regulation of ribosomal RNA2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized.

    In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network.

    Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions.

  • 29.
    Caracciolo, Barbara
    et al.
    Stockholm University, Faculty of Social Sciences, Stress Research Institute. Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Xu, Weili
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Collins, Stephen
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Fratiglioni, Laura
    Stockholm University, Faculty of Social Sciences, Aging Research Center (ARC), (together with KI).
    Cognitive decline, dietary factors and gut-brain interactions2014In: Mechanisms of Ageing and Development, ISSN 0047-6374, E-ISSN 1872-6216, Vol. 136, p. 59-69Article in journal (Refereed)
    Abstract [en]

    Cognitive decline in elderly people often derives from the interaction between aging-related changes and age-related diseases and covers a large spectrum of clinical manifestations, from intact cognition through mild cognitive impairment and dementia. Epidemiological evidence supports the hypothesis that modifiable lifestyle-related factors are associated with cognitive decline, opening new avenues for prevention. Diet in particular has become the object of intense research in relation to cognitive aging and neurodegenerative disease. We reviewed the most recent findings in this rapidly expanding field. Some nutrients, such as vitamins and fatty acids, have been studied longer than others, but strong scientific evidence of an association is lacking even for these compounds. Specific dietary patterns, like the Mediterranean diet, may be more beneficial than a high consumption of single nutrients or specific food items. A strong link between vascular risk factors and dementia has been shown, and the association of diet with several vascular and metabolic diseases is well known. Other plausible mechanisms underlying the relationship between diet and cognitive decline, such as inflammation and oxidative stress, have been established. In addition to the traditional etiological pathways, new hypotheses, such as the role of the intestinal microbiome in cognitive function, have been suggested and warrant further investigation.

  • 30.
    Carlenor, Elisabeth
    Stockholm University.
    Mitochondrial nicotinamide nucleotide transhydrogenase: studies of the mammalian and plant enzymes with special reference to the beef heart and potato transhydrogenases1990Doctoral thesis, comprehensive summary (Other academic)
  • 31.
    Carlsson, Mikael A.
    et al.
    Stockholm University, Faculty of Science, Department of Zoology.
    Enell, Lina E.
    Stockholm University, Faculty of Science, Department of Zoology.
    Nässel, Dick R.
    Stockholm University, Faculty of Science, Department of Zoology.
    Distribution of short neuropeptide F and its receptor in neuronal circuits related to feeding in larval Drosophila2013In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 353, no 3, p. 511-523Article in journal (Refereed)
    Abstract [en]

    Four forms of short neuropeptide F (sNPF1-4), derived from the gene snpf, have been identified in Drosophila and are known to act on a single G-protein-coupled receptor (sNPFR). Several functions have been suggested for sNPFs in Drosophila, including the regulation of feeding and growth in larvae, the control of insulin signalling and the modulation of neuronal circuits in adult flies. Furthermore, sNPF has been shown to act as a nutritional state-dependent neuromodulator in the olfactory system. The role of sNPF in the larval nervous system is less well known. To analyse sites of action of sNPF in the larva, we mapped the distribution of sNPF- and sNPFR-expressing neurons. In particular, we studied circuits associated with chemosensory inputs and systems involved in the regulation of feeding, including neurosecretory cell systems and the hypocerebral ganglion. We employed a combination of immunocytochemistry and enhancer trap and promoter Gal4 lines to drive green fluorescent protein. We found a good match between the distribution of the receptor and its ligand. However, several differences between the larval and adult systems were observed. Thus, neither sNPF nor its receptor was found in the olfactory (or other sensory) systems in the larva and cells producing insulin-like peptides did not co-express sNPFR, as opposed to results from adults. Moreover, sNPF was expressed in a subpopulation of Hugin cells (second-order gustatory neurons) only in adult flies. We propose that the differences in sNPF signalling between the developmental stages is explained by differences in their feeding behaviour.

  • 32. Carmona-Gutierrez, Didac
    et al.
    Bauer, Maria Anna
    Zimmermann, Andreas
    Aguilera, Andres
    Austriaco, Nicanor
    Ayscough, Kathryn
    Balzan, Rena
    Bar-Nun, Shoshana
    Barrientos, Antonio
    Belenky, Peter
    Blondel, Marc
    Braun, Ralf J.
    Breitenbach, Michael
    Burhans, William C.
    Büttner, Sabrina
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Graz, Austria.
    Cavalieri, Duccio
    Chang, Michael
    Cooper, Katrina F.
    Corte-Real, Manuela
    Costa, Vitor
    Cullin, Christophe
    Dawes, Ian
    Dengjel, Jorn
    Dickman, Martin B.
    Eisenberg, Tobias
    Fahrenkrog, Birthe
    Fasel, Nicolas
    Frohlich, Kai-Uwe
    Gargouri, Ali
    Giannattasio, Sergio
    Goffrini, Paola
    Gourlay, Campbell W.
    Grant, Chris M.
    Greenwood, Michael T.
    Guaragnella, Nicoletta
    Heger, Thomas
    Heinisch, Juergen
    Herker, Eva
    Herrmann, Johannes M.
    Hofer, Sebastian
    Jimenez-Ruiz, Antonio
    Jungwirth, Helmut
    Kainz, Katharina
    Kontoyiannis, Dimitrios P.
    Ludovico, Paula
    Manon, Stephen
    Martegani, Enzo
    Mazzoni, Cristina
    Megeney, Lynn A.
    Meisinger, Chris
    Nielsen, Jens
    Nystrom, Thomas
    Osiewacz, Heinz D.
    Outeiro, Tiago F.
    Park, Hay-Oak
    Pendl, Tobias
    Petranovic, Dina
    Picot, Stephane
    Polcic, Peter
    Powers, Ted
    Ramsdale, Mark
    Rinnerthaler, Mark
    Rockenfeller, Patrick
    Ruckenstuhl, Christoph
    Schaffrath, Raffael
    Segovia, Maria
    Severin, Fedor F.
    Sharon, Amir
    Sigrist, Stephan J.
    Sommer-Ruck, Cornelia
    Sousa, Maria Joao
    Thevelein, Johan M.
    Thevissen, Karin
    Titorenko, Vladimir
    Toledano, Michel B.
    Tuite, Mick
    Voegtle, F. -Nora
    Westermann, Benedikt
    Winderickx, Joris
    Wissing, Silke
    Woelfl, Stefan
    Zhang, Zhaojie J.
    Zhao, Richard Y.
    Zhou, Bing
    Galluzzi, Lorenzo
    Kroemer, Guido
    Madeo, Frank
    Guidelines and recommendations on yeast cell death nomenclature2018In: Microbial cell, E-ISSN 2311-2638, Vol. 5, no 1, p. 4-31Article, review/survey (Refereed)
    Abstract [en]

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.

  • 33.
    Cavellán, Erica
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    Chromatin remodelling in Pol I and III transcription2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Compaction of chromosomes in the eukaryotic cell is due to interactions between DNA and proteins and interactions between proteins. These two types of interaction form a dynamic structure, known as "chromatin". The condensation of chromatin must be carefully regulated, since the structure is an obstacle for factors that need access to the DNA. An extensive range of components, one group of which is the ATP-dependent chromatin remodel-ling complexes, controls the accessibility of DNA. These complexes have been studied in a variety of eukaryotic systems, and their functions in major events in the cell, such as replication, DNA-repair and transcription have been established, as have their roles in the assembly and maintenance of chromatin. All of the complexes contain a highly conserved ATPase, which belongs to the SWI2/SNF2 family of proteins, one group of which is known as the ISWI proteins. There are two forms of ISWI in human, known as "SNF2h" and "SNF2l".

    We have identified a human SNF2h-assembly, B-WICH, that consists of SNF2h, William’s syndrome transcription factor (WSTF), nuclear myosin (NM1), and a number of additional nuclear proteins including the Myb-binding protein 1a (Myb bp1a), SF3b155/SAP155, the RNA helicase II/Guα, the proto-oncogene Dek, and the Cockayne Syndrome protein B (CSB). The 45S rRNA, 5S rRNA and 7SL RNA are all parts of the B-WICH assembly. The formation of B-WICH depends on active transcription, and is implicated in the regulation of both RNA transcription by both pol I and pol III. The B-WICH provides a link between RNA and the chromatin structure.

  • 34.
    Chai, Zhen
    Stockholm University, Faculty of Science.
    Fever: the role of IL-1, IL-6, CRF and glucocorticoids1996Doctoral thesis, comprehensive summary (Other academic)
  • 35.
    Cheng, Lei
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Brzozowska, Beata
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Warsaw, Poland.
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lundholm, Lovisa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lisowska, Halina
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Jan Kochanowski University, Poland.
    Comet assay reveals an interaction of DNA lesions and impairment of DNA repair in peripheral blood lymphocytes simultaneously exposed to alpha particles and X-raysManuscript (preprint) (Other academic)
    Abstract [en]

    The biological effectiveness of ionising radiation is related to the ionisation density which is defined by the linear energy transfer LET. Radiation quality factors are applied to calculate the equivalent dose in the field of radiation protection and the biologically effective dose in the field of radiotherapy. Additivity is assumed in exposure scenarios where radiations of different qualities are mixed. We have carried out a series of studies on the cytogenetic effect of exposing human peripheral blood lymphocytes to a mixed beam of the high LET alpha radiation and low LET X-rays and could demonstrate that both radiations interact in producing more chromosomal aberrations than expected based on additivity. The aim of the present investigation was to look at the mechanism of the interaction, especially with respect to the question if it is due to an augmented level of initial damage or impaired DNA repair. The level of DNA damage and the kinetics of damage repair was quantified by the alkaline comet assay. The levels of phosphorylated, key DNA damage response (DDR) proteins were also measured by Western blotting. The results revealed that alpha particles and X-rays interact in inducing DNA damage above the level predicted by assuming additivity and that the repair of damage occurs with a delay. Moreover, the activation levels of the key DDR proteins ATM, p53 and DNA PK were highest in cells exposed to mixed beams substantiating the idea exposure to mixed beams presents a challenge to the cellular DNA damage response system. 

  • 36.
    Cheng, Lei
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lisowska, Halina
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wegierek-Ciuk, Aneta
    Stepien, Katarzyna
    Kuszewski, Tomasz
    Lankoff, Anna
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Modulation of radiation-induced cytogenetic damage in human peripheral blood lymphocytes by hypothermia2015In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 793, no SI, p. 96-100Article in journal (Refereed)
    Abstract [en]

    Purpose: Recent studies have shown that low temperature (hypothermia) at exposure can act in a radioprotective manner at the level of cytogenetic damage. The mechanisms of this phenomenon are not understood, but it was suggested to be due to hypothermia-induced perturbations of the cell cycle. The purpose of the present study was to detect whether a reduced frequency of micronuclei is observed in peripheral blood lymphocytes (PBL) irradiated at low temperature and harvested sequentially at 3 time points. Additionally, the level of apoptosis was estimated by microscopic analysis of the MN slides. Materials and methods: Experiments were carried out with blood drawn from three donors at the Stockholm University and from three donors at the Jan Kochanowski University. Prior to irradiation, blood samples were incubated for 20 mm and irradiated at the respective temperature (0 degrees C and 37 degrees C) with gamma rays. Whole blood cultures were set up, cytochalasin B was added after 44h of irradiation and the samples were harvested after 72,96 and 120 h of incubation time. Results and conclusions: The frequency of micronuclei was markedly lower in PBL harvested at 72h, 96 h and 120 h following irradiation at 0 degrees C as compared to 37 degrees C. This indicates that the temperature effect observed in peripheral blood lymphocytes after irradiation is not related to a temporary perturbation of the cell cycle. Also, it is not due to selective elimination of damaged cells by apoptosis.

  • 37.
    Dang, Li
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lisowska, Halina
    Shakeri Manesh, Sara
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Deperas-Kaminska, Marta
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. Jan Kochanowski University, Poland.
    Staaf, Elina
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Brehwens, Karl
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology. Jan Kochanowski University, Poland.
    Radioprotective effect of hypothermia on cells - a multiparametric approach to delineate the mechanisms2012In: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 88, no 7, p. 507-514Article in journal (Refereed)
    Abstract [en]

    Purpose: Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection. Materials and methods: Human lymphoblastoid TK6 cells were incubated for 20 min at 0.8 or 37 degrees C and subsequently exposed to 1 Gy of gamma- or X-rays. The influence of ataxia telangiectasia mutated (ATM)-mediated double-strand break signalling and histone deacetylase-dependent chromatin condensation was investigated using the micronucleus assay. Furthermore, the effect of hypothermia was investigated at the level of phosphorylated histone 2AX (gamma H2AX) foci, clonogenic cell survival and micronuclei in sequentially-harvested cells. Results: The radioprotective effect of hypothermia (called the temperature effect [TE]) was evident only at the level of micronuclei at a single fixation time, was not influenced by the inhibition of ATM kinase activity and completely abolished by the histone deacetylase inhibition. No TE was seen at the level of gamma H2AX foci and cell survival. Conclusions: We suggest that low temperature during irradiation can induce a temporary cell cycle shift, which could lead to a reduced micronucleus frequency. Future experiments focused on cell cycle progression are needed to confirm this hypothesis.

  • 38. Danielsson, Daniel
    et al.
    Brehwens, Karl
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Halle, Martin
    Marczyk, Michal
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Polanska, Joanna
    Munck-Wikland, Eva
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Jan Kochanowski University, Poland.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Influence of genetic background and oxidative stress response on risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer2016In: Head and Neck, ISSN 1043-3074, E-ISSN 1097-0347, Vol. 38, no 3, p. 387-393Article in journal (Refereed)
    Abstract [en]

    Background: Osteoradionecrosis (ORN) of the mandible is a severe complication of head and neck radiotherapy (RT) treatment, where the impact of individual radiosensitivity has been a suggested explanation. Methods: A cohort of patients with stage II/III ORN was compared to matched controls. Blood was collected and irradiated in vitro to study the capacity to handle radiation-induced oxidative stress. Patients were also genotyped for 8 single-nucleotide polymorphisms (SNPs) in genes involved in the oxidative stress response. Results: A difference in 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dG) levels was found between the patient cohorts (p = 0.01). The SNP rs1695 in glutathione s-transferase p1 (GSTP1) was also found to be more frequent in the patients with ORN (p = .02). Multivariate analysis of the clinical and biological factors revealed concomitant brachytherapy plus the 2 biomarkers to be significant factors which influense risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer. Conclusion: The current study indicates that oxidative stress response contributes to individual radiosensitivity and healthy tissue damage caused by RT and may be predicted by biomarker analysis.

  • 39. Danielsson, Frida
    et al.
    Skogs, Marie
    Huss, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Rexhepaj, Elton
    O'Hurley, Gillian
    Klevebring, Daniel
    Ponten, Fredrik
    Gad, Annica K. B.
    Uhlen, Mathias
    Lundberg, Emma
    Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 17, p. 6853-6858Article in journal (Refereed)
    Abstract [en]

    The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.

  • 40.
    Davis, Monica M
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Alvarez, Francisco J
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Ryman, Kicki
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Holm, Åsa A
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Ljungdahl, Per O
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Engström, Ylva
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Wild-Type Drosophila melanogaster as a Model Host to Analyze Nitrogen Source Dependent Virulence of Candida albicans2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 11, p. e27434-Article in journal (Refereed)
    Abstract [en]

    The fungal pathogen Candida albicans is a common cause of opportunistic infections in humans. We report that wild-type Drosophila melanogaster (OrR) flies are susceptible to virulent C. albicans infections and have established experimental conditions that enable OrR flies to serve as model hosts for studying C. albicans virulence. After injection into the thorax, wild-type C. albicans cells disseminate and invade tissues throughout the fly, leading to lethality. Similar to results obtained monitoring systemic infections in mice, well-characterized cph1Δ efg1Δ and csh3Δ fungal mutants exhibit attenuated virulence in flies. Using the OrR fly host model, we assessed the virulence of C. albicans strains individually lacking functional components of the SPS sensing pathway. In response to extracellular amino acids, the plasma membrane localized SPS-sensor (Ssy1, Ptr3, and Ssy5) activates two transcription factors (Stp1 and Stp2) to differentially control two distinct modes of nitrogen acquisition (host protein catabolism and amino acid uptake, respectively). Our results indicate that a functional SPS-sensor and Stp1 controlled genes required for host protein catabolism and utilization, including the major secreted aspartyl protease SAP2, are required to establish virulent infections. By contrast, Stp2, which activates genes required for amino acid uptake, is dispensable for virulence. These results indicate that nutrient availability within infected hosts directly influences C. albicans virulence.

  • 41.
    de Jong, Jasper
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dethlefsen, Olga
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Utilization of fetal and newborn serum to uncover novel regulators of subcutaneous adipocyte differentiationManuscript (preprint) (Other academic)
  • 42.
    de Jong, Jasper M. A.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Larsson, Ola
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    A stringent validation of mouse adipose tissue identity markers2015In: American Journal of Physiology. Endocrinology and Metabolism, ISSN 0193-1849, E-ISSN 1522-1555, Vol. 308, no 12, p. E1085-E1105Article in journal (Refereed)
    Abstract [en]

    The nature of brown adipose tissue in humans is presently debated: whether it is classical brown or of brite/beige nature. The dissimilar developmental origins and proposed distinct functions of the brown and brite/beige tissues make it essential to ascertain the identity of human depots with the perspective of recruiting and activating them for the treatment of obesity and type 2 diabetes. For identification of the tissues, a number of marker genes have been proposed, but the validity of the markers has not been well documented. We used established brown (interscapular), brite (inguinal), and white (epididymal) mouse adipose tissues and corresponding primary cell cultures as validators and examined the informative value of a series of suggested markers earlier used in the discussion considering the nature of human brown adipose tissue. Most of these markers unexpectedly turned out to be noninformative concerning tissue classification (Car4, Cited1, Ebf3, Eva1, Fbxo31, Fgf21, Lhx8, Hoxc8, and Hoxc9). Only Zic1 (brown), Cd137, Epsti1, Tbx1, Tmem26 (brite), and Tcf21 (white) proved to be informative in these three tissues. However, the expression of the brite markers was not maintained in cell culture. In a more extensive set of adipose depots, these validated markers provide new information about depot identity. Principal component analysis supported our single-gene conclusions. Furthermore, Zic1, Hoxc8, Hoxc9, and Tcf21 displayed anteroposterior expression patterns, indicating a relationship between anatomic localization and adipose tissue identity (and possibly function). Together, the observed expression patterns of these validated marker genes necessitates reconsideration of adipose depot identity in mice and humans.

  • 43.
    de Jong, Jasper M. A.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wouters, René T. F.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Boulet, Nathalie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Petrovic, Natasa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The β3-adrenergic receptor is dispensable for browning of adipose tissues2017In: American Journal of Physiology. Endocrinology and Metabolism, ISSN 0193-1849, E-ISSN 1522-1555, Vol. 312, no 6, p. E508-E518Article in journal (Refereed)
    Abstract [en]

    Brown and brite/beige adipocytes are attractive therapeutic targets to treat metabolic diseases. To maximally utilize their functional potential, further understanding is required about their identities and their functional differences. Recent studies with β3-adrenergic receptor knockout mice reported that brite/beige adipocytes, but not classical brown adipocytes, require the β3-adrenergic receptor for cold-induced transcriptional activation of thermogenic genes. We aimed to further characterize this requirement of the β3-adrenergic receptor as a functional distinction between classical brown and brite/beige adipocytes. However, when comparing wild-type and β3-adrenergic receptor knockout mice, we observed no differences in cold-induced thermogenic gene expression (Ucp1, Pgc1a, Dio2 and Cidea) in brown or white (brite/beige) adipose tissues. Irrespective of the duration of the cold exposure or the sex of the mice, we observed no effect of the absence of the β3-adrenergic receptor. Experiments with the β3-adrenergic receptor agonist CL-316,243 verified the functional absence of β3-adrenergic signaling in these knockout mice. The β3-adrenergic receptor knockout model in the present study was maintained on a FVB/N background, whereas earlier reports used C57BL/6 and 129Sv mice. Thus, our data imply background-dependent differences in adrenergic signaling mechanisms in response to cold exposure. Nonetheless, the present data indicate that the β3-adrenergic receptor is dispensable for cold-induced transcriptional activation in both classical brown and, as opposed to earlier studies, brite/beige cells. This should be taken into account in the increasing number of studies on the induction of browning and their extrapolation to human physiology.

  • 44.
    Dinic, Jelena
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Plasma membrane order; the role of cholesterol and links to actin filaments:  2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains.

  • 45.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Plasma membrane order in T cell signalling2009Conference paper (Other academic)
    Abstract [en]

    Plasma membrane nanodomains, referred to as lipid rafts, more ordered than the bulk membrane play an important role in T cell signalling by forming signalling platforms in activated T cells. However, the existence of lipid rafts in resting T cells is contentious. Using laurdan, a membrane probe whose peak emission wavelength depends on the lipid environment, evidence is presented for the existence of ordered nanodomains in resting T cells.

    T cell signalling can be initiated by stimulating the T cell receptor (TCR), crosslinking the lipid raft markers GM1 (sphingolipid) or glycosylphosphatidylinositol (GPI) anchored proteins. The aggregation of lipid raft components induces the same response in Jurkat T cells as the ligation of an antigen to the TCR. Changes in membrane order linked with reorganization of the plasma membrane upon Jurkat T cell activation were followed at 37°C. Fluorescent images were analyzed for generalised polarisation values - a measure of the relative abundance of liquid ordered and liquid disordered domains. TCR patching does not increase the overall membrane order suggesting that membrane domains of high order are brought together in the patches. This supports the existence of small ordered membrane domains in resting T cells that aggregate upon activation. Patching of GM1, the GPI-anchored protein CD59 and the non lipid raft marker CD45 significantly increases the overall membrane order. So does general crosslinking of membrane components with Concanavalin A. Remodelling of the actin cytoskeleton is an integral part of TCR signaling and T cell activation. Disrupting actin polymerization using latrunculin B decreases membrane order and stabilizing actin filaments with jasplakinolide increases membrane order. An increase in membrane order appears to be a general effect of plasma membrane component patching and is likely due to a global induction of actin polymerization at the plasma membrane.

  • 46.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Biverståhl, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 1, p. 298-306Article in journal (Refereed)
    Abstract [en]

    Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

  • 47.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Uppsala universitet, Institutionen för medicinsk cellbiologi.
    Actin filaments at the plasma membrane in live cells cause the formation of ordered lipid domains via phosphatidylinositol 4,5-bisphosphateIn: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137Article in journal (Refereed)
    Abstract [en]

    The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerization decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of phosphatidylinositol 4,5-bisphosphate lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, leads to the formation of ordered domains that is correlated with an increase in cell peripheral actin filaments. In membrane blebs, which are detached from the underlying actin filaments the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form where actin filaments attach to the plasma membrane via phosphatidylinositol 4,5-bisphosphate. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

  • 48.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Uppsala universitet, Institutionen för medicinsk cellbiologi .
    Riehl, Astrid
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    The T cell receptor resides in small ordered plasma membrane domains that aggregate upon T cell activationManuscript (preprint) (Other academic)
    Abstract [en]

    T cell signaling emanates from large lipid raft platforms. Whether lipid rafts form upon T cell receptor (TCR) engagement or exist in resting T cells was the focus of this study. Plasma membrane order was followed in live T cells at 37°C using laurdan to report on lipid packing. Patching of the TCR in both Jurkat and human primary CD4+ T cells resulted in higher fractions of ordered plasma domains in the patches but did not increase the overall membrane order. The TCR colocalized with actin filaments in unstimulated Jurkat T cells and this colocalization was most prominent for cells in G1 phase. Moreover, the TCR located to the nuclear envelope, in addition to the plasma membrane, in cells in S and G2/M phase. Our study suggests that the TCR resides in ordered plasma membrane domains/lipid rafts that are linked to actin filament and aggregate upon T cell activation.

  • 49.
    Domingo Prim, Judit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The exosome and the maintenance of genome integrity2016Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The RNA exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome plays a role in DNA repair. We have shown that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DNA double-strand breaks (DSBs) in Drosophila S2 cells and human HeLa cells exposed to either ionizing radiation or I-PpoI endonuclease cleavage. DIS3, the other catalytic subunit of the nuclear exosome, is also recruited to DSBs, whereas the exosome core subunit EXOSC7 is not. Depletion of different exosome subunits does not interfere with the phosphorylation of the histone variants H2Av (Drosophila) or H2AX (humans), but depletion of RRP6/EXOSC10 impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A–V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway in animal cells. Taken together, our results suggest that a 3’-5’ ribonucleolytic activity is required for efficient DNA repair. 

  • 50. Fergusson, Joannah R.
    et al.
    Smith, Kira E.
    Fleming, Vicki M.
    Rajoriya, Neil
    Newell, Evan W.
    Simmons, Ruth
    Marchi, Emanuele
    Björkander, Sophia
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Kang, Yu-Hoi
    Swadling, Leo
    Kurioka, Ayako
    Sahgal, Natasha
    Lockstone, Helen
    Baban, Dilair
    Freeman, Gordon J.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Davis, Mark M.
    Davenport, Miles P.
    Venturi, Vanessa
    Ussher, James E.
    Willberg, Christian B.
    Klenerman, Paul
    CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages2014In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 9, no 3, p. 1075-1088Article in journal (Refereed)
    Abstract [en]

    The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCR gamma delta+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage.

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