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  • 951. Söderhäll, J. Arvid
    Quinones in lipid membranes and solutions: a computational study of biomolecules2001Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 952.
    Söderqvist, Henrik
    Stockholms universitet.
    Intracellular sorting of an integral membrane protein from the nuclear pore complex1998Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 953.
    Söderström, Mats
    Stockholms universitet.
    Leukotriene C synthase: an enzyme distinct from microsomal and cytosolic glutathione transferases1990Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 954. Tanaka, Gen
    et al.
    Nakase, Ikuhiko
    Fukuda, Yasunori
    Masuda, Ryo
    Oishi, Shinya
    Shimura, Kazuya
    Kawaguchi, Yoshimasa
    Takatani-Nakase, Tomoka
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Okawa, Katsuya
    Matsuoka, Masao
    Fujii, Nobutaka
    Hatanaka, Yasumaru
    Futaki, Shiroh
    CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV2012Ingår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 19, nr 11, s. 1437-1446Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.

  • 955.
    Tarry, Michael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Skaar, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Draheim, Roger R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Production of human tetraspanin proteins in Escherichia coli2012Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 82, nr 2, s. 373-379Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tetraspanins are found in multicellular eukaryotes and are generally thought to act as scaffolding proteins, localizing multiple proteins to a specific region of the cell membrane. Activities for tetraspanins have been identified in several fundamental processes such as motility, cell adhesion, proliferation and viral entry. Tetraspanins are also key players in cancer development and progression. However, structural and biochemical information on tetraspanins is decidely limited, due in no small part to the difficulties associated with expressing eukaryotic membrane proteins. In this study, we have used GFP fusions of a library of human tetraspanin proteins to identify growth conditions for expression in Escherichia coli. Three tetraspanin-GFP proteins could be produced at high enough levels to allow subsequent purification, paving the way for future structural and biochemical studies.

  • 956.
    Teclebrhan, Habtemichael
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Cellular organization of ubiquinone biosynthesis1995Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 957. Tedesco, Sara
    et al.
    Bayat, Narges
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Gabriela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Buque, Xabier
    Aspichueta, Patricia
    Fresnedo, Olatz
    Cristoabl, Susana
    Proteomic and lipidomic analysis of primary mouse hepatocytes exposed tometal and metal oxide nanoparticles2015Ingår i: Journal of Integrated Omics, ISSN 2182-0287, Vol. 5, nr 1Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The global analysis of the cellular lipid and protein content upon exposure to metal and metal oxide nanoparticles (NPs) can provide an overview of the possible impact of exposure. Proteomic analysis has been applied to understand the nanoimpact however the relevance of the alteration on the lipidic profile has been underestimated. In our study, primary mouse hepatocytes were treated with ultra-small (US) TiO2-USNPs as well as ZnO-NPs, CuO-NPs and Ag-NPs. The protein extracts were analysed by 2D-DIGE and quantified by imaging software and the selected differentially expressed proteins were identified by nLC-ESI-MS/MS. In parallel, lipidomic analysis of the samples was performed using thin layer chromatography (TLC) and analyzed by imaging software. Our findings show an overall ranking of the nanoimpact at the cellular and molecular level: TiO2-USNPs<ZnO-NPs<Ag-NPs<CuO-NPs. CuO-NPs and Ag-NPs were cytotoxic while ZnO-NPs and CuO-NPs had oxidative capacity. TiO2-USNPs did not have oxidative capacity and were not cytotoxic.  The most common cellular impact of the exposure was the down-regulation of proteins. The proteins identified were involved in urea cycle, lipid metabolism, electron transport chain, metabolism signaling, cellular structure and we could also identify nuclear proteins. CuO-NPs exposure decreased phosphatidylethanolamine and phosphatidylinositol and caused down-regulation of electron transferring protein subunit beta. Ag-NPs exposure caused increased of total lipids and triacylglycerol and decrease of sphingomyelin. TiO2-USNPs also caused decrease of sphingomyelin as well as up-regulation of ATP synthase and electron transferring protein alfa. ZnO-NPs affected the proteome in a concentration-independent manner with down-regulation of RNA helicase.  ZnO-NPs exposure did not affect the cellular lipids. To our knowledge this work represents the first integrated proteomic and lipidomic approach to study the effect of NPs exposure to primary mouse hepatocytes in vitro.

  • 958.
    Teixeira, Pedro F.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Branca, Rui M.
    Kmiec, Beata
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A Flowchart to Analyze Protease Activity in Plant Mitochondria2015Ingår i: Plant Mitochondria: Methods and Protocols / [ed] James Whelan, Monika W. Murcha, Springer-Verlag New York, 2015, Vol. 1305, s. 123-130Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Proteases are one of the most abundant classes of enzymes and are involved in a plethora of biological processes in many cellular compartments, including the mitochondria. To understand the role of proteases is essential to determine their substrate repertoire, preferably in an in vivo setting. In this chapter we describe general guidelines to analyze protease activity using several strategies, from in-gel analysis to mass spectrometry mapping of the cleavage site(s) and fluorogenic probes that can easily be used in vivo. To exemplify this flowchart, we used the recently characterized organellar oligopeptidase of Arabidopsis (Arabidopsis thaliana), an enzyme that takes part in degradation of short peptides within mitochondria and chloroplasts.

  • 959.
    Teixeira, Pedro F.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kmiec, Beata
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Branca, Rui M. M.
    Murcha, Monika W.
    Byzia, Anna
    Ivanova, Aneta
    Whelan, James
    Drag, Marcin
    Lehtio, Janne
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A multi-step peptidolytic cascade for amino acid recovery in chloroplasts2017Ingår i: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 13, nr 1, s. 15-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Plastids (including chloroplasts) are subcellular sites for a plethora of proteolytic reactions, required in functions ranging from protein biogenesis to quality control. Here we show that peptides generated from pre-protein maturation within chloroplasts of Arabidopsis thaliana are degraded to amino acids by a multi-step peptidolytic cascade consisting of oligopeptidases and aminopeptidases, effectively allowing the recovery of single amino acids within these organelles.

  • 960.
    Teixeira, Pedro Filipe
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    PII proteins as global regulators of bacterial nitrogen metabolism2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Nitrogen is an essential element to sustain life, being a component of most biological macromolecules. In spite of the abundance of gaseous N2, the availability of nitrogen compounds that can be readily used by most microorganisms is scarce and its production energetically demanding. Due to the central importance of nitrogen metabolism, most microorganisms evolved elaborate mechanisms to ensure efficient regulation, balancing substrate availability, product formation and energy expenditure.

    In most bacteria, many archaea and some plants, the different aspects of nitrogen metabolism are coordinated by members of the PII family of signal transduction proteins, acting as fundamental molecular messengers controlling several cellular processes. In proteobacteria, including the nitrogen fixing organism Rhodospirillum rubrum, these proteins are involved in regulation at different levels: they regulate gene expression, modulating the activity of several transcription factors; they control the flux through the ammonium transport protein (AmtB); they influence the activity of key metabolic enzymes, e.g. glutamine synthetase (GS) and nitrogenase. The signal sensing and integration by these proteins is achieved in two different yet interdependent strategies: allosteric regulation (by the binding of metabolites like ATP, ADP, 2-oxoglutarate) and reversible post-translational modification. Signal integration likely results in different conformations of the proteins, influencing the direct protein-protein interaction with the cellular targets.

    In the present work, using R. rubrum as a model organism, we have studied some aspects of the biochemistry of PII proteins in terms of regulatory interactions with the ammonium transport protein AmtB1 and the adenylyltransferase GlnE (involved in GS regulation). Additionally, we have investigated the post-translational modification of PII proteins, showing for the first time in vivo in addition in vitro selectivity in the modification of different PII proteins.

    Our results contributed to elucidate several new aspects in the regulation by PII proteins and also strengthened the idea that these proteins act as global regulators in the context of bacterial nitrogen metabolism.

  • 961.
    Teixeira, Pedro Filipe
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Processing peptidases in mitochondria and chloroplasts2013Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1833, nr 2, s. 360-370Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Most of the mitochondrial and chloroplastic proteins are nuclear encoded and synthesized in the cytosol as precursor proteins with N-terminal extensions called targeting peptides. Targeting peptides function as organellar import signals, they are recognized by the import receptors and route precursors through the protein translocons across the organellar membranes. After the fulfilled function, targeting peptides are proteolytically cleaved off inside the organelles by different processing peptidases. The processing of mitochondrial precursors is catalyzed in the matrix by the Mitochondrial Processing Peptidase, MPP, the Mitochondrial Intermediate Peptidase, MIP (recently called Octapeptidyl aminopeptidase 1, Oct1) and the Intermediate cleaving peptidase of 55 kDa, Icp55. Furthermore, different inner membrane peptidases (Inner Membrane Proteases, IMPs, Atp23, rhomboids and AAA proteases) catalyze additional processing functions, resulting in intra-mitochondrial sorting of proteins, the targeting to the intermembrane space or in the assembly of proteins into inner membrane complexes. Chloroplast targeting peptides are cleaved off in the stroma by the Stromal Processing Peptidase, SPP. If the protein is further translocated to the thylakoid lumen, an additional thylakoid-transfer sequence is removed by the Thylakoidal Processing Peptidase, TPP. Proper function of the D1 protein of Photosystem II reaction center requires its C-terminal processing by Carboxyterminal processing protease, CtpA. Both in mitochondria and in chloroplasts, the cleaved targeting peptides are finally degraded by the Presequence Protease, PreP. The organellar proteases involved in precursor processing and targeting peptide degradation constitute themselves a quality control system ensuring the correct maturation and localization of proteins as well as assembly of protein complexes, contributing to sustenance of organelle functions. Dysfunctions of several mitochondrial processing proteases have been shown to be associated with human diseases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  • 962.
    Teixeira, Pedro Filipe
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pinho, Catarina Moreira
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Branca, Rui M.
    Lehtio, Janne
    Levine, Rodney L.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    In vitro oxidative inactivation of human presequence protease (hPreP)2012Ingår i: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 53, nr 11, s. 2188-2195Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mitochondrial peptidasome called presequence protease (Prep) is responsible for the degradation of presequences and other unstructured peptides including the amyloid-beta, peptide, whose accumulation may have deleterious effects on mitochondrial function. Recent studies showed that PreP activity is reduced in Alzheimer disease (AD) patients and AD mouse models compared to controls, which correlated with an enhanced reactive oxygen species production in mitochondria. In this study, we have investigated the effects of a biologically relevant oxidant, hydrogen peroxide (H2O2), on the activity of recombinant human PreP (hPreP). H2O2 inhibited hPreP activity in a concentration-dependent manner, resulting in oxidation of amino acid residues (detected by carbonylation) and lowered protein stability. Substitution of the evolutionarily conserved methionine 206 for leucine resulted in increased sensitivity of hPreP to oxidation, indicating a possible protective role of M2O6 as internal antioxidant. The activity of hPreP oxidized at low concentrations of H2O2 could be restored by methionine sulfoxide reductase A (MsrA), an enzyme that localizes to the mitochondrial matrix, suggesting that hPreP constitutes a substrate for MsrA. In summary, our in vitro results suggest a possible redox control of hPreP in the mitochondrial matrix and support the protective role of the conserved methionine 206 residue as an internal antioxidant.

  • 963.
    Teixeira, Pedro Filipe
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Selao, Tiago Toscano
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Henriksson, Veronika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wang, He
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Norén, Agneta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordlund, Stefan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Diazotrophic growth of Rhodospirillum rubrum with 2-oxoglutarate as sole carbon source affects the regulation of nitrogen metabolism as well as the soluble proteome2010Ingår i: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 161, nr 8, s. 651-659Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    2-Oxoglutarate plays a central role as a signal in the regulation of nitrogen metabolism in the phototrophic diazotroph Rhodospirillum rubrum. In order to further study the role of this metabolite, we have constructed an R. rubrum strain that has the capacity to grow on 2-oxoglutarate as sole carbon source, in contrast to wild-type R. rubrum. This strain has the same growth characteristics as wild-type with malate as carbon source, but showed clear metabolic differences when 2-oxoglutarate was used. Among other things, the regulation of nitrogen metabolism is altered, which can be related to different modification profiles of the regulatory PII proteins.

  • 964.
    Teixeira, Pedro
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wang, He
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Stefan, Nordlund
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influnced by the nitrogen source used during growth2010Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 192, nr 5, s. 1463-1466Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and PII modification is dependent on the nitrogen source used for growth, N2 or glutamate, although both lead to nitrogenase derepression.

  • 965.
    ter Beek, Josy
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Krause, Nils
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lachmann, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 42, s. 30626-30635Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

  • 966. Thuy, Tran Thi
    et al.
    Thorsen, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Glycosylation Profiling of Therapeutic Antibodies in Serum Samples Using a Microfluidic CD Platform and MALDI-MS2013Ingår i: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 24, nr 7, s. 1053-1063Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose, and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper, a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic compact-disc (CD) platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified, and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme alpha 1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and, simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel, and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.

  • 967.
    Tjärnberg, Andreas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nordling, Torbjörn E. M.
    Studham, Matthew
    Nelander, Sven
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Swedish eScience Research Center, Sweden.
    Avoiding pitfalls in L-1-regularised inference of gene networks2015Ingår i: Molecular Biosystems, ISSN 1742-206X, E-ISSN 1742-2051, Vol. 11, nr 1, s. 287-296Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Statistical regularisation methods such as LASSO and related L-1 regularised regression methods are commonly used to construct models of gene regulatory networks. Although they can theoretically infer the correct network structure, they have been shown in practice to make errors, i.e. leave out existing links and include non-existing links. We show that L-1 regularisation methods typically produce a poor network model when the analysed data are ill-conditioned, i.e. the gene expression data matrix has a high condition number, even if it contains enough information for correct network inference. However, the correct structure of network models can be obtained for informative data, data with such a signal to noise ratio that existing links can be proven to exist, when these methods fail, by using least-squares regression and setting small parameters to zero, or by using robust network inference, a recent method taking the intersection of all non-rejectable models. Since available experimental data sets are generally ill-conditioned, we recommend to check the condition number of the data matrix to avoid this pitfall of L-1 regularised inference, and to also consider alternative methods.

  • 968.
    Tjärnberg, Andreas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nordling, Torbjörn E. M.
    Studham, Matthew
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Optimal Sparsity Criteria for Network Inference2013Ingår i: Journal of Computational Biology, ISSN 1066-5277, E-ISSN 1557-8666, Vol. 20, nr 5, s. 398-408Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gene regulatory network inference (that is, determination of the regulatory interactions between a set of genes) provides mechanistic insights of central importance to research in systems biology. Most contemporary network inference methods rely on a sparsity/regularization coefficient, which we call zeta (zeta), to determine the degree of sparsity of the network estimates, that is, the total number of links between the nodes. However, they offer little or no advice on how to select this sparsity coefficient, in particular, for biological data with few samples. We show that an empty network is more accurate than estimates obtained for a poor choice of zeta. In order to avoid such poor choices, we propose a method for optimization of zeta, which maximizes the accuracy of the inferred network for any sparsity-dependent inference method and data set. Our procedure is based on leave-one-out cross-optimization and selection of the zeta value that minimizes the prediction error. We also illustrate the adverse effects of noise, few samples, and uninformative experiments on network inference as well as our method for optimization of zeta. We demonstrate that our zeta optimization method for two widely used inference algorithms-Glmnet and NIR-gives accurate and informative estimates of the network structure, given that the data is informative enough.

  • 969.
    Tjärnlund, Anna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut, Avdelningen för immunologi.
    Rodríguez, Ariane
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Cardona, P J
    Guirado, E
    Ivanyi, J
    Singh, M
    Marsh, P.D.
    Williams, A
    Troye-Blomberg, M
    Fernandez, Carmen
    Polymeric Ig receptor knockout mice are more susceptible to mycobacteria infection in the respiratory tract2006Ingår i: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 18, nr 5, s. 807-816Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    It is generally accepted that cellular, and not humoral immunity, plays the crucial role in defense against intracellular bacteria. However, accumulating data indicate the importance of humoral immunity for the defense against a number of intracellular bacteria, including mycobacteria. We have investigated the role of secretory IgA, the main isotype found in mucosal tissues, in protection against mycobacterial infection, using polymeric IgR (pIgR)-deficient mice. Characterization of the humoral response induced after intra-nasal immunizations with the mycobacterial antigen PstS-1 revealed a loss of antigen-specific IgA response in saliva from the knockout mice. IgA level in the bronchoalveolar lavage of knockout mice was similar to wild-type level, although the IgA antibodies must have reached the lumen by other means than pIgR-mediated transport. Infection with Mycobacterium bovis bacillus Calmette–Guérin (BCG) demonstrated that the immunized pIgR−/− mice were more susceptible to BCG infection than immunized wild-type mice, based on higher bacterial loads in the lungs. This was accompanied by a reduced production of both IFN-γ and tumor necrosis factor-alpha (TNF-α) in the lungs. Additionally, the pIgR−/− mice displayed reduced natural resistance to mycobacterial infection proved by significantly higher bacterial growth in their lungs compared with wild-type mice after infection with virulent Mycobacterium tuberculosis. The knockout mice appeared to have a delayed mycobacteria-induced immune response with reduced expression of protective mediators, such as IFN-γ, TNF-α, inducible nitric oxide synthase and regulated upon activation normal T cell sequence, during early infection. Collectively, our results show that actively secreted IgA plays a role in protection against mycobacterial infections in the respiratory tract, by blocking entrance of bacilli into the lungs, in addition to modulation of the mycobacteria-induced pro-inflammatory response.

  • 970.
    Tjörnhammar, Marie-Louise
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Cyclic guanosine 3', 5'-monophosphate: studies on synthesis and efflux from hepatocytes, liver slices and cerebellar slices and on serotonin-mediated cGMP synthesis in human platelets1985Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The present study established the existence of a carrier mediated transport of 3'5' cGMP from rat liver slices, hepatocytes and cerebellar slices in which the cellular levels of 3'5' cGMP were elevated by application of appropriate stimuli and inhibitors of the 3'5' cyclic nucleotide phosphodiesterase. The carrier is specific for 3'5' cGMP as opposed to 3'5' cAMP. The temperature dependent transport is inhibited by probenecid, a known inhibitor of anion transport with IC50 value of 0.5 itiM. Alkylating reagents increase the rate of transport implying the participation of some protein.The molecular mechanism of the 5HT-mediated 5-10 fold increase in 3'5' cGMP levels in human platelets, which unlike other neurotransmitter mediated increases in 3'5' cGMP did not depend on the presence of extracellular Ca++, was investigated. On the basis of the Na+ and Cl- dependence of cGMP response it was concluded that the uptake of 5HT is an obligatory step in stimulation of the synthesis of 3'5' cGMP. Inhibition of the 5HT-mediated increase in 3'5' cGMP by 5HT uptake blocking antidepressant drugs but not by antagonists of 5HT-type 1 or type 2 receptors corroborates this conclusion.The 5HT-mediated increase in 3'5' cGMP has been studied in platelets from depressed patients during the course of electroconvulsive treatment.

  • 971.
    Todde, Guido
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Hovmöller, Sven
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Laaksonen, Aatto
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK). University of Cagliari, Italy; Stellenbosch University, South Africa.
    Mocci, Francesca
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK). University of Cagliari, Italy.
    Glucose oxidase from Penicillium amagasakiense: Characterization of the transition state of its denaturation from molecular dynamics simulations2014Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, nr 10, s. 2353-2363Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glucose oxidase (GOx) is a flavoenzyme having applications in food and medical industries. However, GOx, as many other enzymes when extracted from the cells, has relatively short operational lifetimes. Several recent studies (both experimental and theoretical), carried out on small proteins (or small fractions of large proteins), show that a detailed knowledge of how the breakdown process starts and proceeds on molecular level could be of significant help to artificially improve the stability of fragile proteins. We have performed extended molecular dynamics (MD) simulations to study the denaturation of GOx (a protein dimer containing nearly 1200 amino acids) to identify weak points in its structure and in this way gather information to later make it more stable, for example, by mutations. A denaturation of a protein can be simulated by increasing the temperature far above physiological temperature. We have performed a series of MD simulations at different temperatures (300, 400, 500, and 600 K). The exit from the protein's native state has been successfully identified with the clustering method and supported by other methods used to analyze the simulation data. A common set of amino acids is regularly found to initiate the denaturation, suggesting a moiety where the enzyme could be strengthened by a suitable amino acid based modification.

  • 972.
    Toddo, Stephen
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Engineering membrane proteins for production and topology2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli.

    For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.

  • 973.
    Toddo, Stephen
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Söderström, Bill
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Palombo, Isolde
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Norholm, Morten H. H.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daley, Daniel O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli2012Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 21, nr 10, s. 1571-1576Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.

  • 974.
    Toft, Erica
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Glutathione-binding proteins in the ovary and implications for reproductive toxicology1997Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 975.
    Tracy, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bergqvist, Filip
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ivanova, Elena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jacobsen, Kristin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Iverfeldt, Kerstin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Exposure to the Saturated Free Fatty Acid Palmitate AltersBV-2 Microglia Inflammatory Response2013Ingår i: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, nr 3, s. 805-812Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Elevated levels of free fatty acids (FFAs) in plasma and increased incidence of chronic systemic inflammation are associated with obesity. In the brain, activated microglia are believed to play different roles during inflammation that may either be neuroprotective or promote neurodegeneration. Here, we have investigated the effects of FFAs on microglial response to inflammatory stimuli. Our results indicate that the saturated FFA palmitate on its own induces alternative activation of BV-2 microglia cells. Further, pre-exposure to palmitate changed the response of microglia to lipopolysaccharide (LPS). We show that palmitate affects the mRNA levels of the pro-inflammatory cytokines interleukin-1β and interleukin-6. The transcription factor CCAAT/enhancer-binding protein δ is also affected by pre-exposure to palmitate. Furthermore, the phagocytic activity of microglia was investigated using fluorescent beads. By analyzing the bead uptake by fluorescence-activated cell sorting, we found that palmitate alone, as well as together with LPS, stimulated the phagocytic activity of microglia. Taken together, our results suggest that exposure of microglia to increased levels of free fatty acids may alter the consequences of classical inflammatory stimuli.

  • 976.
    Tryselius, Ylva
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    A molecular characterization of selected cecropins and cysteine proteinases in Drosophila1997Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Insects that have been infected with bacteria produce antibacterial peptides. Among the most potent antibacterial peptides in Drosophila are the cecropins. This work describes the cloning and expression studies of the CecC gene, which completes the picture of the cecropin locus in Drosophila melanogaster. Inducible CecC gene expression has been detected in the fat body and potential hemocytes, by RNase protection and in situ hybridizations. The CecC gene reaches its highest levels in early pupae, where gene expression can also be detected in the hindgut. A conserved region in the promoter regions of the Drosophila cecropin genes has been identified, consisting of three elements. A suitable plasmid for P element-mediated transformation in Drosophila has been constructed, pWimm. By cloning any gene into a pWimm restriction site, it can be put under the tightly regulated CecA2 gene control. CecA2 mediated pWimm expression has been shown to be induced by bacterial infection of transgenic flies. In addition to the previously described expression in the fat body, we detected inducible expression in the female reproductive tract, and constitutive expression in the male reproductive tract.

    This thesis also describes two new, blood cell expressed, cysteine proteinases in Drosophila. Calpains are specific cytosolic endopeptidases, with a putative regulatory function. This thesis presents the cloning of a new calpain, that is expressed in hemocytes, the central nervous system and the midgut. Immunostaining experiments show that the protein is only detected in a subset of the cells that express the mRNA. Differential splicing of CalpA gene transcripts results in two different calpains, of which one lacks a calcium-binding domain. We also present the cloning of a cathepsin-like cyteine proteinase gene, CP1, from Drosophila. We show that hemocytic Drosophila mbn-2 cells express CP1 in high amounts, and the protein is localized to small granules by immunofluorescence experiments. These data indicates that the CP1 might have a role in the degradation of phagocytosed material in Drosophila.

  • 977.
    Turunen, Mikael
    Stockholms universitet.
    Uptake & regulation of ubiquinone & tocopherol2002Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 978.
    Turunen, Mikael
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schedin-Weiss, Sophia
    Defect in fatty acid esterification of dolichol in Niemann-Pick type C1 mouse livers in vivo2007Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1771, nr 4, s. 506-513Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Fatty acid esterification of dolichol and cholesterol in Niemann-Pick type C 1 mouse (Balb/c NIH npcl(-/-)) livers was investigated in response to treatment with peroxisomal proliferators. These inducers have hypolipidemic properties and influence the mevalonate pathway and the intracellular transport of the final products of this biosynthetic route. Such inducers are consequently interesting to use in a disease model with defective intracellular transport of lipids. In wild-type mice, the levels of dolichol and cholesterol found as free alcohols were not changed to any great extent upon treatment with the peroxisomal inducers dehydroepiandrosterone, clofibrate and diethylhexylphtalate. In contrast, the amounts of dolichyl esters increased whereas cholesteryl esters decreased by the same treatments. The rate of enzymatic esterification of dolichol in isolated microsomes was accordingly elevated after 5- to 7-day treatments with the efficient peroxisomal proliferators DEHP and PFOA, while the corresponding esterification of cholesterol was decreased. Upon peroxisomal induction in npcl(-/-) mice, the enzymatic dolichol esterification in vitro increased whereas the low concentration of dolichyl esters remained unchanged. The results thus demonstrate that the induction of fatty acid esterification of dolichol in vivo is impaired in npcl(-/-) mouse liver. It is therefore proposed that the intracellular lipid transport defect in npcl(-/-) mouse liver disables either dolichol and/or the fatty acid from reaching the site of esterification in vivo. This proposal was strengthened by the finding that the amount of dolichol was decreased in an isolated Golgi fraction from npcl(-/-) mice.

  • 979.
    Ullah, Shahid
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Alsberg, Tomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Vestergren, Robin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Berger, Urs
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Determination of perfluoroalkyl carboxylic, sulfonic, and phosphonic acids in food2012Ingår i: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, nr 8, s. 2193-2201Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A sensitive and accurate method was developed and validated for simultaneous analysis of perfluoroalkyl carboxylic acids, sulfonic acids, and phosphonic acids (PFPAs) at low picograms per gram concentrations in a variety of food matrices. The method employed extraction with acetonitrile/water and cleanup on a mixed-mode co-polymeric sorbent (C8 + quaternary amine) using solid-phase extraction. High-performance liquid chromatographic separation was achieved on a C18 column using a mobile phase gradient containing 5 mM 1-methyl piperidine for optimal chromatographic resolution of PFPAs. A quadrupole time-of-flight high-resolution mass spectrometer operating in negative ion mode was used as detector. Method detection limits were in the range of 0.002 to 0.02 ng g(-1) for all analytes. Sample preparation (extraction and cleanup) recoveries at a spiking level of 0.1 ng g(-1) to a baby food composite were in the range of 59 to 98 %. A strong matrix effect was observed in the analysis of PFPAs in food extracts, which was tentatively assigned to sorption of PFPAs to the injection vial in the solvent-based calibration standard. The method was successfully applied to a range of different food matrices including duplicate diet samples, vegetables, meat, and fish samples.

  • 980. Unell, Maria
    et al.
    Nordin, Karolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jernberg, Cecilia
    Stenstrom, John
    Jansson, Janet K.
    Degradation of mixtures of phenolic compounds by Arthrobacter chlorophenolicus A62008Ingår i: Biodegradation, ISSN 0923-9820, E-ISSN 1572-9729, Vol. 19, nr 4, s. 495-505Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In this study the chlorophenol-degrading actinobacterium, Arthrobacter chlorophenolicus A6, was tested for its ability to grow on mixtures of phenolic compounds. During the experiments depletion of the compounds was monitored, as were cell growth and activity. Activity assays were based on bioluminescence output from a luciferase-tagged strain. When the cells were grown on a mixture of 4-chlorophenol, 4-nitrophenol and phenol, 4-chlorophenol degradation apparently was delayed until 4-nitrophenol was almost completely depleted. Phenol was degraded more slowly than the other compounds and not until 4-nitrophenol and 4-chlorophenol were depleted, despite this being the least toxic compound of the three. A similar order of degradation was observed in non-sterile soil slurries inoculated with A. chlorophenolicus. The kinetics of degradation of the substituted phenols suggest that the preferential order of their depletion could be due to their respective pKa values and that the dissociated phenolate ions are the substrates. A mutant strain (T99), with a disrupted hydroxyquinol dioxygenase gene in the previously described 4-chlorophenol degradation gene cluster, was also studied for its ability to grow on the different phenols. The mutant strain was able to grow on phenol, but not on either of the substituted phenols, suggesting a different catabolic pathway for the degradation of phenol by this microorganism.

  • 981. Uusna, Julia
    et al.
    Langel, Kent
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    Toxicity, Immunogenicity, Uptake, and Kinetics Methods for CPPs2015Ingår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. 133-148Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have been utilized as delivery vectors for various payloads, both in vitro and in vivo. Similar issues as for any other drug delivery systems: cytotoxicity and the tendency to induce innate immune response may limit their applications in clinics. Therefore, assessment of cytotoxicity and immunogenicity is an important step toward characterization of applicability of these delivery vehicles. Studying internalization mechanisms and kinetics of CPPs provides important information for the development of novel and more efficient cellular delivery vectors. This chapter describes methods and protocols for investigation of cytotoxicity and immunogenic activities of CPPs in vitro and in vivo as well as methods for studying cellular uptake and internalization kinetics of CPPs. In the first section we describe methods for in vitro cell viability studies and ELISA assay, which allows to measure cytokine release in cell culture media and in blood serum in response to different CPP applications. This chapter also provides a protocol for assessing caspase-1 activity essential for inflammation. In the second section of this chapter, we describe a comprehensive method and protocol for determining the endocytosis mechanisms utilized in CPP uptake by using luciferin-CPP conjugates and endocytosis inhibitors.

  • 982.
    Uvell, Hanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Signaling and transcriptional regulation of antimicrobial peptide genes in Drosophila melanogaster2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In Drosophila, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of Drosophila. We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from Drosophila cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.

  • 983.
    Uzdavinys, Povilas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Establishing the molecular mechanism of sodium/proton exchangers2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na+/H+ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes.

    The goal of my thesis is to establish the molecular basis of ion exchange in Na+/H+ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology.

    In my thesis, I will first present an overview of Na+/H+ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na+/H+ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na+/H+ exchanger (NHA2) in an isolated liposome reconstitution system.

  • 984.
    Vare, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Interstrand Crosslinks - Induction and repair2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    DNA-skadande ämnen är vanligt i cancerbehandling, då snabbt växande celler, såsom cancerceller är betydligt känsligare än normala celler för DNA skador. En grupp av ämnen som vanligen används i cancerbehandling är korsbindare av DNA. Dessa ämnen kommer reagera två gånger med DNA och skapa två bindningar mitt emot varandra. DNA strängen, som består av två delar, måste kunna separeras och kopieras (replikation) på ett tillförlitligt sätt för att cellerna ska kunna dela sig och bli flera. DNA strängen måste också kunna dela sig och bli avläst rätt för att nya proteiner ska kunna bildas (transkription). När korsbindarna har bundit till DNA strängarna, hindrar detta deras separation och därigenom förhindras även avläsningen och kopieringen.  För att göra undersökningarna av DNA korsbindande ämnen ännu lite svårare, så ger korsbindare flera olika typer av skador. Dels kan det bli flera olika typer av korsbindningar, både mellan två DNA-strängar (ICL) vilket är den farligaste och mest svårreparerade typen, men det kan också ske inom samma DNA-sträng (intrastrand crosslink) eller mellan en DNA-sträng och ett protein (DNA-protein crosslink). Korsbindare kan även bilda enbindningsskador (monoaddukt), vilket innebär den bara binder en gång till DNA.

    För att cellen ska kunna överleva, så måste den reparera skadorna och ta bort korsbindningen eller monoaddukten. Hur detta sker i människor är inte helt klarlagt men det verkar som det sker i flera steg. Till att börja med klipps DNA sönder i ena strängen på båda sidorna om korsbindningen, detta gör att den kvarvarande delen av korsbindningen kan böjas bort. Därefter kommer cellen att skapa nytt DNA för att fylla mellanrummet som bildats. Cellen använder sig av den andra DNA strängen som mall för att sätta in rätt DNA baser, men i fallet med korsbindande ämnen så är även den strängen skadad och därför finns det en stor risk för att fel DNA baser sätts in och då uppstår mutationer. Nästa steg är att klippa den kvarvarande delen av korsbindningen, även denna gång skapas ett mellanrum som måste fyllas med nya baser.

    Den första artikeln i avhandlingen handlar om att försöka reda ut om det är ICLen eller monoaddukten som är orsak till olika effekter som påträffas efter behandling med korsbindande ämnen. Det vi fann var att även om det bara var från ICLs som vi kunde mäta en effekt på replikationen, så fick vi nästan lika stark effekt från monoaddukterna, som från ICL, för en av de vanligast använda markörerna (kännetecknen) för båda DNA strängarna var brutna på samma ställe (dubbelstränsbrott). Detta berodde dock inte på att även monoaddukterna skapade dubbelsträngsbrott, utan på att markören vi använde var ospecifik. Vi fann även att även om ICLs har mycket större effekt än monoaddukten på cellens överlevnad m.m., så kan man inte bortse ifrån effekten av monoaddukten och att den troligen har en betydande roll för de korsbindande ämnen som endast ger en liten del ICLs.

    I artikel två har vi utvecklat en ny metod, som gör det möjligt att mäta hur många ICLs som bildas vid en viss dos av de korsbindande ämnen vi undersöker. Vi kan även mäta hur fort ICLerna kan repareras i mänskliga celler med hjälp av metoden. Tack vare en kombination av våra mätningar och med hjälp av datorsimuleringar, kunde vi räkna ut hur många ICLs som bildades per dos för tre vanliga korsbindare. Vi kunde även visa att 50 % av ICLen har påbörjat reparationen och kommit så långt att de var bortklippta från ena stängen inom 3 timmar efter behandlingen.

    I artikel tre undersöker vi vilka proteiner som är inblandade i den tidiga delen av ICL reparationen, alltså fram till och med att celler klipper ut korsbindningen på båda sidorna om skadan i ena strängen. Här visar vi att celler som är defekta i reparationsprotein kallat XPA, har en betydligt långsammare borttagning av ICLer än vad båda normala celler och celler defekta i reparationsprotein XPE har. Vi visar även att detta inte påverkar cellens replikationshastighet, eller har någon effekt på cellens överlevnad.

    Den fjärde artikeln handlar om acetaldehyd, som bildas när alkohol förbränns i kroppen. Acetaldehyd har föreslagits bilda ICL och därför undersökte vi vilka effekter den har på cellerna. Vi visar i den här artikeln att det krävs nysyntes av DNA för att acetaldehyd ska leda till dubbelsträngsbrott. Celler kan reparera dessa dubbelsträngsbrott med hjälp av reparationssystem, som kallas homolog rekombination, men att reparationen ibland blir felaktig.

    I den femte och sista artikeln i avhandligen undersöker vi ett av de vanligast föreslagna proteinen för att sköta klippningen av DNA (ERCC1/XPF) och hur den är inblandad i reparationen av korsbindningar. Vi kan här visa att även det krosbindande ämnet mitomycin C bromsar replikationshastigheter och att ERCC1/XPF är nödvändigt för att kunna fullfölja homolog rekombination av ICLs.

  • 985.
    Vare, Daniel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Groth, Petra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Carlsson, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Johansson, Fredrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Erixon, Klaus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Jenssen, Dag
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cellsIngår i: DNA Repair, ISSN 1568-7864, E-ISSN 1568-7856Artikel i tidskrift (Refereegranskat)
  • 986.
    Vargas Möller-Hergt, Braulio
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The interactome of the yeast mitochondrial ribosome: Organization of mitochondrial post-transcriptional regulation, membrane protein insertion and quality control2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The proteins found in mitochondria originate from two different genetic systems. Most mitochondrial proteins are synthesized in the cytosol and post-translationally imported into the organelle. However, a small subset of mitochondrial proteins is encoded in an organelle-resident genome. Mitochondria contain factors responsible for replication, transcription and, most important for this thesis, synthesis of the mitochondrially encoded proteins. In the course of evolution the mitochondria specific ribosomes were extensively remodeled. The reasons for many of these adaptations are currently not well understood. For example, the mitoribosome is less stable and abundant than its bacterial counterpart. Therefore, I contributed in the development of robust biochemical tools in order to isolate and analyze the intact yeast mitoribosome and interaction partners by mass spectrometry. The results revealed a higher order organization of mitochondrial gene expression in complexes that we termed MIOREX (mitochondrial organization of gene expression). Besides the mitoribosome, MIOREX complexes contain factors involved in all steps of gene expression. This study also established many new ribosomal interaction partners, among them some proteins that were previously completely uncharacterized. In order to study these proteins, I refined the mass spectrometry approach, allowing a subunit-specific assignment of ribosomal interaction partners. The Mrx15 protein was determined by this approach as an interactor of the large subunit. I established that Mrx15 has overlapping functions with the ribosome receptor Mba1. Both proteins are necessary for mitoribosome membrane attachment and co-translational Cox2 membrane insertion. In a subsequent study I found a functional interaction of MRX15 and MBA1 with the regulators of the membrane-bound AAA proteases of the mitochondrial quality control system. Furthermore, the absence of Mrx15 leads to increased, the absence of Mba1 to decreased proteotoxic stress resistance of yeast cells. These results demonstrate an interesting connection between the mitochondrial quality control and membrane insertion machineries, suggesting an early quality control step during the biogenesis of mitochondrially encoded proteins. In addition, we could reveal a subunit-specific interaction of translational activators and client mRNAs with the mitochondrial ribosome. This organization demonstrated how cytochrome b synthesis is pre-organized by specific translational activators independently of the COB mRNA. In summary, the work in this thesis showed how the vast and diverse interactome of the yeast mitoribosome organizes and regulates mitochondrial translation. These regulation mechanisms highlighted many organelle specific features. The work presented here will serve as starting point to design future studies aimed at a better understanding on how mitochondria adapted to organize gene expression inside the organelle.

  • 987.
    Vargas Möller-Hergt, Braulio
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Carlström, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Stephan, Katharina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Imhof, Axel
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria2018Ingår i: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 29, nr 20, s. 2359-2507Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry-based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria.

  • 988. Vargas-Uribe, Mauricio
    et al.
    Rodnin, Mykola V.
    Öjemalm, Karin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Holgado, Aurora
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kyrychenko, Alexander
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Posokhov, Yevgen O.
    Makhatadze, George
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ladokhin, Alexey S.
    Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain2015Ingår i: Journal of Membrane Biology, ISSN 0022-2631, E-ISSN 1432-1424, Vol. 248, nr 3, s. 383-394Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain.

  • 989.
    Vasconcelos, Luís
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Arukuusk, Piret
    Pärnaste, Ly
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Effects of cargo molecules on membrane perturbation caused by transportan10 based cell-penetrating peptides2014Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1838, nr 12, s. 3118-3129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides with the ability to escape endosomes and reach the target are of great value as delivery vectors for different bioactive cargoes and future treatment of human diseases. We have studied two such peptides, NickFect1 and NickFect51, both originated from stearylated transportan10 (PF3). To obtain more insight into the mechanism(s) of peptide delivery and the biophysical properties of an efficient vector system, we investigated the effect of different bioactive oligonucleotide cargoes on peptide-membrane perturbation and peptide structural induction. We studied the membrane interactions of the peptides with large unilamellar vesicles and compared their effects with parent peptides transportan10 and PF3. In addition, cellular uptake and peptide-mediated oligonucleotide delivery were analyzed. Calcein leakage experiments showed that similar to transportan10, NickFect51 caused a significant degree of membrane leakage, whereas NickFect1, similar to PF3, was less membrane perturbing. The results are in agreement with previously published results indicating that NickFect51 is a more efficient endosomal escaper. However, the presence of a large cargo like plasmid DNA inhibited NickFect's membrane perturbation and cellular uptake efficiency of the peptide was reduced. We conclude that the pathway for cellular uptake of peptide complexes is cargo dependent, whereas the endosomal escape efficacy depends on peptide hydrophobicity and chemical structure. For small interfering RNA delivery, NickFect51 appears to be optimal. The biophysical signature shows that the peptide alone causes membrane perturbation, but the cargo complex does not. These two biophysical characteristics of the peptide and its cargo complex may be the signature of an efficient delivery vector system.

  • 990. Verdurmen, Wouter P. R.
    et al.
    Bovee-Geurts, Petra H.
    Wadhwani, Parvesh
    Ulrich, Anne S.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    van Kuppevelt, Toin H.
    Brock, Roland
    Preferential Uptake of L- versus D-Amino Acid Cell-Penetrating Peptides in a Cell Type-Dependent Manner2011Ingår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 18, nr 8, s. 1000-1010Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The use of protease-resistant D-peptides is a prominent strategy for overcoming proteolytic sensitivity in the use of cell-penetrating peptides (CPPs) as delivery vectors. So far, no major differences have been reported for the uptake of L- and D-peptides. Here we report that cationic L-CPPs are taken up more efficiently than their D-counterparts in MC57 fibrosarcoma and He La cells but not in Jurkat T leukemia cells. Reduced uptake of D-peptides co-occurred with persistent binding to heparan sulfates (HS) at the plasma membrane. In vitro binding studies of Land D-peptides with HS indicated similar binding affinities. Our results identify two key events in the uptake of CPPs: binding to HS chains and the initiation of internalization. Only the second event depends on the chirality of the CPP. This knowledge may be exploited for a stereochemistry-dependent preferential targeting of cells.

  • 991.
    Vereshchaga, Yana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hedin, Linnea E
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cristobal, Susana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insertion Properties of Marginally Hydrophobic Helices in the LacY Lactose Permease TransporterManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Transmembrane helices are generally believed to be recognized individually by the translocon based on theirhydrophobicity, but it has been proposed that they could also be recognized as pairs of helices. The fact thatmost transmembrane helices are individually clearly hydrophobic seems to support separate helix insertion,but there are important exceptions where the helices are only borderline hydrophilic, at least according tosequence-based prediction. Conrming these patterns and characterizing their role for insertion of helices isan important part in deciphering membrane protein insertion and folding. Here, we use a combination ofsequence bioinformatics, simplied physical modeling, and experiments to investigate whether helices in theLacY lactose permease transporter are recognized by the translocon, and if not whether helix-helix interactionsmight stabilize their insertion. From the experimentally determined biological hydrophobicity scale, ve out of thetwelve transmembrane segments of LacY are predicted to have low spontaneous insertion, which is qualitativelyconrmed in a simplied simulation model using an implicit membrane environment as well as experimentallyin vitro. For some pairs a small, but signicant, increase in insertion eciency was seen both in the simulationsand in the in vitro system. However, the overall insertion eciency is only marginally increased when pairsof borderline hydrophobic helices are co-inserted, which suggests that translocon-mediated membrane insertionpredominantly recognizes individual helices. It also seems to imply that stabilization of marginally hydrophobichelices - at least for LacY - is a collective eect in the nal folded membrane protein, rather than caused by favorable interactions and hairpin formation during insertion.

  • 992.
    Vestergren, Robin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Ullah, Shahid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Cousins, Ian T.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    Berger, Urs
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för tillämpad miljövetenskap (ITM).
    A matrix effect-free method for reliable quantification of perfluoroalkyl carboxylic acids and perfluoroalkane sulfonic acids at low parts per trillion levels in dietary samples2012Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1237, s. 64-71Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In recent exposure modeling studies diet has been identified as the dominant pathway of human exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS). However, the paucity of highly sensitive and accurate analytical data to support these studies means that their conclusions are open to question. Here a novel matrix effect-free method is described for ultra-trace analysis of perfluoroalkyl carboxylic acids and perfluoroalkane sulfonic acids in dietary samples of varied composition. The method employs ion pair extraction of the analytes into methyl tert-butyl ether and subsequent solid phase extraction clean-up on Florisil and graphitized carbon. The target compounds are separated and detected using ultra performance liquid chromatography coupled to tandem mass spectrometry. Special care was taken to avoid procedural blank contamination and potential contamination sources were elucidated. The performance of the method was validated for five different food test matrices including a duplicate diet sample. Method detection limits in the low to sub pg g(-1) range were obtained for all target analytes, which is 5-100 times more sensitive than previously reported for duplicate diet samples. Total method recoveries were consistently between 50 and 80% for all analytes in all tested food matrices and effects of co-extracted matrix constituents on ionization of the target compounds were found to be negligible. The precision of the method (defined as percentage relative standard deviation) at concentrations close to the respective method limits of quantification was <15% for all analytes. Accurate quantification at ultra-trace levels was demonstrated by laboratory control spike experiments. For the first time the presence of long-chain PFCAs in duplicate diet samples is reported. The method presented here can thus support an improved assessment of human exposure from dietary intake for a range of PFCA and PFSA homologues. Re-analysis of duplicate diet samples, which had been analyzed earlier using another analytical methodology, indicated that dietary intake of PFOA and PFOS may previously have been overestimated.

  • 993. Vicedomini, Riccardo
    et al.
    Vezzi, Francesco
    Scalabrin, Simone
    Arvestad, Lars
    Stockholms universitet, Naturvetenskapliga fakulteten, Numerisk analys och datalogi (NADA).
    Policriti, Alberto
    GAM-NGS: genomic assemblies merger for next generation sequencing2013Ingår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 14, nr Suppl.7, s. S6-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: In recent years more than 20 assemblers have been proposed to tackle the hard task of assembling NGS data. A common heuristic when assembling a genome is to use several assemblers and then select the best assembly according to some criteria. However, recent results clearly show that some assemblers lead to better statistics than others on specific regions but are outperformed on other regions or on different evaluation measures. To limit these problems we developed GAM-NGS (Genomic Assemblies Merger for Next Generation Sequencing), whose primary goal is to merge two or more assemblies in order to enhance contiguity and correctness of both. GAM-NGS does not rely on global alignment: regions of the two assemblies representing the same genomic locus (called blocks) are identified through reads' alignments and stored in a weighted graph. The merging phase is carried out with the help of this weighted graph that allows an optimal resolution of local problematic regions. Results: GAM-NGS has been tested on six different datasets and compared to other assembly reconciliation tools. The availability of a reference sequence for three of them allowed us to show how GAM-NGS is a tool able to output an improved reliable set of sequences. GAM-NGS is also a very efficient tool able to merge assemblies using substantially less computational resources than comparable tools. In order to achieve such goals, GAM-NGS avoids global alignment between contigs, making its strategy unique among other assembly reconciliation tools. Conclusions: The difficulty to obtain correct and reliable assemblies using a single assembler is forcing the introduction of new algorithms able to enhance de novo assemblies. GAM-NGS is a tool able to merge two or more assemblies in order to improve contiguity and correctness. It can be used on all NGS-based assembly projects and it shows its full potential with multi-library Illumina-based projects. With more than 20 available assemblers it is hard to select the best tool. In this context we propose a tool that improves assemblies (and, as a by-product, perhaps even assemblers) by merging them and selecting the generating that is most likely to be correct.

  • 994.
    Vilhjálmsdóttir, Jóhanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Choreography of a proton pump: Studies of charge-transfer reactions in cytochrome c oxidase2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In the last step of cellular respiration, electrons from metabolites are transferred to molecular oxygen, mediated by the enzyme complexes of the respiratory chain. Some of these enzyme complexes couple these redox reactions to formation of an electrochemical proton gradient across the cell membrane. The proton gradient is used e.g. by ATP synthase to drive synthesis of ATP. 

    The terminal enzyme complex in the respiratory chain, cytochrome c oxidase (CytcO), catalyses reduction of O2 to water. In this process it contributes to maintaining the electrochemical proton gradient by two separate mechanisms: (i) by uptake of electrons and protons from the opposite sides of the membrane (for O2 reduction to water). (ii) by proton pumping across the membrane. Protons used in the O2 reduction, as well as protons that are pumped, are taken up through two different proton-uptake pathways, the D and the K pathways. In addition, a third proton-transfer pathway has been suggested for the mitochondrial CytcOs, namely the H pathway. So far, the molecular mechanism by which CytcO pumps protons has not been determined. 

    In this work we have studied proton- and electron-transfer reactions in aa3-type CytcOs, with the aim of understanding the functional design of the proton-pumping machinery in CytcO. First, we studied structural variants of CytcO from the bacterium Rhodobacter (R.) sphaeroides, where an amino-acid at position 425, previously shown to undergo redox-induced conformational changes, was substituted. The results point to a link between redox-induced structural changes and intramolecular proton-transfer rates through the D pathway. Second, we studied the electron distribution in the “activated” oxidized (OH) state of CytcO, by using an electrostatic complex of CytcO and cytochrome c. We also investigated electron-transfer reactions linked to proton pumping in structural variants of CytcO from R. sphaeroides and the yeast Saccharomyces (S.) cerevisiae, with mutations in the proposed D and H proton-uptake pathways. The data indicate that the S. cerevisiae mitochondrial CytcO uses the D pathway for proton uptake and pumping as the R. sphaeroides CytcO. Lastly, we studied reactions linked to proton uptake and pumping in structural variants of CytcO from R. sphaeroides with alterations in both proton-uptake pathways. The data elucidated the mechanism of proton transfer and gating in CytcO.

  • 995.
    Vilhjálmsdóttir, Jóhanna
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Albertsson, Ingrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Proton transfer in uncoupled variants of cytochrome c oxidaseManuskript (preprint) (Övrigt vetenskapligt)
  • 996.
    Vink, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Phosphorylation in the thylakoid membrane - role of substrate activation2002Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Phosphorylation of specific side chains in target proteins by protein kinases is a major means of regulation of cellular processes. In the thylakoid membrane of higher plants, the redox-controlled phosphorylation of photosystem II (PSII) and light-harvesting complex II (LHCII) proteins regulates energy distribution between the two photosystems and PSII protein dynamics. The thylakoid proteins are phosphorylated by several distinct kinases that are subject to different control mechanisms. Regulation of LHCII kinase(s) activation is signalled by the redox-state of the plastoquinone pool and the cytochrome b6/f complex and modulated by the thiol redox-state of the chloroplast. Phosphorylation of PSII core-complex proteins may be achieved by PSII core associated kinase(s) that are regulated by the redox-state of the plastoquinone pool alone without involvement of the cytochrome b6/f complex. The identities of the protein kinases in the thylakoid membrane are still unknown. In this work a kinase enriched fraction obtained from a spinach thylakoid extract by perfusion chromatography is described. This fraction phosphorylates isolated LHCII and the CP43 protein of isolated PSII cores and exhibits a major protein kinase band of 64 kDa.

    This work further demonstrates that, in addition to the well-studied redox-regulation of the protein kinases in the thylakoid membrane, phosphorylation can also be modulated at the substrate level. The exposure of the phosphorylation sites of both the LHCII and the CP43 protein to the respective protein kinases is regulated by light-induced conformational changes. This so-called substrate activation increases the phosphorylation level of both proteins. However, illumination for long times and/or in high light in situ, in the intact thylakoid, induces a dramatic decrease in the phosphorylation level of the LHCII under conditions preventing phosphorylation. The implications of these light-induced conformational changes at the substrate level are discussed in relation to energy distribution between the two photosystems and the dynamics of the thylakoid membrane as a whole. Furthermore, the effect of xantophyll pigments on the light-induced conformational changes and thylakoid protein phosphorylation is presented.

  • 997.
    Vintila, Simina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Botaniska institutionen.
    Selao, Tiago
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Noren, Agneta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bergman, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Botaniska institutionen.
    El-Shehawy, Rehab
    Stockholms universitet, Naturvetenskapliga fakulteten, Botaniska institutionen.
    Characterization of nifH gene expression, modification and rearrangement in Nodularia spumigena strain AV12011Ingår i: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 77, nr 2, s. 449-459Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The annually reoccurring blooms that characterize the surface waters of the Baltic Sea are dominated by filamentous, heterocystous cyanobacteria such as Nodularia spumigena. In a previous study, we have demonstrated that N. spumigena strain AV1 differentiates heterocysts in the absence of detectable nitrogen fixation activity, an unusual physiological trait that is clearly distinct from other well-studied cyanobacteria. To further analyze the uncoupling between these two processes, we analyzed the gene expression and modification of the nitrogenase enzyme (the enzyme responsible for nitrogen fixation) in N. spumigena AV1, as well as in several other N. spumigena strains. Here, we demonstrate the occurrence of two nifH gene copies in N. spumigena strain AV1, only one of which is located in a complete nifHDK cluster and several NifH protein forms. Furthermore, we demonstrate the occurrence of a DNA rearrangement mechanism acting within the nifH gene copy located in the nifHDK cluster and present only in the strains exhibiting the previously reported uncoupling between heterocyst differentiation and nitrogen fixation processes. These data stress the existence of a distinct and complex regulatory circuit related to nitrogen fixation in this ecologically significant bloom-forming cyanobacterium.

  • 998.
    Virgin, Ivar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    On the mechanism and consequences of light-induced D₁ protein degradation in higher plants1992Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The basic source of energy for the whole biosphere is sunlight which is converted tochemical energy by the process of photosynthesis in a wide variety of organisms rangingfrom prokaryotic bacteria to eukaryotic higher plants. It is therefore surprising thatphotosynthetic activity can be reduced when plants are exposed to excessive light,especially under adverse environmental conditions. This phenomenom is denotedphotoinhibition and is considered to be responsible for a reduction of crop plantproductivity. The primary target for photoinhibition is photosystem II (PSII), a keycomponent in the photosynthetic process. During the last five years we have reached aconsiderable understanding of the photinhibition process and the subsequent repairsteps. It has been shown that light stress leads to inhibition of PSII electron transport andirreversible damage and turnover of the reaction center Di protein.

    In this thesis the photoinhibition process is studied in vitro using isolated thylakoidmembranes and highly purified PSII preparations. Detalied molecular mechanisms forthe light induced inactivation and the subsequent Di protein degradation is presented.The main findings can be summarized; (i) The Di protein is involved in the ligation ofthe manganese cluster, (ii) Several sequential events that lead to photoinactivation ofPSII electron transport and Di protein degradation can be distinguished; (iii) Strong lightleads to a fully reduced acceptor side of PSII, eventually leading to double reduction andimpairment of the primary quinone acceptor, Qa; (iv ) Perturbation of the water oxidisingreaction renders PS II highly susceptible to photoinactivation and D1 protein degradation;(v) Isolated thylakoid membranes contain a complete D i protein proteolytic machinery;(vi) The proteolytic machinery, responsible for primary cleavage of the Di protein, isan integral part of the PSII complex; (vii) Light-induced D \ protein degradation involvesa serine type protease; (viii) Di protein degradation leads to gross structural changes inPSÜ assembly including manganese release and migration of polypeptides to the stromaexposed thylakoids; (ix) Recovery from PSII damage must also involve religation andactivation of redox components as well as reassembly of protein subunits.

  • 999.
    Virkki, Minttu T.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Agrawal, Nitin
    Edsbäcker, Elin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Cristobal, Susana
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kauko, Anni
    Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core2014Ingår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, nr 7, s. 981-992Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The folding of most integral membrane proteins follows a two-step process: initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four-helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step, this intermediate is folded into a six-helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: first, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3, and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD-simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process.

  • 1000.
    Virkki, Minttu T.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Peters, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center (SeRC), Sweden.
    Nilsson, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sörensen, Therese
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cristobal, Susana
    Wallner, Björn
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center (SeRC), Sweden.
    The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix2014Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, nr 16, s. 2982-2991Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.

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