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  • 101.
    Hedlund, Sebastian
    Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Expression of B-adrenergic receptors in chicken fetuses2006Självständigt arbete på avancerad nivå (magisterexamen), 20 poäng / 30 hpStudentuppsats
    Abstract [en]

    Chicken fetuses exposed to chronic hypoxia suffer from growth retardation and induces an overall sympathetic activity, including elevation of the concentration of circulating catecholamines. Simultaneously, hypoxic fetuses display a lowered β-adrenoreceptor (βAR) density in myocardial tissue. In vertebrates, β1AR and β2AR are the most important signalling pathways for acute elevation of cardiac performance. The aim of this study was to see how chronic hypoxia affects the level of messenger RNA (mRNA) for the β1AR in the fetal chicken heart at different developmental ages. The broiler chicken is a suitable model organism for studying the progression of heart failure because the fast growth rate requires a large increase in blood perfusion at the end of fetal development. The β1AR sequence of the broiler chicken is 1587 bp and located on chromosome 6. When running a PCR for quantification of the sequence, primers for almost the whole sequence failed (1404 bp) and so did primers of 1193 bp; instead primers of 692 bp of the sequence were used and made quantification possible. Similar results were obtained from both the heart and liver of day 15 fetal chickens. The PCR product was cloned into a TOPO vector and sent for sequencing, to enable the making of a probe for a northern blot analysis of the mRNA in the fetal chicken hearts.

  • 102.
    Hedman, Johannes
    et al.
    Lunds Universitet/Lunds Tekniska Högskola.
    Ansell, Ricky
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Nordgaard, Anders
    Linköpings universitet, Institutionen för datavetenskap, Statistik. Linköpings universitet, Filosofiska fakulteten.
    A ranking index for quality assessment of forensic DNA profiles2010Ingår i: BMC Research Notes, ISSN 1756-0500, Vol. 3, nr 290Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background

    Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms.

    Results

    We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles.

    Conclusions

    The developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification.

  • 103.
    Heinrup, Rebecka
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biologi.
    Evaluation of isobutanol tolerance and gene expression in four different Saccharomyces cerevisiae strains for the development of bio-butanol production2016Självständigt arbete på grundnivå (kandidatexamen), 10,5 poäng / 16 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Today, most transportation fuels are derived from crude oil. However, fossil fuels are limited resources and contribute to climate change, and are therefore not considered as sustainable. Biofuels are highly relevant candidates for replacing fossil fuels and research has gone into butanol as a biofuel. It has a high energy density, is less hygroscopic and can be blended up to 85% with gasoline. The yeast Saccharomyces cerevisiae is considered a good host for bio- butanol production; it produces small amounts of isobutanol naturally through the Ehrlich pathway, is easy to manipulate genetically and can therefore be engineered to produce higher titres of butanol. End-product toxicity, however, is a problem that needs to be solved to make butanol production in S. cerevisiae more effective, since the organism cannot tolerate higher concentrations of butanol than 2%. Four different S. cerevisiae strains were cultivated in 1.5%, 2%, 3% and 4% isobutanol by spot tests and in liquid media to evaluate their tolerance. Gene expression was measured for genes RPN4, RTG1 and ILV2 to examine their up-regulation and relevance in butanol tolerance. S. cerevisiae strain Saflager 34/70 was determined as the most tolerant strain and was able to grow in 2% liquid isobutanol and 3% isobutanol on agar plates. A three-fold up-regulation of RPN4, a transcription factor involved in the regulation of proteasome gene expression, was observed. These results contribute to the progress of genetic engineering of butanol host organisms, which is needed to create a more effective production of butanol as a biofuel. 

  • 104.
    Hemmingsson, Lovisa
    Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Bri2 BRICHOS domain: Eukaryotic expression and importance of strictly conserved cysteine residues2017Självständigt arbete på avancerad nivå (masterexamen), 40 poäng / 60 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Alzheimer’s disease (AD), the most common form of dementia is associated with fibril formation of amyloid-ß peptides (Aß). Aß, proteolytically derived from Aß precursor protein (AßPP), is the major component of amyloid plaques in AD brains. Familial British and Danish dementias (FBD and FDD) share pathological and clinical characteristics with AD, and the underlying mechanisms are associated with amyloid formation of mutant peptides released from the Bri2 protein.

    Bri2 interacts with AßPP and its BRICHOS domain has been shown to delay Aß40 and Aß42 fibril formation and toxicity in vitro and in vivo. This makes Bri2 BRICHOS a promising anti-amyloid chaperone and a potential treatment strategy for AD. Furthermore, Bri2 BRICHOS possesses a general chaperone activity as it suppresses non-fibrillar aggregation of destabilized citrate synthase (CS). Recent findings show that Bri2 BRICHOS produced in E.coli can form different molecular weight assemblies, ranging from monomers to dimers and poly-disperse oligomers. The oligomers inhibit CS aggregation, whereas the monomers and dimers are more efficient against Aß42 fibrillation and neurotoxicity, respectively.

    The work in this thesis shows that similar Bri2 BRICHOS quaternary structures are formed in eukaryotic cells as in E.coli. Larger BRICHOS oligomers were found in cell media, derived from proteolytically processed endogenous Bri2 in SH-SY5Y cells, as well as in human embryonic kidney (HEK293) cells transfected with a Bri2 BRICHOS construct. Recombinant human Bri2 BRICHOS mutants with one or none of the two strictly conserved cysteine residues were studied. All mutant monomers become proteolytically degraded during purification, but form stable oligomers. Single Cys to Ser mutants form stable disulfide-dependent dimers that differ in ability to prevent Aß42 fibrillation, the most stable mutant (C164S) being even more efficient than the wildtype Bri2 BRICHOS dimer. This result suggests that intra or intermolecular disulfide(s) and oligomerization affect Bri2 BRICHOS stability and activity towards Aß42 fibril formation.

  • 105.
    Hemmingsson, Lovisa
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska fakulteten.
    Klasén, Johan
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska fakulteten.
    In vitro studies of Thiopurine S-Methyltransferase: Ligand binding interactions and development of a new enzymatic activity assay for TPMTwt, TPMT*6 and TPMT*82015Självständigt arbete på grundnivå (kandidatexamen), 10 poäng / 15 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity.

    In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX.

    Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.

  • 106.
    Hernandez, Luiza I.
    et al.
    Department of Internal Medicine, University of of Iowa, Iowa City, IA 52242, United States; University of of Iowa, Iowa City, IA 52242, United States.
    Flenker, Katie S.
    Department of Internal Medicine, University of of Iowa, Iowa City, IA 52242, United States; University of of Iowa, Iowa City, IA 52242, United States.
    Hernandez, Frank J
    Department of Internal Medicine, University of of Iowa, Iowa City, IA 52242, United States; University of of Iowa, Iowa City, IA 52242, United States.
    Klingelhutz, Aloysius J.
    Department of Microbiology, University of of Iowa, Iowa City, IA 52242, United States; Department of Radiation Oncology, University of of Iowa, Iowa City, IA 52242, United States; University of of Iowa, Iowa City, IA 52242, United States.
    McNamara, James O.
    Department of Internal Medicine, University of of Iowa, Iowa City, IA 52242, United States; University of of Iowa, Iowa City, IA 52242, United States.
    Giangrande, Paloma H.
    Department of Internal Medicine, University of of Iowa, Iowa City, IA 52242, United States; Department of Radiation Oncology, University of of Iowa, Iowa City, IA 52242, United States; University of of Iowa, Iowa City, IA 52242, United States.
    Methods for evaluating cell-specific, cell-internalizing RNA aptamers2013Ingår i: Pharmaceuticals, ISSN 1424-8247, E-ISSN 1424-8247, Vol. 6, nr 3, s. 295-319Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell- based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate- based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (QUSIM) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

  • 107.
    Hessling, Elin
    Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Comparing the serotonergic system in vertebrates and invertebrates2017Självständigt arbete på grundnivå (kandidatexamen), 10 poäng / 15 hpStudentuppsats (Examensarbete)
    Abstract [en]

    The serotonergic system is involved in a broad range of functions in both vertebrates and invertebrates and is highly conserved across taxa. Serotonin is an important monoamine acting in the brains of humans and animals, and has large and varying influences on many aspects of an individual’s life. For example, in humans, serotonin modulates feelings of happiness and in fruit flies, higher levels of serotonin increase aggression. In humans, an abnormal serotonergic system can result in health issues, such as depression and obsessive compulsive disorders, for which medications have been developed, including selective serotonin reuptake inhibitors (SSRI). Because the serotonin system has a large influence on human health, understanding how it functions is of great interest to researchers. Using comparative studies to explore differences in the serotonin system across taxa can provide insight into the mechanistic details of the system. To investigate if the serotonin system is comparable between vertebrates and invertebrates, a literature study with particular focus on receptors and proteins involved was performed. In addition, this report takes part in an experimental study investigating the effect of the SSRI fluoxetine in Mediterranean field crickets.  Fluoxetine reduced exploration propensity of crickets, which was reversed, compared to what was anticipated and compared to effects seen in vertebrates. The literature review suggests that serotonin receptors are quite similar, but that proteins differ more when comparing vertebrates and invertebrates. This offers a likely explanation as to why results of studies on these different groups of animals may differ.

  • 108.
    Heurtel Thuswaldner, Sophie
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Nucleotide-binding Proteins in the Plant Thylakoid Membrane2006Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex.

    In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs.

    The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.

    Delarbeten
    1. Update in nucleotide-dependent processes in plant chloroplasts
    Öppna denna publikation i ny flik eller fönster >>Update in nucleotide-dependent processes in plant chloroplasts
    2008 (Engelska)Ingår i: Current Knowledge in Plant Cell Compartments / [ed] Spetea, C.Thuswaldner, S., Kerala: Research Signpost Publisher , 2008, s. 105-149Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    Chloroplasts are photosynthetically active plastids found in all green plant cells. They have two types of membranes, the double envelope membrane surrounding the organelle and the thylakoid membrane containing the photosynthetic machinery. The envelope membrane represents the interface between the cytosol and chloroplast stroma, whereas the thylakoid membrane is the interface between the stroma and the lumenal space. This chapter attempts to give an update in nucleotide-dependent processes in plant chloroplasts. The current knowledge is that ATP is produced in the light-dependent photosynthetic reactions in the thylakoid membrane, and is used during CO2-fixation in the stroma as well as in the energy-dependent processes occurring on the thylakoid and envelope membranes. There is also increasing evidence that the thylakoid lumen is a chloroplast compartment with an unexpectedly active nucleotide metabolism, expanding its function beyond a bioenergetic perspective. Here we will discuss three distinct classes of chloroplast nucleotide-binding proteins: (i) transporters involved in ATP synthesis, translocation and utilization; (ii) nucleoside diphosphate kinases, involved in conversion of ATP to other nucleotides; and (iii) GTP-binding proteins, using the energy of GTP hydrolysis to drive various processes during chloroplast biogenesis, function and turnover. The main aspects reviewed for each chloroplast protein will be prediction, proteomic and/or individual identification, in vitro biochemical characterization and in planta functional analysis.

    Ort, förlag, år, upplaga, sidor
    Kerala: Research Signpost Publisher, 2008
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-14184 (URN)978-81-308-0104-9 (ISBN)
    Tillgänglig från: 2007-01-15 Skapad: 2007-01-15 Senast uppdaterad: 2013-08-26Bibliografiskt granskad
    2. Identification of an ATP/ADP carrier in the Arabidopsis chloroplast thylakoid membrane. Heterologous expression and functional characterization.
    Öppna denna publikation i ny flik eller fönster >>Identification of an ATP/ADP carrier in the Arabidopsis chloroplast thylakoid membrane. Heterologous expression and functional characterization.
    Visa övriga...
    2006 (Engelska)Artikel i tidskrift (Refereegranskat) Submitted
    Identifikatorer
    urn:nbn:se:liu:diva-14185 (URN)
    Tillgänglig från: 2007-01-15 Skapad: 2007-01-15 Senast uppdaterad: 2010-04-29
    3. Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex
    Öppna denna publikation i ny flik eller fönster >>Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex
    Visa övriga...
    2007 (Engelska)Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, nr 6, s. 500-508Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409–1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins.MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn2+ and Ca2+ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.

    Ort, förlag, år, upplaga, sidor
    Amsterdam: Elsevier, 2007
    Nyckelord
    Photosystem II, PsbO protein, GTPase, Oxygen-evolving complex, D1 protein
    Identifikatorer
    urn:nbn:se:umu:diva-2870 (URN)10.1016/j.bbabio.2006.10.009 (DOI)17223069 (PubMedID)
    Tillgänglig från: 2008-01-07 Skapad: 2008-01-07 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
  • 109.
    Hild Walett, Oliver
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Berlin, Emmanuel
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten.
    Larsson, Johan
    Arvidsson, Sofie
    Fors, Filip
    Gavelius, Marianne
    Genander, Filip
    Granqvist, Johanna
    Lifwergren, Philip
    Sandéhn, Alexandra
    Viksten, Martin
    Wenhov, Irma
    Investigating the function of GroES with hard-to-fold proteins in vivo2019Studentarbete övrigt, 10 poäng / 15 hpStudentuppsats (Examensarbete)
    Abstract [en]

    The use of molecular chaperones can increase the yield of correctly folded proteins. This is especially needed in the expression of proteins non-native to the host organism. This study set out to investigate the function of the chaperone GroES; a component in the GroE-system. The function of this chaperone has only been studied alone in vitro. Here we lay ground to further studies on GroES and its ability to act alone in vivo. GroES was expressed from a plasmid and characterized through its potential to increase the amount of correctly folded proteins. Characterization was mainly done by fluorescence spectroscopy with hard-to-fold proteins linked to fluorescent probes. Results show a very clear increase in fluorescence for most of the substrate proteins tested, indicating that GroES has a significant role in the GroE-system and perhaps outside of it.

  • 110.
    Hombach-Klonisch, Sabine
    et al.
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada .
    Panigrahi, Soumya
    Department of Physiology, University of Manitoba, Winnipeg, Canada; Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada.
    Rashedi, Iran
    Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Seifert, Anja
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada.
    Alberti, Esteban
    Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.
    Pocar, Paola
    Department of Animal Science, Faculty of Veterinary Medicine, University of Milan, Milan, Italy .
    Kurpisz, Maciej
    Institute of Human Genetics, Polish Academy of Science, Poznan, Poland .
    Schulze-Osthoff, Klaus
    Institute of Molecular Medicine, University of Duesseldorf, Duesseldorf, Germany .
    Mackiewicz, Andrzej
    Department of Cancer Immunology, Poznan University of Medical Sciences, and Great-Poland Cancer Center, Poznan, Poland.
    Los, Marek Jan
    BioApplications Enterprises, Winnipeg, MB, Canada.
    Adult stem cells and their trans-differentiation potential-perspectives and therapeutic applications2008Ingår i: Journal of Molecular Medicine, ISSN 0946-2716, E-ISSN 1432-1440, Vol. 86, nr 12, s. 1301-1314Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time. Normal stem cells in the adult organism are responsible for renewal and repair of aged or damaged tissue. Adult stem cells are present in virtually all tissues and during most stages of development. In this review, we introduce the reader to the basic information about the field. We describe selected stem cell isolation techniques and stem cell markers for various stem cell populations. These include makers for endothelial progenitor cells (CD146/MCAM/MUC18/S-endo-1, CD34, CD133/prominin, Tie-2, Flk1/KD/VEGFR2), hematopoietic stem cells (CD34, CD117/c-Kit, Sca1), mesenchymal stem cells (CD146/MCAM/MUC18/S-endo-1, STRO-1, Thy-1), neural stem cells (CD133/prominin, nestin, NCAM), mammary stem cells (CD24, CD29, Sca1), and intestinal stem cells (NCAM, CD34, Thy-1, CD117/c-Kit, Flt-3). Separate section provides a concise summary of recent clinical trials involving stem cells directed towards improvement of a damaged myocardium. In the last part of the review, we reflect on the field and on future developments.

  • 111.
    Hu, Zhang-Jun
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten.
    Yang, Guanqing
    Anhui Univ, Peoples R China.
    Hu, Jiwen
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten.
    Wang, Hui
    Anhui Univ, Peoples R China.
    Eriksson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten.
    Zhang, Ruilong
    Anhui Univ, Peoples R China.
    Zhang, Zhongping
    Anhui Univ, Peoples R China.
    Uvdal, Kajsa
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten.
    Real-time visualizing the regulation of reactive oxygen species on Zn2+ release in cellular lysosome by a specific fluorescent probe2018Ingår i: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 264, s. 419-425Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reactive oxygen species (ROS) regulating the release of free zinc ions (Zn2+) in cellular lysosome is closely related to various pathways of cellular signal transduction, such as inflammation and oxidative stress. Directly visualizing Zn2+ release in lysosome is essential for in-depth understanding these physiological processes, and is still an atelic challenge. In this work, we successfully fabricate a lysosome-specific Zn2+ fluorescent probe and achieve the visualization of ROS-induced Zn2+ release in lysosome of inflammatory cells. The as-prepared probe combines a green fluorophore, an ionophore with five-dentate sites, and a morpholine as the lysosome-specific localization moiety. The fluorescence of the fluorophore in the free probe is suppressed by a photoinduced electron transfer (PET) process from nitrogen atoms in the ionophore. Upon the addition of Zn2+, the fluorescence can be promoted immediately, achieving the real-time detection. Meanwhile, the probe is sensitive and selective to Zn2+, which provides the capability to detect low-concentration of free Zn2+ in lysosomes. Accordingly, the Zn2+ release was clearly observed in lysosome with the increase of ROS levels when the inflammation occurred in living cells. (c) 2018 Published by Elsevier B.V.

    Publikationen är tillgänglig i fulltext från 2020-03-07 12:11
  • 112.
    Hug, H.
    et al.
    Universitäts-Kinderklinik Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany, Universität Münster, Röntgen-Strasse 21, D-48149 Münster, Germany, and Orpegen Pharma, Czerny-Ring 22, D-69115 Heidelberg, Germany .
    Los, Marek Jan
    Department of Immunology and Cell Biology, University of Münster, Münster, Germany.
    Hirt, W.
    Orpegen Pharma, Czerny-Ring 22, D-69115 Heidelberg, Germany .
    Debatin, K. M.
    Universitäts-Kinderklinik Ulm, Prittwitzstrasse 43, D-89075 Ulm, Germany, Universität Münster, Röntgen-Strasse 21, D-48149 Münster, Germany.
    Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells1999Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, nr 42, s. 13906-13911Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Caspases (cysteine aspartate-specific proteases) are a structurally related group of cysteine proteases that cleave peptide bonds following specific recognition sequences. They play a central role in activating apoptosis of vertebrate cells. To measure apoptosis induced by various stimuli and at an early apoptotic stage, caspases are an ideal target. This is especially the case when apoptotic cells have to be analyzed ex vivo before phagocytes remove them. A new and sensitive caspase assay is based on a substrate that contains only aspartate residues linked to rhodamine 110. With this and similar substrates, we are able to detect intracellular caspase activation by flow cytometry after apoptosis induction in intact hematopoetic cell lines.

  • 113.
    Höst, Gunnar E.
    et al.
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Visuell informationsteknologi och applikationer. Linköpings universitet, Tekniska högskolan.
    Schönborn, Konrad J.
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Visuell informationsteknologi och applikationer. Linköpings universitet, Tekniska högskolan.
    Bivall Persson, Petter
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Visuell informationsteknologi och applikationer. Linköpings universitet, Tekniska högskolan.
    Tibell, Lena A.E.
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Visuell informationsteknologi och applikationer. Linköpings universitet, Tekniska högskolan.
    Methods for investigating students’ learning and interaction with a haptic virtual biomolecular model2010Ingår i: Contemporary Science Education Research: International Perspectives / [ed] M.F. Taşar & G. Çakmakcı, Ankara: Pegem Akademi , 2010, s. 115-121Konferensbidrag (Refereegranskat)
    Abstract [en]

    Although immersive haptic virtual technologies are emerging rapidly in modern education, few methods exist for delivering data on the pedagogical merits of such models in the molecular life sciences. This paper reports on a selection of methods that we have used to obtain and analyse data on students’ learning and interaction with a haptic virtual model of protein-ligand docking, previously designed by author PBP. The methods have been developed and employed during four consecutive years in which the model has been part of an advanced biomolecular interactions course. In this regard, we present data-collection methods that include written items, interviews, think-aloud tasks and automated time-stamped logs and, corresponding quantitative and qualitative analytical procedures such as pre/posttest statistical comparisons, word usage analysis and, visualised profiling of students’ interaction with the model. Our results suggest that these methods are useful for generating valuable information on students’ learning gain, changes in conceptual understanding, reasoning processes and patterns of interactivity with the model. Dissemination of such methods could provide an empirical contribution to the dearth of research instruments in this domain. Future research will develop these methodologies to explore the relationship between using the model and students’ conceptual and embodied learning.

  • 114.
    Ihnatko, Robert
    et al.
    Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 45 Bratislava, Slovakia.
    Kubes, M
    Institute of Virology, Slovak Academy of Sciences, Dúbravská cesta 9, 842 45 Bratislava, Slovakia.
    TNF signaling: early events and phosphorylation.2007Ingår i: General Physiology and Biophysics, ISSN 0231-5882, E-ISSN 1338-4325, Vol. 26, nr 3, s. 159-67Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.

  • 115.
    Ihnatko, Robert
    et al.
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Kubes, Miroslav
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Takacova, Martina
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Sedlakova, Olga
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Sedlak, Jan
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Pastorek, Jaromir
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Kopacek, Juraj
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Pastorekova, Silvia
    Centre of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, 845 05 Bratislava, Slovak Republic.
    Extracellular acidosis elevates carbonic anhydrase IX in human glioblastoma cells via transcriptional modulation that does not depend on hypoxia2006Ingår i: International Journal of Oncology, ISSN 1019-6439, Vol. 29, nr 4, s. 1025-33Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Most solid tumors display extracellular acidosis, which only partially overlaps with hypoxia and induces distinct adaptive changes leading to aggressive phenotype. Although acidosis is mainly attributable to excessive production of lactic acid, it also involves carbonic anhydrase (CA) IX-mediated conversion of CO(2) to an extracellular proton and a bicarbonate ion transported to cytoplasm. CA IX is pre-dominantly expressed in tumors with poor prognosis and its transcription and activity are induced by hypoxia. Here we investigated whether low extracellular pH in absence of hypoxia can influence CA IX expression in cell lines derived from glioblastoma, a tumor type particularly linked with acidosis. Our data show that extracellular acidosis increased the level of CA IX protein, mRNA and the activity of minimal CA9 promoter that contains binding sites for HIF-1 and SP-1 transcription factors. Mutation within each of these two biding sites reduced the promoter activity, but did not eliminate the increase by acidosis. Transfection of HIF-1alpha cDNA produced additive inducing effect with acidosis. Normoxic acidosis was accompanied by HIF-1alpha protein accumulation and transiently increased phosphorylation of ERK1/2. Expression of a dominant-negative mutant of ERK2 reduced the CA9 promoter activity in both standard and acidic conditions. Similar result was obtained by inhibitors of MAPK and PI3K pathways, whose combination completely suppressed CA IX expression and abolished induction by acidosis. Altogether, our results suggest that acidosis increases the CA IX expression via a hypoxia-independent mechanism that operates through modulation of the basic CA9 transcriptional machinery.

  • 116.
    Ihnatko, Robert
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten.
    Theodorsson, Elvar
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk kemi.
    Short N-terminal galanin fragments are occurring naturally in vivo2017Ingår i: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 63Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GALL, GAL(2), and GAL(3), binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1.20), galanin(6-20), and tested on coronal rat brain sections competing with I-125-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced I-125-galanin(1-29) but galanin (6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species. Biological significance: This study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments.

  • 117.
    Ingelsson, Björn
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Fristedt, Rikard
    Biophysics of Photosynthesis Physics and Astronomy Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
    Turkina, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Phosphorylation stoichiometry determination in plant photosynthetic membranes.2015Ingår i: Plant Phosphoproteomics: Methods and Protocols / [ed] Waltraud X. Schulze, New York: Springer-Verlag New York, 2015, s. 121-134Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    This chapter describes different strategies for the study of phosphorylation dynamics and stoichiometry in photosynthetic membranes. Detailed procedures for the detection, large-scale identification, and quantification of phosphorylated proteins optimized for plant thylakoid proteins are given.

  • 118.
    Islam, Mohammad Mirazul
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Karolinska Institute, Sweden.
    Ravichandran, Ranjithkumar
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Olsen, D.
    FibroGen Inc, CA 94158 USA.
    Kozak Ljunggren, Monika
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Ögonkliniken US/LiM.
    Lee, Chyan-Jang
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Sensorvetenskap och Molekylfysik. Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten.
    Griffith, May
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Karolinska Institute, Sweden.
    Phopase, Jaywant
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Self-assembled collagen-like-peptide implants as alternatives to human donor corneal transplantation2016Ingår i: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 6, nr 61, s. 55745-55749Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Extracellular matrix proteins like collagen promote regeneration as implants in clinical studies. However, collagens are large and unwieldy proteins, making small functional peptide analogs potentially ideal substitutes. Self-assembling collagen-like-peptides conjugated with PEG-maleimide were assembled into hydrogels. When tested pre-clinically as corneal implants in mini-pigs, they promoted cell and nerve regeneration, forming neo-corneas structurally and functionally similar to natural corneas.

  • 119.
    Jain, Mayur Vilas
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Shareef, Ahmad
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Likus, Wirginia
    Department of Human Anatomy, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland.
    Cieślar-Pobuda, Artur
    Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland.
    Ghavami, Saeid
    Department of Human Anatomy and Cell Science, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada.
    Łos, Marek J.
    Department of Pathology, Pomeranian Medical University, Szczecin, Poland / LinkoCare Life Sciences AB, Linköping, Sweden.
    Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins2016Ingår i: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, nr 15, s. 20953-20965Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Micro-RNAs (miRs) represent an innovative class of genes that act as regulators of gene expression. Recently, the aberrant expression of several miRs has been associated with different types of cancers. In this study, we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co-transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301-inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K-signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway-mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway.

  • 120.
    Jan Anton Koster, L.
    et al.
    Eindhoven University of Technology, Netherlands; University of Groningen, Netherlands.
    Kemerink, Martijn
    Eindhoven University of Technology, Netherlands.
    Wienk, Martijn M.
    Eindhoven University of Technology, Netherlands.
    Maturova, Klara
    Eindhoven University of Technology, Netherlands.
    Janssen, Rene A. J.
    Eindhoven University of Technology, Netherlands.
    Quantifying Bimolecular Recombination Losses in Organic Bulk Heterojunction Solar Cells2011Ingår i: Advanced Materials, ISSN 0935-9648, E-ISSN 1521-4095, Vol. 23, nr 14, s. 1670-+Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present a new experimental technique that affords direct quantification of the fraction of charge carriers lost in poly(3-hexylthiophene): fullerene solar cells by bimolecular recombination. Depending on annealing conditions up to 17% of carriers recombine bimolecularly under solar illumination. We explain our findings with a closed analytical expression for the photocurrent generated by an organic solar cell.

  • 121.
    Jansson, Lennie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi.
    Purification and refolding of a novel dipeptidyl peptidase III2019Självständigt arbete på grundnivå (kandidatexamen), 10,5 poäng / 16 hpStudentuppsats (Examensarbete)
    Abstract [en]

    There is a continuous search for novel enzymes to complement the abilities of today’s commercially available enzyme and find tailor-fit alternatives to suit the diverse array of bio-based industries. One application could be to increase biogas yield by finding substrate degrading proteases that can be added to the anaerobic digestion process and survive degradation themselves. A novel enzyme identified as a hypothetical dipeptidyl peptidase III, a zinc dependent metallo-protease, was found by a metaproteogenomics approach to be produced by the microorganisms of a thermophilic biogas process. The aim of this study was to express and refold a recombinant variant of the novel DPP III to its active form after production in inclusion bodies in Escherichia Coli. Assaying of refolding conditions was performed by stepwise dialysis and drip dilution. Nine attempts were performed based on findings in literature, although no other variant of DPP III has earlier been successfully refolded from inclusion bodies. The study resulted in a limited set of conditions of temperature, volumes, metal ions, salts and other additives being tested in the refolding buffers. Enzyme refolding and activation was monitored by the hydrolysis of the DPP III fluorescent substrate Arg-Arg β-naphthylamide trihydrochloride, alongside with measurements of protein concentration and SDS-PAGE. The novel DPP III was successfully purified but no definite strategy of producing correctly folded protein was found.

  • 122.
    Jansson, Sandra
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik.
    Regulation of non-specific lipid transfer proteins in abiotically stressed Physcomitrella patens2011Självständigt arbete på grundnivå (kandidatexamen), 15 poäng / 22,5 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Non-specific lipid transfer proteins is a large and diverse protein family found in plants, with roles in biological systems ranging from long distance signaling to plant pathogen defense. Little is known about the roles of nsLTPs, but recent studies have cast some light on the issue, among other things proposing that they may be involved in the cutice formation on land-living liverworts, mosses and non-seedbearing plants. Increased cuticle formation is thought to be a part of a plants defense system against stress. In this experiment, the expression of nsLTPs type G in the moss Physcomitrella patens was examined by qRT-PCR on cDNA synthesized from already existing mRNA samples from moss under different abiotic stresses. The different stresses were UV-light, salt (ion toxicity), heavy metal, cold drought, plant hormone and osmosis. House-keeping gene P. patens beta-tubuline 1 was used as reference and relative expression analysis was performed. The study showed a general down-regulation of PpLTPg's in the abiotically stressed samples, and the possible coupled regulatory response of PpLTPg3 and PpLTPg5. The results imply that the PpLTPg's in Physcomitrella patens could be connected to biological processes that cease during stress, or that they worl through negative feedback to support plant defense against stress.

  • 123.
    Jinnelöv, Anders
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Investigation of small molecules binding to UDP-galactose 4'-epimerase: A validated drug target for Trypanosoma brucei, the parasite responsible for African Sleeping Sickness.2009Självständigt arbete på avancerad nivå (masterexamen), 30 poäng / 45 hpStudentuppsats (Examensarbete)
    Abstract [en]

    African sleeping sickness is a parasitic infection spread by the protozoan parasite Trypanosoma brucei, and drugs used today are toxic and painful. Galactose metabolism is essential for the survival of T. brucei and without a functional UDP galactose 4’ epimerase (GalE) galactose starvation occurs and cell death will follow. In this Master thesis project two assays observing binding of small molecules to TbGalE has been investigated in attempt to establish an assay that in the future could be used for screening for drugs.

    TbGalE was biotinylated through the Pinpoint Xa vector and expressed in E. coli cells. The protein was successfully immobilized to a Streptavidin chip for Surface Plasmon Resonance experiments and the binding of the substrates UDP-galactose and UDP-glucose was observed. Unfortunately, the assay was not optimal for screening due to low signal response. However, the established protocol for expressing biotinylated proteins that bind to Streptavidin surfaces could be used in further experiments with TbGalE and other drug targets for African sleeping sickness.

    The fluorescent sugar nucleotide analogue UDPAmNS, which is a known inhibitor for E. coli GalE, was synthesised and purified and then used to establish a displacement assay. IC50 of UDPAmNS against TbGalE was determined and a synergic effect in fluorescence between the protein and the inhibitor was proven. Further, evidence for a reduction in fluorescence by displacing UDPAmNS with UDP was obtained. This reduction in fluorescence was also shown by a predicted cofactor inhibitor. The IC50 against TbGalE for this compound was determined before the displacement assay, which showed that the cofactor inhibitor, at least partly, binds to the active site of TbGalE. The UDPAmNS displacement assay could have the potential of becoming a robust screening assay for TbGalE, in the effort to find a better drug for African sleeping sickness.

  • 124.
    Johansson, Anna
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Dependence-induced changes in opioid-receptor gene expression2013Självständigt arbete på avancerad nivå (masterexamen), 30 poäng / 45 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Using drugs such as alcohol and morphine among others can be addictive in some individuals, and progress into a substance abuse disorder. The mesolimbic dopaminergic system (MD-system) is involved in the reward process during the development of drug addiction. The MD-system is critical for survival and affects different behaviors in both man and animal. Neurochemical pathways drive for instance physical activity, food intake, love and reproduction and are part of the natural reward process involved partly in the release of dopamine (DA) into frontal lobes. Within the MD-system opioid receptors throughout the brain are affected by drug intake, and activation of these receptors modulate DA-release in brain regions involved in reward-behavior. The aim of this study was to evaluate gene expression of MOR and DOR within the endogenous opioid system (EO-system) in relation to voluntary physical activity, a natural reinforcer. Further on investigations of the drug alcohol

    was compared to the natural reinforcer sucrose using voluntary consumption.

    For both experiments qRT-PCR was used to measure mRNA levels of MOR and DOR from brain areas of interest. We found a small significant up regulation in NAc, PFC and VTA but for DOR in VTA a down regulation in gene expression of physical exercising mice. Additionally these two different genes

    OPRM1- and the OPRD1- gene are down regulated in VTA and NAc due to alcohol- and sugar-intake. This implicate that the natural reward system and their ORs point in the direction of earlier findings; the opioid receptors have a key role in regulate alcohol intake and the natural rewarding stimuli as food intake.

  • 125.
    Johansson, Johannes D
    et al.
    ICFO - Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology.
    Mireles, Miguel
    ICFO - Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology.
    Morales-Dalmau, Jordi
    ICFO - Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology.
    Farzam, Parisa
    ICFO - Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology.
    Martínez-Lozano, Mar
    Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology, Bellvitge Biomedical Research Institute –IDIBELL.
    Casanovas, Oriol
    Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology, Bellvitge Biomedical Research Institute –IDIBELL.
    Durduran, Turgut
    ICFO - Institut de Ciències Fotòniques, The Barcelona Institute of Science and Technology.
    Scanning, non-contact, hybrid broadband diffuse optical spectroscopy and diffuse correlation spectroscopy system.2016Ingår i: Biomedical Optics Express, ISSN 2156-7085, E-ISSN 2156-7085, Vol. 7, nr 2, s. 481-498Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A scanning system for small animal imaging using non-contact, hybrid broadband diffuse optical spectroscopy (ncDOS) and diffuse correlation spectroscopy (ncDCS) is presented. The ncDOS uses a two-dimensional spectrophotometer retrieving broadband (610-900 nm) spectral information from up to fifty-seven source-detector distances between 2 and 5 mm. The ncDCS data is simultaneously acquired from four source-detector pairs. The sample is scanned in two dimensions while tracking variations in height. The system has been validated with liquid phantoms, demonstrated in vivo on a human fingertip during an arm cuff occlusion and on a group of mice with xenoimplanted renal cell carcinoma.

  • 126.
    Johansson, Mikaela
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Metaproteogenomics-guided enzyme discovery: Targeted identification of novel proteases in microbial communities2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Industrial biotechnology is a large and growing industry as it is part of establishing a “greener” and more sustainable bioeconomy-based society. Using enzymes as biocatalysts is a viable alternative to chemicals and energy intense industrial processes and is en route to a more sustainable industry. Enzymes have been used in different areas for ages and are today used in many industrial processes such as biofuels production, food industry, tanning, chemical synthesis, pharmaceuticals etc. Enzymes are today a billion-dollar industry in itself and the demand for novel catalysts for various present and future processes of renewable resources are high and perfectly in line with converting to a more sustainable society.

    Most enzymes used in industry today have been identified from isolated and pure cultured microorganisms with identified desirable traits and enzymatic capacities. However, it is known that less than 1% of all microorganisms can be can be obtained in pure cultures. Thus, if we were to rely solely on pure culturing, this would leave the 99% of the microorganisms that constitutes the “microbial dark matter” uninvestigated for their potential in coding for and producing valuable novel enzymes. Therefore, to investigate these “unculturable” microorganisms for novel and valuable enzymes, pure-culture independent methods are needed.

    During the last two decades there has been a fast and extensive development in techniques and methods applicable for this purpose. Especially important has been the advancements made in mass spectrometry for protein identification and next generation sequencing of DNA. With these technical developments new research fields of proteomics and genomics have been developed, by which the complete protein complement of cells (the proteome) and all genes (the genome) of organisms can be investigated. When these techniques are applied to microbial communities these fields of research are known as meta-proteomics and meta-genomics.

    However, when applied to complex microbial communities, difficulties different from those encountered in their original usage for analysis of single multicellular organisms or cell linages arises, and when used independently both methods have their own limitations and bottlenecks. In addition, both metaproteomics and metagenomics are largely non-targeting techniques. Thus, if the purpose is still to - somewhat contradictory – use these non-targeting methods for targeted identification of novel enzymes with certain desired activities and properties from within microbial communities, special measures need to be taken.

    The work presented in this thesis describes the development of a method that combines

    metaproteomics and metagenomics (i.e. metaproteogenomics) for the targeted discovery of novel enzymes with desired activities, and their correct coding genes, from within microbial communities. Thus, what is described is a method that can be used to circumvent the pure-culturing problem so that a much larger fraction of the microbial dark matter can be specifically investigated for the identification of novel valuable enzymes.

    Delarbeten
    1. Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state
    Öppna denna publikation i ny flik eller fönster >>Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state
    2016 (Engelska)Ingår i: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, nr 18, s. 7989-8002Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Novel enzymes that are stable in diverse conditions are intensively sought because they offer major potential advantages in industrial biotechnology, and microorganisms in extreme environments are key sources of such enzymes. However, most potentially valuable enzymes are currently inaccessible due to the pure culturing problem of microorganisms. Novel metagenomic and metaproteomic techniques that circumvent the need for pure cultures have theoretically provided possibilities to identify all genes and all proteins in microbial communities, but these techniques have not been widely used to directly identify specific enzymes because they generate vast amounts of extraneous data. In a first step towards developing a metaproteomic approach to pinpoint targeted extracellular hydrolytic enzymes of choice in microbial communities, we have generated and analyzed the necessary conditions for such an approach by the use of a methanogenic microbial community maintained on a chemically defined medium. The results show that a metabolic steady state of the microbial community could be reached, at which the expression of the targeted hydrolytic enzymes were suppressed, and that upon enzyme induction a distinct increase in the targeted enzyme expression was obtained. Furthermore, no cross talk in expression was detected between the two focal types of enzyme activities under their respective inductive conditions. Thus, the described approach should be useful to generate ideal samples, collected before and after selective induction, in controlled microbial communities to clearly discriminate between constituently expressed proteins and extracellular hydrolytic enzymes that are specifically induced, thereby reducing the analysis to only those proteins that are distinctively up-regulated.

    Ort, förlag, år, upplaga, sidor
    Springer, 2016
    Nyckelord
    Microbial community; Enzyme discovery; Metaproteomics; Biogas; Cellulase; Protease
    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:liu:diva-131888 (URN)10.1007/s00253-016-7547-z (DOI)000382008000017 ()27115757 (PubMedID)
    Anmärkning

    Funding Agencies|Swedish Research Council [621-2009-4150]; InZymes Biotech AB

    Tillgänglig från: 2016-10-13 Skapad: 2016-10-11 Senast uppdaterad: 2018-05-15
    2. Assessment of sample preparation methods for metaproteomics of extracellular proteins
    Öppna denna publikation i ny flik eller fönster >>Assessment of sample preparation methods for metaproteomics of extracellular proteins
    2017 (Engelska)Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 516, s. 23-36Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Enzyme discovery in individual strains of microorganisms is compromised by the limitations of pure culturing. In principle, metaproteomics allows for fractionation and study of different parts of the protein complement but has hitherto mainly been used to identify intracellular proteins. However, the extracellular environment is also expected to comprise a wealth of information regarding important proteins. An absolute requirement for metaproteomic studies of protein expression, and irrespective of downstream methods for analysis, is that sample preparation methods provide clean, concentrated and representative samples of the protein complement. A battery of methods for concentration, extraction, precipitation and resolubilization of proteins in the extracellular environment of a constructed microbial community was assessed by means of 2D gel electrophoresis and image analysis to elucidate whether it is possible to make the extracellular protein complement available for metaproteomic analysis. Most methods failed to provide pure samples and therefore negatively influenced protein gel migration and gel background clarity. However, one direct precipitation method (TCA-DOC/acetone) and one extraction/precipitation method (phenol/methanol) provided complementary high quality 2D gels that allowed for high spot detection ability and thereby also spot detection of less abundant extracellular proteins.

    Ort, förlag, år, upplaga, sidor
    Elsevier, 2017
    Nyckelord
    Enzyme discovery, Microbial community, Metaproteome, Extracellular, Sample preparation, 2D gel electrophoresis
    Nationell ämneskategori
    Analytisk kemi Biokatalys och enzymteknik
    Identifikatorer
    urn:nbn:se:liu:diva-132902 (URN)10.1016/j.ab.2016.10.008 (DOI)000388056800005 ()27742212 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet, 621-2009-4150
    Anmärkning

    Funding agencies: Swedish Research Council [621-2009-4150]; Tekniska Verken i Linkoping AB; InZymes Biotech AB

    Tillgänglig från: 2016-12-01 Skapad: 2016-12-01 Senast uppdaterad: 2018-05-15Bibliografiskt granskad
  • 127.
    Johnston, James B.
    et al.
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Navaratnam, Sri
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Pitz, Marshall W.
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Maniate, Jerry M.
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Wiechec, Emilia
    Manitoba Institute of Cell Biology, CancerCare Manitoba.
    Baust, Heinrich
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Gingerich, Joel
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Skliris, Georgios P.
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Murphy, Leigh C.
    University of Manitoba,Winnipeg, MB,R3E 0V9, Canada.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Targeting the EGFR pathway for cancer therapy2006Ingår i: Current Medicinal Chemistry, ISSN 0929-8673, E-ISSN 1875-533X, Vol. 13, nr 29, s. 3483-3492Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Clinical studies have shown that HER-2/Neu is over-expressed in up to one-third of patients with a variety of cancers, including B-cell acute lymphoblastic leukemia (B-ALL), breast cancer and lung cancer, and that these patients are frequently resistant to conventional chemo-therapies. Additionally, in most patients with multiple myeloma, the malignant cells over-express a number of epidermal growth factor receptors (EGFR)s and their ligands, HB-EGF and amphiregulin, thus this growth-factor family may be an important aspect in the patho-biology of this disease. These and other, related findings have provided the rationale for the targeting of the components of the EGFR signaling pathways for cancer therapy. Below we discuss various aspects of EGFR-targeted therapies mainly in hematologic malignancies, lung cancer and breast cancer. Beside novel therapeutic approaches, we also discuss specific side effects associated with the therapeutic inhibition of components of the EGFR-pathways. Alongside small inhibitors, such as Lapatinib (Tykerb, GW572016), Gefitinib (Iressa, Z131839), and Erlotinib (Tarceva, OSI-774), a significant part of the review is also dedicated to therapeutic antibodies (e.g.: Trastuzumab / Herceptin, Pertuzumab / Omnitarg / rhuMab-2C4, Cetuximab / Erbitux / IMC-C225, Panitumumab / Abenix / ABX-EGF, and also ZD6474). In addition, we summarize, both current therapy development driven by antibody-based targeting of the EGFR-dependent signaling pathways, and furthermore, we provide a background on the history and the development of therapeutic antibodies.

  • 128.
    Jonsson, Josefine
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Effect of voluntary exercise on BDNF/TrkB gene expression and alcohol intake.2012Självständigt arbete på avancerad nivå (masterexamen), 30 poäng / 45 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Voluntary wheel running is rewarding and believed to activate the same brain reward system as in alcohol and drug addiction. Brain-derived neurotrophic factor (BDNF), a well-known growth factor widely expressed in the brain, is modulated by both voluntary exercise and alcohol consumption. The aim of this study was to evaluate how voluntary exercise affects the expression levels of BDNF and its receptor TrkB in brain regions involved in positive and negative reinforcement. Additionally we wanted to evaluate the effect it may have on alcohol drinking behaviors in C57BL/6 mice, a mouse model which are naturally prone for engaging in voluntary exercise and voluntary alcohol consumption.

    We found a small upregulation in DG and CA1 after three weeks of exercise, confirming findings by others, and a significant 3-fold downregulation of BDNF in NAc after both three weeks of exercise and exercise followed by a five week period of either ethanol intake or not. Interestingly, we here show a significant 100-fold increase in BDNF after exercise and a 120-fold increase after both exercise and alcohol consumption in amygdala, a region involved in regulation of anxiety-related behavior and negative reinforcement. Additionally a slightly lower 10-fold increase in BDNF was seen after exercise and a 15-fold increase after exercise followed by ethanol in prefrontal cortex, a structure contributing to reward-related behavior. Behaviorally, we could not either directly following exercise or at five weeks post-exercise detect any significant effect of wheel-running on depression-related behavior. However, we did find that exercise significantly increased the alcohol intake.

  • 129.
    Kalkat, Manpreet
    et al.
    Univ Toronto, Canada; Univ Hlth Network, Canada.
    Resetca, Diana
    Univ Toronto, Canada; Univ Hlth Network, Canada.
    Lourenco, Corey
    Univ Toronto, Canada; Univ Hlth Network, Canada.
    Chan, Pak-Kei
    Univ Hlth Network, Canada.
    Wei, Yong
    Univ Hlth Network, Canada; Struct Genom Consortium, Canada.
    Shiah, Yu-Jia
    Ontario Inst Canc Res, Canada.
    Vitkin, Natasha
    Univ Hlth Network, Canada.
    Tong, Yufeng
    Struct Genom Consortium, Canada; Univ Toronto, Canada.
    Sunnerhagen, Maria
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Done, Susan J.
    Univ Toronto, Canada; Univ Hlth Network, Canada.
    Boutros, Paul C.
    Univ Toronto, Canada; Ontario Inst Canc Res, Canada.
    Raught, Brian
    Univ Toronto, Canada; Univ Hlth Network, Canada.
    Penn, Linda Z.
    Univ Toronto, Canada; Univ Hlth Network, Canada.
    MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis2018Ingår i: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 72, nr 5, s. 836-+Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transforming members of the MYC family (MYC, MYCL1, and MYCN) encode transcription factors containing six highly conserved regions, termed MYC homology boxes (MBs). By conducting proteomic profiling of the MB interactomes, we demonstrate that half of the MYC interactors require one or more MBs for binding. Comprehensive phenotypic analyses reveal that two MBs, MBO and MBII, are universally required for transformation. MBII mediates interactions with acetyltransferase-containing complexes, enabling histone acetylation, and is essential for MYC-dependent tumor initiation. By contrast, MBO mediates interactions with transcription elongation factors via direct binding to the general transcription factor TFIIF. MBO is dispensable for tumor initiation but is a major accelerator of tumor growth. Notably, the full transforming activity of MYC can be restored by co-expression of the non-transforming MBO and MBII deletion proteins, indicating that these two regions confer separate molecular functions, both of which are required for oncogenic MYC activity.

  • 130.
    Kanan, Tarek
    et al.
    Bahcesehir Univ, Turkey.
    Kanan, Duaa
    Bahcesehir Univ, Turkey.
    Erol, Ismail
    Bahcesehir Univ, Turkey; Gebze Tech Univ, Turkey.
    Yazdi, Samira
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelning för neurobiologi. Linköpings universitet, Medicinska fakulteten. Max Planck Inst Dynam and Complex Tech Syst, Germany.
    Stein, Matthias
    Max Planck Inst Dynam and Complex Tech Syst, Germany.
    Durdagi, Serdar
    Bahcesehir Univ, Turkey; Bahcesehir Univ, Turkey.
    Targeting the NF-kappa B/I kappa B alpha complex via fragment-based E-Pharmacophore virtual screening and binary QSAR models2019Ingår i: Journal of Molecular Graphics and Modelling, ISSN 1093-3263, E-ISSN 1873-4243, Vol. 86, s. 264-277Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nuclear factor-kappa B (NF-kappa B) transcription factors represent a conserved family of proteins that regulate not only immune cells, but also heart cells, glial cells and neurons, playing a fundamental role in various cellular processes. Due to its dysregulation in certain cancer types as well as in chronic inflammation and autoimmune diseases, it has recently been appreciated as an important therapeutic target. The aim of this study was to investigate the binding pocket of NF-kappa B (p50/p65) heterodimer complex in association with NF-kappa B inhibitor I kappa B alpha to identify potent ligands via fragment-based e-pharmacophore screening. The ZINC Clean Fragments (similar to 2 million) and the Schrodingers medically relevant Glide fragments library (similar to 670) were used to create the e-pharmacophore models at the potential binding site which was validated by site mapping. Glide/HTVS docking was conducted followed by re-docking of the top 20% fragments by Glide/SP and Glide/XP protocols. The top-85000 Glide XP-docked fragments were used to generate the e-pharmacophore hypotheses. The Otava small molecule library (similar to 260000 drug-like molecules) and 85 known NF-kappa B inhibitors were additionally screened against the derived e-pharmacophore models. The top-1000 high-scored molecules, which were well aligned to the e-pharmacophore models, from the Otava small molecule library, were then docked into the binding pocket. Finally, the selected 88 hit molecules and the 85 known inhibitors were analyzed by the MetaCore/MetaDrug (TM) platform, which uses developed binary QSAR models for therapeutic activity prediction as well as pharmacokinetic and toxicity profile predictions of screening molecules. Ligand selection criteria led to the refinement of 3 potent hit molecules using molecular dynamics (MD) simulations to better investigate their structural and dynamical profiles. The selected hit molecules had a low toxicity and a significant therapeutic potential for heart failure, antiviral activity, asthma and depression, all conditions in which NF-kappa B plays a critical role. These hit ligands were also structurally stable at the NE-kappa B/I kappa B alpha complex as per the MD simulations and MM/GBSA analysis. Two of these ligands (Otava IDs: 1426436 and 6248112) showed stronger binding and therefore are hypothesized to be more potent. The identification of new potent NF-kappa B/I kappa B alpha inhibitors may thus present a novel therapy for inflammation-mediated conditions as well as cancer, facilitating more efficient research, and leading the way to future drug development efforts. (C) 2018 Elsevier Inc. All rights reserved.

  • 131.
    Kannisto, Kristina
    Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Heat-sensitive TRP channels detected in pancreatic beta cells by microfluorometry and western blot2007Självständigt arbete på avancerad nivå (magisterexamen), 20 poäng / 30 hpStudentuppsats
    Abstract [en]

    Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot.

    Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein.

    Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells.

    Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.

  • 132.
    Karlsson, Jan Olof G
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten.
    Ignarro, Louis J
    Department of Molecular and Medical Pharmacology, University of California, USA.
    Lundström, Ingemar
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biosensorer och bioelektronik. Linköpings universitet, Tekniska fakulteten.
    Jynge, Per
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten.
    Almén, Torsten
    Department of Diagnostic Radiology, Lund University.
    Calmangafodipir [Ca4Mn(DPDP)5], mangafodipir (MnDPDP) and MnPLED with special reference to their SOD mimetic and therapeutic properties2015Ingår i: Drug Discovery Today, ISSN 1359-6446, E-ISSN 1878-5832, Vol. 20, nr 4, s. 411-421Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) participate in pathological tissue damage. Mitochondrial manganese superoxide dismutase (MnSOD) normally keeps ROS and RNS in check. During development of mangafodipir (MnDPDP) as a magnetic resonance imaging (MRI) contrast agent, it was discovered that MnDPDP and its metabolite manganese pyridoxyl ethyldiamine (MnPLED) possessed SOD mimetic activity. MnDPDP has been tested as a chemotherapy adjunct in cancer patients and as an adjunct to percutaneous coronary intervention in patients with myocardial infarctions, with promising results. Whereas MRI contrast depends on release of Mn2+, the SOD mimetic activity depends on Mn2+ that remains bound to DPDP or PLED. Calmangafodipir [Ca4Mn(DPDP)5] is stabilized with respect to Mn2+ and has superior therapeutic activity. Ca4Mn(DPDP)5 is presently being explored as a chemotherapy adjunct in a clinical multicenter Phase II study in patients with metastatic colorectal cancer.

  • 133.
    Karlsson, Markus
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet.
    Oxidative stress-related damage of retinal pigment epithelial cells: possible protective properties of autophagocytosed iron-binding proteins2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Oxidative stress is a major pathogenic factor in the development of age-related macular degeneration (AMD), which is the most common cause of severe central visual impairment in the elderly population in the western world.

    It is believed that the degenerative process starts in the retinal pigment epithelium (RPE). The post-mitotic RPE is a single layer of pigmented cells located behind the photoreceptors – rods and cones – of the retina. Daily, the RPE cells phagocytose and recycle the expended tips of the photoreceptor outer segments. This heavy phagocytic burden leads to substantial oxidative stress in the cells, which is further enhanced by intense illumination and a high oxygen tension. A hallmark of early AMD is a progressive build-up of the non-degradable age pigment lipofuscin (LF) in lysosomes of the RPE. LF accumulation hampers phagocytosis and autophagy in the RPE, resulting in increased amounts of cellular debris in and around the cells. This decreases the function and viability of both RPE cells and photoreceptors.

    Iron is known to accumulate in the retina with increasing age, particularly in AMDaffected eyes, and amplifies oxidative stress by acting as a potent catalyst in the generation of hydroxyl radicals. These highly reactive radicals contribute to LF formation and may, if abundantly present, also directly damage lysosomal membranes. The subsequent leakage of degrading enzymes to the cytosol initiates cell death via apoptosis or necrosis.

    In this thesis, we have investigated the oxidative stress response of human RPE (ARPE-19) cells compared to murine J774 cells, another type of lysosome-rich cells with a high phagocytic capacity. The ARPE-19 cells were found to be extremely resistant to oxidative stress and tolerated exposure to single doses of H2O2 in concentrations up to 150 times higher than the J774 cells before lysosomal rupture and ensuing cell death occurred. This resistance was increased even further when the cells were protected with a potent iron chelator that prevents redox-active iron to participate in hydroxyl radical generation. Both cell lines were shown to be equally effective in degrading H2O2 and seem to contain comparable amounts of total as well as intralysosomal iron. Therefore, we reasoned that the insensitivity of ARPE-19 cells to H2O2 exposure might be related to a mechanism which keeps their intralysosomal iron bound in a non redox-active form. This theory was supported by our finding of very high basal expression levels of metallothionein (MT), heat shock-protein 70 (HSP70) and ferritin (FT) in ARPE-19 cells compared to J774 cells. All of these proteins have previously been shown to possess potent iron-binding properties. The ARPE-19 cells were also shown to have a higher basal rate of autophagy. SiRNA-mediated attenuation of MT, HSP70 and FT levels in the ARPE-19 cells resulted, to some degree, in an increased sensitivity to H2O2 treatment. Furthermore, a human cell stress array showed several other stress-related proteins to be up-regulated in ARPE-19 cells.

    Additionally, we evaluated the commonly used, but frequently misinterpreted, H2DCF test for oxidative stress. It was demonstrated that oxidation of H2DCF into fluorescent DCF mainly reflects relocation to the cytosol of lysosomal iron and mitochondrial cytochrome c, rather than being the result of some poorly defined “general” oxidative stress.

    In conclusion, our results indicate that the extreme resistance to oxidative stress exhibited by the ARPE-19 cells might be related to a high continuous autophagic influx of iron-binding proteins into the lysosomal compartment. Before being degraded, such proteins will temporarily keep intralysosomal iron bound in a non redox-active form, thereby inhibiting hydroxyl radical formation. This may partly explain why RPE cells, in spite of their exposed location and heavy burden of phagocytosis, usually manage to survive and evade significant LF accumulation until late in life.

    Delarbeten
    1. ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron
    Öppna denna publikation i ny flik eller fönster >>ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron
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    2009 (Engelska)Ingår i: AUTOPHAGY, ISSN 1554-8627, Vol. 5, nr 4, s. 494-501Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Normal retinal pigment epithelial (RPE) cells are postmitotic, long-lived and basically not replaced. Daily, they phagocytose substantial amounts of lipid-rich material (photoreceptor outer segment discs), and they do so in the most oxygenated part of the body-the retina. One would imagine that this state of affairs should be associated with a rapid formation of the age pigment lipofuscin (LF). However, LF accumulation is slow and reaches significant amounts only late in life when, if substantial, it often coincides with or causes age-related macular degeneration. LF formation occurs inside the lysosomal compartment as a result of iron-catalyzed peroxidation and polymerization. This process requires phagocytosed or autophagocytosed material under degradation, but also the presence of redox-active low mass iron and hydrogen peroxide. To gain some information on how RPE cells are able to evade LF formation, we investigated the response of immortalized human RPE cells (ARPE-19) to oxidative stress with/without the protection of a strong iron-chelator. The cells were found to be extremely resistant to hydrogen peroxide-induced lysosomal rupture and ensuing cell death. This marked resistance to oxidative stress was not explained by enhanced degradation of hydrogen peroxide, but to a certain extent further increased by the potent lipophilic iron chelator STH. The cells were also able to survive, and even replicate, at high concentrations of SIH and showed a high degree of basal autophagic flux. We hypothesize that RPE cells have a highly developed capacity to keep lysosomal iron in a nonredox-active form, perhaps by pronounced autophagy of iron-binding proteins in combination with an ability to rapidly relocate low mass iron from the lysosomal compartment.

    Nyckelord
    age-related macular degeneration, hydrogen peroxide, iron, iron chelation, lipofuscin, lysosomal stability, lysosomes, macrophage, oxidative stress, retinal pigment epithelial cells
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-18569 (URN)10.4161/auto.5.4.7961 (DOI)
    Tillgänglig från: 2009-06-01 Skapad: 2009-06-01 Senast uppdaterad: 2014-10-24
    2. What does the commonly used DCF test for oxidative stress really show?
    Öppna denna publikation i ny flik eller fönster >>What does the commonly used DCF test for oxidative stress really show?
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    2010 (Engelska)Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 428, nr 2, s. 183-90Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

    Nyckelord
    age-related macular degeneration (AMD), 2', 7'-dichlorofluorescein (DCF), lysosome, oxidative stress, reactive oxygen species (ROS), transition metal.
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-56466 (URN)10.1042/BJ20100208 (DOI)20331437 (PubMedID)
    Tillgänglig från: 2010-05-17 Skapad: 2010-05-17 Senast uppdaterad: 2017-12-12
    3. Autophagy of iron-binding proteins may contribute to the oxidative stress resistance of ARPE-19 cells
    Öppna denna publikation i ny flik eller fönster >>Autophagy of iron-binding proteins may contribute to the oxidative stress resistance of ARPE-19 cells
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    2013 (Engelska)Ingår i: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 116, s. 359-365Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The objective of this study was to elucidate possible reasons for the remarkable resistance of human retinal pigment epithelial (RPE) cells to oxidative stress. Much oxidative damage is due to hydrogen peroxide meeting redox-active iron in the acidic and reducing lysosomal environment, resulting in the production of toxic hydroxyl radicals that may oxidize intralysosomal content, leading to lipofuscin (LF) formation or, if more extensive, to permeabilization of lysosomal membranes. Formation of LF is a risk factor for age-related macular degeneration (AMD) and known to jeopardize normal autophagic rejuvenation of vital cellular biomolecules. Lysosomal membrane permeabilization causes release of lysosomal content (redox-active iron, lytic enzymes), which may then cause cell death. Total cellular and lysosomal low-mass iron of cultured, immortalized human RPE (ARPE-19) cells was compared to that of another professional scavenger cell line, J774, using atomic absorption spectroscopy and the cytochemical sulfide-silver method (SSM). It was found that both cell lines contained comparable levels of total as well as intralysosomal iron, suggesting that the latter is mainly kept in a non-redox-active state in ARPE-19 cells. Basal levels and capacity for upregulation of the iron-binding proteins ferritin, metallothionein and heat shock protein 70 were tested in both cell lines using immunoblotting. Compared to J774 cells, ARPE-19 cells were found to contain very high basal levels of all these proteins, which could be even further upregulated following appropriate stimulation. These findings suggest that a high basal expression of iron-binding stress proteins, which during their normal autophagic turnover in lysosomes may temporarily bind iron prior to their degradation, could contribute to the unusual oxidative stress-resistance of ARPE-19 cells. A high steady state influx of such proteins into lysosomes would keep the level of lysosomal redox-active iron permanently low. This, in turn, should delay intralysosomal accumulation of LF in RPE cells, which is known to reduce autophagic turnover as well as uptake and degradation of worn out photoreceptor tips. This may explain why severe LF accumulation and AMD normally do not develop until fairly late in life, in spite of RPE cells being continuously exposed to high levels of oxygen and light, as well as large amounts of lipid-rich material.

    Ort, förlag, år, upplaga, sidor
    Elsevier, 2013
    Nyckelord
    oxidative stress, ARPE-19, retinal pigment epithelium, iron, metallothionein, HSP70, ferritin, age-related macular degeneration
    Nationell ämneskategori
    Cell- och molekylärbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-102718 (URN)10.1016/j.exer.2013.10.014 (DOI)000327562500041 ()
    Anmärkning

    Funding Agencies|Crown Princess Margaretas Foundation for the Visually Handicapped||Edvin Jordan Foundation for Ophthalmological Research||Linkoping University Hospital Research Fund (ALF)||

    Tillgänglig från: 2013-12-19 Skapad: 2013-12-19 Senast uppdaterad: 2018-01-11
    4. Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress
    Öppna denna publikation i ny flik eller fönster >>Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress
    2016 (Engelska)Ingår i: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 94, nr 6, s. 556-565Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Purpose

    Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress.

    Methods

    The sensitivity of ARPE-19 cells to H2O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4, heat shock or FeCl3, as well as siRNA-mediated downregulation of the same proteins.

    Results

    Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2O2. This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins.

    Conclusion

    The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low.

    Ort, förlag, år, upplaga, sidor
    Wiley-Blackwell Publishing Inc., 2016
    Nyckelord
    age-related macular degeneration, ARPE-19, ferritin, HSP70, iron metallothionein, oxidative stress, retinal pigment epithelium
    Nationell ämneskategori
    Klinisk medicin
    Identifikatorer
    urn:nbn:se:liu:diva-111557 (URN)10.1111/aos.13076 (DOI)000383520800034 ()
    Anmärkning

    At the time for thesis presentation publication was in status: Manuscript

    Funding agencies:  Crown Princess Margaretas Foundation for the Visually Handicapped; Edvin Jordan Foundation for Ophthalmological Research; Linkoping University Hospital Research Fund (ALF)

    Tillgänglig från: 2014-10-24 Skapad: 2014-10-24 Senast uppdaterad: 2017-12-05Bibliografiskt granskad
  • 134.
    Klonisch, Thomas
    et al.
    Department of Human Anatomy and Cell Sciences, Winnipeg, Canada.
    Wiechec, Emilia
    Manitoba Institute of Cell Biology, CancerCare Manitoba; Department of Human Genetics, University of Aarhus, Aarhus, Denmark.
    Hombach-Klonisch, Sabine
    Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, R3E 0W3, MB, Canada.
    Ande, Sudharsana R.
    Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, R3E 0V9, MB, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, R3E 0W3, MB, Canada.
    Wesselborg, Sebastian
    Department of Internal Medicine I, University of Tübingen, D-72076 Tübingen, Germany.
    Schulze-Osthoff, Klaus
    Interfaculty Institute for Biochemistry, University of Tübingen, Germany.
    Los, Marek Jan
    University of Manitoba, Winnipeg, Canada.
    Cancer stem cell markers in common cancers - therapeutic implications2008Ingår i: Trends in Molecular Medicine, ISSN 1471-4914, E-ISSN 1471-499X, Vol. 14, nr 10, s. 450-460Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.

  • 135.
    Koon Lim, Seng
    et al.
    Nanyang Technology University, Singapore.
    Sandén, Camilla
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Selegård, Robert
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Liedberg, Bo
    Nanyang Technology University, Singapore.
    Aili, Daniel
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, nr 21123, s. 1-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

  • 136.
    Koulikovska, Marina
    et al.
    Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten.
    Rafat, Mehrdad
    Linköpings universitet, Institutionen för medicinsk teknik, Biomedicinsk instrumentteknik. Linköpings universitet, Tekniska fakulteten. Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Medicinska fakulteten. LinkoCare Life Sciences AB, Linköping, Sweden.
    Petrovski, Goran
    University of Debrecen, Debrecen, Hungary; University of Szeged, Szeged, Hungary.
    Veréb, Zoltán
    University of Debrecen, Debrecen, Hungary; University of Szeged, Szeged, Hungary.
    Akhtar, Saeed
    Department of Optometry, College of Applied Medicine, King Saud University, Riyadh, Saudi Arabia.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Ögonkliniken US/LiM.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Ögonkliniken US/LiM.
    Enhanced Regeneration of Corneal Tissue Via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique2015Ingår i: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 21, nr 5-6, s. 1116-1130Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.

  • 137.
    Krzemieniecki, Krzysztof
    et al.
    Department of Chemotherapy, M. Sklodowska-Curie Oncology Center, 31-115, Krakow, Poland .
    Szpyt, Elzbieta
    Bonifratrow Monastyr Hospital, 31-061, Krakow, Poland.
    Rashedi, Iran
    Department of Medical Genetics, Tehran University of Medical Sciences, 14155-6447, Tehran, Iran; Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada .
    Gawron, Katarzyna
    Department of General Biology, Silesian Medical School, 41-808, Zabrze, Poland.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Targeting of solid tumors and blood malignancies by antibody-based therapies - EGFR-pathway as an example2006Ingår i: Central European Journal of Biology, ISSN 1895-104X, E-ISSN 1644-3632, Vol. 1, nr 2, s. 167-182Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A well-coordinated interaction between extracellular signals and intracellular response forms the basis of life within multicellular organisms, with growth factors playing a crucial role in these interactions. Discoveries in recent years have shown that components of the Epidermal Growth Factor (EGF) signaling system have frequently been used by cancer cells to autonomously provide survival and proliferation signals. The main focus of this review is the ErbB epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases including ErbB1/EGFR, ErbB2/HER2/neu, ErbB3/HER3, and ErbB4/HER4 as therapeutic targets. Since the ErbB receptor family regulates cell proliferation through the Ras-mitogen-activated protein kinase (RAS/MAPK) pathway, and cell survival and transformation through the phosphatidylinositol 3-kinase (PI3K/AKT) pathway, pharmacological targeting of these pathways is also discussed. We will also address the clinical studies that have been conducted to evaluate antibody-based therapies mostly on solid tumors and hematologic malignancies. (c) Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.

  • 138.
    Krzyaniak, Matthew D.
    et al.
    University of Alabama, AL 35487 USA; Northwestern University, IL 60208 USA.
    Cruce, Alex
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska fakulteten. University of Alabama, AL 35487 USA.
    Vennam, Preethi
    University of Alabama, AL 35487 USA.
    Lockart, Molly
    University of Alabama, AL 35487 USA.
    Berka, Vladimir
    University of Texas Health Science Centre Houston, TX 77030 USA.
    Tsai, Ah-Lim
    University of Texas Health Science Centre Houston, TX 77030 USA.
    Bowman, Michael K.
    University of Alabama, AL 35487 USA.
    The tetrahydrobiopterin radical interacting with high- and low-spin heme in neuronal nitric oxide synthase - A new indicator of the extent of NOS coupling2016Ingår i: FREE RADICAL BIOLOGY AND MEDICINE, ISSN 0891-5849, Vol. 101, s. 367-377Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Reaction intermediates trapped during the single-turnover reaction of the neuronal ferrous nitric oxide synthase oxygenase domain (Fe(II)nNOS(OX)) show four EPR spectra of free radicals. Fully-coupled nNOSOX with cofactor (tetrahydrobiopterin, BH4) and substrate (L-arginine) forms the typical BH4 cation radical with an EPR spectrum similar to 4.0 mT wide and hyperfine tensors similar to reports for a biopterin cation radical in inducible NOSOX (iNOS(OX)). With excess thiol, nNOSox lacking BH4 and L-arg is known to produce superoxide. In contrast, we find that nNOSOX with BH4 but no L-arg forms two radicals with rather different, fast (similar to 250 mu s at 5 K) and slower (similar to 500 mu s at 20 K), electron spin relaxation rates and a combined similar to 7.0 mT wide EPR spectrum. Rapid freeze-quench CW- and pulsed-EPR measurements are used to identify these radicals and their origin. These two species are the same radical with identical nuclear hyperfine couplings, but with spin-spin couplings to high-spin (4.0 mT component) or low-spin (7.0 mT component) Fe(III) heme. Uncoupled reactions of nNOS leave the enzyme in states that can be chemically reduced to sustain unregulated production of NO and reactive oxygen species in ischemia-reperfusion injury. The broad EPR signal is a convenient indicator of uncoupled nNOS reactions producing low-spin Fe(III) heme.

  • 139.
    Kulikovska, Marina
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Hälsouniversitetet.
    Corneal stromal cell responses to traumatic wounds and topical treatments2015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Background. The cornea has unique anatomic, cellular, molecular, and functional features that lead to important mechanistic differences in the process of repair in comparison with what occurs in skin and other organs. The first observable stromal response in corneal wound healing is keratocyte apoptosis. Shortly thereafter, remaining keratocytes in adjacent areas obtain a fibroblastic phenotype and begin to proliferate and to migrate, transforming into myofibroblasts, a phenotype associated with remodeling of stromal collagen. Return to normalcy following wound healing includes elimination of myofibroblasts and restoration of the quiescent state of the keratocytes. Often, however, a wound healing response results in the persistence of myofibroblasts and their subsequent production of fibrous scar tissue.

    Aims. The overall aim is to understand the role of keratocytes, and their phenotypic variations in a cornea subjected to various types of trauma or treatments. More specific aims are to define expression pattern of alpha-smooth muscle actin (α-SMA) and chaperonin containing T-complex polypeptide 1 (CCT) in ultraviolet radiation wound model, to evaluate the effect of biglycan and platelet rich plasma (PRP) treatment during wound healing after corneal incision, and to characterize the structure of the bioengineered porcine construct and its interaction with stromal cells after implantation.

    Methods. CCT and α-SMA expression level was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in rabbit corneas subjected to ultraviolet radiation (UVR). Effect of biglycan and PRP on keratocyte phenotype and survival was evaluated by immunohistochemistry, and real time PCR using rat corneas after  incisional wounding. Bioengineered porcine construct (BPC) was implanted into rabbit corneas using femtosecond laser-enabled intrastromal keratoplasty (FLISK) and characterized by means of immunohistochemistry, electron microscopy, and in vivo confocal microscopy (IVCM).

    Results and conclusions. In a mild wound, the expression of α-SMA mRNA is followed by expression of mRNA of at least one subunit of the complex folding α-SMA. At protein level, α-SMA is detected in the front line of repopulating keratocytes. Expression levels for both mRNAs decline as the stroma repopulation process progresses.

    Biglycan appears to accelerate corneal wound healing in vivo by modulating myofibroblast apoptosis, resulting in removal of myofibroblasts that may otherwise compromise corneal transparency.

    PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor rich formulation may lead to myofibroblast accumulation by modulation of the TGF-β pathway.

    A cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate normal regenerative healing of the corneal stroma.

    Taken together, results present an interesting possibility to combine BPC implantation and topical biglycan treatment to improve surgical outcome in future studies.

    Delarbeten
    1. The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate, α-smooth muscle actin, during corneal wound healing
    Öppna denna publikation i ny flik eller fönster >>The expression pattern of the subunit of chaperonin containing T-complex polypeptide 1 and its substrate, α-smooth muscle actin, during corneal wound healing
    2005 (Engelska)Ingår i: Acta Ophthalmologica Scandinavica, ISSN 1395-3907, E-ISSN 1600-0420, Vol. 83, nr 5, s. 543-548Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Purpose: This study was designed to demonstrate the expression of the chaperonin containing T-complex polypeptide 1 (CCT) and α-smooth muscle actin (α-SMA), in normal corneas and corneas treated with ultraviolet radiation (UVR). The wound model chosen is previously characterized, the injury is mild and the cornea heals to transparency. Methods: Rabbit corneas were exposed to UVR at the dose producing keratitis. The corneas were allowed to heal for up to 5 days and the paraffin-embedded tissue specimens were double stained and examined morphologically and immunohistochemically. Expression of CCT and α-SMA genes was investigated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Results: There was a front of repopulating keratocytes that showed positive staining for α-SMA after 3 days. The α-SMA mRNA was already strongly expressed after 1 day, whereas the expression level of CCT was increased after 2 days. After 5 days the levels were decreased. By this time the stroma was partly repopulated by keratocytes. Conclusion: In a mild wound, the expression of α-SMA mRNA is followed by expression of mRNA of at least one subunit of the complex folding α-SMA. At protein level, α-SMA is detected in the front line of repopulating keratocytes. Expression levels for both mRNAs decline as the stroma repopulation process progresses. Copyright © Acta Ophthalmol Scand 2005.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-29987 (URN)10.1111/j.1600-0420.2005.00482.x (DOI)15425 (Lokalt ID)15425 (Arkivnummer)15425 (OAI)
    Tillgänglig från: 2009-10-09 Skapad: 2009-10-09 Senast uppdaterad: 2017-12-13
    2. Platelet Rich Plasma Prolongs Myofibroblast Accumulation in Corneal Stroma with Incisional Wound
    Öppna denna publikation i ny flik eller fönster >>Platelet Rich Plasma Prolongs Myofibroblast Accumulation in Corneal Stroma with Incisional Wound
    2015 (Engelska)Ingår i: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 40, nr 11, s. 1102-1110Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Purpose: The purpose of this study was to determine whether platelet rich plasma (PRP) has an effect on corneal stromal cells in a rat model of wound healing following corneal incision. Materials and Methods: The effect of PRP on corneal wound healing in vivo was investigated in a corneal incision wound model in rats. 40 rats were wounded by deep corneal incision, and treated with either topically administered PRP (20 rats) or sodium chloride (20 rats). At 4 hours and 1, 3, and 5 days after incision, α-smooth muscle actin (α SMA), SMAD2 and SMAD3 expression and apoptosis in stromal cells were evaluated by immunohistochemistry, and IL-1β mRNA expression was evaluated by real time PCR.

    Results: PRP treated corneas exhibited reduced stromal cell apoptosis at day 3 and day 5 (p = 0.038, and <0.001, respectively) relative to controls. Interleukin-1β mRNA expression, however, was unchanged in PRP treated corneas relative to controls. Topical PRP treatment resulted in a higher proportion of αSMA-positive myofibroblasts recruited to the wound site relative to control corneas. PRP did not affect activation of SMAD2 but activation of SMAD3 was significantly reduced at day 1 (p=0.001) and dramatically increased at day 5 (p=0.032).

    Conclusions: PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor rich formulation may lead to myofibroblast accumulation by modulation of the TGF-β pathway.

    Ort, förlag, år, upplaga, sidor
    Taylor & Francis, 2015
    Nyckelord
    Platelet rich plasma, corneal wound healing, α-smooth muscle actin, apoptosis, keratocytes
    Nationell ämneskategori
    Cell- och molekylärbiologi Medicinsk bioteknologi
    Identifikatorer
    urn:nbn:se:liu:diva-114698 (URN)10.3109/02713683.2014.978478 (DOI)000369891500004 ()
    Anmärkning

    Funding agencies:Swedish Research Council, Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse, County Council of Ostergotland 

    Vid tiden för disputation förelåg publikationen endast som manuskript

    Tillgänglig från: 2015-03-03 Skapad: 2015-03-03 Senast uppdaterad: 2018-01-22Bibliografiskt granskad
    3. Topical Biglycan Modulates Stromal Cell Apoptosis in Corneal Incisional Wound Model
    Öppna denna publikation i ny flik eller fönster >>Topical Biglycan Modulates Stromal Cell Apoptosis in Corneal Incisional Wound Model
    2015 (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Purpose: The purpose of this study was to determine whether exogenous topicallyapplied biglycan has an effect on corneal stromal cells during wound healing.

    Methods: Enzyme-linked immunosorbent assay (ELISA) was used to determine the effect of biglycan on cell survival in vitro following IL-1β induced cell death. In a corneal incisional wound model, 40 rats were wounded and treated with either topically administered biglycan or sodium chloride (sham control). At 4 hours and 1, 2, and 5 days after incision, α-smooth muscle actin (SMA) expression and apoptosis in stromal cells were evaluated by immunohistochemistry.

    Results: In vitro, biglycan significantly enhanced IL-1β-induced apoptosis of myofibroblasts (p = 0.038), but not corneal fibroblasts. Biglycan treated corneas exhibited reduced stromal cell apoptosis at 4 hours, day 1 and day 5 (p = 0.012, 0.040, and 0.048, respectively) and increased apoptosis at day 3 (p = 0.003) relative to controls. In wounded corneas, biglycan appeared to promote early accumulation of myofibroblasts and initiate an earlier subsequent apoptosis of these cells, relative to controls.

    Conclusion: Biglycan appears to accelerate corneal wound healing in vivo by modulating myofibroblast apoptosis, resulting in removal of myofibroblasts that may otherwise compromise corneal transparency.

    Nyckelord
    Corneal wound healing; biglycan; keratocytes; IL-1β; α-SMA
    Nationell ämneskategori
    Cell- och molekylärbiologi Medicinsk bioteknologi
    Identifikatorer
    urn:nbn:se:liu:diva-114697 (URN)
    Tillgänglig från: 2015-03-03 Skapad: 2015-03-03 Senast uppdaterad: 2018-01-22
    4. Enhanced Regeneration of Corneal Tissue Via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique
    Öppna denna publikation i ny flik eller fönster >>Enhanced Regeneration of Corneal Tissue Via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique
    Visa övriga...
    2015 (Engelska)Ingår i: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 21, nr 5-6, s. 1116-1130Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Severe shortage of donor corneas for transplantation, particularly in developing countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.

    Ort, förlag, år, upplaga, sidor
    Mary Ann Liebert, 2015
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-114699 (URN)10.1089/ten.tea.2014.0562 (DOI)000350549500025 ()25412075 (PubMedID)
    Tillgänglig från: 2015-03-03 Skapad: 2015-03-03 Senast uppdaterad: 2018-01-22Bibliografiskt granskad
  • 140.
    Kuruvilla, Jacob
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Proteomics as a multifaceted tool in medicine and environmental assessment2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Proteomics is evolving as a multi-faceted tool for addressing various biochemical and biomedical queries in the field of scientific research. This involves various stages, ranging from sample preparation to data analysis and biological interpretation. Sample preparation involves isolating proteins from the sample source, purifying and digesting them to initiate shotgun proteomics. Shotgun proteomics identifies proteins by bottom-up proteomic approaches where proteins are identified from the fragmentation spectra of their own peptides.

    Paper I: deals with the simplification of functional characterization for nanoparticles intended for use in biomedicine. Proteomics was constructive in differentiating and semi-quantifying the surface of protein corona. This could be beneficial in predicting the interactions between nanoparticles and a biological entity like the cell or a receptor protein and provide initial valuable information related to targeting, uptake and safety.

    Paper II: deals with understanding effects of TiO2 nanoparticles on endothelial cells. A combinatorial approach, involving transcriptomics and proteomics was used to identify aberrations in the permeability and integrity of endothelial cells and tissues. Our study also investigated the correlation of size and how they motivated a differential cellular response. In case of intravenous entry for nanoparticles in targeted drug delivery systems, endothelial cells are the first barrier encountered by these drug carriers. This evaluation involving endothelial cell response could be very instrumental during the designing of NP based drug delivery systems.

    Paper III: Pharmaceuticals and its metabolites could be very hazardous, especially if its disposal is not managed properly. Since water bodies are the ultimate sink, these chemicals could end up there, culminating in toxicity and other ‘mixture effects’ in combination with other factors. To evaluate the effects of the pharmaceutical, propranolol and climatic factors like low salinity conditions, a microcosm exposure was designed and shotgun proteomics helped understand its impact on mussel gills. In this study too, a combination of transcriptomics and proteomics unveiled molecular mechanisms altered in response to stressors, both individually and in combination.

    Paper IV: An interplay of various factors like EBF1 and PAX5 determines B-cell lineage and commitment. This might have been materialized by direct and transient proteinprotein interactions. A unique method called BioID helped screen relevant interactions in living cells by the application of a promiscuous biotin ligase enzyme capable of tagging proteins through biotinylation based on a proximity radius. Biotinylation of endogenous proteins enabled their selective isolation by exploiting the high affinity of biotin and streptavidin on streptavidin coated agarose beads, leading to their identification by mass spectrometry. The biotinylated proteins were potential candidate interactors of EBF1 and PAX5, which were later confirmed by sequencing techniques like ChIP-Seq, ATAC seq, and visualization techniques like proximity ligation assay (PLA).

    Delarbeten
    1. Surface proteomics on nanoparticles, a step to simplify the rapid prototyping of nanoparticles
    Öppna denna publikation i ny flik eller fönster >>Surface proteomics on nanoparticles, a step to simplify the rapid prototyping of nanoparticles
    2017 (Engelska)Ingår i: Nanoscale Horizons, ISSN 2055-6756, nr 1, s. 55-64Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Engineered nanoparticles for biomedical applications requireincreasing effectiveness in targeting specific cells while preservingnon-target cell’s safety. We developed a surface proteomicsmethod for a rapid and systematic analysis of the interphasebetween the nanoparticle protein corona and the targeting cellsthat could implement the rapid prototyping of nanomedicines.Native nanoparticles entering in a protein-rich liquid mediaquickly form a macromolecular structure called protein corona.This protein structure defines the physical interaction betweennanoparticles and target cells. The surface proteins compose thefirst line of interaction between this macromolecular structureand the cell surface of a target cell. We demonstrated that SUSTU(SUrface proteomics, Safety, Targeting, Uptake) provides aqualitative and quantitative analysis from the protein coronasurface. With SUSTU, the spatial dynamics of the protein coronasurface can be studied. Data from SUSTU would ascertain thenanoparticle functionalized groups exposed at destiny that couldcircumvent preliminary in vitro experiments. Therefore thismethod could implement the analysis of nanoparticle targetingand uptake capability and could be integrated into a rapidprototyping strategy which is a major challenge in nanomaterialscience. Data are available via ProteomeXchange with identifierPXD004636.

    Ort, förlag, år, upplaga, sidor
    Royal Society of Chemistry, 2017
    Nyckelord
    nanoparticle, protein corona, mass spectrometry, surface proteomics, targeting, rapid prototyping, nanomedicine
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-132406 (URN)10.1039/c6nh00162a (DOI)000391450000006 ()
    Projekt
    Nanoimpact; nanoparticles and rapid prototyping
    Tillgänglig från: 2016-11-09 Skapad: 2016-11-09 Senast uppdaterad: 2018-04-17Bibliografiskt granskad
    2. Shotgun proteomics to unravel marine mussel (Mytilus edulis) response to long-term exposure to low salinity and propranolol in a Baltic Sea microcosm
    Öppna denna publikation i ny flik eller fönster >>Shotgun proteomics to unravel marine mussel (Mytilus edulis) response to long-term exposure to low salinity and propranolol in a Baltic Sea microcosm
    Visa övriga...
    2016 (Engelska)Ingår i: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 137, s. 97-106Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Pharmaceuticals, among them the β-adrenoreceptor blocker propranolol, are an important group of environmental contaminants reported in European waters. Laboratory exposure to pharmaceuticals on marine species has been performed without considering the input of the ecosystem flow. To unravel the ecosystem response to long-term exposure to propranolol we have performed long-term exposure to propranolol and low salinity in microcosms. We applied shotgun proteomic analysis to gills of Mytilus edulis from those Baltic Sea microcosms and identified 2071 proteins with a proteogenomic strategy. The proteome profiling patterns from the 587 highly reproductive proteins among groups define salinity as a key factor in the mussel´s response to propranolol. Exposure at low salinity drives molecular mechanisms of adaptation based on a decrease in the abundance of several cytoskeletal proteins, signalling and intracellular membrane trafficking pathway combined with a response towards the maintenance of transcription and translation. The exposure to propranolol combined with low salinity modulates the expression of structural proteins including cilia functions and decrease the expression membrane protein transporters. This study reinforces the environment concerns of the impact of low salinity in combination with anthropogenic pollutants and anticipate critical physiological conditions for the survival of the blue mussel in the northern areas.

    Ort, förlag, år, upplaga, sidor
    Elsevier, 2016
    Nyckelord
    Mytilus edulis, shotgun proteomics, propranolol, low salinity, environmental monitoring, climate change
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-124213 (URN)10.1016/j.jprot.2016.01.010 (DOI)000374368800010 ()
    Forskningsfinansiär
    Vetenskapsrådet
    Anmärkning

    Funding agencies: Swedish Research Council-Natural Science; VR-NT; Carl Trygger Foundation; Oscar and Lilli Lamms Minne Foundation; Angpanneforening Research Foundation; Magnus Bergsvall Foundation; IKERBASQUE; Basque Foundation for Science; VINNOVA; County Council of Oste

    Tillgänglig från: 2016-01-22 Skapad: 2016-01-22 Senast uppdaterad: 2017-11-30Bibliografiskt granskad
  • 141.
    Kuruvilla, Jacob
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Farinha, Ana Paula
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Bayat, Narges
    Department of Biochemistry and Biophysics, Arrhenius laboratories, Stockholm University, Stockholm, Sweden.
    Cristobal, Susana
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. IKERBASQUE, Basque Foundation for Science, Department of Physiology, Faculty of Medicine and Dentristy, University of the Basque Country, Leioa, Spain.
    Surface proteomics on nanoparticles, a step to simplify the rapid prototyping of nanoparticles2017Ingår i: Nanoscale Horizons, ISSN 2055-6756, nr 1, s. 55-64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Engineered nanoparticles for biomedical applications requireincreasing effectiveness in targeting specific cells while preservingnon-target cell’s safety. We developed a surface proteomicsmethod for a rapid and systematic analysis of the interphasebetween the nanoparticle protein corona and the targeting cellsthat could implement the rapid prototyping of nanomedicines.Native nanoparticles entering in a protein-rich liquid mediaquickly form a macromolecular structure called protein corona.This protein structure defines the physical interaction betweennanoparticles and target cells. The surface proteins compose thefirst line of interaction between this macromolecular structureand the cell surface of a target cell. We demonstrated that SUSTU(SUrface proteomics, Safety, Targeting, Uptake) provides aqualitative and quantitative analysis from the protein coronasurface. With SUSTU, the spatial dynamics of the protein coronasurface can be studied. Data from SUSTU would ascertain thenanoparticle functionalized groups exposed at destiny that couldcircumvent preliminary in vitro experiments. Therefore thismethod could implement the analysis of nanoparticle targetingand uptake capability and could be integrated into a rapidprototyping strategy which is a major challenge in nanomaterialscience. Data are available via ProteomeXchange with identifierPXD004636.

  • 142.
    Larsson, Caroline
    et al.
    Linköpings universitet, Utbildningsvetenskap. Linköpings universitet, Institutionen för samhälls- och välfärdsstudier, Lärande, Estetik, Naturvetenskap (LEN).
    Höst, Gunnar
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Medie- och Informationsteknik. Linköpings universitet, Tekniska högskolan.
    Olson, Arthur
    Tibell, Lena
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Medie- och Informationsteknik. Linköpings universitet, Tekniska högskolan.
    Using a Dynamic Physical Model to help Students Visualize the Process of Self-assembly2009Konferensbidrag (Refereegranskat)
  • 143.
    Leijon, Sara
    Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Molecular characterization of cholinergic vestibular and olivocochlear efferent neurons in the rodent brainstem.2010Självständigt arbete på avancerad nivå (masterexamen), 40 poäng / 60 hpStudentuppsats (Examensarbete)
    Abstract [en]

    The neural code from the inner ear to the brain is dynamically controlled by central nervous efferent feedback to the audio-vestibular epithelium. Although such efference provides the basis for a cognitive control of our hearing and balance, we know surprisingly little about this feedback system. This project has investigated the applicability of a transgenic mouse model, expressing a fluorescent protein under the choline-acetyltransferase (ChAT) promoter, for targeting the cholinergic audio-vestibular efferent neurons in the brainstem. It was found that the mouse model is useful for targeting the vestibular efferents, which are fluorescent, but not the auditory efferents, which are not highlighted. This model enables, for the first time, physiological studies of the vestibular efferent neurons and their synaptic inputs. We next assessed the expression of the potassium channel family Kv4, known to generate transient potassium currents upon depolarization. Such potassium currents are found in auditory efferent neurons, but it is not known whether Kv4 subunits are expressed in these neurons. Moreover, it is not known if Kv4 is present and has a function in the vestibular efferent neurons. Double labelling with anti-ChAT and anti-Kv4.2 or Kv4.3 demonstrates that the Kv4.3 subunits are abundantly expressed in audio-vestibular efferents, thus indicating that this subunit is a large contributor to the excitability and firing properties of the auditory efferent neurons, and most probably also for the vestibular efferent neurons. In addition, we also unexpectedly found a strong expression of Kv4.3 in principal cells of the superior olive, the neurons which are important for sound localization.

  • 144.
    LeVine III, Harry
    et al.
    University of Kentucky, Lexington, USA.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Reporters of amyloid structural polymorphism2014Ingår i: Bio-nanoimaging: protein misfolding & aggregation / [ed] Vladimir N. Uversky, Yuri L. Lyubchenko, London: Academic Press, 2014, s. 69-79Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    Misfolding of proteins induced by environmental conditions or by the presence of destabilizing mutations often triggers squestration (aggresome formation) or cellular removal (unfolded protein response (UPR), autophagy) intracellular responses. Extracellular aggregates are phagocytosed or endocytosed by macrophages in the periphery and microglia and astrocytes in the brain and central nervous system. In some cases a highly stable altemative assembly structure, an amyloid fibril, is formed that is highly resistant to degadation and thus accumulates.

  • 145.
    Li, Wei
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Johnson, Henrik
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Experimentell patologi. Linköpings universitet, Hälsouniversitetet.
    Yuan, Xi-Ming
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Experimentell patologi. Linköpings universitet, Hälsouniversitetet.
    Jonasson, Lena
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för kardiovaskulär medicin. Linköpings universitet, Hälsouniversitetet.
    7beta-hydroxycholesterol induces natural killer cell death via oxidative lysosomal destabilization2009Ingår i: Free radical research, ISSN 1071-5762, E-ISSN 1029-2470, Vol. 43, nr 11, s. 1072-1079Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Peripheral natural killer (NK) cells are reduced in patients with coronary artery disease and highly susceptible to apoptosis induced by oxidized lipids including 7beta-hydroxycholesterol (7betaOH) in vitro. The present study aimed to further explore the mechanisms behind 7betaOH-mediated cytotoxicity to human NK cells. Human NK cells were purified and treated with 7betaOH in different concentrations and times. Cell death, lysosomal and mitochondrial permeabilization and reactive oxygen species (ROS) production were then analysed. The 7betaOH induced time and dose dependent apoptosis and necrosis in human NK cells, which was preceded by loss of lysosomal integrity and enhanced ROS production. At later time points, the mitochondrial membrane permeability in 7betaOH-treated cells was significantly increased. The findings indicate that 7betaOH induces human NK cell death through early lysosomal permeabilization and consequent oxidative stress. The data further suggest that 7betaOH may induce immune disturbances in clinical settings such as atherosclerosis.

  • 146.
    Liang, Wenzhao
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin. Jilin University, Peoples R China.
    Ward, Liam
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin.
    Karlsson, Helen
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin. Linköpings universitet, Medicinska fakulteten.
    Ljunggren, Stefan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin. Linköpings universitet, Medicinska fakulteten.
    Li, Wei
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Barn- och ungdomskliniken i Linköping.
    Lindahl, Mats
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten.
    Yuan, Ximing
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Region Östergötland, Hjärt- och Medicincentrum, Arbets- och miljömedicin. Linköpings universitet, Medicinska fakulteten.
    Distinctive proteomic profiles among different regions of human carotid plaques in men and women2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, nr 26231Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The heterogeneity of atherosclerotic tissue has limited comprehension in proteomic and metabolomic analyses. To elucidate the functional implications, and differences between genders, of atherosclerotic lesion formation we investigated protein profiles from different regions of human carotid atherosclerotic arteries; internal control, fatty streak, plaque shoulder, plaque centre, and fibrous cap. Proteomic analysis was performed using 2-DE with MALDI-TOF, with validation using nLC-MS/MS. Protein mapping of 2-DE identified 52 unique proteins, including 15 previously unmapped proteins, of which 41 proteins were confirmed by nLC-MS/MS analysis. Expression levels of 18 proteins were significantly altered in plaque regions compared to the internal control region. Nine proteins showed site-specific alterations, irrespective of gender, with clear associations to extracellular matrix remodelling. Five proteins display gender-specific alterations with 2-DE, with two alterations validated by nLC-MS/MS. Gender differences in ferritin light chain and transthyretin were validated using both techniques. Validation of immunohistochemistry confirmed significantly higher levels of ferritin in plaques from male patients. Proteomic analysis of different plaque regions has reduced the effects of plaque heterogeneity, and significant differences in protein expression are determined in specific regions and between genders. These proteomes have functional implications in plaque progression and are of importance in understanding gender differences in atherosclerosis.

  • 147.
    Liin, Sara
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelning för neurobiologi. Linköpings universitet, Medicinska fakulteten.
    Yazdi, Samira
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelning för neurobiologi. Linköpings universitet, Medicinska fakulteten.
    Ramentol, Rosamary
    Univ Miami, FL 33136 USA.
    Barro-Soria, Rene
    Univ Miami, FL 33136 USA.
    Larsson, H. Peter
    Univ Miami, FL 33136 USA.
    Mechanisms Underlying the Dual Effect of Polyunsaturated Fatty Acid Analogs on Kv7.12018Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 24, nr 11, s. 2908-2918Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Polyunsaturated fatty acid (PUFA) analogs represent a new class of potential anti-arrhythmic K(V)7.1 and K(V)7.1+KCNE1 channel activators. In this study, we describe dual independent activating effects of negatively charged PUFA analogs on K(V)7.1 and K(V)7.1+KCNE1 that are dependent on discrete channel motifs. PUFA analogs are critically dependent on K326 in S6 of K(V)7.1 to increase the maximum conductance and critically dependent on specific S4 arginines in K(V)7.1 to shift the voltage dependence of channel opening toward negative voltages. Our findings provide insights into how K(V)7.1+KCNE1 activators may interact electrostatically both with the pore domain and the voltage-sensing domain to augment channel activity. We believe that the molecular understanding of how PUFA analogs induce dual independent activating effects is an important step toward the development of effective anti-arrhythmic drugs that target K(V)7.1 channels.

  • 148.
    Lindberg, Lina
    Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Evaluation of Different Enzymes and Yeasts, and Their Impact on Bioethanol Production Based on Debranned Wheat2009Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Bioethanol is a fuel of tomorrow, and progress in the use of enzymes and reduction of non-fermentable materials by debranning will probably be a part to make it more economical with low environmental impact.

     

    Ethanol production based on debranned wheat was optimized in this study by batch experiments as well as continuous experiments in laboratory scale. Enzymes from Novozymes and Genencor were compared and no significant differences were discovered between the different set of enzymes. The yeast strains Ethanol Red and AmyloFerm were compared with traditional baker’s yeast and baker’s yeast were surprisingly the fastest to ferment, but Ethanol Red had higher viability during fermentation. Protease addition during saccharification does not seem to improve fermentation with baker’s yeast. Prolonged liquefaction and saccharification time does probably not have any large impact on glucose yield. The continuous lab-scale process has a potential to be a realistic model but the stirring has to be improved and the pipe diameter increased.

  • 149.
    Lindgren, Petter
    et al.
    Department of Biological Agents, Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Uemå, Sweden.
    Myrtennäs, Kerstin
    Department of Biological Agents, Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden..
    Forsman, Mats
    Department of Biological Agents, Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Johansson, Anders
    Department of Clinical Microbiology and Molecular Infection Medicine Sweden (MIMS), Umeå University, Sweden.
    Stenberg, Per
    Department of Biological Agents, Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden and Department of Ecology and Environmental Science (EMG), Umeå University, Sweden.
    Nordgaard, Anders
    Linköpings universitet, Institutionen för datavetenskap, Statistik och maskininlärning. Linköpings universitet, Filosofiska fakulteten. Swedish Police Auhtority, National Forensic Centre (NFC).
    Ahlinder, Jon
    Department of Biological Agents, Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
    A likelihood ratio-based approach for improved source attribution in microbiological forensic investigations2019Ingår i: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Forensic Science International, ISSN 0379-0738, Vol. 302, artikel-id 109869Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A common objective in microbial forensic investigations is to identify the origin of a recovered pathogenic bacterium by DNA sequencing. However, there is currently no consensus about how degrees of belief in such origin hypotheses should be quantified, interpreted, and communicated to wider audiences. To fill this gap, we have developed a concept based on calculating probabilistic evidential values for microbial forensic hypotheses. The likelihood-ratio method underpinning this concept is widely used in other forensic fields, such as human DNA matching, where results are readily interpretable and have been successfully communicated in juridical hearings. The concept was applied to two case scenarios of interest in microbial forensics: (1) identifying source cultures among series of very similar cultures generated by parallel serial passage of the Tier 1 pathogen Francisella tularensis, and (2) finding the production facilities of strains isolated in a real disease outbreak caused by the human pathogen Listeria monocytogenes. Evidence values for the studied hypotheses were computed based on signatures derived from whole genome sequencing data, including deep-sequenced low-frequency variants and structural variants such as duplications and deletions acquired during serial passages. In the F. tularensis case study, we were able to correctly assign fictive evidence samples to the correct culture batches of origin on the basis of structural variant data. By setting up relevant hypotheses and using data on cultivated batch sources to define the reference populations under each hypothesis, evidential values could be calculated. The results show that extremely similar strains can be separated on the basis of amplified mutational patterns identified by high-throughput sequencing. In the L. monocytogenes scenario, analyses of whole genome sequence data conclusively assigned the clinical samples to specific sources of origin, and conclusions were formulated to facilitate communication of the findings. Taken together, these findings demonstrate the potential of using bacterial whole genome sequencing data, including data on both low frequency SNP signatures and structural variants, to calculate evidence values that facilitate interpretation and communication of the results. The concept could be applied in diverse scenarios, including both epidemiological and forensic source tracking of bacterial infectious disease outbreaks.

  • 150.
    Link, Verena M
    et al.
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA; Faculty of Biology, Division of Evolutionary Biology, Ludwig-Maximilian University of Munich, Munich, Germany.
    Duttke, Sascha H
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Chun, Hyun B
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Holtman, Inge R
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA; Department of Neuroscience, Section Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
    Westin, Emma
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Hoeksema, Marten A
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Abe, Yohei
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Skola, Dylan
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Romanoski, Casey E
    Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA.
    Tao, Jenhan
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Fonseca, Gregory J
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Troutman, Ty D
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Spann, Nathanael J
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Strid, Tobias
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Sakai, Mashito
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Yu, Miao
    Ludwig Institute for Cancer Research, La Jolla, CA, USA.
    Hu, Rong
    Ludwig Institute for Cancer Research, La Jolla, CA, USA.
    Fang, Rongxin
    Ludwig Institute for Cancer Research, La Jolla, CA, USA.
    Metzler, Dirk
    Faculty of Biology, Division of Evolutionary Biology, Ludwig-Maximilian University of Munich, Munich, Germany.
    Ren, Bing
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA; Ludwig Institute for Cancer Research, La Jolla, CA, USA.
    Glass, Christopher K
    Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA; Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
    Analysis of Genetically Diverse Macrophages Reveals Local and Domain-wide Mechanisms that Control Transcription Factor Binding and Function.2018Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 173, nr 7, s. 1796-1809.e17, artikel-id S0092-8674(18)30511-7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.

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