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  • 251. Molnar, Matyas
    et al.
    Friberg, Peter
    Fu, Ying
    Brisslert, Mikael
    Adams, Michael
    Chen, Yun
    Effects of Quantum Dot Labeling on Endothelial Progenitor Cell Function and Viability2010In: Cell MedicineArticle in journal (Refereed)
    Abstract [en]

    Endothelial progenitor cells (EPC) play an important role in repairing damaged endothelium. An effective imaging method for in vivo tracking of EPCs is essential for understanding EPC-based cell therapy. Fluorescent quantum dots (QDs) have attractive optical characteristics such as extreme brightness and photostability. QDs are currently being investigated as probes for stem cell labeling; however, there is concern about whether QDs can be used safely. We investigated whether quantum dot (QD) labeling would influence EPC viability and function. Rat bone marrow-derived EPCs were cultured and characterized. The cells were labeled with near-infrared-emitting, carboxyl-coated QDs (8 nM) for 24 h. QD labeling efficiency was higher than 97%. Using WST-1 assay, we showed that the viability of the QD-labeled EPCs was not different from that of the control EPCs. Moreover, QD labeling did not influence the ability of EPCs to form capillary tubes on Matrigel and to migrate. The percentage of QD-positive cells decreased with time, probably due to the rapid division of EPCs. These data suggest that the carboxyl-coated QD705 can be useful for labeling EPCs without interrupting their viability and functions.

  • 252.
    Molnár, Mátyás
    KTH, School of Biotechnology (BIO).
    Quantum dot biomarkers for tracking endothelial progenitor cells and activated endothelium2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The development of new visualization techniques is essential in the field of bioimaging to gain deeper knowledge about various medical and biological events at the cellular and molecular levels. Quantum dots (QDs) are semiconductor nanocrystals which are opening new perspectives in biotechnology due to their powerful optical properties and high stability.  Utilization of QDs instead of commonly used organic dyes in labeling techniques adds several advantages, such as longer signal observation time of the biological samples; convenient multicolor labeling; deep tissue and organ tracking. Furthermore, more information can be obtained about the environment where QDs are located by characterizing the optical behavior of the QDs.

    In this thesis work carboxyl coated QDs were used to label endothelial progenitor cells (EPCs). EPCs play an important role in cardiovascular diseases and the repair of the damaged endothelium, therefore an efficient in vivo tracking method is essential to understand the therapeutic potential of these cells. Our findings show an effective, cytoplasmic labeling of EPCs. Cell viability assay shows that QDs are not toxic to the cells in the concentration and incubation time tested in the study and QDs does not change the cell functions.

    Furthermore commercially available QDs were functionalized with VCAM-1 binding peptide (VCAM-1BP) to specifically detect activated endothelial cells and activated endothelial wall in the aorta. Endothelial wall of the arteries are activated with VCAM-1 in the early stages of atherosclerosis, therefore early detection of this chronic disease may be performed with VCAM-1BP functionalized QDs. We found that VCAM-1BP functionalized QDs were able to detect the activated endothelium specifically.

    The optical behavior of the QDs in different conditions and bio-systems were also characterized. It was found that several circumstances like different coating ligands can modify the fluorescence of the QDs. We observed that QD fluorescence intensity changes with different concentrations of Na+ and K+. We also found that significant blue-shift in QD peak emission occurs when QDs are internalized by cells and when functionalized QDs interact with cell surface molecule.

    This thesis work provides efficient labeling techniques for cells and tissues with QDs and important knowledge about the optical behavior of QDs in bio-systems. These results give further perspectives to the future application of QD biomarkers as tracking agent and optical sensors for the different environmental changes in bio-systems.

    List of papers
    1. Molecular imaging of inflammation-activated endothelium using bio-conjugated quantum dots
    Open this publication in new window or tab >>Molecular imaging of inflammation-activated endothelium using bio-conjugated quantum dots
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Bioimaging, Quantum dots, Regenerative medicine, Nanotechnology
    National Category
    Medical Engineering Nano Technology Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-182951 (URN)
    Available from: 2012-10-19 Created: 2012-10-19 Last updated: 2012-10-19
    2. Significant blue-shift of luminescence peak from VCAM-1-binding peptide-functionalized quantum dots upon binding with VCAM-1
    Open this publication in new window or tab >>Significant blue-shift of luminescence peak from VCAM-1-binding peptide-functionalized quantum dots upon binding with VCAM-1
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Bioimaging, Confocal microscopy, Quantum dots, Regenerative medicine, Nanotechnology
    National Category
    Nano Technology Medical Engineering Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-182950 (URN)
    Available from: 2012-10-19 Created: 2012-10-19 Last updated: 2012-10-19
    3. Effects of Quantum Dot Labeling on Endothelial Progenitor Cell Function and Viability
    Open this publication in new window or tab >>Effects of Quantum Dot Labeling on Endothelial Progenitor Cell Function and Viability
    Show others...
    2010 (English)In: Cell MedicineArticle in journal (Refereed) Published
    Abstract [en]

    Endothelial progenitor cells (EPC) play an important role in repairing damaged endothelium. An effective imaging method for in vivo tracking of EPCs is essential for understanding EPC-based cell therapy. Fluorescent quantum dots (QDs) have attractive optical characteristics such as extreme brightness and photostability. QDs are currently being investigated as probes for stem cell labeling; however, there is concern about whether QDs can be used safely. We investigated whether quantum dot (QD) labeling would influence EPC viability and function. Rat bone marrow-derived EPCs were cultured and characterized. The cells were labeled with near-infrared-emitting, carboxyl-coated QDs (8 nM) for 24 h. QD labeling efficiency was higher than 97%. Using WST-1 assay, we showed that the viability of the QD-labeled EPCs was not different from that of the control EPCs. Moreover, QD labeling did not influence the ability of EPCs to form capillary tubes on Matrigel and to migrate. The percentage of QD-positive cells decreased with time, probably due to the rapid division of EPCs. These data suggest that the carboxyl-coated QD705 can be useful for labeling EPCs without interrupting their viability and functions.

    Keywords
    Endothelial progenitor cells, Quantum dots, Cell labeling, Migration, Capillary tube formation
    National Category
    Nano Technology Cell Biology Medical Engineering
    Identifiers
    urn:nbn:se:uu:diva-182949 (URN)10.3727/215517910X451603 (DOI)
    Available from: 2012-10-19 Created: 2012-10-19 Last updated: 2012-10-19
    4. Optical characterization of colloidal CdSe quantum dots in endothelial progenitor cells.
    Open this publication in new window or tab >>Optical characterization of colloidal CdSe quantum dots in endothelial progenitor cells.
    2010 (English)In: Journal of Nanobiotechnology, E-ISSN 1477-3155, Vol. 8, no 1, p. 2-Article in journal (Refereed) Published
    Abstract [en]

    We have quantitatively analyzed the confocal spectra of colloidal quantum dots (QDs) in rat endothelial progenitor cells (EPCs) by using Leica TCS SP5 Confocal Microscopy System. Comparison of the confocal spectra of QDs located inside and outside EPCs revealed that the interaction between the QDs and EPCs effectively reduces the radius of the exciton confinement inside the QDs so that the excitonic energy increases and the QD fluorescence peak blueshifts. Furthermore, the EPC environment surrounding the QDs shields the QDs so that the excitation of the QDs inside the cells is relatively weak, whereas the QDs outside the cells can be highly excited. At high excitations, the occupation of the ground excitonic state in the QD outside the cells becomes saturated and high-energy states excited, resulting in a large relaxation energy and a broad fluorescence peak. This permits, in concept, to use QD biomarkers to monitor EPCs by characterizing QD fluorescence spectra.

    Keywords
    Quantum dots, Confocal microscopy, Endothelial progenitor cells, Regenerative medicine
    National Category
    Physical Sciences Biological Sciences Nano Technology
    Identifiers
    urn:nbn:se:uu:diva-182929 (URN)10.1186/1477-3155-8-2 (DOI)20205887 (PubMedID)
    Available from: 2012-10-19 Created: 2012-10-18 Last updated: 2024-02-15
    5. Effects of K+ and Na+ ions on the fluorescence of colloidal CdSe/CdS and CdSe/ZnS quantum dots
    Open this publication in new window or tab >>Effects of K+ and Na+ ions on the fluorescence of colloidal CdSe/CdS and CdSe/ZnS quantum dots
    Show others...
    2011 (English)In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 155, p. 823-830Article in journal (Refereed) Published
    Abstract [en]

    In this work we studied the effects of K+ and Na+ ions on fluorescence properties of the colloidal quantum dots (QDs). It was found that the fluorescence intensity was enhanced when low concentration of ions was introduced to QD solutions, while it became weakened when ion concentrations reached their physiological levels in many bio environments. Such fluorescence changes can be attributed to interactions between QD surface ligands and ions as well as the Coulomb potential of ions that displaces the wave functions of the electron and hole confined inside the QD. These results are important for understanding the influence of different biological environments, such as extracellular and intracellular compartments, on optical properties of colloidal QDs.

    Keywords
    Colloidal quantum dots (QDs), Fluorescence properties of QDs, Electron and hole confinements in QDs, Effects of K<sup>+</sup> and Na<sup>+</sup> ions
    National Category
    Biological Sciences Chemical Sciences Physical Sciences Nano Technology
    Identifiers
    urn:nbn:se:uu:diva-182931 (URN)10.1016/j.snb.2011.01.055 (DOI)
    Available from: 2012-10-19 Created: 2012-10-18 Last updated: 2017-12-07
    6. Role of surface ligands in optical properties of colloidal CdSe/CdS quantum dots
    Open this publication in new window or tab >>Role of surface ligands in optical properties of colloidal CdSe/CdS quantum dots
    Show others...
    2011 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 13, no 13, p. 5848-5854Article in journal (Refereed) Published
    Abstract [en]

    In order to study the role of surface ligands in determining optical properties of colloidal quantum dots (QDs), we have selectively fabricated and studied CdSe/CdS core-shell QDs with strongly confined electron and hole states attached with commonly used surface ligands. Optical properties, viz. absorption and fluorescence of these QDs, are characterized from which salient changes have been observed for different ligand substitutions which, through theoretical analysis, can be associated with electronic structure properties of the QD-ligand composite systems, in particular localization of wave functions of electrons and holes in the QDs and the band matching of the HOMO-LUMO gap of the ligands. The findings can be utilized to facilitate the understanding and optimization of properties of QD biomarkers with functionalizing surface ligands for targeting cellular objects.

    National Category
    Chemical Sciences Nano Technology Physical Sciences
    Identifiers
    urn:nbn:se:uu:diva-182928 (URN)
    Available from: 2012-10-19 Created: 2012-10-18 Last updated: 2017-12-07
  • 253. Moren, Anita
    et al.
    Bellomo, Claudia
    Tsubakihara, Yutaro
    Kardassis, Dimitri
    Mikulits, Wolfgang
    Heldin, Carl-Henrik
    Moustakas, Aristidis
    LXRα limits the pro-fibrotic action of TGFβ in liver cancer-associated fibroblastsManuscript (preprint) (Other academic)
  • 254.
    Mutvei, Anders P.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Weill Cornell Med, Sandra & Edward Meyer Canc Ctr, Belfer Res Bldg,413 E 69th St, New York, NY 10021 USA.;Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Nobels Vag 16,KI Solna Campus Karolinska Inst, SE-17177 Stockholm, Sweden..
    Nagiec, Michal J.
    Weill Cornell Med, Sandra & Edward Meyer Canc Ctr, Belfer Res Bldg,413 E 69th St, New York, NY 10021 USA..
    Hamann, Jens C.
    Weill Cornell Med, Sandra & Edward Meyer Canc Ctr, Belfer Res Bldg,413 E 69th St, New York, NY 10021 USA..
    Kim, Sang Gyun
    Weill Cornell Med, Sandra & Edward Meyer Canc Ctr, Belfer Res Bldg,413 E 69th St, New York, NY 10021 USA..
    Vincent, C. Theresa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. NYU, Sch Med, Dept Microbiol, New York, NY 10016 USA..
    Blenis, John
    Weill Cornell Med, Sandra & Edward Meyer Canc Ctr, Belfer Res Bldg,413 E 69th St, New York, NY 10021 USA..
    Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 1416Article in journal (Refereed)
    Abstract [en]

    The kinase mTOR complex 1 (mTORC1) promotes cellular growth and is frequently dysregulated in cancers. In response to nutrients, mTORC1 is activated on lysosomes by Rag and Rheb guanosine triphosphatases (GTPases) and drives biosynthetic processes. How limitations in nutrients suppress mTORC1 activity remains poorly understood. We find that when amino acids are limited, the Rap1-GTPases confine lysosomes to the perinuclear region and reduce lysosome abundance, which suppresses mTORC1 signaling. Rap1 activation, which is independent of known amino acid signaling factors, limits the lysosomal surface available for mTORC1 activation. Conversely, Rap1 depletion expands the lysosome population, which markedly increases association between mTORC1 and its lysosome-borne activators, leading to mTORC1 hyperactivity. Taken together, we establish Rap1 as a critical coordinator of the lysosomal system, and propose that aberrant changes in lysosomal surface availability can impact mTORC1 signaling output.

    Download full text (pdf)
    FULLTEXT01
  • 255.
    Naboulsi, Rakan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Swedish Univ Agr Sci, Dept Anim Breeding & Genet, S-75007 Uppsala, Sweden.;Texas A&M Univ, Dept Vet Integrat Biosci, College Stn, TX 77843 USA..
    Younis, Shady
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Ain Shams Univ, Dept Anim Breeding & Genet, Cairo 11241, Egypt.;Stanford Univ, Dept Med, Div Immunol & Rheumatol, Stanford, CA 94305 USA..
    ZBED6 regulates Igf2 expression partially through its regulation of miR483 expression2021In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 19484Article in journal (Refereed)
    Abstract [en]

    The expression of Igf2 in mammals shows a complex regulation involving multiple promoters and epigenetic mechanisms. We previously identified a novel regulatory mechanism based on the interaction between the transcriptional factor ZBED6 and Igf2 intron. Disruption of the ZBED6-Igf2 interaction leads to a dramatic up-regulation of IGF2 expression postnatally. In the current study we characterize an additional layer of regulation involving miR483 encoded by another Igf2 intron. We found a highly significant up-regulation of miR483 expression when the ZBED6-Igf2 axis is disrupted in transgenic mice. Furthermore, CRISPR/Cas9 mediated knock-out of miR483 in C2C12 myoblast cells, both wild-type and cells with disrupted ZBED6-Igf2 axis (Igf2(dGGCT)), resulted in down-regulation of Igf2 expression and a reduced proliferation rate. This was further validated using miR483 mimics and inhibitors. RNA-seq analysis revealed a significant enrichment of genes involved in the PI3K-Akt signaling pathway among genes down-regulated in miR483(-/-) cells, including Igf2 down-regulation. The opposite pattern was observed in Igf2(dGGCT) cells, where Igf2 is up-regulated. Our data suggest a positive feedback between miR483 and Igf2 promoter activity, strongly affecting how ZBED6 controls Igf2 expression in various cell types.

    Download full text (pdf)
    FULLTEXT01
  • 256. Nag, Shalini
    et al.
    Larsson, Mårten
    Institute of Molecular and Cell Biology, A*STAR, Singapore.
    Robinson, Robert C
    Burtnick, Leslie D
    Gelsolin: the tail of a molecular gymnast2013In: Cytoskeleton (Hoboken, N.J.), ISSN 1949-3592, Vol. 70, no 7, p. 360-384Article, review/survey (Refereed)
    Abstract [en]

    Gelsolin superfamily members are Ca(2+) -dependent, multidomain regulators of the actin cytoskeleton. Calcium binding activates gelsolin by inducing molecular gymnastics (large-scale conformational changes) that expose actin interaction surfaces by releasing a series of latches. A specialized tail latch has distinguished gelsolin within the superfamily. Active gelsolin exhibits actin filament severing and capping, and actin monomer sequestering activities. Here, we analyze a combination of sequence, structural, biophysical and biochemical data to assess whether the molecular plasticity, regulation and actin-related properties of gelsolin are also present in other superfamily members. We conclude that all members of the superfamily will be able to transition between a compact conformation and a more open form, and that most of these open forms will interact with actin. Supervillin, which lacks the severing domain 1 and the F-actin binding-site on domain 2, is the clear exception. Eight calcium-binding sites are absolutely conserved in gelsolin, adseverin, advillin and villin, and compromised to increasing degrees in CapG, villin-like protein, supervillin and flightless I. Advillin, villin and supervillin each contain a potential tail latch, which is absent from CapG, adseverin and flightless I, and ambiguous in villin-like protein. Thus, calcium regulation will vary across the superfamily. Potential novel isoforms of the superfamily suggest complex regulation at the gene, transcript and protein levels. We review animal, clinical and cellular data that illuminate how the regulation of molecular flexibility in gelsolin-like proteins permits cells to exploit the force generated from actin polymerization to drive processes such as cell movement in health and disease.

  • 257. Nag, Shalini
    et al.
    Ma, Qing
    Wang, Hui
    Chumnarnsilpa, Sakesit
    Lee, Wei Lin
    Larsson, Mårten
    Kannan, Balakrishnan
    Hernandez-Valladares, Maria
    Burtnick, Leslie D
    Robinson, Robert C
    Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 33, p. 13713-8Article in journal (Refereed)
    Abstract [en]

    Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca(2+) and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca(2+) locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.

  • 258.
    Nahálková, Jarmila
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The protein-interaction network with functional roles in tumorigenesis, neurodegeneration, and aging2016In: Molecular and Cellular Biochemistry, ISSN 0300-8177, E-ISSN 1573-4919, Vol. 423, no 1-2, p. 187-196Article, review/survey (Refereed)
    Abstract [en]

    The present review summarizes the knowledge about a protein-interaction network, which includes proteins with significant functions in the mechanisms of aging and age-related diseases. All the detected interacting proteins TPPII, p53, MYBBP1A, CDK2 and SIRT7, SIRT6, and CD147 are suitable for the development of antitumor therapeutics and treatments for diseases of aging. TPPII and SIRT6 directly affect glucose metabolism which drive malignant growth. In addition, SIRT6 activators are attractive candidates for Alzheimer's disease (AD) due to the protection effect of SIRT6 overexpression from DNA damage. TPPII activity exhibits a decreasing effect on mTOR signaling, and its requirement for the degradation of A beta peptides in the human fibroblasts suggests that it has dual functions in tumorigenesis and AD-related pathology. Likewise, the direct promotion of the invasiveness of breast epithelial cells and the contribution to the A beta degradation by stimulating the matrix metalloproteinases production suggest a double functional role for CD147. An association of the partial portion of cellular CD147 to gamma-secretase further supports the functional relation to AD pathology. The animal and cellular models with downregulated or knockout TPPII, p53, SIRT6, SIRT7, and MYBBP1A expression levels illustrate similar functions of the interacting proteins. They demonstrate similar effects on the length of life span, premature aging, and lipid metabolism. The presented protein-interaction network is relevant to the discoveries of the mechanisms of tumorigenesis, aging, and neurodegeneration.

  • 259.
    Nazir, Madiha
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Senkowski, Wojciech
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nyberg, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Blom, Kristin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Edqvist, Per-Henrik D
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Andersson, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Gustafsson, Mats G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Fryknäs, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Targeting tumor cells based on Phosphodiesterase 3A expression2017In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 361, no 2, p. 308-315Article in journal (Refereed)
    Abstract [en]

    We and others have previously reported a correlation between high phosphodiesterase 3 A (PDE3A) expression and selective sensitivity to phosphodiesterase (PDE) inhibitors. This indicates that PDE3A could serve both as a drug target and a biomarker of sensitivity to PDE3 inhibition. In this report, we explored publicly available mRNA gene expression data to identify cell lines with different PDE3A expression. Cell lines with high PDE3A expression showed marked in vitro sensitivity to PDE inhibitors zardaverine and quazinone, when compared with those having low PDE3A expression. Immunofluorescence and immunohistochemical stainings were in agreement with PDE3A mRNA expression, providing suitable alternatives for biomarker analysis of clinical tissue specimens. Moreover, we here demonstrate that tumor cells from patients with ovarian carcinoma show great variability in PDE3A protein expression and that level of PDE3A expression is correlated with sensitivity to PDE inhibition. Finally, we demonstrate that PDE3A is highly expressed in subsets of patient tumor cell samples from different solid cancer diagnoses and expressed at exceptional levels in gastrointestinal stromal tumor (GIST) specimens. Importantly, vulnerability to PDE3 inhibitors has recently been associated with co-expression of PDE3A and Schlafen family member 12 (SLFN12). We here demonstrate that high expression of PDE3A in clinical specimens, at least on the mRNA level, seems to be frequently associated with high SLFIV12 expression. In conclusion, PDE3A seems to be both a promising biomarker and drug target for individualized drug treatment of various cancers.

  • 260.
    Nebie, Ouada
    et al.
    Taipei Med Univ, Coll Biomed Engn, Grad Inst Biomed Mat & Tissue Engn, 250 Wu Xing St, Taipei 11031, Taiwan..
    Barro, Lassina
    Taipei Med Univ, Coll Biomed Engn, Int PhD Program Biomed Engn, Taipei, Taiwan..
    Wu, Yu-Wen
    Taipei Med Univ, Coll Biomed Engn, Grad Inst Biomed Mat & Tissue Engn, 250 Wu Xing St, Taipei 11031, Taiwan..
    Knutson, Folke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Buee, Luc
    Univ Lille, CHU Lille, INSERM, U1172,Lille Neurosci & Cognit, Lille, France..
    Devos, David
    Univ Lille, CHU Lille, INSERM, U1172,Lille Neurosci & Cognit, Lille, France..
    Peng, Chih-Wei
    Taipei Med Univ, Coll Biomed Engn, Int PhD Program Biomed Engn, Taipei, Taiwan.;Taipei Med Univ, Coll Biomed Engn, Sch Biomed Engn, Taipei, Taiwan..
    Blum, David
    Univ Lille, CHU Lille, INSERM, U1172,Lille Neurosci & Cognit, Lille, France..
    Burnouf, Thierry
    Taipei Med Univ, Coll Biomed Engn, Grad Inst Biomed Mat & Tissue Engn, 250 Wu Xing St, Taipei 11031, Taiwan.;Taipei Med Univ, Coll Biomed Engn, Int PhD Program Biomed Engn, Taipei, Taiwan.;Taipei Med Univ, Int PhD Program Cell Therapy & Regenerat Med, Taipei, Taiwan.;Taipei Med Univ, PhD Program Mind Brain & Consciousness, Taipei, Taiwan..
    Heat-treated human platelet pellet lysate modulates microglia activation, favors wound healing and promotes neuronal differentiation in vitro2021In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 32, no 2, p. 226-237Article in journal (Refereed)
    Abstract [en]

    The neurorestorative efficacy of human platelet lysates in neurodegenerative disorders is still under investigation. Platelets prepared from standard and pathogen reduced platelet concentrates were pelletized, washed, concentrated, and subjected to freeze-thawing. The lysate was heated to 56 degrees C for 30 min and characterized. Toxicity was evaluated using SH-SY5Y neuroblastoma, BV-2 microglial, and EA-hy926 endothelial cells. Inflammatory activity was tested by examining tumor necrosis factor (TNF) and cyclooxygenase (COX)-2 expressions by BV-2 microglia with or without stimulation by lipopolysaccharides (LPS). The capacity to stimulate wound healing was evaluated by a scratch assay, and the capacity to differentiate SH-SY5Y into neurons was also examined. Platelet lysates contained a range of neurotrophins. They were not toxic to SH-SY5Y, EA-hy926, or BV-2 cells, did not induce the expression of TNF or COX-2 inflammatory markers by BV-2 microglia, and decreased inflammation after LPS stimulation. They stimulated the wound closure in the scratch assay and induced SH-SY5Y differentiation as revealed by the increased length of neurites as well as beta 3-tubulin and neurofilament staining. These data confirm the therapeutic potential of platelet lysates in the treatment of disorders of the central nervous system and support further evaluation as novel neurorestorative biotherapy in preclinical models.

  • 261.
    Nebie, Ouada
    et al.
    Taipei Med Univ, Coll Biomed Engn, Grad Inst Biomed Mat & Tissue Engn, 250 Wu Xing St, Taipei 11031, Taiwan.
    Devos, David
    Univ Lille, Inserm, F-59000 Lille, France.
    Vingtdeux, Valerie
    CHU Lille, Lille Neurosci & Cognit Degenerat & Vasc Cogn Dis, UMRS1171, F-59000 Lille, France.
    Barro, Lassina
    Taipei Med Univ, Coll Biomed Engn, Int PhD Program Biomed Engn, Taipei, Taiwan.
    Devedjian, Jean-Christophe
    Jonneaux, Aurelie
    Univ Lille, Inserm, F-59000 Lille, France.
    Chou, Ming-Li
    Taipei Med Univ, Coll Biomed Engn, Grad Inst Biomed Mat & Tissue Engn, 250 Wu Xing St, Taipei 11031, Taiwan;St Antoine Hosp, INSERM UMRS 938 CdR St Antoine, Paris, France.
    Bordet, Regis
    Univ Lille, Inserm, F-59000 Lille, France.
    Buee, Luc
    Knutson, Folke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Blum, David
    CHU Lille, Lille Neurosci & Cognit Degenerat & Vasc Cogn Dis, UMRS1171, F-59000 Lille, France.
    Burnouf, Thierry
    Taipei Med Univ, Coll Biomed Engn, Grad Inst Biomed Mat & Tissue Engn, 250 Wu Xing St, Taipei 11031, Taiwan;Taipei Med Univ, Int PhD Program Cell Therapy & Regenerat Med, Taipei, Taiwan.
    The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates2019In: Journal of Biomedical Science, ISSN 1021-7770, E-ISSN 1423-0127, Vol. 26, article id 89Article in journal (Refereed)
    Abstract [en]

    Background: Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson's disease models.

    Methods: Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (-80 degrees C/37 degrees C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot.

    Results: Platelet lysates contained the expected level of total proteins (ca. 7-14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death.

    Conclusion: Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.

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  • 262.
    Ngamjariyawat, Anongnad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Thammasat Univ, Fac Med, Dept Preclin Sci, Div Anat, Pathum Thani, Thailand..
    Cen, Jing
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Said, Romain
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Incedal, Ceren
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine2023In: Frontiers in Endocrinology, E-ISSN 1664-2392, Vol. 14, article id 1060675Article in journal (Refereed)
    Abstract [en]

    Introduction: High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions.

    Methods: Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied.

    Results: We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P-3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents.

    Discussion: It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

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  • 263.
    Nguyen, Phuoc My
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gandasi, Nikhil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xie, Beichen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sugahara, Sari
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Univ Tokyo, Grad Sch Pharmaceut Sci, Lab Hlth Chem, Tokyo, Japan.
    Xu, Yingke
    Zhejiang Univ, Dept Biomed Engn, Key Lab Biomed Engn,Minist Educ, Zhejiang Prov Key Lab Cardiocerebral Vasc Detect, Hangzhou, Zhejiang, Peoples R China.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    The PI(4)P phosphatase Sac2 controls insulin granule docking and release2019In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 218, no 11, p. 3714-3729Article in journal (Refereed)
    Abstract [en]

    Insulin granule biogenesis involves transport to, and stable docking at, the plasma membrane before priming and fusion. Defects in this pathway result in impaired insulin secretion and are a hallmark of type 2 diabetes. We now show that the phosphatidylinositol 4-phosphate phosphatase Sac2 localizes to insulin granules in a substrate-dependent manner and that loss of Sac2 results in impaired insulin secretion. Sac2 operates upstream of granule docking, since loss of Sac2 prevented granule tethering to the plasma membrane and resulted in both reduced granule density and number of exocytic events. Sac2 levels correlated positively with the number of docked granules and exocytic events in clonal beta cells and with insulin secretion in human pancreatic islets, and Sac2 expression was reduced in islets from type 2 diabetic subjects. Taken together, we identified a phosphoinositide switch on the surface on insulin granules that is required for stable granule docking at the plasma membrane and impaired in human type 2 diabetes.

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  • 264.
    Nicholson, Anna M.
    et al.
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Olpe, Cora
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England;Cambridge Stem Cell Inst, Wellcome Trust Med Res Council, Cambridge, England.
    Hoyle, Alice
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Thorsen, Ann-Sofie
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Rus, Teja
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Colombe, Mathilde
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Brunton-Sim, Roxanne
    Norwich Res Pk BioRepository, James Watson Rd, Norwich NR4 7UQ, Norfolk, England.
    Kemp, Richard
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Marks, Kate
    St James Univ Hosp, Pathol & Tumour Biol, Level 4,Wellcome Trust Brenner Bldg,Beckett St, Leeds LS9 7TF, W Yorkshire, England.
    Quirke, Phil
    St James Univ Hosp, Pathol & Tumour Biol, Level 4,Wellcome Trust Brenner Bldg,Beckett St, Leeds LS9 7TF, W Yorkshire, England.
    Malhotra, Shalini
    CUHFT, Dept Histopathol, Box 235, Cambridge, England.
    ten Hoopen, Rogier
    CUHFT, Dept Histopathol, Box 235, Cambridge, England.
    Ibrahim, Ashraf
    CUHFT, Dept Histopathol, Box 235, Cambridge, England.
    Lindskog, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Myers, Meagan B.
    US FDA, Div Genet & Mol Toxicol, Natl Ctr Toxicol Res, HFT 120,3900 NCTR Rd, Jefferson, AR 72079 USA.
    Parsons, Barbara
    US FDA, Div Genet & Mol Toxicol, Natl Ctr Toxicol Res, HFT 120,3900 NCTR Rd, Jefferson, AR 72079 USA.
    Tavare, Simon
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Wilkinson, Mark
    Norwich Res Pk BioRepository, James Watson Rd, Norwich NR4 7UQ, Norfolk, England.
    Morrissey, Edward
    Univ Oxford, John Radcliffe Hosp, MRC Weatherall Inst Mol Med, Oxford OX3 9DS, England.
    Winton, Douglas J.
    Canc Res UK Cambridge Inst, Li Ka Shing Ctr, Robinson Way, Cambridge CB2 0RE, England.
    Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium2018In: Cell Stem Cell, ISSN 1934-5909, E-ISSN 1875-9777, Vol. 22, no 6, p. 909-918.e8Article in journal (Refereed)
    Abstract [en]

    We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.

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  • 265.
    Niklasson, Mia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergström, Tobias
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Sundström, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nyberg, Frida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Haglund, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Westermark, Bengt
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.
    Segerman, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab. Natl Vet Inst, Dept Microbiol, Uppsala, Sweden.
    Segerman, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology. Uppsala Univ, Dept Immunol Genet & Pathol, Sci Life Lab, Rudbeck Lab, Uppsala, Sweden;Uppsala Univ, Uppsala Univ Hosp, Dept Med Sci Canc Pharmacol & Computat Med, Uppsala, Sweden.
    Mesenchymal transition and increased therapy resistance of glioblastoma cells is related to astrocyte reactivity2019In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 249, no 3, p. 295-307Article in journal (Refereed)
    Abstract [en]

    Grade IV astrocytoma/glioblastoma multiforme (GBM) is essentially incurable, partly due to its heterogenous nature, demonstrated even within the glioma-initiating cell (GIC) population. Increased therapy resistance of GICs is coupled to transition into a mesenchymal (MES) cell state. The GBM MES molecular signature displays a pronounced inflammatory character and its expression vary within and between tumors. Herein, we investigate how MES transition of GBM cells relates to inflammatory responses of normal astroglia. In response to CNS insults astrocytes enter a reactive cell state and participate in directing neuroinflammation and subsequent healing processes. We found that the MES signature show strong resemblance to gene programs induced in reactive astrocytes. Likewise, astrocyte reactivity gene signatures were enriched in therapy-resistant MES-like GIC clones. Variable expression of astrocyte reactivity related genes also largely defined intratumoral GBM cell heterogeneity at the single-cell level and strongly correlated with our previously defined therapy-resistance signature (based on linked molecular and functional characterization of GIC clones). In line with this, therapy-resistant MES-like GIC secreted immunoregulatory and tissue repair related proteins characteristic of astrocyte reactivity. Moreover, sensitive GIC clones could be made reactive through long-term exposure to the proinflammatory cytokine interleukin 1 beta (IL1 beta). IL1 beta induced a slow MES transition, increased therapy resistance, and a shift in DNA methylation profile towards that of resistant clones, which confirmed a slow reprogramming process. In summary, GICs enter through MES transition a reactive-astrocyte-like cell state, connected to therapy resistance. Thus, from a biological point of view, MES GICs would preferably be called 'reactive GICs'. The ability of GBM cells to mimic astroglial reactivity contextualizes the immunomodulatory and microenvironment reshaping abilities of GBM cells that generate a tumor-promoting milieu. This insight will be important to guide the development of future sensitizing therapies targeting treatment-resistant relapse-driving cell populations as well as enhancing the efficiency of immunotherapies in GBM. (c) 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  • 266.
    Nilsson, Ola B.
    et al.
    Stockholm Univ, Ctr Biomembrane Res, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Hedman, Rickard
    Stockholm Univ, Ctr Biomembrane Res, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Marino, Jacopo
    Univ Munich, CiPS M, Gene Ctr, D-81377 Munich, Germany.;Univ Munich, CiPS M, Ctr Integrated Prot Sci Munich, D-81377 Munich, Germany..
    Wickles, Stephan
    Univ Munich, CiPS M, Gene Ctr, D-81377 Munich, Germany.;Univ Munich, CiPS M, Ctr Integrated Prot Sci Munich, D-81377 Munich, Germany..
    Bischoff, Lukas
    Univ Munich, CiPS M, Gene Ctr, D-81377 Munich, Germany.;Univ Munich, CiPS M, Ctr Integrated Prot Sci Munich, D-81377 Munich, Germany..
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Mueller-Lucks, Annika
    Stockholm Univ, Ctr Biomembrane Res, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Trovato, Fabio
    Penn State Univ, Dept Chem, University Pk, PA 16802 USA..
    Puglisi, Joseph D.
    Stanford Univ, Sch Med, Dept Biol Struct, Stanford, CA 94305 USA.;Stanford Univ, Sch Med, Stanford Magnet Resonance Lab, Stanford, CA 94305 USA..
    O'Brien, Edward P.
    Penn State Univ, Dept Chem, University Pk, PA 16802 USA..
    Beckmann, Roland
    Univ Munich, CiPS M, Gene Ctr, D-81377 Munich, Germany.;Univ Munich, CiPS M, Ctr Integrated Prot Sci Munich, D-81377 Munich, Germany..
    von Heijne, Gunnar
    Stockholm Univ, Ctr Biomembrane Res, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.;Stockholm Univ, Sci Life Lab, S-17121 Solna, Sweden..
    Cotranslational Protein Folding inside the Ribosome Exit Tunnel2015In: Cell Reports, E-ISSN 2211-1247, Vol. 12, no 10, p. 1533-1540Article in journal (Refereed)
    Abstract [en]

    At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of co-translational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

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  • 267.
    Niss, Frida
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Using Optogenetics and Fictive Locomotion to Investigate the Effects of Inhibiting Renshaw Cells on Normal Locomotion in P3 Mice2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The circuit of recurring inhibition between motor neurons and Renshaw cells in the spinal cord has been known for around 70 years, though no determined function has been outlined as of yet. Renshaw cells are thought to be part of the central pattern generator in the spinal cord establishing them as an important part of the animal’s locomotive properties. In this study we aimed to investigate the role of Renshaw cells in locomotion with the help of optogenetics and electrophysiology. Halorhodopsin was inserted into the genome of mice and driven to expression with Cre recombinase in Renshaw cells. The spinal cord of P3 mice was extracted and by inducing fictive locomotion with appropriate neurotransmitters we could inhibit the Renshaw cells in action with a green laser, opening the halorhodopsin channels for Cl- ions. In previous experiments where the ability of Renshaw cells to release inhibitory neurotransmitters was inactivated, no effect was observed in either behavioral experiments or electrophysiological experiments. In a system where the effect of Renshaw cells was knocked out acutely with optogenetics there was no discernible change in fictive locomotion cycle length, frequency or amplitude. Nor was there an effect on alternation. The access of light to the Renshaw cells area might have been limited during the experiment considering the angle of light delivery and strength of the laser. Furthermore, the maturity of Renshaw cells at P3, the exclusive ability of the marker used to target Renshaw cells and the observed nature of neonatal inhibitory neurons acting as excitatory neurons could all be called into question about whether they contributed to these results or not.   

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  • 268.
    Nissbeck, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Determining the oligomeric structure of PARN2012Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Poly(A)-specific ribonuclease (PARN) is a deadenylase that degrades the poly(A) tail of eukaryotic mRNA. PARN also interacts with the 5’-cap structure of the mRNA. The binding of the cap structure enhances the deadenylation rate. PARN has previously been described as a dimer. We have studied PARN with size exclusion chromatography to investigate the oligomeric composition and revealed oligomeric compositions of PARN that are larger than dimeric PARN. Deadenylation assays have been used to measure the cap stimulated activity of PARN. The deadenylation assays showed that the cap stimulated activity of PARN correlated with the abundance of oligomers corresponding in size to tetrameric PARN. We present a model for tetrameric PARN and propose a mechanistic model for how the cap stimulates PARN mediated deadenylation.

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  • 269.
    Niu, Mengyao
    et al.
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA..
    Steffan, Breanne N.
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA..
    Fischer, Gregory J.
    Univ Wisconsin, Dept Genet, Madison, WI 53706 USA..
    Venkatesh, Nandhitha
    Univ Wisconsin, Dept Plant Pathol, Madison, WI 53706 USA..
    Raffa, Nicholas L.
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA..
    Wettstein, Molly A.
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA..
    Bok, Jin Woo
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA..
    Greco, Claudio
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA..
    Zhao, Can
    Univ Manchester, Div Infect Immun & Resp Med, Manchester Fungal Infect Grp, Manchester, Lancs, England..
    Berthier, Erwin
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA.;Univ Wisconsin, Dept Biomed Engn, Madison, WI USA..
    Oliw, E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Beebe, David
    Univ Wisconsin, Dept Biomed Engn, Madison, WI USA.;Univ Wisconsin, Dept Pathol & Lab Med, Madison, WI USA..
    Bromley, Michael
    Univ Manchester, Div Infect Immun & Resp Med, Manchester Fungal Infect Grp, Manchester, Lancs, England..
    Keller, Nancy P.
    Univ Wisconsin, Dept Med Microbiol & Immunol, Madison, WI 53706 USA.;Univ Wisconsin Madison, Dept Bacteriol, Madison, WI 53706 USA..
    Fungal oxylipins direct programmed developmental switches in filamentous fungi2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 5158Article in journal (Refereed)
    Abstract [en]

    Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

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  • 270.
    Norrby, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Can Feeding Amyloid Fibrils Trigger Amyloid Formation in C. Elegans?2023Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Amyloidosis are diseases caused by misfolded proteins that have transformed into extracellular insoluble amyloid fibrils. One type of amyloidosis is AA amyloidosis caused by AA amyloid and diseases connected are Rheumatoid arthritis and Tuberculosis. C. Elegans is a nematode used as a model organism and in this experiment. They are transgenic and express GFP (green fluorescent protein), a probe that mark the body-wall muscle cells in order to be visible in fluorescence microscopes. 

    Worms expressing AA45 and AA76 as well as a GFP control were fed with E. Coli OP50 and amyloid AA896 or Lin100. One control group was only fed with OP50. Three different aspects were researched: the size of the worms, their movements and a confocal microscope was used to detect amyloid. Neither the size of the worms nor the movements seemed to be linked to AA amyloid formation. However, amyloid were detected in worms expressing AA45 and AA76 but not in the GFP control when studied through a confocal microscope. 

    In this research it is shown that worms fed with amyloid fibrils and expressing AA45 or AA76 start forming amyloid in the body-wall muscle. This can be of much help in future research in order to make simpler diagnostical methods which can reduce the time for patients to start treatment and improve chances of survival or living with the disease with less complications.

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  • 271.
    Norreen-Thorsen, Marthe
    et al.
    Arctic Univ Norway, Translat Vasc Res, Dept Clin Med, N-9019 Tromso, Norway.
    Struck, Eike Christopher
    Arctic Univ Norway, Translat Vasc Res, Dept Clin Med, N-9019 Tromso, Norway.
    Oling, Sofia
    Arctic Univ Norway, Translat Vasc Res, Dept Clin Med, N-9019 Tromso, Norway.
    Zwahlen, Martin
    Royal Inst Technol KTH, Dept Prot Sci, Sci Life Lab, S-17121 Stockholm, Sweden.
    Von Feilitzen, Kalle
    Royal Inst Technol KTH, Dept Prot Sci, Sci Life Lab, S-17121 Stockholm, Sweden.
    Odeberg, Jacob
    Arctic Univ Norway, Translat Vasc Res, Dept Clin Med, N-9019 Tromso, Norway.;Royal Inst Technol KTH, Dept Prot Sci, Sci Life Lab, S-17121 Stockholm, Sweden.;Univ Hosp North Norway UNN, N-9019 Tromso, Norway.;Karolinska Univ Hosp, Coagulat Unit, Dept Hematol, S-17176 Stockholm, Sweden.
    Lindskog, Cecilia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Pontén, Fredrik
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Uhlen, Mathias
    Royal Inst Technol KTH, Dept Prot Sci, Sci Life Lab, S-17121 Stockholm, Sweden.
    Dusart, Philip James
    Arctic Univ Norway, Translat Vasc Res, Dept Clin Med, N-9019 Tromso, Norway.;Royal Inst Technol KTH, Dept Prot Sci, Sci Life Lab, S-17121 Stockholm, Sweden.;Karolinska Inst, Clin Chem & Blood Coagulat Res, Dept Mol Med & Surg, S-17176 Stockholm, Sweden.;Karolinska Univ Hosp, Karolinska Univ Lab, Clin Chem, S-17176 Stockholm, Sweden.
    Butler, Lynn Marie
    Arctic Univ Norway, Translat Vasc Res, Dept Clin Med, N-9019 Tromso, Norway.;Royal Inst Technol KTH, Dept Prot Sci, Sci Life Lab, S-17121 Stockholm, Sweden.;Karolinska Inst, Clin Chem & Blood Coagulat Res, Dept Mol Med & Surg, S-17176 Stockholm, Sweden.;Karolinska Univ Hosp, Karolinska Univ Lab, Clin Chem, S-17176 Stockholm, Sweden.
    A human adipose tissue cell-type transcriptome atlas2022In: Cell Reports, E-ISSN 2211-1247, Vol. 40, no 2, article id 111046Article in journal (Refereed)
    Abstract [en]

    The importance of defining cell-type-specific genes is well acknowledged. Technological advances facilitate high-resolution sequencing of single cells, but practical challenges remain. Adipose tissue is composed pri-marily of adipocytes, large buoyant cells requiring extensive, artefact-generating processing for separation and analysis. Thus, adipocyte data are frequently absent from single-cell RNA sequencing (scRNA-seq) data -sets, despite being the primary functional cell type. Here, we decipher cell-type-enriched transcriptomes from unfractionated human adipose tissue RNA-seq data. We profile all major constituent cell types, using 527 visceral adipose tissue (VAT) or 646 subcutaneous adipose tissue (SAT) samples, identifying over 2,300 cell-type-enriched transcripts. Sex-subset analysis uncovers a panel of male-only cell-type-enriched genes. By resolving expression profiles of genes differentially expressed between SAT and VAT, we identify mesothelial cells as the primary driver of this variation. This study provides an accessible method to profile cell-type-enriched transcriptomes using bulk RNA-seq, generating a roadmap for adipose tissue biology.

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  • 272.
    Ntefidou, Maria
    et al.
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Eklund, D. Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Physiology and Environmental Toxicology.
    Le Bail, Aude
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Schulmeister, Sylwia
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Scherbel, Franziska
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Brandl, Lisa
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Doerfler, Wolfgang
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Eichstaedt, Chantal
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Bannmueller, Anna
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Ljung, Karin
    Swedish Univ Agr Sci, Umeå Plant Sci Ctr, Dept Forest Genet & Plant Physiol, S-90183 Umeå, Sweden..
    Kost, Benedikt
    Univ Erlangen Nurnberg, Dept Biol, Cell Biol, D-91058 Erlangen, Germany..
    Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells2023In: Cell Reports, E-ISSN 2211-1247, Vol. 42, no 2, article id 112130Article in journal (Refereed)
    Abstract [en]

    RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain -contain-ing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hor-mone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remark-ably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in medi-ating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

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  • 273.
    Odqvist, Lina
    et al.
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Jevnikar, Zala
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Riise, Rebecca
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Oberg, Lisa
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Rhedin, Magdalena
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Leonard, Dag
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Yrlid, Linda
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Jackson, Sonya
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Mattsson, Johan
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Nanda, Sambit
    Univ Dundee, MRC Prot Phosphorylat & Ubiquitylat Unit, Dundee, Scotland.
    Cohen, Philip
    Univ Dundee, MRC Prot Phosphorylat & Ubiquitylat Unit, Dundee, Scotland.
    Knebel, Axel
    Univ Dundee, MRC Prot Phosphorylat & Ubiquitylat Unit, Dundee, Scotland.
    Arthur, Simon
    Univ Dundee, Sch Life Sci, Div Immunol & Cell Signaling, Dundee, Scotland.
    Thorn, Kristofer
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Svenungsson, Elisabet
    Karolinska Inst, Dept Med, Rheumatol Unit, Stockholm, Sweden.
    Jonsen, Andreas
    Lund Univ, Skane Univ Hosp, Dept Clin Sci Lund, Rheumatol, Lund, Sweden.
    Gunnarsson, Iva
    Karolinska Inst, Dept Med, Rheumatol Unit, Stockholm, Sweden.
    Tandre, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Alexsson, Andrei
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kastbom, Alf
    Linkoping Univ, Dept Rheumatol, Linkoping, Sweden;Linkoping Univ, Dept Clin & Expt Med, Linkoping, Sweden.
    Rantapaa-Dahlqvist, Solbritt
    Umea Univ, Med Fak, Dept Publ Hlth & Clin Med Rheumatol, Umea, Sweden.
    Eloranta, Maija-Leena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bengtsson, Anders
    Lund Univ, Skane Univ Hosp, Dept Clin Sci Lund, Rheumatol, Lund, Sweden.
    Johansson, Patrik
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Discovery Sci, Molndal, Sweden.
    Sandling, Johanna K.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology.
    Sjowall, Christopher
    Linkoping Univ, Dept Rheumatol, Linkoping, Sweden;Linkoping Univ, Dept Clin & Expt Med, Linkoping, Sweden.
    Rönnblom, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Collins, Barry
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden.
    Vaarala, Outi
    AstraZeneca R&D Gothenburg, BioPharmaceut R&D, Res & Early Dev, Resp Inflammat & Autoimmune, Molndal, Sweden;MedImmune LLC, Resp Inflammat & Autoimmun Dept, Gaithersburg, MD 20878 USA.
    Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil extracellular traps in systemic lupus erythematosus2019In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 78, no 10, p. 1363-1370Article in journal (Refereed)
    Abstract [en]

    Objectives

    Genetic variations in TNFAIP3 (A20) deubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-kappa B but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis.

    Methods

    CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926.

    Results

    Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-kappa B signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes.

    Conclusions

    We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.

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  • 274.
    Olsson, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cedervall, Jessica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The pro-inflammatory role of platelets in cancer2018In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 29, no 6, p. 569-573Article in journal (Refereed)
    Abstract [en]

    Thrombosis is a frequent issue in cancer patients. Tumor-induced platelet activation and coagulation does not only constitute a significant risk for thrombosis, but also contribute to tumor progression by promoting critical processes such as angiogenesis and metastasis. In addition to their role in hemostasis, platelets are increasingly recognized as regulators of inflammation. By modulating the immune system, platelets regulate several aspects of cancer-associated pathology. Platelets influence the inflammatory response in cancer by affecting the activation status of the endothelium and by recruiting leukocytes to primary and metastatic tumor sites, as well as to distant organs unaffected by tumor growth. Furthermore, platelets participate in the formation of neutrophil extracellular traps, which can promote metastasis, thrombosis, and contribute to organ failure. In this review, we discuss the role of platelets as coordinators of the immune system during malignant disease and the potential of targeting platelets to prevent cancer-associated pathology.

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  • 275.
    Omar-Hmeadi, Muhmmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Regulation of docking and priming in pancreatic α- and β-cells2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The secretion of islet hormones from endocrine cells of the pancreas plays vital roles in maintaining glucose homeostasis. Dysfunction of these cells leads to diabetes, a devastating metabolic disorder affecting millions worldwide, but underlying mechanisms remain poorly understood. In hyperglycemic conditions, β-cells secrete insulin, whereas α-cells secrete an increased amount of glucagon in hypoglycemic conditions. Both insulin and glucagon are stored in secretory granules preceding their release by regulated exocytosis. This process involves several steps, including tethering, docking, priming, and finally, a fusion of the granules with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and phosphoinositides (PIs) drive pancreatic hormone exocytosis and secretion, which follows a biphasic time course. Biphasic secretion is thought to reflect the vastly different release probabilities of individual granules, but direct evidence for this is still lacking.  Therefore, this thesis investigates exocytosis in the two main pancreatic cell types with a particular focus on preceding steps docking and priming, to identify rate-limiting steps in health and type-2 diabetes (T2D). Our data indicated that granule docking is critical for sustained secretion in α- and β-cells. Glucagon granule exocytosis had a U-shaped sensitivity to glucose in both healthy and T2D α-cells. However, T2D α-cells exhibited a marginal decrease in exocytosis, as well as docking, and they were markedly insensitive to somatostatin and insulin. T2D β-cells reduced exocytosis dramatically, and docking was compromised and no longer responsive to glucose, which correlated with reduced insulin secretion and elevated donor HbA1c. These results were further strengthened by the finding that expression of a group of genes that are involved explicitly in granule docking was reduced (by RNAseq of islets from over 200 human donors), and overexpression of the corresponding proteins increased granule docking in human β-cells.

    We further aimed to study the basis for the recruitment of these proteins to the docking site. Here we tested the hypothesis that highly charged lipids mainly PIs act as a hotspot to interact with SNARE proteins that initiate docking. We showed the homogenous distribution of all PIs markers in the plasma membrane, with no PIs microdomains at the exocytotic site during granule docking. However, rapid and local PI(4,5)P2 signaling at fusion sites was crucial for stabilizing fusion pore by binding to proteins related to the release site. These results suggested a role of PI(4,5)P2 in priming and fusion regulation rather than docking. Overall, this work gives new insights into the mechanisms underlying pancreatic hormone secretion in both healthy and diabetic conditions.

    List of papers
    1. Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
    Open this publication in new window or tab >>Glucose-Dependent Granule Docking Limits Insulin Secretion and Is Decreased in Human Type 2 Diabetes
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    2018 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 27, no 2, p. 470-478Article in journal (Refereed) Published
    Abstract [en]

    Glucose-stimulated insulin secretion is biphasic, with a rapid first phase and a slowly developing sustained second phase; both are disturbed in type 2 diabetes (T2D). Biphasic secretion results from vastly different release probabilities of individual insulin granules, but the morphological and molecular basis for this is unclear. Here, we show that human insulin secretion and exocytosis critically depend on the availability of membrane-docked granules and that T2D is associated with a strong reduction in granule docking. Glucose accelerated granule docking, and this effect was absent in T2D. Newly docked granules only slowly acquired release competence; this was regulated by major signaling pathways, but not glucose. Gene expression analysis indicated that key proteins involved in granule docking are downregulated in T2D, and overexpression of these proteins increased granule docking. The findings establish granule docking as an important glucose-dependent step in human insulin secretion that is dysregulated in T2D.

    Keywords
    GLP-1, biphasic secretion, dense core vesicle, docking, exocytosis, genome-wide association, insulin secretion, priming, somatostatin, type 2 diabetes
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-341518 (URN)10.1016/j.cmet.2017.12.017 (DOI)000424465200021 ()29414688 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Diabetes AssociationSwedish Society for Medical Research (SSMF)The Swedish Brain FoundationNovo NordiskErnfors Foundation
    Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2019-08-02Bibliographically approved
    2. Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes
    Open this publication in new window or tab >>Paracrine control of α-cell glucagon exocytosis is compromised in human type-2 diabetes
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-389793 (URN)
    Available from: 2019-07-27 Created: 2019-07-27 Last updated: 2019-08-02
    3. PtdIns(4,5)P2 is not required for secretory granule docking
    Open this publication in new window or tab >>PtdIns(4,5)P2 is not required for secretory granule docking
    2018 (English)In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854, Vol. 19, no 6, p. 436-445Article in journal (Refereed) Published
    Abstract [en]

    Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co-clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high-resolution total internal reflection imaging of EGFP-labeled PtdIns markers or syntaxin-1 at secretory granule release sites in live insulin-secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin-1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin-1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P-2) by recruitment of a 5-phosphatase strongly inhibited Ca2+-dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin-1. Cell permeabilization by -toxin or formaldehyde-fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P-2 accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin-1 at the release site.

    Keywords
    exocytosis, insulin, live cell imaging, phosphoinositides, PtdIns(4, 5)P-2, syntaxin clustering, vesicle docking
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-356858 (URN)10.1111/tra.12562 (DOI)000432037000005 ()29542271 (PubMedID)
    Funder
    Swedish Research CouncilSwedish Child Diabetes FoundationSwedish Society for Medical Research (SSMF)Novo NordiskThe Swedish Brain FoundationErnfors Foundation
    Available from: 2018-08-15 Created: 2018-08-15 Last updated: 2019-08-02Bibliographically approved
    4. Fusion pore regulation by transient local generation of PI(4,5)P2 in pancreatic in β-cells
    Open this publication in new window or tab >>Fusion pore regulation by transient local generation of PI(4,5)P2 in pancreatic in β-cells
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-389794 (URN)
    Available from: 2019-07-27 Created: 2019-07-27 Last updated: 2019-08-02
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  • 276.
    Omar-Hmeadi, Muhmmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Insulin granule biogenesis and exocytosis2021In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 78, no 5, p. 1957-1970Article, review/survey (Refereed)
    Abstract [en]

    Insulin is produced by pancreatic beta-cells, and once released to the blood, the hormone stimulates glucose uptake and suppresses glucose production. Defects in both the availability and action of insulin lead to elevated plasma glucose levels and are major hallmarks of type-2 diabetes. Insulin is stored in secretory granules that form at the trans-Golgi network. The granules undergo extensive modifications en route to their release sites at the plasma membrane, including changes in both protein and lipid composition of the granule membrane and lumen. In parallel, the insulin molecules also undergo extensive modifications that render the hormone biologically active. In this review, we summarize current understanding of insulin secretory granule biogenesis, maturation, transport, docking, priming and eventual fusion with the plasma membrane. We discuss how different pools of granules form and how these pools contribute to insulin secretion under different conditions. We also highlight the role of the beta-cell in the development of type-2 diabetes and discuss how dysregulation of one or several steps in the insulin granule life cycle may contribute to disease development or progression.

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  • 277. Onyango, I
    et al.
    Hjälm, G
    Larsson, M
    Rask, L
    Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes.1999In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 247, no 1, p. 9-16Article in journal (Refereed)
    Abstract [en]

    The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.

  • 278. Ortiz, Conrad
    et al.
    Caja, Laia
    Fundacio Inst Invest Biomed Bellvitge IDIBELL, Barcelona, Spain.
    Sancho, Patricia
    Bertran, Esther
    Fabregat, Isabel
    Inhibition of the EGF receptor blocks autocrine growth and increases the cytotoxic effects of doxorubicin in rat hepatoma cells: role of reactive oxygen species production and glutathione depletion2008In: Biochemical pharmacology, ISSN 1873-2968, Vol. 75, no 10, p. 1935-1945Article in journal (Refereed)
    Abstract [en]

    FaO rat hepatoma cells show increased levels of the epidermal growth factor receptor (EGFR) ligands, when compared with adult normal hepatocytes, and higher activity of the TNF-alpha converting enzyme (TACE/ADAM17), which is required for EGFR ligand proteolysis and activation. In this work we have analysed the consequences of inhibiting the EGFR in FaO rat hepatoma cells, focusing the attention on autocrine growth and protection from apoptosis. Results have indicated that FaO cells show overactivation of the EGFR pathway, which induces basal growth (in the absence of serum) and protection from pro-apoptotic agents, such as doxorubicin, generating drug resistance. Treatment of cells with the combination of doxorubicin and the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478, a potent and specific inhibitor of EGFR tyrosine kinase) potently inhibits autocrine growth and induces apoptosis. The apoptotic effect correlates with high expression and activation of the pro-apoptotic Bax and decreased transcript and protein levels of the anti-apoptotic Mcl-1 and Bcl-x(L). Furthermore, the combination of AG1478 and doxorubicin induces reactive oxygen species (ROS) production and glutathione depletion in FaO cells, coincident with up-regulation of the NADPH oxidase NOX4 and down-regulation of the gamma-glutamylcysteine synthetase (gamma-GCS), a key regulatory enzyme of the glutathione synthesis. Incubation of cells with glutathione ethyl ester attenuates the apoptosis induced by the combination of doxorubicin and AG1478, which indicates that glutathione depletion is required for an efficient cell death. In conclusion, targeting EGFR combined with other conventional pro-apoptotic drugs should potentially be effective in antineoplastic therapy towards liver cancer.

  • 279.
    Ortsäter, Henrik
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
    Disturbed Islet Function and Alterations in Islet Protein Expression2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.

    List of papers
    1. Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets
    Open this publication in new window or tab >>Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets
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    2000 In: Diabetologia, ISSN 0012-186, Vol. 43, no 10, p. 1313-1318Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-93449 (URN)
    Available from: 2005-09-16 Created: 2005-09-16Bibliographically approved
    2. Contribution of glycolytic and mitochondrial pathways in glucose-induced changes in islet respiration and insulin secretion
    Open this publication in new window or tab >>Contribution of glycolytic and mitochondrial pathways in glucose-induced changes in islet respiration and insulin secretion
    2002 In: Pflügers Archiv European Journal of Physiology, ISSN 0031-6768, Vol. 444, no 4, p. 506-512Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-93450 (URN)
    Available from: 2005-09-16 Created: 2005-09-16Bibliographically approved
    3. Protein profiling and identification of differentially expressed islet proteins: Oleate-induced lowering of chaperone protein peptidyl-prolyl isomerase B
    Open this publication in new window or tab >>Protein profiling and identification of differentially expressed islet proteins: Oleate-induced lowering of chaperone protein peptidyl-prolyl isomerase B
    In: Journal of Proteomic ResearchArticle in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-93451 (URN)
    Available from: 2005-09-16 Created: 2005-09-16Bibliographically approved
    4. Protein expression patterns in mice fed high fat and high sucrose diet
    Open this publication in new window or tab >>Protein expression patterns in mice fed high fat and high sucrose diet
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-93452 (URN)
    Available from: 2005-09-16 Created: 2005-09-16 Last updated: 2010-01-13Bibliographically approved
    5. Regulation of 11β-hydroxysteroid dehydrogenase type 1 and glucose-stimulated insulin secretion in pancreatic islet of Langerhans
    Open this publication in new window or tab >>Regulation of 11β-hydroxysteroid dehydrogenase type 1 and glucose-stimulated insulin secretion in pancreatic islet of Langerhans
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    2005 In: Diabetes/Metabolism Research and Reviews, Vol. 21, no 4, p. 359-366Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-93453 (URN)
    Available from: 2005-09-16 Created: 2005-09-16Bibliographically approved
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  • 280.
    Oscarsson, Emelie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. ORF Genetics.
    Optimization of Recombinant Growth Factor Extraction and Purification from Transgenic Barley2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The substitution of traditionally produced meat, one of the largest contributors to climate change, for cell-cultured meat would greatly favor the conservation of wildlife and environmental integrity. Cell-cultured meat is expensive, growth factors in the cell culture media contributing with over 95% of the total production cost. 

    ORF genetics offers recombinant growth factors produced by transgenic barley plantswhich is a perfect host to reduce protein production cost and provide endotoxin-free products. Efficient target protein extraction and purification are crucial steps in the protein processing and finding conditions to achieve higher growth factor yields are the main drivers of optimization experiments.In this study, extraction of two growth factors, human PDGF-BB (Platelet-derived growth factor BB) and bovine FGF basic (Fibroblast growth factor basic) were optimized and in case of the human PDGF-BB, a DoE (design of experiment) based experimental screening was performed to improve magnetic bead-based affinity purification. Before the magnetic separation, a preliminary affinity resin screening was performed with IMAC columns chelated with different metals to reveal which metal binds the 6xHis-tag containing human PDGF-BB protein with the highest affinity and to use the corresponding magnetic bead in the screening experiments.

    Through a series of extraction experiments 50 mM Kpi, 500 mM NaCl, 10% glycerol, 5 mM MgCl2, pH 7.5 was found to be the optimal buffer for human PDGF-BB extraction and 50 mM Kpi, 500 mM NaCl, 10 mM BeMeOH, pH 7.5 optimal for bovine FGFb extraction. Affinity resin screening of human PDGF-BB with IMAC columns proved nickel to be the metal which binds human PDGF-BB with a 6xHis-tag with the highest affinity. Design of Experiment (DoE) software, JMP, was used to optimize Nickel- NTA type magnetic bead separation of human PDGF-BB by identifying the significant factors in the process. The screening design and the statistical analysis determining the imidazole concentration during washing, the bead:seed ratio and binding time to be the most significant factors contributing to the human PDGF-BB yield.

    These optimization designs and experimental setups can be built upon for further growth factor purification optimization, and contributing to cost effective production of growth factors and helping cell-cultured meat to the global food market.

    The full text will be freely available from 2029-06-07 13:23
  • 281.
    Othman, Hemn
    et al.
    Univ Putra Malaysia, Fac Vet Med, Upm Serdang, Selangor, Malaysia.;Univ Sulaimani, Coll Pharm, Sulaymaniyah, Kurdistan Regio, Iraq..
    Rahman, Heshu
    Univ Sulaimani, Coll Med, Sulaymaniyah, Kurdistan Regio, Iraq.;Komar Univ Sci & Technol, Coll Hlth Sci, Sulaymaniyah, Kurdistan Regio, Iraq..
    Mohan, Syam
    Jazan Univ, Subst Abuse & Toxicol Res Ctr, Jazan, Saudi Arabia..
    Aziz, Sadat
    Univ Sulaimani, Coll Vet Med, Sulaymaniyah, Kurdistan Regio, Iraq..
    Marif, Hardi
    Univ Sulaimani, Coll Vet Med, Sulaymaniyah, Kurdistan Regio, Iraq..
    Ford, Dianne
    Northumbria Univ, Fac Hlth & Life Sci, Newcastle Upon Tyne, Tyne & Wear, England..
    Abdulsamad, Nozlena
    Univ Sains Malaysia, Integrat Med Cluster, Inst Perubatan Pergigian Termaju IPPT, Sains BERTAM, Kepala Batas, Pulau Pinang, Malaysia..
    Amin, Kawa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Lung- allergy- and sleep research. Univ Sulaimani, Coll Med, Sulaymaniyah, Kurdistan Regio, Iraq.
    Abdullah, Rasedee
    Univ Putra Malaysia, Fac Vet Med, Upm Serdang, Selangor, Malaysia..
    Antileukemic Effect of Palladium Nanoparticles Mediated by White Tea (Camellia sinensis) Extract In Vitro and in WEHI-3B-Induced Leukemia In Vivo2020In: Evidence-based Complementary and Alternative Medicine, ISSN 1741-427X, E-ISSN 1741-4288, Vol. 2020, article id 8764096Article in journal (Refereed)
    Abstract [en]

    This study investigated thein vivoantileukemic activity of palladium nanoparticles (Pd@W.tea-NPs) mediated by white tea extract in a murine model. The cell viability effect of Pd@W.tea-NPs, "blank" Pd nanoparticles, and white tea extract alone was determined in murine leukemia WEHI-3B cells and normal mouse fibroblasts (3T3 cells). Apoptotic and cell cycle arrest effects of Pd@W.tea-NPs in WEHI-3B cells were evaluated. The effects of Pd@W.tea-NPs administered orally to leukemic mice at 50 and 100 mg/kg daily over 28 days were evaluated. Pd@W.tea-NPs reduced the viability of WHEI-3B cells with IC(50)7.55 mu g/ml at 72 h. Blank Pd nanoparticles and white tea extract alone had smaller effects on WHEI-3B viability and on normal fibroblasts. Pd@W.tea-NPs increased the proportion of Annexin V-positive WHEI-3B cells and induced G2/M cell cycle arrest. Leukemic cells in the spleen were reduced by Pd@W.tea-NPs with an increase in Bax/Bcl-2 and cytochrome-C protein and mRNA levels indicating the activation of the mitochondrial apoptotic pathway. These effects replicated the effects of ATRA and were not observed using blank Pd nanoparticles. Pd@W.tea-NPs afford therapeutic efficacy against leukemia likely to pivot on activation of the mitochondrial pathway of apoptotic signaling and hence appear attractive potential candidates for development as a novel anticancer agent.

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  • 282.
    Pabis, Anna
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily2016In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 37, p. 14-21Article in journal (Refereed)
    Abstract [en]

    Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity.

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  • 283.
    Page, Donna J.
    et al.
    Univ Manchester, Fac Biol Med & Hlth, Michael Smith Bldg,Oxford Rd, Manchester M13 9PT, Lancs, England;Manchester Metropolitan Univ, Sch Healthcare Sci, Manchester M1 5GD, Lancs, England.
    Thuret, Raphael
    Univ Manchester, Fac Biol Med & Hlth, Michael Smith Bldg,Oxford Rd, Manchester M13 9PT, Lancs, England.
    Venkatraman, Lakshmi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Boston Univ, Biomed Engn Dept, 610 Commonwealth Ave, Boston, MA 02215 USA.
    Takahashi, Tokiharu
    Univ Manchester, Fac Biol Med & Hlth, Michael Smith Bldg,Oxford Rd, Manchester M13 9PT, Lancs, England.
    Bentley, Katie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Boston Univ, Biomed Engn Dept, 610 Commonwealth Ave, Boston, MA 02215 USA;Harvard Med Sch, Beth Israel Deaconess Med Ctr, Ctr Vasc Biol Res, Boston, MA 02215 USA;Francis Crick Inst, Cellular Adapt Behav Lab, Midland Rd, London NW1 1AT, England;Kings Coll London, Fac Nat & Math Sci, Dept Informat, Strand Campus, London WC2B 4BG, England.
    Herbert, Shane P.
    Univ Manchester, Fac Biol Med & Hlth, Michael Smith Bldg,Oxford Rd, Manchester M13 9PT, Lancs, England.
    Positive Feedback Defines the Timing, Magnitude, and Robustness of Angiogenesis2019In: Cell Reports, E-ISSN 2211-1247, Vol. 27, no 11, p. 3139-3151.e5Article in journal (Refereed)
    Abstract [en]

    Angiogenesis is driven by the coordinated collective branching of specialized leading "tip" and trailing "stalk" endothelial cells (ECs). While Notch-regulated negative feedback suppresses excessive tip selection, roles for positive feedback in EC identity decisions remain unexplored. Here, by integrating computational modeling with in vivo experimentation, we reveal that positive feedback critically modulates the magnitude, timing, and robustness of angiogenic responses. In silico modeling predicts that positivefeedback-mediated amplification of VEGF signaling generates an ultrasensitive bistable switch that underpins quick and robust tip-stalk decisions. In agreement, we define a positive-feedback loop exhibiting these properties in vivo, whereby Vegf-induced expression of the atypical tetraspanin, tm4sf18, amplifies Vegf signaling to dictate the speed and robustness of EC selection for angiogenesis. Consequently, tm4sf18 mutant zebrafish select fewer motile ECs and exhibit stunted hypocellular vessels with unstable tip identity that is severely perturbed by even subtle Vegfr attenuation. Hence, positive feedback spatiot-emporally shapes the angiogenic switch to ultimately modulate vascular network topology.

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  • 284.
    Paivandy, Aida
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Melo, Fabio R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sellin, Mikael E.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production2019In: Cell Death Discovery, E-ISSN 2058-7716, Vol. 5, article id 95Article in journal (Refereed)
    Abstract [en]

    Mast cells contribute to the pathology of allergic and other disorders. Strategies to interfere with harmful mast cell-related activities are therefore warranted. Previously we established a principle for inducing selective apoptosis of mast cells, by the use of lysosomotropic agents that cause secretory granule permeabilization, leading to production of reactive oxygen species (ROS). However, the mechanism of ROS production has not been known. Here we addressed this issue. Live microscopy analysis showed that the secretory granules comprise major subcellular compartments for ROS production in response to mefloquine. As further signs for the primary involvement of secretory granules, both ROS production and cell death was blunted in mast cells lacking serglycin, a secretory granule-restricted proteoglycan. Inhibition of granule acidification caused an essentially complete blockade of granule permeabilization, ROS production and cell death in response to mefloquine. ROS production was also attenuated in the presence of an iron chelator, and after inhibition of either granzyme B or the ERK1/2 MAP kinase signaling pathway. Together, our findings reveal that the mast cell secretory granules constitute major sites for ROS production in mast cells subjected to lysosomotropic challenge. Moreover, this study reveals a central role for granule acidification in ROS generation and the pro-apoptotic response triggered downstream of secretory granule permeabilization.

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  • 285.
    Panara, Virginia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Monteiro, Rui
    Univ Birmingham, Inst Canc & Genom Sci, Coll Med & Dent Sci, Birmingham, England.;Univ Birmingham, Birmingham Ctr Genome Biol, Birmingham, England..
    Koltowska, Katarzyna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Epigenetic Regulation of Endothelial Cell Lineages During Zebrafish Development-New Insights From Technical Advances2022In: Frontiers in Cell and Developmental Biology, E-ISSN 2296-634X, Vol. 10, article id 891538Article, review/survey (Refereed)
    Abstract [en]

    Epigenetic regulation is integral in orchestrating the spatiotemporal regulation of gene expression which underlies tissue development. The emergence of new tools to assess genome-wide epigenetic modifications has enabled significant advances in the field of vascular biology in zebrafish. Zebrafish represents a powerful model to investigate the activity of cis-regulatory elements in vivo by combining technologies such as ATAC-seq, ChIP-seq and CUT&Tag with the generation of transgenic lines and live imaging to validate the activity of these regulatory elements. Recently, this approach led to the identification and characterization of key enhancers of important vascular genes, such as gata2a, notch1b and dll4. In this review we will discuss how the latest technologies in epigenetics are being used in the zebrafish to determine chromatin states and assess the function of the cis-regulatory sequences that shape the zebrafish vascular network.

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  • 286.
    Panda, Rishab
    et al.
    Indian Inst Sci Educ & Res Mohali, Dept Chem Sci, Mohali, Punjab, India..
    Panda, Pritam Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory. Karolinska Inst, Inst Environm Med, Div Immunol & Chron Dis, Stockholm, Sweden..
    Krishnamoorthy, Janarthanan
    Jimma Univ, Jimma Inst Technol, Sch Biomed Engn, Jimma 378, Ethiopia..
    Kar, Rajiv K.
    Indian Inst Technol Guwahati, Jyoti & Bhupat Mehta Sch Hlth Sci & Technol, Gauhati 781039, Assam, India..
    Network analysis of chromophore binding site in LOV domain2023In: Computers in Biology and Medicine, ISSN 0010-4825, E-ISSN 1879-0534, Vol. 161, article id 106996Article in journal (Refereed)
    Abstract [en]

    Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.

  • 287.
    Pano, Filip
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    The role of mast cell tryptase in regulating breast cancer growth2023Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    The full text will be freely available from 2025-03-10 19:07
  • 288.
    Parks, Luke
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. University of Michigan, Department of Radiation Oncology.
    Nascent RNA sequencing of unperturbed newly divided cells2017Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Establishing a definitive cell cycle progression has been one of the fundamental aims of cellular biology. Its importance lies in gaining insight into the basic processes of life as well as the functions of mutant cell cycle pathways in promoting cancer by replication deficiencies and loss of checkpoint control. Currently used methods to control cell cycle and synchronize cells, function by halting cell cycle progression. Such harsh methods are detrimental to the cell and insufficient to provide an accurate reflection of the cell cycle. This study focused on replicating and confirming the efficiency of a technique developed by Helmstetter, called the “Baby Machine,” that can produce new born cells with little to no perturbations. Using this in conjunction with a short pulse RNA labelling technique, called Bru-seq, allowed the capture and RNA sequencing of synchronized cells and its nascent RNA. Here we show the first glimpse into the transcriptional profile of newly divided cells as well as novel rapid exon splicing and transcription read-through processes.

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    Parks, Thesis
  • 289.
    Patrick, Michael
    et al.
    Univ Wisconsin Madison, Dept Med Genet, Madison, WI USA..
    Dennis, Patrick P.
    Howard Hughes Med Inst, Ashburn, VA USA..
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bremer, Hans
    Univ Texas Dallas, Dept Mol & Cell Biol, Dallas, TX 75230 USA..
    Free RNA polymerase in Escherichia coli2015In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 119, p. 80-91Article in journal (Refereed)
    Abstract [en]

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [R-f] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [R-f] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [R-f](rel), can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [R-f](rel) in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia col cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [R-f] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [R-f].

  • 290.
    Pauls, Dennis
    et al.
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany.;Univ Leipzig, Inst Biol, Dept Anim Physiol, Talstr 33, D-04103 Leipzig, Germany..
    Selcho, Mareike
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany.;Univ Leipzig, Inst Biol, Dept Anim Physiol, Talstr 33, D-04103 Leipzig, Germany..
    Raederscheidt, Johanna
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany..
    Amatobi, Kelechi M.
    Univ Wurzburg, Bioctr, Julius von Sachs Inst, Pharmaceut Biol, Julius von Sachs Pl 2, D-97082 Wurzburg, Germany..
    Fekete, Agnes
    Univ Wurzburg, Bioctr, Julius von Sachs Inst, Pharmaceut Biol, Julius von Sachs Pl 2, D-97082 Wurzburg, Germany..
    Krischke, Markus
    Univ Wurzburg, Bioctr, Julius von Sachs Inst, Pharmaceut Biol, Julius von Sachs Pl 2, D-97082 Wurzburg, Germany..
    Hermann-Luibl, Christiane
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany..
    Ozbek-Unal, Ayten Gizem
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany..
    Ehmann, Nadine
    Univ Leipzig, Inst Biol, Dept Anim Physiol, Talstr 33, D-04103 Leipzig, Germany..
    Itskov, Pavel M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Schiöth: Functional Pharmacology. Easy Behav, Rua Sao Tome & Principe 23, P-2765282 Estoril, Portuga.
    Kittel, Robert J.
    Univ Leipzig, Inst Biol, Dept Anim Physiol, Talstr 33, D-04103 Leipzig, Germany..
    Helfrich-Foerster, Charlotte
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany..
    Kuehnlein, Ronald P.
    Karl Franzens Univ Graz, Inst Mol Biosci, Humboldtstr 50, A-8010 Graz, Austria.;BioTechMed Graz, A-8010 Graz, Austria.;Karl Franzens Univ Graz, Field Excellence BioHlth, Humboldtstr 50, A-8010 Graz, Austria..
    Mueller, Martin J.
    Univ Wurzburg, Bioctr, Julius von Sachs Inst, Pharmaceut Biol, Julius von Sachs Pl 2, D-97082 Wurzburg, Germany..
    Wegener, Christian
    Univ Wurzburg, Bioctr, Theodor Boveri Inst, Neurobiol & Genet, D-97074 Wurzburg, Germany..
    Endocrine signals fine-tune daily activity patterns in Drosophila2021In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 31, no 18, p. 4076-+Article in journal (Refereed)
    Abstract [en]

    Animals need to balance competitive behaviors to maintain internal homeostasis. The underlying mechanisms are complex but typically involve neuroendocrine signaling. Using Drosophila, we systematically manipulated signaling between energy-mobilizing endocrine cells producing adipokinetic hormone (AKH), octopaminergic neurons, and the energy-storing fat body to assess whether this neuroendocrine axis involved in starvation-induced hyperactivity also balances activity levels under ad libitum access to food. Our results suggest that AKH signals via two divergent pathways that are mutually competitive in terms of activity and rest. AKH increases activity via the octopaminergic system during the day, while it prevents high activity levels during the night by signaling to the fat body. This regulation involves feedback signaling from octopaminergic neurons to AKH-producing cells (APCs). APCs are known to integrate a multitude of metabolic and endocrine signals. Our results add a new facet to the versatile regulatory functions of APCs by showing that their output contributes to shape the daily activity pattern under ad libitum access to food.

  • 291.
    Pavlovic, Natasa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Rani, Bhavna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Heindryckx, Femke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Platelets as Key Factors in Hepatocellular Carcinoma2019In: Cancers, ISSN 2072-6694, Vol. 11, no 7, article id 1022Article, review/survey (Refereed)
    Abstract [en]

    Hepatocellular carcinoma (HCC) is a primary liver cancer that usually develops in the setting of chronic inflammation and liver damage. The hepatic microenvironment plays a crucial role in the disease development, as players such as hepatic stellate cells, resident liver macrophages (Kupffer cells), endothelial cells, extracellular matrix, and a variety of immune cells interact in highly complex and intertwined signaling pathways. A key factor in these cross-talks are platelets, whose role in cancer has gained growing evidence in recent years. Platelets have been reported to promote HCC cell proliferation and invasion, but their involvement goes beyond the direct effect on tumor cells, as they are known to play a role in pro-fibrinogenic signaling and the hepatic immune response, as well as in mediating interactions between these factors in the stroma. Anti-platelet therapy has been shown to ameliorate liver injury and improve the disease outcome. However, platelets have also been shown to play a crucial role in liver regeneration after organ damage. Therefore, the timing and microenvironmental setting need to be kept in mind when assessing the potential effect and therapeutic value of platelets in the disease progression, while further studies are needed for understanding the role of platelets in patients with HCC.

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  • 292.
    Pedersen, Julia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Evolution of acetylcholine receptors and study of the anatomy of the mouse brain reward system2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis work is divided in two parts. In the first part, I make use of the transgenic TRPV1-Cre mouse line as a tool to investigate the midbrain ventral tegmental area (VTA). By using a ChR2-EYFP construct, detailed mapping of connectivity shows that TRPV1-Cre VTA neurons innervate many brain areas such as the prefrontal cortex (PFC), ventral pallidum, bed nucleus of stria terminalis and lateral habenula. Interestingly, a mainly excitatory subcircuit from the VTA to PFC in the TRPV1-Cre mouse was identified which suggests a fast modulatory mechanism of the PFC by a VTA subpopulation. These results are discussed in the light of behavioral and neurophysiological literature. In the second part, the evolution of the vertebrate acetylcholine (ACh) receptor gene families in relation to the whole genome duplications (WGDs), also called 1R and 2R, was investigated. The nicotinic ACh receptors (nAChRs) form a complex gene family, where the members have evolved with varying rates. Our analyses combined phylogeny, intron positions and chromosomal synteny in order to elucidate the nAChR evolution in relation to the vertebrate WGDs. We found that ten ancestral nAChR genes were present prior to the WGDs. 1R and 2R then expanded this set to 19 genes, of which 16 are present in mammals today. The teleost specific WGD, 3R, further expanded the repertoire into 31 genes, of which 27 genes are present in zebrafish. The muscarinic ACh receptors (mAChRs) on the other hand form a smaller receptor family. Using the same approach, our analyses show that there were two ancestral genes present prior to the WGDs, expanding to five genes following 1R and 2R. In zebrafish, all genes retained duplicates in 3R resulting in ten mAChR genes present today. Our analyses also showed that four mAChR teleost genes have gained introns, some up to six introns. The evolutionary analyses of the receptor gene families show that all vertebrate duplication events in the AChR families, except for two among the nAChR genes, occurred through 1R, 2R and 3R, displaying the substantial impact of the WGDs on the evolution of the AChR genes.

    List of papers
    1. The connectivity of the TRPV1-Cre mouse line as a useful tool for exploring the function of a VTA subpopulation
    Open this publication in new window or tab >>The connectivity of the TRPV1-Cre mouse line as a useful tool for exploring the function of a VTA subpopulation
    (English)Manuscript (preprint) (Other academic)
    National Category
    Cell Biology Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-353920 (URN)
    Available from: 2018-06-18 Created: 2018-06-18 Last updated: 2018-06-18
    2. Evolution of vertebrate nicotinic acetylcholine receptors
    Open this publication in new window or tab >>Evolution of vertebrate nicotinic acetylcholine receptors
    2019 (English)In: BMC Evolutionary Biology, E-ISSN 1471-2148, Vol. 19, article id 38Article in journal (Refereed) Published
    Abstract [en]

    Background

    Many physiological processes are influenced by nicotinic acetylcholine receptors (nAChR), ranging from neuromuscular and parasympathetic signaling to modulation of the reward system and long-term memory. Due to the complexity of the nAChR family and variable evolutionary rates among its members, their evolution in vertebrates has been difficult to resolve. In order to understand how and when the nAChR genes arose, we have used a broad approach of analyses combining sequence-based phylogeny, chromosomal synteny and intron positions.

    Results

    Our analyses suggest that there were ten subunit genes present in the vertebrate predecessor. The two basal vertebrate tetraploidizations (1R and 2R) then expanded this set to 19 genes. Three of these have been lost in mammals, resulting in 16 members today. None of the ten ancestral genes have kept all four copies after 2R. Following 2R, two of the ancestral genes became triplicates, five of them became pairs, and three seem to have remained single genes. One triplet consists of CHRNA7, CHRNA8 and the previously undescribed CHRNA11, of which the two latter have been lost in mammals but are still present in lizards and ray-finned fishes. The other triplet consists of CHRNB2, CHRNB4 and CHRNB5, the latter of which has also been lost in mammals. In ray-finned fish the neuromuscular subunit gene CHRNB1 underwent a local gene duplication generating CHRNB1.2. The third tetraploidization in the predecessor of teleosts (3R) expanded the repertoire to a total of 31 genes, of which 27 remain in zebrafish. These evolutionary relationships are supported by the exon-intron organization of the genes.

    Conclusion

    The tetraploidizations explain all gene duplication events in vertebrates except two. This indicates that the genome doublings have had a substantial impact on the complexity of this gene family leading to a very large number of members that have existed for hundreds of millions of years.

    National Category
    Evolutionary Biology Cell Biology Biochemistry and Molecular Biology Bioinformatics and Systems Biology
    Identifiers
    urn:nbn:se:uu:diva-353919 (URN)10.1186/s12862-018-1341-8 (DOI)000457255700002 ()30700248 (PubMedID)
    Funder
    The Swedish Brain FoundationCarl Tryggers foundation
    Available from: 2018-06-18 Created: 2018-06-18 Last updated: 2024-01-17Bibliographically approved
    3. Evolution of the muscarinic acetylcholine receptors in vertebrates
    Open this publication in new window or tab >>Evolution of the muscarinic acetylcholine receptors in vertebrates
    2018 (English)In: eNeuro, E-ISSN 2373-2822, Vol. 5, no 5, article id e0340-18.2018Article in journal (Refereed) Published
    Abstract [en]

    The family of muscarinic acetylcholine receptors (mAChRs) consists of five members in mammals, encoded by theCHRM1-5 genes. The mAChRs are G-protein-coupled receptors, which can be divided into the following two subfamilies: M2 and M4 receptors coupling to Gi/o; and M1, M3, and M5 receptors coupling to Gq/11. However, despite the fundamental roles played by these receptors, their evolution in vertebrates has not yet been fully described. We have combined sequence-based phylogenetic analyses with comparisons of exon–intron organi- zation and conserved synteny in order to deduce the evolution of the mAChR receptors. Our analyses verify the existence of two ancestral genes prior to the two vertebrate tetraploidizations (1R and 2R). After these events, one gene had duplicated, resulting in CHRM2 and CHRM4; and the other had triplicated, forming the CHRM1,CHRM3, and CHRM5 subfamily. All five genes are still present in all vertebrate groups investigated except theCHRM1 gene, which has not been identified in some of the teleosts or in chicken or any other birds. Interestingly, the third tetraploidization (3R) that took place in the teleost predecessor resulted in duplicates of all five mAChR genes of which all 10 are present in zebrafish. One of the copies of the CHRM2 and CHRM3 genes and bothCHRM4 copies have gained introns in teleosts. Not a single separate (nontetraploidization) duplicate has been identified in any vertebrate species. These results clarify the evolution of the vertebrate mAChR family and reveal a doubled repertoire in zebrafish, inviting studies of gene neofunctionalization and subfunctionalization.

    Keywords
    acetylcholine, G-protein-coupled receptor, gene duplication, muscarinic, tetraploidization, zebrafish
    National Category
    Neurology
    Research subject
    Neuroscience
    Identifiers
    urn:nbn:se:uu:diva-371818 (URN)10.1523/ENEURO.0340-18.2018 (DOI)000456897300002 ()30564629 (PubMedID)
    Funder
    Carl Tryggers foundation
    Note

    De två första författarna delar förstaförfattarskapet.

    Available from: 2019-01-02 Created: 2019-01-02 Last updated: 2020-11-16Bibliographically approved
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  • 293.
    Pedersen, Julia E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    The connectivity of the TRPV1-Cre mouse line as a useful tool for exploring the function of a VTA subpopulationManuscript (preprint) (Other academic)
  • 294.
    Pedersen, Julia E.
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Bergqvist, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larhammar, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Evolution of vertebrate nicotinic acetylcholine receptors2019In: BMC Evolutionary Biology, E-ISSN 1471-2148, Vol. 19, article id 38Article in journal (Refereed)
    Abstract [en]

    Background

    Many physiological processes are influenced by nicotinic acetylcholine receptors (nAChR), ranging from neuromuscular and parasympathetic signaling to modulation of the reward system and long-term memory. Due to the complexity of the nAChR family and variable evolutionary rates among its members, their evolution in vertebrates has been difficult to resolve. In order to understand how and when the nAChR genes arose, we have used a broad approach of analyses combining sequence-based phylogeny, chromosomal synteny and intron positions.

    Results

    Our analyses suggest that there were ten subunit genes present in the vertebrate predecessor. The two basal vertebrate tetraploidizations (1R and 2R) then expanded this set to 19 genes. Three of these have been lost in mammals, resulting in 16 members today. None of the ten ancestral genes have kept all four copies after 2R. Following 2R, two of the ancestral genes became triplicates, five of them became pairs, and three seem to have remained single genes. One triplet consists of CHRNA7, CHRNA8 and the previously undescribed CHRNA11, of which the two latter have been lost in mammals but are still present in lizards and ray-finned fishes. The other triplet consists of CHRNB2, CHRNB4 and CHRNB5, the latter of which has also been lost in mammals. In ray-finned fish the neuromuscular subunit gene CHRNB1 underwent a local gene duplication generating CHRNB1.2. The third tetraploidization in the predecessor of teleosts (3R) expanded the repertoire to a total of 31 genes, of which 27 remain in zebrafish. These evolutionary relationships are supported by the exon-intron organization of the genes.

    Conclusion

    The tetraploidizations explain all gene duplication events in vertebrates except two. This indicates that the genome doublings have had a substantial impact on the complexity of this gene family leading to a very large number of members that have existed for hundreds of millions of years.

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  • 295.
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Studies of Giardia-host interactions: role of cysteine-rich surface proteins.2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Giardia intestinalis is a eukaryotic parasite that colonizes the small intestine of humans and animals causing the diarrheal disease known as giardiasis. This parasite is not invasive and does not internalize into host cells but it rather attaches to the brush border surface of the small intestine disrupting the epithelial barrier. Giardia causes around 280 million symptomatic infections in humans every year, while it can also cause chronic and asymptomatic infections. Giardiasis is a multifactorial disease but only few factors that directly contribute in the pathogenesis and virulence of the disease have been identified. G. intestinalis has eight genetic groups, but only two of them (A and B) are known to infect humans.

    In this thesis, whole genome sequencing was performed for two human assemblage A isolates (AS175 and AS98) and were compared to assemblage A isolate WB genome (Paper I). Genome-wide variations were identified among the three isolates including isolate-specific coding sequences and high level of nucleotide diversity of multi-gene families such as VSPs and HCMPs.

    We further used an in vitro model for parasite interaction with host intestinal epithelial cells (IECs) to study the interplay between Giardia and the human host. We have identified the major Giardia excretory-secretory products (ESPs) released by two Giardia isolates (WB and GS) when they interact with the Caco-2 IECs (Paper II). Wide changes in the transcriptome (Paper III) and the proteome (Paper IV) of the parasite (WB isolate) and the host IECs have been studied giving us a further understanding of the parasite-host interactions. An understudied gene family (HCMPs) was studied and further characterized during interactions in both RNA and protein level (Paper III, IV).

    In conclusion, the thesis has provided a further understanding of Giardia-host interactions in vitro and the molecular mechanisms involved.

    List of papers
    1. Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates
    Open this publication in new window or tab >>Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates
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    2015 (English)In: BMC Genomics, E-ISSN 1471-2164, Vol. 16, article id 697Article in journal (Refereed) Published
    Abstract [en]

    Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

    National Category
    Genetics Microbiology
    Identifiers
    urn:nbn:se:uu:diva-264039 (URN)10.1186/s12864-015-1893-6 (DOI)000361093400009 ()26370391 (PubMedID)
    Funder
    Swedish Research Council FormasSwedish Research Council
    Available from: 2015-10-06 Created: 2015-10-05 Last updated: 2024-01-17Bibliographically approved
    2. Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells
    Open this publication in new window or tab >>Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells
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    2017 (English)In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 11, no 12, article id e0006120Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND:

    Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known.

    METHODOLOGY:

    To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro.

    FINDINGS:

    The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis.

    CONCLUSIONS:

    This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of host and parasite ESPs during interactions and reveals the intricate cellular responses that can explain disease mechanisms and attenuated inflammatory responses during giardiasis.

    National Category
    Analytical Chemistry Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-338331 (URN)10.1371/journal.pntd.0006120 (DOI)000419108500030 ()29228011 (PubMedID)
    Available from: 2018-01-08 Created: 2018-01-08 Last updated: 2019-04-19Bibliographically approved
    3. High Cysteine Proteins are up-regulated during Giardia-host cell interaction.
    Open this publication in new window or tab >>High Cysteine Proteins are up-regulated during Giardia-host cell interaction.
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    Giardia, HCMPs, interactions, host-parasite interactions, infection, parasite, RNA-seq
    National Category
    Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-381936 (URN)
    Available from: 2019-04-16 Created: 2019-04-16 Last updated: 2019-04-19
    4. Proteome analyses of Giardia–host cell interactions in vitro.
    Open this publication in new window or tab >>Proteome analyses of Giardia–host cell interactions in vitro.
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Giardia, host-parasite interactions, parasite, infections, proteomics
    National Category
    Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-381937 (URN)
    Available from: 2019-04-16 Created: 2019-04-16 Last updated: 2019-04-19
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  • 296.
    Peirasmaki, Dimitra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Xu, Feifei
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ferella, Marcela
    Karolinska Inst, Sci Life Lab SciLifeLab, Eukaryot Single Cell Genom Platform, Solna, Sweden..
    Campos, Sara
    Max Planck Inst Infect Biol, Dept Mol Biol, Berlin, Germany..
    Liu, Jingyi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Svärd, Staffan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala Univ, Dept Cell & Mol Biol, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab SciLifeLab, Uppsala, Sweden..
    High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated DuringGiardia-Host Cell Interactions2020In: Frontiers in Genetics, E-ISSN 1664-8021, Vol. 11, article id 913Article in journal (Refereed)
    Abstract [en]

    Giardia intestinaliscolonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used anin vitromodel of the parasite's interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) inGiardia, which might relate to the establishment of infection and disease induction.Giardiatrophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles duringGiardia-host cell interactions.

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  • 297.
    Peirasmaki, Dimitra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Xu, Feifei
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Ferella, Marcela
    Eukaryotic Single Cell Genomics Platform, Karolinska Institute, SciLifeLab, Sweden.
    Campos, Sara
    Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Liu, Jingyi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    High Cysteine Proteins are up-regulated during Giardia-host cell interaction.Manuscript (preprint) (Other academic)
  • 298.
    Peirasmaki, Dimitra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Xia, Dong
    The Royal Veterinary College, London, United Kingdom.
    Attree, Elizabeth
    The Royal Veterinary College, London, United Kingdom.
    Ferella, Marcela
    Eukaryotic Single Cell Genomics Platform, Karolinska Institute, SciLifeLab, Sweden.
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wastling, Jonathan
    Faculty of Natural Sciences, University of Keele, Newcastle-under-Lyme, United Kingdom.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Proteome analyses of Giardia–host cell interactions in vitro.Manuscript (preprint) (Other academic)
  • 299.
    Peleli, Maria
    et al.
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Zollbrecht, Christa
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Montenegro, Marcelo F.
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Hezel, Michael
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Zhong, Jianghong
    Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Persson, Erik G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Holmdahl, Rikard
    Karolinska Inst, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Weitzberg, Eddie
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Lundberg, Jon O.
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Carlström, Mattias
    Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Enhanced XOR activity in eNOS-deficient mice Effects on the nitrate-nitrite-NO pathway and ROS homeostasis2016In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 99, p. 472-484Article in journal (Refereed)
    Abstract [en]

    Xanthine oxidoreductase (XOR) is generally known as the final enzyme in purine metabolism and as a source of reactive oxygen species (ROS). In addition, this enzyme has been suggested to mediate nitric oxide (NO) formation via reduction of inorganic nitrate and nitrite. This NO synthase (NOS)-independent pathway for NO generation is of particular importance during certain conditions when NO bioavailability is diminished due to reduced activity of endothelial NOS (eNOS) or increased oxidative stress, including aging and cardiovascular disease. The exact interplay between NOS- and XOR-derived NO generation is not fully elucidated yet. The aim of the present study was to investigate if eNOS deficiency is associated with changes in XOR expression and activity and the possible impact on nitrite, NO and ROS homeostasis. Plasma levels of nitrate and nitrite were similar between eNOS deficient (eNOS(-/-)) and wildtype (wt) mice. XOR activity was upregulated in eNOS(-/-) compared with wt, but not in nNOS(-/-), iNOS(-/-) or wt mice treated with the non-selective NOS inhibitor L-NAME. Following an acute dose of nitrate, plasma nitrite increased more in eNOS(-/-) compared with wt, and this augmented response was abolished by the selective XOR inhibitor febuxostat. Livers from eNOS(-/-) displayed higher nitrite reducing capacity compared with wt, and this effect was attenuated by febuxostat. Dietary supplementation with nitrate increased XOR expression and activity, but concomitantly reduced superoxide generation. The latter effect was also seen in vitro after nitrite administration. Treatment with febuxostat elevated blood pressure in eNOS(-/-), but not in wt mice. A high dose of dietary nitrate reduced blood pressure in na ve eNOS(-/-) mice, and again this effect was abolished by febuxostat. In conclusion, eNOS deficiency is associated with an upregulation of XOR facilitating the nitrate-nitrite-NO pathway and decreasing the generation of ROS. This interplay between XOR and eNOS is proposed to play a significant role in NO homeostasis and blood pressure regulation.

  • 300.
    Pellegrini, Paola
    et al.
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden.;VLVBio, Hastholmsvagen 32, S-13130 Nacka, Sweden..
    Selvaraju, Karthik
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden..
    Faustini, Elena
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden..
    Mofers, Arjan
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden..
    Zhang, Xiaonan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Ternerot, Jens
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden..
    Schubert, Alice
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden..
    Linder, Stig
    Karolinska Inst, Dept Oncol Pathol, S-17176 Stockholm, Sweden..
    D'Arcy, Padraig
    Linkoping Univ, Dept Biomed & Clin Sci, S-58183 Linkoping, Sweden..
    Induction of ER Stress in Acute Lymphoblastic Leukemia Cells by the Deubiquitinase Inhibitor VLX15702020In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 13, article id 4757Article in journal (Refereed)
    Abstract [en]

    The proteasome is a validated target of cancer therapeutics. Inhibition of proteasome activity results in the activation of the unfolded protein response (UPR) characterized by phosphorylation of eukaryotic initiation factor 2 alpha (eIF2 alpha), global translational arrest, and increased expression of the proapoptotic CHOP (C/EBP homologous protein) protein. Defects in the UPR response has been reported to result in altered sensitivity of tumor cells to proteasome inhibitors. Here, we characterized the effects of the deubiquitinase (DUB) inhibitor VLX1570 on protein homeostasis, both at the level of the UPR and on protein translation, in acute lymphoblastic leukemia (ALL). Similar to the 20S inhibitor bortezomib, VLX1570 induced accumulation of polyubiquitinated proteins and increased expression of the chaperone Grp78/Bip in ALL cells. Both compounds induced cleavage of PARP (Poly (ADP-ribose) polymerase) in ALL cells, consistent with induction of apoptosis. However, and in contrast to bortezomib, VLX1570 treatment resulted in limited induction of the proapoptotic CHOP protein. Translational inhibition was observed by both bortezomib and VLX1570. We report that in distinction to bortezomib, suppression of translation by VXL1570 occurred at the level of elongation. Increased levels of Hsc70/Hsp70 proteins were observed on polysomes following exposure to VLX1570, possibly suggesting defects in nascent protein folding. Our findings demonstrate apoptosis induction in ALL cells that appears to be uncoupled from CHOP induction, and show that VLX1570 suppresses protein translation by a mechanism distinct from that of bortezomib.

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