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  • 201.
    Chantzi, Efthymia
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin.
    Jarvius, Malin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin. Uppsala Univ, In Vitro Syst Pharmacol Facil, SciLifeLab Drug Discovery & Dev, Uppsala, Sweden.
    Niklasson, Mia
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Neuroonkologi.
    Segerman, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Neuroonkologi.
    Gustafsson, Mats G
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin.
    COMBImage: a modular parallel processing framework for pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies2018Inngår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 19, artikkel-id 453Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Large-scale pairwise drug combination analysis has lately gained momentum in drug discovery and development projects, mainly due to the employment of advanced experimental-computational pipelines. This is fortunate as drug combinations are often required for successful treatment of complex diseases. Furthermore, most new drugs cannot totally replace the current standard-of-care medication, but rather have to enter clinical use as add-on treatment. However, there is a clear deficiency of computational tools for label-free and temporal image-based drug combination analysis that go beyond the conventional but relatively uninformative end point measurements.

    Results: COMBImage is a fast, modular and instrument independent computational framework for in vitro pairwise drug combination analysis that quantifies temporal changes in label-free video microscopy movies. Jointly with automated analyses of temporal changes in cell morphology and confluence, it performs and displays conventional cell viability and synergy end point analyses. The image processing algorithms are parallelized using Google's MapReduce programming model and optimized with respect to method-specific tuning parameters. COMBImage is shown to process time-lapse microscopy movies from 384-well plates within minutes on a single quad core personal computer.This framework was employed in the context of an ongoing drug discovery and development project focused on glioblastoma multiforme; the most deadly form of brain cancer. Interesting add-on effects of two investigational cytotoxic compounds when combined with vorinostat were revealed on recently established clonal cultures of glioma-initiating cells from patient tumor samples. Therapeutic synergies, when normal astrocytes were used as a toxicity cell model, reinforced the pharmacological interest regarding their potential clinical use.

    Conclusions: COMBImage enables, for the first time, fast and optimized pairwise drug combination analyses of temporal changes in label-free video microscopy movies. Providing this jointly with conventional cell viability based end point analyses, it could help accelerating and guiding any drug discovery and development project, without use of cell labeling and the need to employ a particular live cell imaging instrument.

  • 202.
    Charkoftaki, Georgia
    et al.
    Yale Univ, Yale Sch Publ Hlth, Dept Environm Hlth Sci, New Haven, CT USA.
    Rattray, Nicholas J. W.
    Yale Univ, Yale Sch Publ Hlth, Dept Environm Hlth Sci, New Haven, CT USA.
    Andrén, Per E.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Caprioli, Richard M.
    Vanderbilt Univ, Sch Med, Dept Biochem, Nashville, TN USA; Vanderbilt Univ, Sch Med, Mass Spectrometry Res Ctr, Nashville, TN USA.
    Castellino, Steve
    GSK, Dept Bioimaging Platform Sci & Technol, King Of Prussia, PA USA.
    Duncan, Mark W.
    Biodesix Inc, Boulder, CO USA.
    Goodwin, Richard J. A.
    AstraZeneca, IMED Biotech Unit, Pathol Drug Safety & Metab, Cambridge, England.
    Schey, Kevin L.
    Vanderbilt Univ, Sch Med, Dept Biochem, Nashville, TN USA; Vanderbilt Univ, Sch Med, Dept Ophthalmol & Visual Sci, Nashville, TN USA.
    Shahidi-Latham, Sheerin K.
    Genentech Inc, Dept Drug Metab & Pharmacokinet, San Francisco, CA USA.
    Veselkov, Kirill A.
    Imperial Coll London, Dept Surg & Canc, Fac Med, Computat & Syst Med, London, England.
    Johnson, Caroline H.
    Yale Univ, Yale Sch Publ Hlth, Dept Environm Hlth Sci, New Haven, CT USA; Yale Univ, Yale Sch Med, Yale Canc Ctr, New Haven, CT USA.
    Vasiliou, Vasilis
    Yale Univ, Yale Sch Publ Hlth, Dept Environm Hlth Sci, New Haven, CT USA; Yale Univ, Yale Sch Med, Yale Canc Ctr, New Haven, CT USA; Yale Univ, Yale Sch Med, Dept Ophthalmol & Visual Sci, New Haven, CT USA.
    Yale School of Public Health Symposium on tissue imaging mass spectrometry: illuminating phenotypic heterogeneity and drug disposition at the molecular level2018Inngår i: HUMAN GENOMICS, ISSN 1473-9542, Vol. 12, artikkel-id 10Artikkel i tidsskrift (Annet vitenskapelig)
  • 203.
    Charpentier, Emmanuelle
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Doudna, Jennifer A.
    Biotechnology: rewriting a genome2013Inngår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 495, nr 7439, s. 50-51Artikkel i tidsskrift (Annet vitenskapelig)
  • 204.
    Charpentier, Emmanuelle
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Richter, Hagen
    van der Oost, John
    White, Malcolm F.
    Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity2015Inngår i: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, nr 3, s. 428-441Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

  • 205.
    Chaudhari, Aditi
    et al.
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden.
    Krumlinde, Daniel
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden / Scientific Solutions, Stockholm, Sweden.
    Lundqvist, Annika
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden.
    Akyurek, Levent M
    Department of Medical Chemistry and Cell Biology, University of Gothenburg, Sweden.
    Bandaru, Sashidhar
    Department of Medical Chemistry and Cell Biology, University of Gothenburg, Sweden.
    Skalen, Kristina
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden.
    Stahlman, Marcus
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden.
    Boren, Jan
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden.
    Wettergren, Yvonne
    Department of Surgery, University of Gothenburg, Sweden.
    Ejeskär, Katarina
    Högskolan i Skövde, Forskningscentrum för Systembiologi. Högskolan i Skövde, Institutionen för hälsa och lärande. Department of Medical and Clinical Genetics, University of Gothenburg, Sweden.
    Sopasakis, Victoria Rotter
    Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden.
    p110 alpha Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110 alpha Kinase Activity2015Inngår i: Molecular and Cellular Biology, ISSN 0270-7306, Vol. 35, nr 19, s. 3258-3273Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.

  • 206.
    Chen, Lin
    et al.
    Tsinghua Univ, Yuquan Hosp, Beijing 100040, Peoples R China..
    Ao, Qiang
    China Med Univ, Dept Tissue Engn, Shenyang 110122, Peoples R China..
    Sharma, Hari Shanker
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper, Anestesiologi och intensivvård.
    Wang, Aijun
    Univ Calif Davis, Med Ctr, Surg Bioengn Lab, Dept Surg, Sacramento, CA 95817 USA..
    Feng, Shiqing
    Tianjin Med Univ, Tianjin, Peoples R China..
    Neurorestoratologic Strategies and Mechanisms in the Nervous System2015Inngår i: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, artikkel-id 163170Artikkel i tidsskrift (Annet vitenskapelig)
  • 207.
    Chen, Xiaomei
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap. Univ Michigan, Coll Pharm, Dept Pharmaceut Sci, 428 Church St, Ann Arbor, MI 48109 USA..
    Slättengren, Tim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    de Lange, Elizabeth C. M.
    Leiden Acad Ctr Drug Res, Dept Pharmacol, Leiden, Netherlands..
    Smith, David E.
    Univ Michigan, Coll Pharm, Dept Pharmaceut Sci, 428 Church St, Ann Arbor, MI 48109 USA..
    Hammarlund-Udenaes, Margareta
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Revisiting atenolol as a low passive permeability marker2017Inngår i: Fluids and Barriers of the CNS, ISSN 2045-8118, E-ISSN 2045-8118, Vol. 14, artikkel-id 30Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Atenolol, a hydrophilic beta blocker, has been used as a model drug for studying passive permeability of biological membranes such as the blood-brain barrier (BBB) and the intestinal epithelium. However, the extent of S-atenolol (the active enantiomer) distribution in brain has never been evaluated, at equilibrium, to confirm that no transporters are involved in its transport at the BBB.

    Methods: To assess whether S-atenolol, in fact, depicts the characteristics of a low passive permeable drug at the BBB, a microdialysis study was performed in rats to monitor the unbound concentrations of S-atenolol in brain extracellular fluid (ECF) and plasma during and after intravenous infusion. A pharmacokinetic model was developed, based on the microdialysis data, to estimate the permeability clearance of S-atenolol into and out of brain. In addition, the nonspecific binding of S-atenolol in brain homogenate was evaluated using equilibrium dialysis.

    Results: The steady-state ratio of unbound S-atenolol concentrations in brain ECF to that in plasma (i.e., -K-p,K-uu,K-brain) was 3.5% +/- 0.4%, a value much less than unity. The unbound volume of distribution in brain -(V-u,V- brain) of S-atenolol was also calculated as 0.69 +/- 0.10 mL/g brain, indicating that S-atenolol is evenly distributed within brain parenchyma. Lastly, equilibrium dialysis showed limited nonspecific binding of S-atenolol in brain homogenate with an unbound fraction -(f(u, brain)) of 0.88 +/- 0.07.

    Conclusions: It is concluded, based on -K-p,K-uu,K-brain being much smaller than unity, that S-atenolol is actively effluxed at the BBB, indicating the need to re-consider S-atenolol as a model drug for passive permeability studies of BBB transport or intestinal absorption.

  • 208. Chen, Yun
    et al.
    Molnar, Matyas
    Li, Li
    Friberg, Peter
    Gan, Li-Ming
    Brismar, Hjalmar
    Fu, Ying
    Characterization of VCAM-1-Binding Peptide-Functionalized Quantum Dots for Molecular Imaging of Inflamed Endothelium2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 12, s. e83805-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Inflammation-induced activation of endothelium constitutes one of the earliest changes during atherogenesis. New imaging techniques that allow detecting activated endothelial cells can improve the identification of persons at high cardiovascular risk in early stages. Quantum dots (QDs) have attractive optical properties such as bright fluorescence and high photostability, and have been increasingly studied and developed for bio-imaging and bio-targeting applications. We report here the development of vascular cell adhesion molecule-1 binding peptide (VCAM-1 binding peptide) functionalized QDs (VQDs) from amino QDs. It was found that the QD fluorescence signal in tumor necrosis factor (TNF-) treated endothelial cells in vitro was significantly higher when these cells were labeled with VQDs than amino QDs. The VQD labeling of TNF--treated endothelial cells was VCAM-1 specific since pre-incubation with recombinant VCAM-1 blocked cells' uptake of VQDs. Our ex vivo and in vivo experiments showed that in the inflamed endothelium, QD fluorescence signal from VQDs was also much stronger than that of amino QDs. Moreover, we observed that the QD fluorescence peak was significantly blue-shifted after VQDs interacted with aortic endothelial cells in vivo and in vitro. A similar blue-shift was observed after VQDs were incubated with recombinant VCAM-1 in tube. We anticipate that the specific interaction between VQDs and VCAM-1 and the blue-shift of the QD fluorescence peak can be very useful for VCAM-1 detection in vivo.

  • 209. Chen, Zhen
    et al.
    Mori, Wakana
    Deng, Xiaoyun
    Cheng, Ran
    Ogasawara, Daisuke
    Zhang, Genwei
    Schafroth, Michael A.
    Dahl, Kenneth
    Fu, Hualong
    Hatori, Akiko
    Shao, Tuo
    Zhang, Yiding
    Yamasaki, Tomoteru
    Zhang, Xiaofei
    Rong, Jian
    Yu, Qngzhen
    Hu, Kuan
    Fujinaga, Masayuki
    Xie, Lin
    Kumata, Katsushi
    Gou, Yuancheng
    Chen, Jingjin
    Gu, Shuyin
    Bao, Liang
    Wang, Lu
    Collier, Thomas Lee
    Vasdev, Neil
    Shao, Yihan
    Ma, Jun-An
    Cravatt, Benjamin F.
    Fowler, Christopher
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Josephson, Lee
    Zhang, Ming-Rong
    Liang, Steven H.
    Design, Synthesis, and Evaluation of Reversible and Irreversible Monoacylglycerol Lipase Positron Emission Tomography (PET) Tracers Using a "Tail Switching" Strategy on a Piperazinyl Azetidine Skeleton2019Inngår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 62, nr 7, s. 3336-3353Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Monoacylglycerol lipase (MAGL) is a senile hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the endocannabinoid system (eCB). Selective inhibition of MAGL has emerged as a potential therapeutic approach for the treatment of diverse pathological conditions, including chronic pain, inflammation, cancer, and neurodegeneration. Herein, we disclose a novel array of reversible and irreversible MAGL inhibitors by means of "tail switching" on a piperazinyl azetidine scaffold. We developed a lead irreversible-binding MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with C-11 or F-18. [C-11]8 ([C-11]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible radioligands [C-11]17 ([C-11]PAD) and [F-18]37 ([F-18]MAGL-4-11) also demonstrated excellent in vivo binding specificity toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission tomography tracers with tunability in reversible and irreversible binding mechanisms.

  • 210. Cheng, Ran
    et al.
    Mori, Wakana
    Ma, Longle
    Alhouayek, Mireille
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Hatori, Akiko
    Zhang, Yiding
    Ogasawara, Daisuke
    Yuan, Gengyang
    Chen, Zhen
    Zhang, Xiaofei
    Shi, Hang
    Yamasaki, Tomoteru
    Xie, Lin
    Kumata, Katsushi
    Fujinaga, Masayuki
    Nagai, Yuji
    Minamimoto, Takafumi
    Svensson, Mona
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Wang, Lu
    Du, Yunfei
    Ondrechen, Mary Jo
    Vasdev, Neil
    Cravatt, Benjamin F.
    Fowler, Christopher
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap.
    Zhang, Ming-Rong
    Liang, Steven H.
    In Vitro and in Vivo Evaluation of C-11-Labeled Azetidinecarboxylates for Imaging Monoacylglycerol Lipase by PET Imaging Studies2018Inngår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, nr 6, s. 2278-2291Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing endogenous cannabinoid ligand 2-arachidonoyglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation, and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including C-11-carbonylation and spirocyclic iodonium ylide (SCIDY) radio fluorination. The lead compound [C-11]10 (MAGL-0519) demonstrated high specific binding and selectivity in vitro and in vivo. We also observed unexpected washout kinetics with these irreversible radiotracers, in which in vivo evidence for turnover of the covalent residue was unveiled between MAGL and azetidine carboxylates. This work may lead to new directions for drug discovery and PET tracer development based on azetidine carboxylate inhibitor scaffold.

  • 211.
    Chermenina, Maria
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pokrzywa, Malgorzata
    Antti, Henrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Strömberg, Ingrid
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Single injection of small-molecule amyloid accelerator results in cell death of nigral dopamine neurons in mice2015Inngår i: Parkinson's Disease, ISSN 2090-8083, E-ISSN 2042-0080, Vol. 1, artikkel-id 15024Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The assembly process of a-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson´s disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of a-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of a-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and follwed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substansia nigra of normal and a-synuclein knockout mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed singificant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in a-synuclein knock-out mice injected with accelerator intor the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson´s disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice.

  • 212.
    Chi, Celestine
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Post-synaptic Density Disc Large Zo-1 (PDZ) Domains: From Folding and Binding to Drug Targeting2010Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles.

    In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions.

    I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding.

    Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction.

    Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

  • 213.
    Chi, Celestine N.
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Bach, Anders
    University of Copenhagen.
    Engström, Åke
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Strømgaard, Kristian
    University of Copenhagen.
    Lundström, Patrik
    University of Linköping.
    Ferguson, Neil
    4UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin.
    Jemth, Per
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Conformational change and non-canonical interactions in the complex between human papillomavirus E6 protein and Synapse-Associated Protein 97Inngår i: Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The E6 protein of human papillomavirus exhibits complex interaction patterns with several host proteins and their roles in HPV mediated oncogensis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein SAP97. All potential binding sites in SAP97 can bind E6 with low micromolar affinity. Unexpectedly,binding is not mutually exclusive and all three PDZ domains can simultaneously bind E6. Intriguingly, the quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, despite having a doubled mass, suggesting that a conformational change occurs in the PDZ region upon E6 binding. Further, using NMR, we discovered a new mode of interaction between E6 and PDZ, as a subset of residues distal to the binding pocket in the PDZ2 domain was found to exhibit non-canonical interactions with the E6 protein suggesting that a large proportion of the proteins surface defines binding specificity

  • 214.
    Ching, Rosanna C.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap.
    Kingham, Paul J.
    Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB).
    The role of exosomes in peripheral nerve regeneration2015Inngår i: Neural Regeneration Research, ISSN 1673-5374, E-ISSN 1876-7958, Vol. 10, nr 5, s. 743-747Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Peripheral nerve injuries remain problematic to treat, with poor functional recovery commonly observed. Injuries resulting in a nerve gap create specific difficulties for axonal regeneration. Approaches to address these difficulties include autologous nerve grafts (which are currently the gold standard treatment) and synthetic conduits, with the latter option being able to be impregnated with Schwann cells or stem cells which provide an appropriate micro-environment for neuronal regeneration to occur. Transplanting stem cells, however, infers additional risk of malignant transformation as well as manufacturing difficulties and ethical concerns, and the use of autologous nerve grafts and Schwann cells requires the sacrifice of a functioning nerve. A new approach utilizing exosomes, secreted extracellular vesicles, could avoid these complications. In this review, we summarize the current literature on exosomes, and suggest how they could help to improve axonal regeneration following peripheral nerve injury.

  • 215. Chiruvella, Kishore K
    et al.
    Rajaei, Naghmeh
    Jonna, Venkateswara Rao
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Åstrom, Stefan U
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 21671Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  • 216.
    Chiruvella, Kishore K.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Rajaei, Naghmeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jonna, Venkateswara Rao
    Hofer, Anders
    Åström, Stefan U.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 21671Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  • 217.
    Cholujová, Dana
    et al.
    Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, Bratislava, Slovakia.
    Jakubíková, Jana
    Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, Bratislava, Slovakia.
    Kubeš, Miroslav
    Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
    Arendacká, Barbora
    Institute of Measurement Science, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
    Sapák, Michal
    Institute of Immunology, Medical Faculty of Comenius University, Sasinkova 4, Bratislava, Slovakia.
    Ihnatko, Robert
    Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
    Sedlák, Ján
    Laboratory of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, Bratislava, Slovakia.
    Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays2008Inngår i: Immunobiology, ISSN 0171-2985, E-ISSN 1878-3279, Vol. 213, nr 8, s. 629-640Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive 51chromium (51Cr) release assay is a “gold standard” for assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3h and 90min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland–Altman statistical method was applied to measure true agreement for all CAM–51Cr, CFSE–51Cr, DiO–51Cr and MTG–51Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard 51Cr release assay. Considering linear relationships between data obtained with four fluorochromes and 51Cr release assay as well as linear regression analysis with R2=0.9393 value for CAM–51Cr pair, we found the CAM assay to be the most closely related to the 51Cr assay.

  • 218.
    Choong, Ferdinand
    et al.
    Karolinska Institutet, Stockholm, Sweden.
    Bäck, Marcus
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Fahlen, Sara
    Karolinska Institutet, Stockholm, Sweden.
    Johansson, Leif B. G.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Melican, Keira
    Karolinska Institutet, Stockholm, Sweden.
    Rhen, Mikael
    Karolinska Institutet, Stockholm, Sweden.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Richter-Dahlfors, Agneta
    Karolinska Institutet, Stockholm, Sweden.
    Real-time opto-tracing of curli and cellulose in live Salmonella biofilms using conjugated oligothiophenes2016Inngår i: npj Biofilms and Microbiomes, ISSN 2055-5008, Vol. 2, artikkel-id 16024Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Extracellular matrix (ECM) is the protein- and polysaccharide-rich backbone of bacterial biofilms that provides a defensive barrier in clinical, environmental and industrial settings. Understanding the dynamics of biofilm formation in native environments has been hindered by a lack of research tools. Here we report a method for simultaneous, real-time, in situ detection and differentiation of the Salmonella ECM components curli and cellulose, using non-toxic, luminescent conjugated oligothiophenes (LCOs). These flexible conjugated polymers emit a conformation-dependent fluorescence spectrum, which we use to kinetically define extracellular appearance of curli fibres and cellulose polysaccharides during bacterial growth. The scope of this technique is demonstrated by defining biofilm morphotypes of Salmonella enterica serovars Enteritidis and Typhimurium, and their isogenic mutants in liquid culture and on solid media, and by visualising the ECM components in native biofilms. Our reported use of LCOs across a number of platforms, including intracellular cellulose production in eukaryotic cells and in infected tissues, demonstrates the versatility of this optotracing technology, and its ability to redefine biofilm research.

  • 219.
    Chotteau, Veronique
    et al.
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Wang, Jingjiao
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Tolf, Erika
    KTH, Skolan för bioteknologi (BIO), Bioprocessteknik.
    Wicklund, Linn
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Hovatta, Outi
    Karolinska Institutet, Department of Clinical Science.
    Marutle, Amelia
    Karolinska Institute, Department of Neurobiology, Care Sciences and Society.
    Comparison of cultivation in Techne spinner, Bellco spinner, shake flask and T-flask of human embryonic stem cells2010Inngår i: Proceedings of the SBE's Second International Conference on Stem Cell Engineering, 2010Konferansepaper (Annet vitenskapelig)
    Abstract [en]

    The recent progress in regenerative medicine indicates that pluripotent human embryonic stem cells (hESCs) may hold great potential providing cellular models for drug development and screening, modelling diseases as well as aid in the development of future cell-based therapies for neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Crucial to the success of generating specialized cell populations, is an understanding of the mechanisms, which influence the control of cell growth and differentiation by extrinsic and intrinsic factors. Nowadays, a limitation for the use of hESCs is the lack of proliferation methods in large scale. The purpose of the present work was to study several cultivation systems, which could potentially provide large-scale cultivation processes suitable for human therapy applications. Pluripotent human embryonic stem cells (hESCs), isolated from the inner cell mass of the blastocyst, were cultivated undifferentiated as embryoids bodies, i.e. large spherical aggregates of cells, in absence of serum and feeder layer. The cell growth and culture behavior in T-falsk, orbitally agitated shake flask, Bellco stirred spinner and Techne stirred spinner were observed. In Bellco spinner, the cells were agitated by a rotating impeller providing a movement comparable to stirred bioreactors. In Techne spinner, a slow and gentle orbital movement provided by a rotating bulb-ended stirrer maintained the cells in suspension. The design of this latter spinner allowed lower shear stress in comparison to Bellco spinner and shake flask. It was observed that the cell growth was fastest in Techne spinner followed by cultivation in T-flask and then cultivation in shake flask. Cultivating in Bellco spinner resulted in embryoid dissociation and viability decrease after 14 days. A larger number of single cells, i.e. cells not growing in aggregates, was observed in the static T-flask culture compared to the agitated systems, i.e. shake flask, Bellco spinner or Techne spinner. Probably the agitation promoted the spontaneous aggregation of the cells in spheres. In particular the Techne spinner allowed the most perfect spherical form among the different compared systems. Finally it was observed that hypoxia with 4 % oxygen concentration improved significantly the growth in Techne spinner or T-flask in comparison with normoxia with 21 % oxygen concentration. It was concluded that cultivation in Techne spinner under hypoxia was the most favorable condition among the ones studied here. The agitation provided by Techne spinner improved the cell growth in comparison with static system (T-flask). However using the other agitated systems, shake flask and Bellco spinner, was not comparably beneficial to the cell growth and viability, probably due to the higher shear stress of these systems compared to Techne spinner.

  • 220.
    Christoffersson, Jonas
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    Aronsson, Christopher
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Jury, Michael
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Selegård, Robert
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Aili, Daniel
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Mandenius, Carl-Fredrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device2018Inngår i: Biofabrication, ISSN 1758-5082, E-ISSN 1758-5090, Vol. 11, nr 1, s. 1-13, artikkel-id 015013Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.

  • 221.
    Christoffersson, Jonas
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    Meier, Florian
    Boehringer Ingelheim Pharma GmbH and Co. KG, Nonclinical Drug Safety Germany, D-88397 Biberach an der Riss, Germany.
    Kempf, Henning
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Schwanke, Kristin
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Coffee, Michelle
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Beilmann, Mario
    Boehringer Ingelheim Pharma GmbH and Co. KG, Nonclinical Drug Safety Germany, D-88397 Biberach an der Riss, Germany.
    Zweigerdt, Robert
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
    Mandenius, Carl-Fredrik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska fakulteten.
    A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device2018Inngår i: Bioengineering, E-ISSN 2306-5354, Vol. 5, nr 2, s. 1-13, artikkel-id 36Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

  • 222.
    Chumnarnsilpa, Sakesit
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    The Structural Basis of the Control of Actin Dynamics by the Gelsolin Superfamily Proteins2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Rearrangement of the actin cytoskeleton occurs in a variety of cellular processes and structures and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins (GSPs) control actin organization by severing filaments, capping filament ends and bundling filaments. Structural changes within the GSPs are key in controling their functions. This thesis is aimed in understanding the activation mechanisms of the C-terminal halves of GSPs through investigating the atomic structures of gelsolin, adseverin and villin. X-ray crystallography was used to determine the structures of C-terminal fragments of these 3 proteins. The results demonstrate that: 1) The structure of the activated form of the C-terminal half of gelsolin displays an open conformation, with the actin-binding site on gelsolin domain 4 (G4) fully exposed and all three type-II calcium binding sites (CBS) occupied. Neither actin nor the type-I calcium, which is normally sandwiched between actin and G4, is required to achieve this conformation. 2) Calcium ions at both type-I and type-II CBSs of gelsolin were exchangable within the crystals. Extraction of calcium ions from the CBSs triggered local conformation changes which we speculate are the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. 3) The long helix of G6 in the calcium-bound structure is similar to the helix of calcium-free isolated villin domain 6 (V6). 4) The conformation of the C-terminal half of adseverin in the active state is similar to that of gelsolin. These results suggest that the C-terminal halves of GSPs are activated before forming a complex with actin. The activation involves straightening the helix of domain 6 which is a key component in the global conformation changes of C-terminal halves of these proteins. The results also suggest that a calcium ion may bind to the type-I CBS on domain 4 of the active conformation of GSPs concurrently with forming the complex with actin, hence, stabilizing the GSP:actin complex.

     

  • 223.
    Chumnarnsilpa, Sakesit
    et al.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Loonchanta, Anantasak
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Xue, Bo
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Robinson, Robert C.
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Calcium ion exchange in crystalline gelsolin2006Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 357, nr 3, s. 773-782Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ion-binding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb3+, results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ion-binding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange.

  • 224.
    Chylinski, Krzysztof
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Max F. Perutz Laboratories, University of Vienna, Austria .
    Makarova, Kira S.
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Koonin, Eugene V.
    Classification and evolution of type II CRISPR-Cas systems2014Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, nr 10, s. 6091-6105Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.

  • 225. Claudio, Sustmann
    et al.
    Dickopf, Steffen
    Regula, Jörg T.
    Kettenberger, Hubert
    Mølhøj, Michael
    Gassner, Christian
    Weininger, Diana
    Fenn, Sebastian
    Manigold, Tobias
    Kling, Lothar
    Künkele, Klaus-Peter
    Schwaiger, Manfred
    Bossenmaier, Birgit
    Griese, Julia J.
    Hopfner, Karl-Peter
    Graff-Meyer, Alexandra
    Stahlberg, Henning
    Ringler, Philippe
    Lauer, Matthias E.
    Brinkmann, Ulrich
    Schaefer, Wolfgang
    Klein, Christian
    DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity2019Inngår i: mAbs, ISSN 1942-0862, E-ISSN 1942-0870Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

  • 226.
    Clausson, Carl-Magnus
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Making Visible the Proximity Between Proteins2014Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.

    In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.  

    In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.

    The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.

    The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

  • 227. Clendenen, Tess V
    et al.
    Koenig, Karen L
    Arslan, Alan A
    Lukanova, Annekatrin
    Berrino, Franco
    Gu, Yian
    Hallmans, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Näringsforskning.
    Idahl, Annika
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk vetenskap, Obstetrik och gynekologi.
    Krogh, Vittorio
    Lokshin, Anna E
    Lundin, Eva
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Muti, Paola
    Marrangoni, Adele
    Nolen, Brian M
    Ohlson, Nina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Shore, Roy E
    Sieri, Sabina
    Zeleniuch-Jacquotte, Anne
    Factors associated with inflammation markers, a cross-sectional analysis2011Inngår i: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 56, nr 3, s. 769-778Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Epidemiological studies have reported associations between circulating inflammation markers and risk of chronic diseases. It is of interest to examine whether risk factors for these diseases are associated with inflammation. We conducted a cross-sectional analysis to evaluate whether reproductive and lifestyle factors and circulating vitamin D were associated with inflammation markers, including C-reactive protein, cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-13, TNFα), and cytokine modulators (IL-1RA, sIL-1RII, sIL-2Ra, sIL-4R, sIL-6R, sTNF-R1/R2), among 616 healthy women. We confirmed associations of several inflammation markers with age and BMI. We also observed significantly higher levels of certain inflammation markers in postmenopausal vs. premenopausal women (TNFα, sIL-1RII, sIL-2Ra), with increasing parity (IL-12p40), and with higher circulating 25(OH) vitamin D (IL-13) and lower levels among current users of non-steroidal anti-inflammatory drugs (NSAIDs) (IL-1β, IL-2, IL-10, IL-12p70, and IL-12p40), current smokers (IL-4, IL-13, IL-12p40), and women with a family history of breast or ovarian cancer (IL-4, IL-10, IL-13). Our findings suggest that risk factors for chronic diseases (age, BMI, menopausal status, parity, NSAID use, family history of breast and ovarian cancer, and smoking) are associated with inflammation markers in healthy women.

  • 228. Clyne, N
    et al.
    Wibom, R
    Havu, N
    Hultman, E
    Lins, L-E
    Pehrsson, S K
    Persson, Bengt L.
    Högskolan i Kalmar, Naturvetenskapliga institutionen.
    Rydström, J
    The effect of cobalt on mitochondrial ATP production and cellular morphology in the rat myocardium and skeletal muscle1990Inngår i: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 50, s. 153-159Artikkel i tidsskrift (Fagfellevurdert)
  • 229.
    Collier, Brian
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Posttranscriptional Regulation of Human Papillomavirus Type 16 Late mRNAs2002Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The lifecycle of the human papillomavirus (HPV) is strictly linked to the programmed differentiation of the host cell it infects. Production of late proteins, which are used for the production of the major and minor capsid proteins, is only seen upon terminal differentiation of the epithelium.

    The regulation of late gene expression appears to be a multi-faceted process in which transcription, splicing, polyadenylation, mRNA stability and translation seem to play functional roles.

    In this study, we have identified and examined the roles of cis-acting negative elements present on the late mRNAs and trans-acting factors, which interact specifically with these sequences.

    The HPV-16 L2 mRNA has been shown to contain negative elements in both the 5’ and 3’ halves of the gene. The 5’ negative element had a destabilizing effect on the mRNA, while the element in the 3’end affected the level of protein production. The 3’ element was used in RNA-protein binding studies to identify proteins, which specifically interact. We identified three proteins, hnRNP K and the Poly(rC) binding proteins 1 and 2, interacting with the 3’ negative element. The binding of these proteins was further shown to inhibit the translation of HPV-16 L2 mRNA in vitro.

    The HPV-16 L1 mRNA also contains multiple negative elements, which appear to be restricted to the first 514 nucleotides (nts) of the L1 gene. The strongest negative element was located in the first 129 nucleotides and inhibition appears to be localised to the nucleus.

    Mutation of the negative elements in the first 514 nts, while retaining an open reading frame (ORF) that has the ability to produce L1 protein, resulted in loss of inhibition and production of L1 mRNA and protein. Furthermore, negative elements were also located in the ORFs of HPV 5, 6b, 18, 45, and 56.

    The usage of rare codons in certain genes has been suggested to affect translational efficiency.

    We wished to demonstrate that codon usage did not contribute to the lack of L1 protein production. Alterations of rare codons in the entire L1 ORF increased the translational efficiency of L1 protein 2-fold. Interestingly, RNA analysis of an L1 ORF with mutations in the first 514nts showed the appearance of an approximate 700 bp RNA species. RT-PCR analysis and sequencing demonstrated that this was an mRNA polyadenylated at an internal poly (A) site within the L1 ORF. Mutation of this poly (A) site, results in all L1 mRNA being polyadenylated at the late poly (A) site, downstream of L1.

    In conclusion, this study demonstrates that HPV-16 late gene expression is regulated at many levels, in particular translational inhibition mediated by RNA binding protein and mRNA stability. It remains to be seen if the use of the alternative poly (A) site within the L1 ORF also plays a role in regulating L1 expression.

  • 230. Coltrini, Daniela
    et al.
    Di Salle, Emanuela
    Ronca, Roberto
    Belleri, Mirella
    Testini, Chiara
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi.
    Presta, Marco
    Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR2012Inngår i: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209Artikkel i tidsskrift (Fagfellevurdert)
  • 231. Cooper, Helen M.
    et al.
    Yang, Yang
    Ylikallio, Emil
    Khairullin, Rafil
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia.
    Woldegebriel, Rosa
    Lin, Kai-Lan
    Euro, Liliya
    Palin, Eino
    Wolf, Alexander
    Trokovic, Ras
    Isohanni, Pirjo
    Kaakkola, Seppo
    Auranen, Mari
    Lönnqvist, Tuula
    Wanrooij, Sjoerd
    Tyynismaa, Henna
    ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia2017Inngår i: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 26, nr 8, s. 1432-1443Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

  • 232.
    Costa Felicissimo, Viviane
    KTH, Skolan för bioteknologi (BIO), Teoretisk kemi (stängd 20110512).
    Infrared - X-ray pump probe spectroscopy2005Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using the two technologies combined in pump probe experiments. Three main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding on the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; and the development of theory, with sample applications, for infrared X-ray pump probe spectroscopy - the main theme of the thesis.

    Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found that the X-ray photoelectron spectrum of the water dimer differs strongly from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. Our simulations of infrared X-ray pump-probe spectra were carried out using wave packet propagation techniques.

    The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-ray pump probe spectroscopy - are examined in detail in two sample applications - on the NO molecule and on the dynamics of proton transfer in core ionized water dimer. It is found that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential. This results in a phase dependence of the X-ray pump probe spectra. A proper choice of the delay time of the X-ray pulse allows to directly observe the X-ray transition in the proton transfered well of the core excited potential.

  • 233.
    Costa, Tiago
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College, London, UK.
    Francis, Monika K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Region Västerbotten.
    Farag, Salah
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edgren, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Measurement of Yersinia translocon pore formation in erythrocytes2019Inngår i: Pathogenic Yersinia: methods and protocols / [ed] Viveka Vadyvaloo and Matthew B. Lawrenz, New York, NY, U.S.A.: Humana Press, 2019, s. 211-229Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.

  • 234.
    Crona, Joakim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper.
    Charting the Genetic Landscape and Clonal Architectures of Pheochromocytoma2014Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Genotypic and phenotypic inter patient heterogeneity characterize pheochromocytoma and paraganglioma (PPGL). Up to 60% of PPGL are associated with either somatic or germline mutations in at least 14 established disease causing genes. Consequently, a comprehensive screening test for PPGL patients utilizing standard techniques is not feasible and in the diagnostic approach, multiple different phenotype guided gene prioritization protocols have been utilized. This may result in misdiagnosis, especially in patients with sporadic presentation. Diagnostic testing of somatic mutations in tumour material is not performed due to the lack of actionable results.

    The aims of this study were, (1) to investigate the use of novel sequencing techniques in a clinical application, (2) to discover novel PPGL disease causing loci using novel sequencing techniques, (3) to characterize a large cohort of PPGL for mutations in known disease causing genes and to analyse corresponding genotype-phenotype correlations, (4) to dissect the molecular and genetic landscape of MEN2 PPGL and (5) to determine the clonal architecture and heterogeneity within, and in-between matched PPGL.

    For these purposes we studied PPGL tumours from a total of 96 patients using targeted and/or whole exome enrichment, capillary and high throughput sequencing as well as genome wide array based genotyping. Novel bioinformatics pipelines were constructed for raw data processing and downstream interpretation. Quantitative PCR, western blot and immunohistochemistry were utilized in order to characterize molecular traits. Selected experimental findings were correlated to patient phenotype.

    We conclude that novel sequencing techniques could be utilized in clinical genetic screening of patients with PPGL. Somatic gain-of-function mutations in H-RAS are likely to contribute to disease pathogenesis. Analysing tumour DNA for somatic mutations in disease causing genes could provide relevant clinical information and have an impact on patient management. Concomitant mutations in PPGL may occur in exceptional cases and have a substantial impact on tumour biology and patient phenotype. And finally genetic heterogeneity is present between and within a majority of PPGL tumours.

  • 235.
    Crona, Joakim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper.
    Concurrent somatic VHL deletion and point mutation in a Multiple Endocrine 2 Neoplasia type  metastatic pheochromocytoma.Manuskript (preprint) (Annet vitenskapelig)
  • 236.
    Crona, Joakim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för kirurgiska vetenskaper.
    Spatio-temporal heterogeneity characterize the genetic landscape of pheochromocytoma and defines early events in tumorigenesis.Manuskript (preprint) (Annet vitenskapelig)
  • 237.
    Cros, Olivier
    Linköpings universitet, Institutionen för medicinsk teknik. Linköpings universitet, Tekniska fakulteten.
    Structural properties of the mastoid using image analysis and visualization2017Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The mastoid, located in the temporal bone, houses an air cell system whose cells have a variation in size that can go far below current conventional clinical CT scanner resolution. Therefore, the mastoid air cell system is only partially represented in a CT scan. Where the conventional clinical CT scanner lacks level of minute details, micro-CT scanning provides an overwhelming amount of ne details. The temporal bone being one of the most complex in the human body, visualization of micro-CT scanning of this boneawakens the curiosity of the experimenter, especially with the correct visualization settings.

    This thesis first presents a statistical analysis determining the surface area to volume ratio of the mastoid air cell system of human temporal bone, from micro-CT scanning using methods previously applied for conventional clinical CT scans. The study compared current results with previous studies, with successive downsampling the data down to a resolution found in conventional clinical CT scanning. The results from the statistical analysis showed that all the small mastoid air cells, that cannot be detected in conventional clinical CT scans, do heavily contribute to the estimation of the surface area, and in consequence to the estimation of the surface area to volume ratio by a factor of about 2.6. Such a result further strengthens the idea of the mastoid to play an active role in pressure regulation and gas exchange.

    Discovery of micro-channels through specific use of a non-traditional transfer function was then reported, where a qualitative and a quantitative pre-analysis were performed and reported. To gain more knowledge about these micro-channels, a local structure tensor analysis was applied where structures are described in terms of planar, tubular, or isotropic structures. The results from this structural tensor analysis suggest these microchannels to potentially be part of a more complex framework, which hypothetically would provide a separate blood supply for the mucosa lining the mastoid air cell system.

    The knowledge gained from analysing the micro-channels as locally providing blood to the mucosa, led to the consideration of how inflammation of the mucosa could impact the pneumatization of the mastoid air cell system. Though very primitive, a 3D shape analysis of the mastoid air cell system was carried out. The mastoid air cell system was first represented in a compact form through a medial axis, from which medial balls could be used. The medial balls, representative of how large the mastoid air cells can be locally, were used in two complementary clustering methods, one based on the size diameter of the medial balls and one based on their location within the mastoid air cell system. From both quantitative and qualitative statistics, it was possible to map the clusters based on pre-defined regions already described in the literature, which opened the door for new hypotheses concerning the effect of mucosal inflammation on the mastoid pneumatization.

    Last but not least, discovery of other structures, previously unreported in the literature, were also visually observed and briefly discussed in this thesis. Further analysis of these unknown structures is needed.

  • 238.
    Dagälv, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Role of Heparan Sulfate N-sulfation in Mouse Embryonic Development2010Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Heparan sulfate (HS) is a sulfated glycosaminoglycan expressed by all cells in the body. It is found at the cell surface and in the extracellular matrix where it binds a large amount of various ligands including growth factors and morphogens. HS is important for building up morphogen gradients during embryonic development and to act as coreceptors for signaling molecules. Many different Golgi enzymes are involved in the biosynthesis of HS. It is known that some of these enzymes interact with each other but not how the whole biosynthesis machinery works or how the cell regulates the structure of the HS that it produces.

    In this thesis, cells and mice deficient in two of these biosynthetic enzymes, glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST1) and the isoform NDST2 have been studied. NDSTs perform the first modifications during biosynthesis where they replace N-acetyl groups on N-acetyl-glucosamine units with sulfate groups. It is known that deficiency of NDST1 is lethal, while lack of NDST2 only results in abnormal connective tissue type mast cells. Here it is shown that deficiency of both NDST1 and NDST2 is embryonically lethal. The embryonic stem (ES) cells extracted from the inner cell mass of double knockout blastocysts show in addition an impaired differentiation capacity compared to wild-type ES cells and fail completely to differentiate into cardiac muscle cells which NDST1-/-, NDST2-/- and wild-type ES cells all do.

    Cultured mast cells that lack NDST2 produce heparin that is low-sulfated compared to wild-type HS. To our surprise, we could show that mast cells deficient in NDST1 instead produce a more highly sulfated heparin than wild-type cells. We use a model that predicts that the biosynthesis enzymes work together in a multienzyme complex, the GAGosome, to explain our results. We hypothesize that NDST1 has a higher affinity for the GAGosome than NDST2 which only in the absence of NDST1 gets incorporated into the enzyme complex. When all GAGosomes contain NDST2, a more highly sulfated glycosaminoglycan chain will be synthesized.

    A splice variant of NDST1, NDST1S, has also been studied. We could show that NDST1S lacks enzyme activity but that it probably has the capacity to incorporate into GAGosomes. Overexpression of NDST1S results in altered structure of the HS produced by the cells. We speculate that expression of the splice variant during development may be one way to regulate HS structure.

  • 239.
    Dagälv, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Holmborn, Katarina
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Eriksson, Inger
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Ringvall, Maria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lack of both lethality and defective in vitro differentiation of embryonic stem cells N-deacetylase/N-sulfotransferase 1 and 2 causes early embryonicManuskript (preprint) (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    NDSTs (N-deacetylase/N-sulfotransferases) are enzymes responsible for N-sulfation during heparan sulfate and heparin biosynthesis. While lack of NDST2 results in defective mast cells and NDST1 deficiency causes neonatal death and lung, skeletal and brain defects, lack of both isoforms is not compatible with embryonic development. We here show that NDST1/2-/- embryos die before E6.5 and that embryos dissected out at E5.5 lack parts of the embryo/extraembryonic tissue. Consistent with their in vivo behavior, in vitro cultured NDST1/2 deficient embryos displayed impaired ability of inner cell mass proliferation. In addition, markers for all the three germ layers had a disturbed expression pattern in isolated NDST1/2 deficient embryonic stem (ES) cells. Characterization of heparan sulfate (HS) structure in control ES cells and in ES cells lacking NDST1, NDST2 or both NDST1 and NDST2 revealed big differences. As expected, control cells synthesized HS with the highest degree of sulfation closely followed by HS from NDST2-/- cells, which in turn was more sulfated than HS produced by NDST1-/- cells. HS from NDST1/2-/- cells was almost devoid of sulfate groups. Notably, lack of one NDST isoform did not result in increased expression of any of the others. While all cell types except the NDST1/2-/- cells produced HS with a higher degree of sulfation when allowed to differentiate for 8 days, HS from control cells was still more heavily sulfated than that produced by NDST2-/- cells followed by the HS of NDST1-/- cells. The increase in sulfation was paralleled by increased expression of NDST transcripts and could also be recorded as increased N-sulfotransferase activity of cell lysates. While NDST1/2 deficient ES cells were unable to differentiate into beating cardiomyocytes all NDST1-/- and control embryoid bodies had started to beat after 4 days of culture. Surprisingly, NDST2 deficiency resulted in delayed cardiomyocyte differentiation.

  • 240.
    Dagälv, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Åbrink, Magnus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Kjellén, Lena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Cell surface mast cell proteoglycans identified as heparin-substituted syndecan-2Manuskript (preprint) (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    Connective tissue type mast cells isolated from the peritoneal cavity of mice and then cultured in vitro have been used to answer the question if one cell at a given time point can synthesize heparan sulfate chains with different structure. Characterization of cell surface proteoglycans made by the cells demonstrated that they were identical to syndecan-2, substituted with heparin chains. Ion exchange chromatography showed that the syndecan heparin chains behaved identically as heparin chains recovered from serglycin, inside the cells. This was also the case when mast cells from NDST2 deficient mice were studied. This time, syndecan-2 as well as serglycin derived polysaccharide chains had a lower but identical charge density. We conclude that mast cells only synthesize one kind of heparan sulfate/heparin chain at a time and that polysaccharide chains of identical structure will be found at the cell surface and inside the cell.

  • 241.
    Dahl, Göran
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för biokemi och organisk kemi.
    Kinetic studies of NS3 and NS5B from Hepatitis C virus: Implications and applications for drug discovery2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV.

    By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided.

    Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease.

    This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays.

    Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor.

    Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen.

    The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.

     

    Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

  • 242.
    Dahlsson Leitao, Charles
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Rinne, S. S.
    Mitran, B.
    Vorobyeva, A.
    Andersson, Ken Gösta
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Tolmachev, V.
    Ståhl, Stefan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Löfblom, John
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Proteinvetenskap.
    Orlova, A.
    Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68 Ga-Labeled Tracers2019Inngår i: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, nr 5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)₃-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)₃-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)₃-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)₃-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)₃-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

  • 243.
    Danielson, U. Helena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Molecular Interaction Analysis for Discovery of Drugs Targeting Enzymes and for Resolving Biological Function2015Inngår i: Multifaceted Roles Of Crystallography In Modern Drug Discovery / [ed] Scapin, G; Patel, D; Arnold, E, 2015, s. 223-240Konferansepaper (Fagfellevurdert)
    Abstract [en]

    Analysis of molecular interactions using surface plasmon resonance (SPR) biosensor technology has become a powerful tool for discovery of drugs targeting enzymes and resolving biological function. A major advantage of this technology over other methods for interaction analysis is that it can provide the kinetic details of interactions. This is a consequence of the time resolution of the analysis, which allows individual kinetic rate constants as well as affinities to be determined. A less commonly recognized feature of this technology is that it can reveal the characteristics of more complex mechanisms, e.g. involving multiple steps or conformations of the target or ligand, as well as the energetics, thermodynamics and forces involved.

  • 244.
    Danielsson Thorell, Helena
    et al.
    Karlstads universitet, Institutionen för kemi.
    Karlsson, Jan
    Karlstads universitet, Institutionen för kemi.
    Portelius, Erik
    Karlstads universitet, Institutionen för kemi.
    Nilsson, Thomas
    Karlstads universitet, Institutionen för kemi.
    Cloning, characterisation, and expression of a novel gene encoding chlorite dismutase from Ideonella dechloratans2002Inngår i: Biochimica et Biophysica Acta, Gene Structure and Expression, ISSN 0167-4781, E-ISSN 1879-2634, Vol. 1577, nr 1, s. 445-451Artikkel i tidsskrift (Fagfellevurdert)
  • 245.
    Darmanis, Spyros
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Cui, Tao
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Onkologisk endokrinologi.
    Drobin, Kimi
    KTH - Royal Institute of Technology, Stockholm, Sweden.
    Li, Su-Chen
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Onkologisk endokrinologi.
    Öberg, Kjell
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Onkologisk endokrinologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH - Royal Institute of Technology, Stockholm, Sweden.
    Schwenk, Jochen M.
    KTH - Royal Institute of Technology, Stockholm, Sweden.
    Giandomenico, Valeria
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Onkologisk endokrinologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Identification of Candidate Serum Proteins for Classifying Well-Differentiated Small Intestinal Neuroendocrine Tumors2013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 11, s. e81712-Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background

    Patients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NET) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required.

    Materials and methods

    Suspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases.

    Results

    A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification.

    Conclusions

    We propose new potential protein biomarker candidates for classifying WD-SI-NET at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NET and their eventual use in diagnostics.

  • 246. Das, Sarbashis
    et al.
    Yennamalli, Ragothaman M
    Vishnoi, Anchal
    Gupta, Parul
    Bhattacharya, Alok
    Single-nucleotide variations associated with Mycobacterium tuberculosis KwaZulu-Natal strains.2009Inngår i: Journal of Biosciences, ISSN 0250-5991, E-ISSN 0973-7138, Vol. 34, nr 3, s. 397-404Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The occurrence of drug resistance in Mycobacterium tuberculosis, the aetiological agent of tuberculosis (TB), is hampering the management and control of TB in the world. Here we present a computational analysis of recently sequenced drug-sensitive (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. Single-nucleotide variations (SNVs) were identified in a pair-wise manner using the anchor-based whole genome comparison (ABWGC) tool and its modified version. For this analysis, four fully sequenced genomes of different strains of M. tuberculosis were taken along with three KwaZulu-Natal (KZN) strains isolated from South Africa including one XDR and one MDR strain. KZN strains were compared with other fully sequenced strains and also among each other. The variations were analysed with respect to their biological influence as a result of either altered structure or synthesis. The results suggest that the DR phenotype may be due to changes in a number of genes. The database on KZN strains can be accessed through the website http://mirna.jnu.ac.in/mgdd/.

  • 247.
    Dassie, Justin P.
    et al.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Hernandez, Luiza I.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Thomas, Gregory S.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Long, Matthew E.
    Molecular and Cellular Biology Program, University of of Iowa, Iowa City, IA, United States; Inflammation Program, University of of Iowa, Iowa City, IA, United States.
    Rockey, William M.
    Department of Radiation Oncology, University of of Iowa, Iowa City, IA, United States.
    Howell, Craig A.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Chen, Yani
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Hernandez, Frank J
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Liu, Xiu Y.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Wilson, Mary E.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States; Department of Microbiology, University of of Iowa, Iowa City, IA, United States; Veterans Affairs Medical Center, University of of Iowa, Iowa City, IA, United States.
    Allen, Lee-Ann
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States; Molecular and Cellular Biology Program, University of of Iowa, Iowa City, IA, United States; Inflammation Program, University of of Iowa, Iowa City, IA, United States; Department of Microbiology, University of of Iowa, Iowa City, IA, United States; Veterans Affairs Medical Center, University of of Iowa, Iowa City, IA, United States.
    Vaena, Daniel A.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States.
    Meyerholz, David K.
    Department of Pathology, University of of Iowa, Iowa City, IA, United States.
    Giangrande, Paloma H.
    Department of Internal Medicine, University of of Iowa, 375 Newton Rd, 5202 MERF, Iowa City, IA, United States; Molecular and Cellular Biology Program, University of of Iowa, Iowa City, IA, United States; Department of Radiation Oncology, University of of Iowa, Iowa City, IA, United States.
    Targeted inhibition of prostate cancer metastases with an RNA aptamer to prostate-specific membrane antigen2014Inngår i: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 22, nr 11, s. 1910-1922Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-targeted therapies (smart drugs), which selectively control cancer cell progression with limited toxicity to normal cells, have been developed to effectively treat some cancers. However, many cancers such as metastatic prostate cancer (PC) have yet to be treated with current smart drug technology. Here, we describe the thorough preclinical characterization of an RNA aptamer (A9g) that functions as a smart drug for PC by inhibiting the enzymatic activity of prostate-specific membrane antigen (PSMA). Treatment of PC cells with A9g results in reduced cell migration/invasion in culture and metastatic disease in vivo. Importantly, A9g is safe in vivo and is not immunogenic in human cells. Pharmacokinetic and biodistribution studies in mice confirm target specificity and absence of non-specific on/off-target effects. In conclusion, these studies provide new and important insights into the role of PSMA in driving carcinogenesis and demonstrate critical endpoints for the translation of a novel RNA smart drug for advanced stage PC. © The American Society of Gene amp; Cell Therapy.

  • 248. Daumke, Oliver
    et al.
    Lundmark, Richard
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vallis, Yvonne
    Martens, Sascha
    Butler, P Jonathan G
    McMahon, Harvey T
    Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling2007Inngår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 449, nr 7164, s. 923-927Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.

  • 249.
    de Boeck, Miriam
    et al.
    Leiden Univ, Med Ctr, Dept Mol Cell Biol, NL-2300 RC Leiden, Netherlands.;Leiden Univ, Med Ctr, Canc Gemon Ctr Netherlands, NL-2300 RC Leiden, Netherlands..
    Cui, Chao
    Leiden Univ, Med Ctr, Dept Mol Cell Biol, NL-2300 RC Leiden, Netherlands.;Leiden Univ, Med Ctr, Canc Gemon Ctr Netherlands, NL-2300 RC Leiden, Netherlands..
    Mulder, Aat A.
    Leiden Univ, Med Ctr, Electron Microscopy Sect, Dept Mol Cell Biol, NL-2300 RC Leiden, Netherlands..
    Jost, Carolina R.
    Leiden Univ, Med Ctr, Electron Microscopy Sect, Dept Mol Cell Biol, NL-2300 RC Leiden, Netherlands..
    Ikeno, Souichi
    Showa Pharmaceut Univ, Lab Biochem, Tokyo 1948543, Japan..
    ten Dijke, Peter
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten. Leiden Univ, Med Ctr, Dept Mol Cell Biol, NL-2300 RC Leiden, Netherlands.;Leiden Univ, Med Ctr, Canc Gemon Ctr Netherlands, NL-2300 RC Leiden, Netherlands..
    Smad6 determines BMP-regulated invasive behaviour of breast cancer cells in a zebrafish xenograft model2016Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 24968Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The transforming growth factor-beta (TGF-beta) family is known to play critical roles in cancer progression. While the dual role of TGF-beta is well described, the function of bone morphogenetic proteins (BMPs) is unclear. In this study, we established the involvement of Smad6, a BMP-specific inhibitory Smad, in breast cancer cell invasion. We show that stable overexpression of Smad6 in breast cancer MCF10A M2 cells inhibits BMP signalling, thereby mitigating BMP6-induced suppression of mesenchymal marker expression. Using a zebrafish xenograft model, we demonstrate that overexpression of Smad6 potentiates invasion of MCF10A M2 cells and enhances the aggressiveness of breast cancer MDA-MB-231 cells in vivo, whereas a reversed phenotype is observed after Smad6 knockdown. Interestingly, BMP6 pre-treatment of MDA-MB-231 cells induced cluster formation at the invasive site in the zebrafish. BMP6 also stimulated cluster formation of MDA-MB-231 cells co-cultured on Human Microvascular Endothelial Cells (HMEC)-1 in vitro. Electron microscopy illustrated an induction of cell-cell contact by BMP6. The clinical relevance of our findings is highlighted by a correlation of high Smad6 expression with poor distant metastasis free survival in ER-negative cancer patients. Collectively, our data strongly indicates the involvement of Smad6 and BMP signalling in breast cancer cell invasion in vivo.

  • 250.
    de Oliveira, Felipe
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Landegren, Nils
    Department of Medicine, Solna (MedS), K2, Karolinska Institute, Stockholm, Sweden .
    Muthelo, Phathutshedzo
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Åke, Lernmark
    Department of Clinical Sciences, Skåne University Hospital SUS, Lund University, Malmö, Sweden.
    Landegren, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Kamali-Moghaddam, Masood
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
    Autoimmunity detection via proximity assaysManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Since autoantibodies are recognized as valuable biomarkers for clinical diagnostics and prognostics in autoimmune diseases such as Stiff Person Syndrome (SPS) and Type 1 diabetes, detection of such markers at improved sensitivity and specificity could be of significant interest. In addition, as proximity assays have been shown to offer highly sensitive and specific detection of multiple proteins, the technique could be expanded to applications for autoimmunity detection. In the present study, we have applied the newly developed proximity ligation assay with rolling circle amplification (PLARCA), and proximity extension assay (PEA) for the detection of GADA, autoantibodies specific for glutamic acid decarboxylase 65 (GAD65). Through the use of oligonucleotide conjugated autoantigen GAD65 and anti-human antibodies, as proximity probes, we were able to apply these proximity assays to detect GADA in a set of SPS patient samples. In summary, we have applied and established both PLARCA and PEA, as a proof of concept, for the use of the specific and sensitive autoimmune detection.

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