Prof. Agnieszka Chacińska

email: a.chacinska@dev.dev.cent.uw.edu.pl/en
phone: +48 22 55 43639
room: 04.105
administration: Director
Laboratory of Mitochondrial Biogenesis (Group Leader)

Education and degrees:
2014 Professor of Biological Sciences, nomination by the President of The Republic of Poland
2008 Dr hab., Institute of Biochemistry and Biophysics, Warsaw, Poland
1994-2000 PhD in Biochemistry, Institute of Biochemistry and Biophysics, Warsaw, Poland
1993 Master Degree in Molecular Biology, University of Warsaw
1988-1993 Study of Biology, University of Warsaw, Poland

Research experience and appointments:
since 10.2017 Director of “Regenerative Mechanisms for Health”, International Research Agenda Unit (“ReMedy”)
since 03.2017 Director of the Centre of New Technologies, University of Warsaw
since 10.2016 Visiting Professor at the University Medical Center Göttingen, Germany
2009-2017 Professor and Leader of the Laboratory of Mitochondrial Biogenesis at the International Institute of Molecular and Cell Biology in Warsaw, Poland
2015-2016 Deputy Director of Development at the International Institute of Molecular and Cell Biology in Warsaw, Poland
2008-2009 Associate Member of Excellence Cluster BIOSS–Centre for Biological Signalling Studies, University of Freiburg, Germany
2007-2010 Member of the Board, Collaborative Research Centre 746 (SFB 746), Germany
2007-2010 Project Leader in the Collaborative Research Centre 746 (SFB 746), Germany
2004-2009 Group Leader, Institute for Biochemistry and Molecular Biology, University of Freiburg, Germany
2001-2004 Postdoctoral Fellow in the laboratory of Prof. Nikolaus Pfanner, University of Freiburg, Germany
1999 Visiting scientist in the laboratory of Dr Sabine Rospert, Max Planck Research Unit, Halle, Germany
1996-1998 Visiting scientist in the laboratory of Prof. Gottfried Schatz, Biozentrum, University of Basel, Switzerland
1994-2000 Doctoral research with Prof. Magdalena Boguta, Institute of Biochemistry and Biophysics, Warsaw, Poland
Memberships and Awards:
2017 Leon Marchlewski Medal of The Committee on Molecular Biology of the Cell, Polish Academy of Science for outstanding contribution to the development of molecular biology, cell biology, biochemistry and biophysics
2017 Individual Award from the Rector of University of Warsaw
2016 Corresponding Member of the Polish Academy of Science
2016 Award from the Prime Minister of Poland for scientific achievements
2016 Elected EMBO member
2016 Laureate together with Peter Rehling of Nicolaus Copernicus Polish-German Research Award given jointly by the Foundation for Polish Science (FNP) and the German Research Foundation (DFG)
2015  Award from the Minister of Science and Higher Education for scientific achievements that led to the title of Professor
2015 Award from the President of Polish Academy of Science for scientific achievement
2009 Laureate of the Welcome Programme from the Foundation for Polish Science
2009 EMBO Installation Grantee
2008 Eugen-Graetz Prize for research, University of Freiburg
2001-2003 Long-term FEBS fellowship
2001 Award for the Ph. D. thesis, Institute of Biochemistry and Biophysics,
1997 Grant for young scientists from the Polish State Committee for Scientific Research
1996 Short-term FEBS fellowship


Publications without CeNT affiliation: 

  • Wasilewski, M., Chojnacka, K., Chacinska, A. (2017). Protein trafficking at the crossroads to mitochondria. Biochim Biophys Acta, 1864(1), 125-137.
  • Wasilewski, M., Chojnacka, K., Chacinska, A. (2016). Białka mitochondrialne – import, eksport, degradacja. Postępy Biochemii, 62(2), 94-102.
  • Callegari, S., Richter, F., Chojnacka, K., Jans, D.C., Lorenzi, I., Pacheu-Grau, D., Jakobs, S., Lenz, C., Urlaub, H., Dudek, J., Chacinska, A., Rehling, P. (2016). TIM29 is a subunit of the human carrier translocase required for protein transport. FEBS Lett, 590(23), 4147-4158.
  • Wrobel, L., Sokol, A.M., Chojnacka, M., and Chacinska, A. (2016). The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly. Sci Rep., 6, 27484.
  • Topf, U., Wrobel, L., and Chacinska, A. (2016). Chatty Mitochondria: Keeping Balance in Cellular Protein Homeostasis. Trends Cell Biol, 26(8), 577-86.
  • Turakhiya, U., von der Malsburg, K., Gold, V.A., Guiard, B., Chacinska, A., van der Laan, M., Ieva, R. (2016). Protein Import by the Mitochondrial Presequence Translocase in the Absence of a Membrane Potential. J Mol Biol, 428, 1041-52.
  • Wrobel, L., Topf, U., Bragoszewski, P., Wiese, S., Sztolsztener, M.E., Oeljeklaus, S., Varabyova, A., Lirski, M., Chroscicki, P., Mroczek, S., Januszewicz, E., Dziembowski, A., Koblowska, M., Warscheid, B., Chacinska, A. (2015). Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature, 524, 485-488.
  • Keatinge, M., Bui, H., Menke, A., Chen, Y.C., Sokol, A.M., Bai, Q., Ellett, F., Da Costa, M., Burke, D., Gegg, M., Trollope, L., Payne, T., McTighe, A., Mortiboys, H., de Jager, S., Nuthhall, H., Kuo, M.S., Fleming, A., Schapira, A.H., Renshaw, S.A., Highley, J.R., Chacinska, A., Panula, P., Burton, E.A., O’Neill, M.J., Bandmann, O. (2015). Glucocerebrosidase 1 deficient Danio rerio mirror key pathological aspects of human Gaucher disease and provise evidence of early microglial activation preceding alpha-synuclein-independent neuronal cell death. Hum Mol Genet, 24(23), 6640-52.
  • Sakowska P, Jans DC, Mohanraj K, Riedel D, Jakobs S, Chacinska A. The oxidation status of Mic19 regulates MICOS assembly. 2015. Mol Cell Biol, 35, 4222-4237.
  • Bragoszewski, P., Wasilewski, M., Sakowska, P., Gornicka, A., Böttinger, L., Qiu, J., Wiedemann, N., Chacinska, A. (2015). Retro-translocation of mitochondrial intermembrane space proteins. Proc Natl Acad Sci U S A. 112, 7713-7718.
  • Barchiesi, A., Wasilewski, M., Chacinska, A., Tell, G., Vascotto, C. (2015). Mitochondrial translocation of APE1 relies on the MIA pathway. Nucleic Acids Res. 43, 5451-5464.
  • Chojnacka, M., Gornicka, A., Oeljeklaus, S., Warscheid, B., Chacinska, A. (2015). Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System. J Biol Chem. 290, 15304-15312.
  • Ieva, R., Schrempp, S.G., Opaliński, L., Wollweber, F., Höß, P., Heißwolf, A.K., Gebert, M., Zhang, Y., Guiard, B., Rospert, S., Becker, T., Chacinska, A., Pfanner, N., van der Laan, M. (2014). Mgr2 functions as lateral gatekeeper for preprotein sorting in the mitochondrial inner membrane. Mol Cell 56, 641-652.
  • Gornicka, A., Bragoszewski, P., Chroscicki, P., Wenz, L.S., Schulz, C., Rehling, P., Chacinska, A. (2014). A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria . Mol Biol Cell 25, 3999-4009.
  • Melin, J., Schulz, C., Wrobel, L., Bernhard, O., Chacinska, A., Jahn, O., Schmidt, B., Rehling, P. (2014). Presequence recognition by the tom40 channel contributes to precursor translocation into the mitochondrial matrix. Mol Cell Biol 34, 3473-3485.
  • Sokol, A.M., Sztolsztener, M.E., Wasilewski, M., Heinz, E., Chacinska, A. (2014). Mitochondrial protein translocases for survival and wellbeing. FEBS Lett. 588, 2484-2485.
  • Pfanner, N., van der Laan, M., Amati, P., Capaldi, R.A., Caudy, A.A., Chacinska, A., Darshi, M., Deckers, M., Hoppins, S., Icho, T., Jakobs, S., Ji, J., Kozjak-Pavlovic, V., Meisinger, C., Odgren, P.R., Park, S.K., Rehling, P., Reichert, A.S., Sheikh, M.S., Taylor, S.S., Tsuchida, N., van der Bliek, A.M., van der Klei, I.J., Weissman, J.S., Westermann, B., Zha, J., Neupert, W., Nunnari, J. (2014). Uniform nomenclature for the mitochondrial contact site and cristae organizing system. J Cell Biol 204, 1083-1086.
  • Qiu, J., Wenz, L.S., Zerbes, R.M., Oeljeklaus, S., Bohnert, M., Stroud, D.A., Wirth, C., Ellenrieder, L., Thornton, N., Kutik, S., Wiese, S,, Schulze-Specking, A., Zufall, N., Chacinska, A., Guiard, B., Hunte, C., Warscheid, B., van der Laan, M., Pfanner, N., Wiedemann, N., and Becker, T. (2013). Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation. Cell 154, 596-608.
  • Varabyova, A., Topf, U., Kwiatkowska, P., Wrobel, L., Kaus-Drobek, A., and Chacinska, A. (2013). Mia40 and MINOS act in parallel with Ccs1 in the biogenesis of mitochondrial Sod1. FEBS J 280, 4943-4959.
  • Bragoszewski, P., Gornicka, A., Sztolsztener, M.E., and Chacinska, A. (2013). The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins. Mol Cell Biol 33, 2136-48.
  • Wrobel, L., Trojanowska, A., Sztolsztener, M.E., and Chacinska, A. (2013). Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria.  Mol Biol Cell 24, 543-554.
  • Varabyova, A., Stojanovski, D., and Chacinska, A. (2013). Mitochondrial protein homeostasis. IUBMB Life 65, 191-201.
  • Sztolsztener, M.E., Brewinska, A., Guiard, B., and Chacinska, A. (2013). Disulfide bond formation: sulfhydryl oxidase ALR controls mitochondrial biogenesis of human MIA40. Traffic 14, 309-320.
  • Böttinger, L., Gornicka, A., Czerwik, T., Bragoszewski, P., Loniewska-Lwowska, A., Schulze-Specking, A., Truscott, K.N., Guiard, B., Milenkovic, D., and Chacinska, A. (2012). In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins. Mol Biol Cell 23, 3957-3969.
  • Bohnert, M., Wenz, L.S., Zerbes, R.M., Horvath, S.E., Stroud, D.A., von der Malsburg, K., Muller, J.M., Oeljeklaus, S., Perschil, I., Warscheid, B., Chacinska, A., Veenhuis, M., van der Klei, I.J., Daum, G., Wiedemann, N., Becker, T., Pfanner, N., van der Laan, M. (2012). Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane. Mol Biol Cell 23, 3948-3956.
  • Stojanovski, D., Bragoszewski, P., and Chacinska, A. (2012). The MIA pathway: A tight bond between protein transport and oxidative folding in mitochondria. Biochim Biophys Acta 1823, 1142-1150.
  • von der Malsburg, K., Müller, J.M., Bohnert, M., Oeljeklaus, S., Kwiatkowska, P., Becker, T., Loniewska-Lwowska, A., Wiese, S., Rao, S., Milenkovic, D., Hutu, D.P., Zerbes, R.M., Schulze-Specking, A., Meyer, H.E., Martinou, J.C., Rospert, S., Rehling, P., Meisinger, C., Veenhuis, M., Warscheid, B., van der Klei, I.J., Pfanner ,N.*, Chacinska, A.*, van der Laan, M. (2011). Dual Role of Mitofilin in Mitochondrial Membrane Organization and Protein Biogenesis. Developmental Cell, 21, 694-707. (*co-corresponding author)
  • Becker, T., Wenz, L.S., Krüger, V., Lehmann, W., Müller, J.M., Goroncy, L., Zufall, N., Lithgow, T., Guiard, B., Chacinska, A., Wagner, R., Meisinger, C., Pfanner, N. (2011). The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins. J Cell Biol 194, 387-95.
  • Schulz, C., Lytovchenko, O., Melin, J., Chacinska, A., Guiard, B., Neumann, P., Ficner, R., Jahn, O., Schmidt, B., Rehling, P. (2011). Tim50’s presequence receptor domain is essential for signal driven transport across the TIM23 complex. J Cell Biol 195, 643-56.
  • Chacinska, A., van der Laan, M., Mehnert, C.S., Guiard, B., Mick, D.U., Hutu, D.P., Truscott, K.N., Wiedemann, N., Meisinger, C., Pfanner, N., Rehling, P. (2010). Distinct forms of mitochondrial TOM-TIM supercomplexes define signal-dependent states of preprotein sorting. Mol Cell Biol 30, 307-318.
  • Voegtle, F. N., Schmidt, O., Chacinska, A., Pfanner, N., Meisinger, C. (2010). Native techniques for analysis of mitochondrial protein import. Methods Mol Biol 619, 425-436.
  • Chacinska, A., Koehler, C.M., Milenkovic, D., Lithgow, T., Pfanner, N. (2009). Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628-644.
  • Milenkovic, D., Ramming, T., Müller, J.M., Wenz, L.S., Gebert, N., Schulze-Specking, A., Stojanovski, D., Rospert, S., Chacinska, A. (2009). Identification of the signal directing Tim9 and Tim10 into the intermembrane space of mitochondria. Mol Biol Cell 20, 2530-2539.
  • Baker, M.J., Webb, C.T., Stroud, D.A., Palmer, C.S., Frazier, A.E., Guiard, B., Chacinska, A., Gulbis, J.M., Ryan M. (2009). Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import. Mol Biol Cell 20, 769-779.
  • Stojanovski, D., Milenkovic, D., Müller, J.M., Gabriel, K., Schulze-Specking, A., Baker, M.J., Ryan, M.T., Guiard, B., Pfanner, N.,* Chacinska, A.* (2008). Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase. J Cell Biol 183, 195-202. (*co-corresponding author)
  • Chacinska*, A., Guiard*, B., Müller, J.M., Schulze-Specking, A., Gabriel, K., Kutik S., Pfanner N. (2008). Mitochondrial biogenesis: switching the sorting pathways of the intermembrane space receptor Mia40. J Biol Chem 283, 29723-29729. (*equal contribution).
  • Müller, J.M., Milenkovic, D., Guiard, B., Pfanner, N., Chacinska, A. (2008). Precursor oxidation by Mia40 and Erv1 promotes vectorial transport of proteins into the mitochondrial intermembrane space. Mol Biol Cell 19, 226-236.
  • Hutu, D.P., Guiard, B., Chacinska, A., Becker, D., Pfanner, N., Rehling, P., van der Laan, M. (2008). Mitochondrial protein import motor: differential role of Tim44 in the recruitment of Pam17 and J-complex to the presequence translocase. Mol Biol Cell 19, 2642-2649.
  • Gebert, N., Chacinska, A., Wagner, K., Guiard, B., Koehler, C.M., Rehling, P., Pfanner, N., Wiedemann, N. (2008). Assembly of the three small Tim proteins precedes docking to the mitochondrial carrier translocase. EMBO Rep 9, 548-554.
  • Stojanovski, D., Müller, J.M., Milenkovic, D., Guiard, B., Pfanner, N., Chacinska, A. (2008). The MIA system for protein import into the mitochondrial intermembrane space. Biochim Biophys Acta 1783, 610-617.
  • Milenkovic, D., Gabriel, K., Guiard, B., Schulze-Specking, A., Pfanner, N., Chacinska, A. (2007). Biogenesis of the essential Tim9-Tim10 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermemembrane space receptor Mia40. J Biol Chem. 282, 22472-22480.
  • Milenkovic, D., Müller, J.M.M., Stojanovski, D., Pfanner, N., Chacinska, A. (2007). Diverse mechanisms and machineries for import of mitochondrial proteins.  Biol Chem 388, 891-897.
  • Meisinger, C., Pfannschmidt, S., Rissler, M., Milenkovic, D., Becker, t., Stojanovski, D., Youngman, M.J., Jensen, R.E., Chacinska, A., Guiard, B., Pfanner, N., Wiedemann, N. (2007). The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria. EMBO J 26, 2229-2239.
  • Sanjuán Szklarz, L.K., Kozjak-Pavlovic, V., Vogtle, F.N., Chacinska, A., Milenkovic, D., Vogel, S., Durr, M., Westermann, B., Guiard, B., Martinou, J.C., Borner, C., Pfanner, N., and Meisinger, C. (2007). Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins. J Mol Biol 368, 44-54.
  • Gabriel, K., Milenkovic, D., Chacinska, A., Muller, J., Guiard, b., Pfanner, N., and Meisinger, C. (2007). Novel mitochondrial intermembrane space proteins as substrates of the MIA import pathway. J Mol Biol 365, 612-620.
  • Wiedemann, N., Pfanner, N., and Chacinska, A. (2006). Chaperoning through the mitochondrial intermembrane space. Mol Cell 21, 145-148.
  • Meinecke, M., Wagner, R., Kovermann, P., Guiard, B., Mick, D.U., Hutu, D.P., Voos, W., Truscott, K.N., Chacinska, A., Pfanner, N., and Rehling, P. (2006). Tim50 maintains the permeability barrier of the mitochondrial inner membrane. Science 312, 1523-1526.
  • Albrecht, R., Rehling, P., Chacinska, A., Brix, J., Cadamuro, S.A., Volkmer, R., Guiard, B., Pfanner, N., Zeth, K. (2006). Tim21 binding domain connects the preprotein translocase of both mitochondrial membranes. EMBO Rep 7, 1233-1238.
  • Rospert, S., and Chacinska, A. (2006). Distinct yet linked: chaperone networks in the eukaryotic cytosol. Genome Biol 7, 208.
  • Chacinska, A.*, Lind, M.*, Frazier, A.E., Dudek, J., Meisinger, C., Geissler, A., Sickmann, A., Meyer, H.E., Truscott, K.N., Guiard, B., Pfanner, N., and Rehling, P. (2005). Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell 120, 817-829 (*equal contribution).
  • Rissler, M., Wiedemann, N., Pfannschmidt, S., Gabriel, K., Guiard, B., Pfanner, N., and Chacinska, A. (2005). The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins. J Mol Biol 353, 485-492.
  • van der Laan, M.*, Chacinska, A.*, Lind, M., Perschil, I., Sickmann, A., Meyer, H.E., Guiard, B., Meisinger, C., Pfanner, N., and Rehling, P. (2005). Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor. Mol Cell Biol 25, 7449-7458 (*equal contribution).
  • Sanjuán Szklarz, L.K., Guiard, B., Rissler, M., Wiedemann, N., Kozjak, V., van der Laan, M., Lohaus, C., Marcus, K., Meyer, H.E., Chacinska, A., Pfanner, N., and Meisinger, C. (2005). Inactivation of the mitochondrial heat shock protein Zim17 leads to aggregation of matrix Hsp70s followed by pleiotropic effects on morphology and protein biogenesis. J Mol Biol 351, 206-218.
  • Chacinska, A., Pfannschmidt, S., Wiedemann, N., Kozjak, V., Sanjuán Szklarz, L.K., Schulze-Specking, A., Truscott, K.N., Guiard, B., Meisinger, C., and Pfanner, N. (2004). Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins. EMBO J 23, 3735-3746.
  • Chacinska, A., and Rehling, P. (2004). Moving proteins from the cytosol into mitochondria. Biochem Soc Trans 32, 774-776.
  • Meisinger, C., Rissler, M., Chacinska, A., Sanjuán Szklarz, L.K., Milenkovic, D., Kozjak, V., Schönfisch, B., Lohaus, C., Meyer, H.E., Yaffe, M.P., Guiard, B., Wiedemann, N., and Pfanner, N. (2004). The mitochondrial morphology protein Mdm10 functions in assembly of the preprotein translocase of the outer membrane. Developmental Cell 7, 61-71.
  • Towpik, J., Chacinska, A., Ciesla, M., Ginalski, K., and Boguta, M. (2004). Mutations in yeast MRF1 gene encoding mitochondrial release factor inhibit translation on mitochondrial ribosomes. J Biol Chem 279, 14096-14103.
  • Chacinska, A., Rehling, P., Guiard, B., Frazier, A.E., Schulze-Specking, A., Pfanner, N., Voos, W., and Meisinger, C. (2003). Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM-TIM-preprotein supercomplex. EMBO J 22, 5370-5381.
  • Frazier, A.E., Chacinska, A., Truscott, K.N., Guiard, B., Pfanner, N., and Rehling, P. (2003). Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space. Mol Cell Biol 23, 7818-7828.
  • Sickmann, A., Reinders, J., Wagner, Y., Joppich, C. Zahedi, R., Meyer, H.E., Schönfisch, B., Perschil, I., Chacinska, A., Guiard, B., Rehling, P., Pfanner, N., and Meisinger, C. (2003). The proteome of Saccharomyces cerevisiae mitochondria. Proc Natl Acad Sci USA 100, 13207-13212.
  • Wiedemann, N., Kozjak, V., Chacinska, A., Schönfisch, B., Rospert, S., Ryan, M.T., Pfanner, N., and Meisinger, C. (2003). Machinery for protein sorting and assembly in the mitochondrial outer membrane. Nature 424, 565-571.
  • Geissler, A.*, Chacinska, A.*, Truscott, K.N., Wiedemann, N., Brandner, K., Sickmann, A., Meyer, H.E., Meisinger, C., Pfanner, N., and Rehling, P. (2002). The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell 111, 507-518 (*equal contribution).
  • Chacinska, A., Pfanner, N., and Meisinger, C. (2002). How mitochondria import hydrophilic and hydrophobic proteins. Trends Cell Biol 12, 299-303.
  • Pfanner, N. and Chacinska, A. (2002). The mitochondrial import machinery: preprotein-conducting channels with binding sites for presequences. Biochim Biophys Acta 1592, 15-24.
  • Chacinska, A., Szczesniak, B., Kochneva-Pervukhova, N. V., Kushnirov, V.V., Ter-Avanesyan, M.D., and Boguta, M. (2001). Ssb1 chaperone is a [PSI+] prion-curing factor. Curr Genet 39, 62-67.
  • Chacinska, A., Boguta, M., Krzewska, J. and Rospert, S. (2000). Prion-dependent switching between respiratory competence and deficiency in the yeast nam9-1 mutant. Mol Cell Biol 20, 7220-9.
  • Chacinska, A., Boguta, M. (2000). Coupling of mitochondrial translation with the formation of respiratory complexes in yeast mitochondria. Acta Biochim Pol 47, 973-91.
  • Boguta, M., Chacinska, A., Murawski, M. and Szczesniak, B. (1997). Expression of the yeast NAM9 gene coding for mitochondrial ribosomal protein. Acta Biochim Pol 44, 251-8.
  • Gora, M., Chacinska, A., Rytka, J. and Labbe-Bois, R. (1996). Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae. Biochimie 78, 144-52.