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Niss, F., Piñero-Paez, L., Zaidi, W., Hallberg, E. & Ström, A.-L. (2022). Key Modulators of the Stress Granule Response TIA1, TDP-43, and G3BP1 are Altered by Polyglutamine Expanded ATXN7. Molecular Neurobiology, 59(8), 5236-5251
Open this publication in new window or tab >>Key Modulators of the Stress Granule Response TIA1, TDP-43, and G3BP1 are Altered by Polyglutamine Expanded ATXN7
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2022 (English)In: Molecular Neurobiology, ISSN 0893-7648, E-ISSN 1559-1182, Vol. 59, no 8, p. 5236-5251Article in journal (Refereed) Published
Abstract [en]

Spinocerebellar ataxia type 7 (SCA7) and other polyglutamine (polyQ) diseases are caused by expansions of polyQ repeats in disease-specific proteins. Aggregation of the polyQ proteins resulting in various forms of cellular stress, that could induce the stress granule (SG) response, is believed to be a common pathological mechanism in these disorders. SGs can contribute to cell survival but have also been suggested to exacerbate disease pathology by seeding protein aggregation. In this study, we show that two SG-related proteins, TDP-43 and TIA1, are sequestered into the aggregates formed by polyQ-expanded ATXN7 in SCA7 cells. Interestingly, mutant ATXN7 also localises to induced SGs, and this association altered the shape of the SGs. In spite of this, neither the ability to induce nor to disassemble SGs, in response to arsenite stress induction or relief, was affected in SCA7 cells. Moreover, we could not observe any change in the number of ATXN7 aggregates per cell following SG induction, although a small, non-significant, increase in total aggregated ATXN7 material could be detected using filter trap. However, mutant ATXN7 expression in itself increased the speckling of the SG-nucleating protein G3BP1 and the SG response. Taken together, our results indicate that the SG response is induced, and although some key modulators of SGs show altered behaviour, the dynamics of SGs appear normal in the presence of mutant ATXN7. 

Keywords
Neurodegeneration, Spinocerebellar ataxia type 7, TDP-43, G3BP1, TIA1, Stress granules
National Category
Neurosciences Biochemistry and Molecular Biology Cell and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-204298 (URN)10.1007/s12035-022-02888-2 (DOI)000809308500003 ()35689166 (PubMedID)2-s2.0-85131678584 (Scopus ID)
Funder
Magnus Bergvall Foundation, 2017-02212Åhlén-stiftelsen, mA6h17, mA2h18, 193051
Available from: 2022-04-29 Created: 2022-04-29 Last updated: 2022-09-28Bibliographically approved
Niss, F., Bergqvist, C., Ström, A.-L. & Hallberg, E. (2022). Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC). In: Martin W. Goldberg (Ed.), The Nuclear Pore Complex: Methods and Protocols (pp. 151-160). New York: Humana Press Inc.
Open this publication in new window or tab >>Monitoring of Chromatin Organization at the Nuclear Pore Complex, Inner Nuclear Membrane, and Nuclear Interior in Live Cells by Fluorescence Ratiometric Imaging of Chromatin (FRIC)
2022 (English)In: The Nuclear Pore Complex: Methods and Protocols / [ed] Martin W. Goldberg, New York: Humana Press Inc. , 2022, p. 151-160Chapter in book (Refereed)
Abstract [en]

The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein “progerin,” which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.

Place, publisher, year, edition, pages
New York: Humana Press Inc., 2022
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 2502
Keywords
Chromatin, Fluorescence ratiometric, Live imaging, Nuclear membrane, Nuclear pore complex, histone, lamin A, cell nucleus, cell nucleus membrane, fluorescence, genetics, HeLa cell line, heterochromatin, human, metabolism, nuclear pore, HeLa Cells, Histones, Humans, Lamin Type A, Nuclear Envelope
National Category
Cell and Molecular Biology Biological Sciences
Identifiers
urn:nbn:se:su:diva-206355 (URN)10.1007/978-1-0716-2337-4_10 (DOI)35412237 (PubMedID)2-s2.0-85128080535 (Scopus ID)978-1-0716-2336-7 (ISBN)978-1-0716-2337-4 (ISBN)
Available from: 2022-06-14 Created: 2022-06-14 Last updated: 2022-09-23Bibliographically approved
Niss, F., Zaidi, W., Hallberg, E. & Ström, A.-L. (2021). Polyglutamine expanded Ataxin-7 induces DNA damage and alters FUS localization and function. Molecular and Cellular Neuroscience, 110, Article ID 103584.
Open this publication in new window or tab >>Polyglutamine expanded Ataxin-7 induces DNA damage and alters FUS localization and function
2021 (English)In: Molecular and Cellular Neuroscience, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 110, article id 103584Article in journal (Refereed) Published
Abstract [en]

Polyglutamine (polyQ) diseases, such as Spinocerebellar ataxia type 7 (SCA7), are caused by expansions of polyQ repeats in disease specific proteins. The sequestration of vital proteins into aggregates formed by polyQ proteins is believed to be a common pathological mechanism in these disorders. The RNA-binding protein FUS has been observed in polyQ aggregates, though if disruption of this protein plays a role in the neuronal dysfunction in SCA7 or other polyQ diseases remains unclear. We therefore analysed FUS localisation and function in a stable inducible PC12 cell model expressing the SCA7 polyQ protein ATXN7. We found that there was a high degree of FUS sequestration, which was associated with a more cytoplasmic FUS localisation, as well as a decreased expression of FUS regulated mRNAs. In contrast, the role of FUS in the formation of gamma H2AX positive DNA damage foci was unaffected. In fact, a statistical increase in the number of gamma H2AX foci, as well as an increased trend of single and double strand DNA breaks, detected by comet assay, could be observed in mutant ATXN7 cells. These results were further corroborated by a clear trend towards increased DNA damage in SCA7 patient fibroblasts. Our findings suggest that both alterations in the RNA regulatory functions of FUS, and increased DNA damage, may contribute to the pathology of SCA7.

Keywords
Neurodegeneration, Polyglutamine disease, RNA-binding protein, FUS
National Category
Cell and Molecular Biology Biological Sciences
Identifiers
urn:nbn:se:su:diva-192819 (URN)10.1016/j.mcn.2020.103584 (DOI)000613264700001 ()33338633 (PubMedID)
Available from: 2021-05-01 Created: 2021-05-01 Last updated: 2022-04-29Bibliographically approved
Bergqvist, C., Niss, F., Figueroa, R. A., Beckman, M., Maksel, D., Jafferali, M. H., . . . Hallberg, E. (2019). Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1. Nucleic Acids Research, 47(9), Article ID e49.
Open this publication in new window or tab >>Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1
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2019 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 9, article id e49Article in journal (Refereed) Published
Abstract [en]

In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.

National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:su:diva-168660 (URN)10.1093/nar/gkz123 (DOI)000473756300001 ()
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590Stiftelsen Olle Engkvist Byggmästare
Available from: 2019-05-02 Created: 2019-05-02 Last updated: 2022-04-29Bibliographically approved
Larsson, V. J., Jafferali, M. H., Vijayaraghavan, B., Figueroa, R. A. & Hallberg, E. (2018). Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp1. Journal of Cell Science, 131(8), Article ID jcs211664.
Open this publication in new window or tab >>Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp1
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2018 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 131, no 8, article id jcs211664Article in journal (Refereed) Published
Abstract [en]

We have investigated a possible role of the inner nuclear membrane protein, Samp1, in the mitotic machinery. Live cell imaging showed that Samp1aYFP distributed as filamentous structures in the mitotic spindle, partially co-localising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistently, mitotic spindles in Samp1 depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes which were rescued by overexpression of Samp1aYFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and HAUS6 of the Augmin complex in live cells. The levels of Haus6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of Haus6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.

Keywords
Samp1, nuclear membrane, mitotic spindle, gamma tubulin, Augmin, Cancer
National Category
Cell Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-154091 (URN)10.1242/jcs.211664 (DOI)000440536400010 ()
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590Stiftelsen Olle Engkvist Byggmästare
Available from: 2018-03-14 Created: 2018-03-14 Last updated: 2022-03-23Bibliographically approved
Vijayaraghavan, B., Figueroa, R. A., Bergqvist, C., Gupta, A. J., Sousa, P. & Hallberg, E. (2018). RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin. Biochimica et Biophysica Acta - Biomembranes, 1860(6), 1326-1334
Open this publication in new window or tab >>RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin
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2018 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, no 6, p. 1326-1334Article in journal (Refereed) Published
Abstract [en]

Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope.

Keywords
Muscular dystrophy, Nuclear membrane, Samp1, Emerin, Ran, FRAP
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-157708 (URN)10.1016/j.bbamem.2018.03.001 (DOI)000432758400009 ()29510091 (PubMedID)
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2022-03-23Bibliographically approved
Mattioli, E., Columbaro, M., Jafferali, M. H., Schena, E., Hallberg, E. & Lattanzi, G. (2018). Samp1 Mislocalization in Emery-Dreifuss Muscular Dystrophy. Cells, 7(10), Article ID 170.
Open this publication in new window or tab >>Samp1 Mislocalization in Emery-Dreifuss Muscular Dystrophy
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2018 (English)In: Cells, E-ISSN 2073-4409, Vol. 7, no 10, article id 170Article in journal (Refereed) Published
Abstract [en]

LMNA linked-Emery-Dreifuss muscular dystrophy (EDMD2) is a rare disease characterized by muscle weakness, muscle wasting, and cardiomyopathy with conduction defects. The mutated protein lamin A/C binds several nuclear envelope components including the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the inner nuclear membrane protein Samp1 (Spindle Associated Membrane Protein 1). Considering that Samp1 is upregulated during muscle cell differentiation and it is involved in nuclear movement, we hypothesized that it could be part of the protein platform formed by LINC proteins and prelamin A at the myotube nuclear envelope and, as previously demonstrated for those proteins, could be affected in EDMD2. Our results show that Samp1 is uniformly distributed at the nuclear periphery of normal human myotubes and committed myoblasts, but its anchorage at the nuclear poles is related to the presence of farnesylated prelamin A and it is disrupted by the loss of prelamin A farnesylation. Moreover, Samp1 is absent from the nuclear poles in EDMD2 myotubes, which shows that LMNA mutations associated with muscular dystrophy, due to reduced prelamin A levels in muscle cell nuclei, impair Samp1 anchorage. Conversely, SUN1 pathogenetic mutations do not alter Samp1 localization in myotubes, which suggests that Samp1 lies upstream of SUN1 in nuclear envelope protein complexes. The hypothesis that Samp1 is part of the protein platform that regulates microtubule nucleation from the myotube nuclear envelope in concert with pericentrin and LINC components warrants future investigation. As a whole, our data identify Samp1 as a new contributor to EDMD2 pathogenesis and our data are relevant to the understanding of nuclear clustering occurring in laminopathic muscle.

Keywords
Emery-Dreifuss Muscular Dystrophy type 2 (EDMD2), Samp1 (NET5), prelamin A, LINC complex, myonuclear positioning
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-162934 (URN)10.3390/cells7100170 (DOI)000448818800028 ()30326651 (PubMedID)
Available from: 2018-12-18 Created: 2018-12-18 Last updated: 2022-03-23Bibliographically approved
Bergqvist, C., Jafferali, M. H., Gudise, S., Markus, R. & Hallberg, E. (2017). An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells. Stem Cell Research, 23, 33-38
Open this publication in new window or tab >>An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells
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2017 (English)In: Stem Cell Research, ISSN 1873-5061, E-ISSN 1876-7753, Vol. 23, p. 33-38Article in journal (Refereed) Published
Abstract [en]

The ability of iPSCs (induced pluripotent stem cells) to generate any cell type in the body makes them valuable tools for cell replacement therapies. However, differentiation of iPSCs can be demanding, slowand variable. During differentiation chromatin is re-organized and silent dense heterochromatin becomes tethered to the nuclear periphery by processes involving the nuclear lamina and proteins of the INM(inner nuclearmembrane). The INM protein, Samp1 (Spindle AssociatedMembrane Protein 1) interacts with Lamin A/C and the INMprotein Emerin, which has a chromatin binding LEM(Lap2-Emerin-Man1)-domain. In this paperweinvestigate if Samp1 can play a role in the differentiation of iPSCs. Samp1 levels increased as differentiating iPSCs started to express Lamin A/C. Interestingly, even under pluripotent culturing conditions, ectopic expression of Samp1 induced a rapid differentiation of iPSCs, ofwhich some expressed the neuronal marker beta III-tubulin already after 6 days. This suggests that Samp1 is involved in early differentiation of iPSCs and could potentially be explored as a tool to promote progression of the differentiation process.

Keywords
Nuclear membrane, Nuclear envelope, Induced pluripotent stem cells, Neuronal differentiation, Regenerative medicine
National Category
Medical Engineering Environmental Biotechnology Cell Biology
Identifiers
urn:nbn:se:su:diva-147935 (URN)10.1016/j.scr.2017.06.008 (DOI)000410958100004 ()28668644 (PubMedID)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2022-03-23Bibliographically approved
Shimoji, M., Figueroa, R. A., Neve, E., Maksel, D., Imreh, G., Morgenstern, R. & Hallberg, E. (2017). Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1. Biochimica et Biophysica Acta - Biomembranes, 1859(2), 238-244
Open this publication in new window or tab >>Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1
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2017 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, no 2, p. 238-244Article in journal (Refereed) Published
Abstract [en]

Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.

Keywords
Subcellular targeting, Dual distribution, Glutathione transferase
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-140296 (URN)10.1016/j.bbamem.2016.11.014 (DOI)000392772100011 ()27913278 (PubMedID)
Available from: 2017-03-13 Created: 2017-03-13 Last updated: 2022-02-28Bibliographically approved
Thanisch, K., Song, C., Engelkamp, D., Koch, J., Wang, A., Hallberg, E., . . . Solovei, I. (2017). Nuclear envelope localization of LEMD2 is developmentally dynamic and lamin A/C dependent yet insufficient for heterochromatin tethering. Differentiation, 94, 58-70
Open this publication in new window or tab >>Nuclear envelope localization of LEMD2 is developmentally dynamic and lamin A/C dependent yet insufficient for heterochromatin tethering
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2017 (English)In: Differentiation, ISSN 0301-4681, E-ISSN 1432-0436, Vol. 94, p. 58-70Article in journal (Refereed) Published
Abstract [en]

Peripheral heterochromatin in mammalian nuclei is tethered to the nuclear envelope by at least two mechanisms here referred to as the A- and B-tethers. The A-tether includes lamins A/C and additional unknown components presumably INM protein(s) interacting with both lamins A/C and chromatin. The B tether includes the inner nuclear membrane (INM) protein Laurin B-receptor, which binds B-type lamins and chromatin. Generally, at least one of the tethers is always present in the nuclear envelope of mammalian cells. Deletion of both causes the loss of peripheral heterochromatin and consequently inversion of the entire nuclear architecture, with this occurring naturally in rod photoreceptors of nocturnal mammals. The tethers are differentially utilized during development, regulate gene expression in opposite manners, and play an important role during cell differentiation. Here we aimed to identify the unknown chromatin binding component(s) of the A-tether. We analyzed 10 mouse tissues by immunostaining with antibodies against 7 INM proteins and found that every cell type has specific, although differentially and developmentally regulated, sets of these proteins. In particular, we found that INM protein LEMD2 is concomitantly expressed with A-type lamins in various cell types but is lacking in inverted nuclei of rod cells. Truncation or deletion of Lmna resulted in the downregulation and mislocalization of LEMD2, suggesting that the two proteins interact and pointing at LEMD2 as a potential chromatin binding mediator of the A-tether. Using nuclei of mouse rods as an experimental model lacking peripheral heterochromatin, we expressed a LEMD2 transgene alone or in combination with lamin C in these cells and observed no restoration of peripheral heterochromatin in either case. We conclude that in contrary to the B-tether, the A-tether has a more intricate composition and consists of multiple components that presumably vary, at differing degrees of redundancy, between cell types and differentiation stages.

Keywords
Nuclear envelope, Nuclear architecture, LEMD proteins, LEMD2, Sampl, Transgenic rod cells
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-142701 (URN)10.1016/j.diff.2016.12.002 (DOI)000397078300006 ()28056360 (PubMedID)
Available from: 2017-05-05 Created: 2017-05-05 Last updated: 2022-02-28Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2092-457x

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