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Publications (10 of 13) Show all publications
Khoonsari, P. E., Häggmark, A., Lonnberg, M., Mikus, M., Kilander, L., Lannfelt, L., . . . Shevchenko, G. (2016). Analysis of the Cerebrospinal Fluid Proteome in Alzheimer's Disease. PLoS ONE, 11(3), Article ID e0150672.
Open this publication in new window or tab >>Analysis of the Cerebrospinal Fluid Proteome in Alzheimer's Disease
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, article id e0150672Article in journal (Refereed) Published
Abstract [en]

Alzheimer's disease is a neurodegenerative disorder accounting for more than 50% of cases of dementia. Diagnosis of Alzheimer's disease relies on cognitive tests and analysis of amyloid beta, protein tau, and hyperphosphorylated tau in cerebrospinal fluid. Although these markers provide relatively high sensitivity and specificity for early disease detection, they are not suitable for monitor of disease progression. In the present study, we used label-free shotgun mass spectrometry to analyse the cerebrospinal fluid proteome of Alzheimer's disease patients and non-demented controls to identify potential biomarkers for Alzheimer's disease. We processed the data using five programs (DecyderMS, Maxquant, OpenMS, PEAKS, and Sieve) and compared their results by means of reproducibility and peptide identification, including three different normalization methods. After depletion of high abundant proteins we found that Alzheimer's disease patients had lower fraction of low-abundance proteins in cerebrospinal fluid compared to healthy controls (p<0.05). Consequently, global normalization was found to be less accurate compared to using spiked-in chicken ovalbumin for normalization. In addition, we determined that Sieve and OpenMS resulted in the highest reproducibility and PEAKS was the programs with the highest identification performance. Finally, we successfully verified significantly lower levels (p<0.05) of eight proteins (A2GL, APOM, C1QB, C1QC, C1S, FBLN3, PTPRZ, and SEZ6) in Alzheimer's disease compared to controls using an antibody-based detection method. These proteins are involved in different biological roles spanning from cell adhesion and migration, to regulation of the synapse and the immune system.

National Category
Neurosciences
Identifiers
urn:nbn:se:kth:diva-185066 (URN)10.1371/journal.pone.0150672 (DOI)000371990100049 ()26950848 (PubMedID)2-s2.0-84961572493 (Scopus ID)
Note

QC 20160415

Available from: 2016-04-15 Created: 2016-04-11 Last updated: 2018-01-10Bibliographically approved
Fredolini, C., Byström, S., Pin, E., Edfors, F., Tamburro, D., Iglesias, M. J., . . . Schwenk, J. M. (2016). Immunocapture strategies in translational proteomics. Expert Review of Proteomics, 13(1), 83-98
Open this publication in new window or tab >>Immunocapture strategies in translational proteomics
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2016 (English)In: Expert Review of Proteomics, ISSN 1478-9450, E-ISSN 1744-8387, Vol. 13, no 1, p. 83-98Article, review/survey (Refereed) Published
Abstract [en]

Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient profiling to assays supporting personalized treatments. In recent years, integrated strategies to couple and combine antibodies with mass spectrometry-based proteomic efforts have emerged, allowing for novel possibilities in basic and clinical research. Described in this review are some of the field's current and emerging immunocapture approaches from an affinity proteomics perspective. Discussed are some of their advantages, pitfalls and opportunities for the next phase in clinical and translational proteomics.

Place, publisher, year, edition, pages
Taylor & Francis, 2016
Keywords
Antibodies, immunocapture, mass spectrometry, protein enrichment, sandwich assays, SRM
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-180965 (URN)10.1586/14789450.2016.1111141 (DOI)000367351000001 ()26558424 (PubMedID)
Note

QC 20160128

Available from: 2016-01-28 Created: 2016-01-26 Last updated: 2017-11-30Bibliographically approved
Häggmark, A., Schwenk, J. M. & Nilsson, P. (2016). Neuroproteomic profiling of human body fluids. PROTEOMICS - Clinical Applications, 10(4), 485-502
Open this publication in new window or tab >>Neuroproteomic profiling of human body fluids
2016 (English)In: PROTEOMICS - Clinical Applications, ISSN 1862-8346, E-ISSN 1862-8354, Vol. 10, no 4, p. 485-502Article, review/survey (Refereed) Published
Abstract [en]

Analysis of protein expression and abundance provides a possibility to extend the current knowledge on disease-associated processes and pathways. The human brain is a complex organ and dysfunction or damage can give rise to a variety of neurological diseases. Although many proteins potentially reflecting disease progress are originating from brain, the scarce availability of human tissue material has lead to utilization of body fluids such as cerebrospinal fluid and blood in disease-related research. Within the most common neurological disorders, much effort has been spent on studying the role of a few hallmark proteins in disease pathogenesis but despite extensive investigation, the signatures they provide seem insufficient to fully understand and predict disease progress. In order to expand the view the field of neuroproteomics has lately emerged alongside developing technologies, such as affinity proteomics and mass spectrometry, for multiplexed and high-throughput protein profiling. Here, we provide an overview of how such technologies have been applied to study neurological disease and we also discuss some important considerations concerning discovery of disease-associated profiles.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2016
Keywords
Affinity proteomics, Biomarkers, Body fluids, Mass spectrometry, Neurological disease, Neuroproteomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-187186 (URN)10.1002/prca.201500065 (DOI)000373947200016 ()26286680 (PubMedID)2-s2.0-84962760658 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20160518

Available from: 2016-05-18 Created: 2016-05-18 Last updated: 2017-11-30Bibliographically approved
Sjöstedt, E., Fagerberg, L., Hallström, B. M., Häggmark, A., Mitsios, N., Nilsson, P., . . . Mulder, J. (2015). Defining the Human Brain Proteome Using Transcriptomics and Antibody-Based Profiling with a Focus on the Cerebral Cortex. PLoS ONE, 10(6), Article ID UNSP e0130028.
Open this publication in new window or tab >>Defining the Human Brain Proteome Using Transcriptomics and Antibody-Based Profiling with a Focus on the Cerebral Cortex
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 6, article id UNSP e0130028Article in journal (Refereed) Published
Abstract [en]

The mammalian brain is a complex organ composed of many specialized cells, harboring sets of both common, widely distributed, as well as specialized and discretely localized proteins. Here we focus on the human brain, utilizing transcriptomics and public available Human Protein Atlas (HPA) data to analyze brain-enriched (frontal cortex) polyadenylated messenger RNA and long non-coding RNA and generate a genome-wide draft of global and cellular expression patterns of the brain. Based on transcriptomics analysis of altogether 27 tissues, we have estimated that approximately 3% (n=571) of all protein coding genes and 13% (n=87) of the long non-coding genes expressed in the human brain are enriched, having at least five times higher expression levels in brain as compared to any of the other analyzed peripheral tissues. Based on gene ontology analysis and detailed annotation using antibody-based tissue micro array analysis of the corresponding proteins, we found the majority of brain-enriched protein coding genes to be expressed in astrocytes, oligodendrocytes or in neurons with molecular properties linked to synaptic transmission and brain development. Detailed analysis of the transcripts and the genetic landscape of brainenriched coding and non-coding genes revealed brain-enriched splice variants. Several clusters of neighboring brain-enriched genes were also identified, suggesting regulation of gene expression on the chromatin level. This multi-angle approach uncovered the brainenriched transcriptome and linked genes to cell types and functions, providing novel insights into the molecular foundation of this highly specialized organ.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-170972 (URN)10.1371/journal.pone.0130028 (DOI)000356329900125 ()26076492 (PubMedID)
Note

QC 20150713

Available from: 2015-07-13 Created: 2015-07-13 Last updated: 2018-10-17Bibliographically approved
Häggmark, A. (2015). Neuroproteomic profiling of human body fluids. (Doctoral dissertation). Stockholm: KTH Royal Institute of Technology
Open this publication in new window or tab >>Neuroproteomic profiling of human body fluids
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis provides results from affinity based studies where human body fluids were profiled to find markers for neurological diseases. Both proteins and autoantibodies were analysed using microarray technologies that can profile hundreds of analytes and hundreds of samples in parallel using small sample volumes. A central element in this work was to develop and apply new methods to study cerebrospinal fluid (CSF), which is the fluid in direct contact with the brain. CSF contains proteins reflecting the physiological state of the central nervous system and therefore offers a unique insight into proteins associated to neurological disorders. As a complement to CSF, bloodderived samples such as serum and plasma, were also investigated as these represent potential sources of disease related proteins. The work presented here summarises the development of assay protocols to study protein and autoantibodies in CSF and blood using planar and bead-based microarrays.

In Paper I, an antibody-based protocol was developed to enable multiplexed protein profiling in CSF. The protocol was then applied for a first analysis within multiple sclerosis (MS) patients. In Paper II, the results were further evaluated in additional CSF as well as plasma samples. Based on the CSF analysis we found two proteins associated to MS; GAP43, a protein related to disease progression and SERPINA3, a protein involved in inflammation. In addition, four other proteins; IRF8, METTL14, IL7 and SLC30A7, were found to have altered plasma levels between the patient groups. The expression of these proteins were further investigated by immunofluorescent staining of human brain tissue, revealing differential localisation of proteins in diseased and healthy brain. In Paper III, a study on extensive protein profiling of plasma in the context of another neurodegenerative disorder, amyotrophic lateral sclerosis (ALS), is described. The levels of three proteins, namely NEFM, RGS18 and SCL25A20, were found to be elevated in ALS patients compared to controls. Among these, NEFM also indicated association to disease subtype as the levels were elevated in patients with definite compared to suspected diagnosis.

In addition to antibodies, we also utilised antigens on microarrays to screen for the presence of autoantibodies in body fluids. In Paper IV, a strategy for this analysis was developed using protein fragments and two types of microarrays. This strategy was then applied for profiling of the autoantibody repertoire of MS patients, revealing 51 protein fragments with potential disease relevance. Interestingly, comparison of plasma and CSF samples obtained from the same patients indicated high concordance of antibodies between the two body fluids. In Paper V, a similar strategy was applied to narcolepsy, another neurological disorder. Our investigation of antibodies in serum revealed higher reactivity towards METTL22, NT5C1A and TMEM134 compared to controls in two independent sample materials.

In conclusion, the presented work constitutes a framework of proteomic assays for enhanced exploration of proteins and autoantibodies in neuroscience. Moreover, we have reported identification of several potential disease markers to be further investigated within neurological disorders.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. p. viii, 67
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:2
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-158944 (URN)978-91-7595-402-8 (ISBN)
Public defence
2015-02-06, Rockefellersalen, KI, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20150116

Available from: 2015-01-16 Created: 2015-01-15 Last updated: 2015-01-16Bibliographically approved
Häggmark, A., Hamsten, C., Wiklundh, E., Lindskog, C., Mattsson, C., Andersson, E., . . . Nilsson, P. (2015). Proteomic Profiling Reveals Autoimmune Targets in Sarcoidosis. American Journal of Respiratory and Critical Care Medicine, 191(5), 574-583
Open this publication in new window or tab >>Proteomic Profiling Reveals Autoimmune Targets in Sarcoidosis
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2015 (English)In: American Journal of Respiratory and Critical Care Medicine, ISSN 1073-449X, E-ISSN 1535-4970, Vol. 191, no 5, p. 574-583Article in journal (Refereed) Published
Abstract [en]

Rationale: There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. Objectives: We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. Methods: Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. Measurements and Main Results: Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Lofgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Lofgren syndrome as compared with patients with Lofgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. Conclusions: Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-164459 (URN)10.1164/rccm.201407-1341OC (DOI)000350833900016 ()25608002 (PubMedID)2-s2.0-84923870587 (Scopus ID)
Funder
Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20150422

Available from: 2015-04-22 Created: 2015-04-17 Last updated: 2017-12-04Bibliographically approved
Byström, S., Ayoglu, B., Häggmark, A., Hong, M.-G., Drobin, K., Forsström, B., . . . et al., . (2014). Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis. Journal of Proteome Research, 13(11), 4607-4619
Open this publication in new window or tab >>Affinity Proteomic Profiling of Plasma, Cerebrospinal Fluid, and Brain Tissue within Multiple Sclerosis
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2014 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 11, p. 4607-4619Article in journal (Refereed) Published
Abstract [en]

The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2014
Keywords
antibodies, suspension bead arrays, plasma, CSF, brain tissue, immunofluorescence, multiple sclerosis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-150383 (URN)10.1021/pr500609e (DOI)000344636500012 ()25231264 (PubMedID)2-s2.0-84908890596 (Scopus ID)
Funder
Swedish Research CouncilAFA InsuranceScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20141212

Updated from manuscript to article in journal.

Available from: 2014-09-02 Created: 2014-09-02 Last updated: 2017-12-05Bibliographically approved
Häggmark, A., Mikus, M., Mohsenchian, A., Hong, M.-G., Forsström, B., Gajewska, B., . . . Nilsson, P. (2014). Plasma profiling revelas three proteins associated to amyotrophic lateral sclerosis. Annals of Clinical and Translational Neurology, 1(8), 544-553
Open this publication in new window or tab >>Plasma profiling revelas three proteins associated to amyotrophic lateral sclerosis
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2014 (English)In: Annals of Clinical and Translational Neurology, ISSN 2328-9503, Vol. 1, no 8, p. 544-553Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease leading to muscular paralysis and death within 3-5 years from onset. Currently, there are no reliable and sensitive markers able to substantially shorten the diagnosis delay. The objective of the study was to analyze a large number of proteins in plasma from patients with various clinical phenotypes of ALS in search for novel proteins or protein profiles that could serve as potential indicators of disease.

METHODS: Affinity proteomics in the form of antibody suspension bead arrays were applied to profile plasma samples from 367 ALS patients and 101 controls. The plasma protein content was directly labeled and protein profiles obtained using 352 antibodies from the Human Protein Atlas targeting 278 proteins. A focused bead array was then built to further profile eight selected protein targets in all available samples.

RESULTS: Disease-associated significant differences were observed and replicated for profiles from antibodies targeting the proteins: neurofilament medium polypeptide (NEFM), solute carrier family 25 (SLC25A20), and regulator of G-protein signaling 18 (RGS18).

INTERPRETATION: Upon further validation in several independent cohorts with inclusion of a broad range of other neurological disorders as controls, the alterations of these three protein profiles in plasma could potentially provide new molecular markers of disease that contribute to the quest of understanding ALS pathology.

National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-158941 (URN)10.1002/acn3.83 (DOI)25356426 (PubMedID)
Note

QC 20150115

Available from: 2015-01-15 Created: 2015-01-15 Last updated: 2018-08-23Bibliographically approved
Häggmark, A., Byström, S., Ayoglu, B., Qundos, U., Uhlén, M., Khademi, M., . . . Nilsson, P. (2013). Antibody-based profiling of cerebrospinal fluid within multiple sclerosis. Proteomics, 13(15), 2256-2267
Open this publication in new window or tab >>Antibody-based profiling of cerebrospinal fluid within multiple sclerosis
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2013 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 15, p. 2256-2267Article in journal (Refereed) Published
Abstract [en]

Antibody suspension bead arrays have proven to enable multiplexed and high-throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad-scale protein profiling of CSF within neurological disorders.

Keywords
Antibody microarrays, Biomarker discovery, Cerebrospinal fluid, Multiplexed proteomics technology, Protein arrays, Proteome profiling
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-134062 (URN)10.1002/pmic.201200580 (DOI)000327008300007 ()2-s2.0-84881230169 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceVinnovaKnut and Alice Wallenberg Foundation
Note

QC 20131115

Available from: 2013-11-15 Created: 2013-11-15 Last updated: 2017-12-06Bibliographically approved
Ayoglu, B., Häggmark, A., Khademi, M., Olsson, T., Uhlén, M., Schwenk, J. M. & Nilsson, P. (2013). Autoantibody profiling in multiple sclerosis using arrays of human protein fragments. Molecular & Cellular Proteomics, 12(9), 2657-2672
Open this publication in new window or tab >>Autoantibody profiling in multiple sclerosis using arrays of human protein fragments
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2013 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 9, p. 2657-2672Article in journal (Refereed) Published
Abstract [en]

Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for the identification of biomarkers for autoimmune diseases. Here, a systematic and undirected approach was taken to screen for profiles of IgG in human plasma from 90 individuals with multiple sclerosis related diagnoses. Reactivity pattern of 11,520 protein fragments (representing ̃38% of all human protein encoding genes) were generated on planar protein microarrays built within the Human Protein Atlas. For more than 2,000 antigens IgG reactivity was observed, among which 64% were found only in single individuals. We used reactivity distributions among multiple sclerosis subgroups to select 384 antigens, which were then reevaluated on planar microarrays, corroborated with suspension bead arrays in a larger cohort (n = 376) and confirmed for specificity in inhibition assays. Among the heterogeneous pattern within and across multiple sclerosis subtypes, differences in recognition frequencies were found for 51 antigens, which were enriched for proteins of transcriptional regulation. In conclusion, using protein fragments and complementary high-throughput protein array platforms facilitated an alternative route to discovery and verification of potentially disease-associated autoimmunity signatures, that are now proposed as additional antigens for large-scale validation studies across multiple sclerosis biobanks.

Keywords
autoantibody, immunoglobulin G, adult, aged, article, controlled study, female, human, major clinical study, male, multiple sclerosis, priority journal, protein microarray, transcription regulation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-133813 (URN)10.1074/mcp.M112.026757 (DOI)000330536400021 ()2-s2.0-84884389479 (Scopus ID)
Funder
VinnovaKnut and Alice Wallenberg FoundationAFA InsuranceSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20131204

Available from: 2013-12-04 Created: 2013-11-11 Last updated: 2017-12-06Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-0056-1313

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