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Saber, Amanj
Publications (10 of 12) Show all publications
Saber, A., Hussain, R., Nakka, S. & Hugosson, S. (2019). Effect of Staphylococcus aureus on the NLRP3 inflammasome, caspase-1 and IL-1 Beta expression in the nasal epithelial cells in chronic rhinosinusitis. European Journal of rhinology and Allergy, 2(1), 6-12
Open this publication in new window or tab >>Effect of Staphylococcus aureus on the NLRP3 inflammasome, caspase-1 and IL-1 Beta expression in the nasal epithelial cells in chronic rhinosinusitis
2019 (English)In: European Journal of rhinology and Allergy, E-ISSN 2636-8072, Vol. 2, no 1, p. 6-12Article in journal (Refereed) Published
Abstract [en]

Objective: This study aimed to elucidate the effect of Staphylococcus aureus and budesonide on the mRNA expression and the biologic role (caspase-1 activation and IL-1β secretion) of NLRP3 inflammasome in primary nasal epithelial cells (NECs) in patients with chronic rhinosinusitis (CRS) and healthy controls.

Material and Methods: Brush biopsies isolated from both patients and healthy controls were denoted respectively for our experiments. These were treated with S. aureus strains (four strains) only and in combination with budesonide (0, 10, 100, 1000 nM). NECs treated with only budesonide (0, 10, 100, 1000 nM) and untreated NECs were used as controls. Expression of NLRP3, caspase-1, IL-1β along with NLRC1/2 was analyzed by qPCR. Caspase-1 activity was measured by fluorogenic substrates Ac-YVAD-AMC. Enzyme-linked immunosorbent (ELISA) assay was performed to measure IL-1β production.

Results: The mRNA levels of NLRC1, NLRC2, caspase-1, and IL-1β significantly increased, while NLRP3 demonstrated a trend toward elevation in the CRS group compared to the healthy controls. Infection with S. aureus increased caspase-1 activity and IL-1β secretion. However, treatment with budesonide decreased mRNA expression of NLRC2 and IL-1β secretion.

Conclusion: Increase in the caspase-1 activity and IL-1β levels, due to possible activation of NLRP3 inflammasomes, upon S. aureus infection, may have an important role in the pathogenesis of CRS.

Place, publisher, year, edition, pages
Turkey: Turkish Rhinologic Society, 2019
Keywords
Budesonide, chronic rhinosinusitis, NLRP3, S. aureus
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology
Identifiers
urn:nbn:se:oru:diva-75068 (URN)10.5152/ejra.2019.110 (DOI)
Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-06Bibliographically approved
Saber, A., Nakka, S. S., Hussain, R. & Hugosson, S. (2019). Staphylococcus aureus in chronic rhinosinusitis: the effect on the epithelial chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) and the epithelial sodium channel (ENaC) physiology. Acta Oto-Laryngologica, 139(7), 652-658
Open this publication in new window or tab >>Staphylococcus aureus in chronic rhinosinusitis: the effect on the epithelial chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) and the epithelial sodium channel (ENaC) physiology
2019 (English)In: Acta Oto-Laryngologica, ISSN 0001-6489, E-ISSN 1651-2251, Vol. 139, no 7, p. 652-658Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory disease of the nose and the paranasal sinuses, often associated with an infection by Staphylococcus aureus (S. aureus). Disturbance in the function of ion channels is regarded as an etiological factor for pathogenesis of CRS.

AIMS: The study aims to measure the mRNA expression of the ENaC and CFTR ion channels in nasal epithelial cells (NECs) and to investigate the effect of both the budesonide and S. aureus on these ion channels.

MATERIALS AND METHOD: NECs biopsies obtained from healthy volunteers and patients with CRS. NECs were infected with S. aureus strains and/or budesonide to study the mRNA expression levels of the ENaC and CFTR ion channels.

RESULTS: The mRNA expression level of CFTR was increased while that of ENaC was decreased. S. aureus infection and budesonide treatment induced a significant modulation of ENaC and CFTR ion channels expression.

CONCLUSION: The CFTR and ENaC ion channel physiology are of importance in the pathogenesis of CRS. Exposure to S. aureus infection and treatment with budesonide modulated the mRNA expression of CFTR and ENaC ion channels.

SIGNIFICANCE: Better understanding of the pathophysiology of CRS.

Place, publisher, year, edition, pages
Taylor & Francis, 2019
Keywords
Chronic rhinosinusitis, ENaC and CFTR ion channel expression
National Category
Immunology
Identifiers
urn:nbn:se:oru:diva-74196 (URN)10.1080/00016489.2019.1603513 (DOI)000482254100019 ()31050570 (PubMedID)2-s2.0-85065389518 (Scopus ID)
Note

Funding Agencies:

Research Committee in Region Örebro County  

Foundation for Medical Research at the University Hospital Örebro (Örebro, Sweden) 

Available from: 2019-05-13 Created: 2019-05-13 Last updated: 2020-01-16Bibliographically approved
Saber, A., Nakka, S. S., Hussain, R. & Hugosson, S. (2018). Staphylococcus aureus in chronic rhinosinusitis: The effect on the epithelial chloride channel cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel physiology. In: : . Paper presented at 12th International Symposium on Experimental Rhinology and Immunology of the Nose (SERIN), Ghent, Belgium,October 23-25, 2018.
Open this publication in new window or tab >>Staphylococcus aureus in chronic rhinosinusitis: The effect on the epithelial chloride channel cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel physiology
2018 (English)Conference paper, Poster (with or without abstract) (Refereed)
Abstract [en]

Background and objectives: Chronic rhinosinusitis (CRS) is an inflammatory disease of the nose and the paranasal sinuses, often associated with an infection by Staphylococcus aureus (S. aureus). Disturbance in the function of ion channels is regarded as an etiological factor for pathogenesis of CRS. The study aims to measure the mRNA expression of the ENaC and CFTR ion channels in nasal epithelial cells (NECs) and to investigate the effect of both the budesonide and S. aureus on these ion channels.

Materials and Method: NECs biopsies obtained from healthy volunteers and patients with CRS. NECs were infected with S. aureus strains and/or budesonide to study the mRNA expression levels of the ENaC and CFTR ion channels.

Results: The mRNA expression level of CFTR was increased while that of ENaC was decreased. S. aureus infection and budesonide treatment induced a significant modulation of ENaC and CFTR ion channels expression.

Conclusion: The CFTR and ENaC ion channel physiology are of importance in the pathogenesis of CRS. Exposure to S. aureus infection and treatment with budesonide modulated the mRNA expression of CFTR and ENaC ion channels. The study is an effort for a better understanding of the pathophysiology of CRS. In order to reach further knowledge, studies on functional properties of the ENaC and CFTR ion channels are necessary.

National Category
Otorhinolaryngology
Identifiers
urn:nbn:se:oru:diva-69521 (URN)
Conference
12th International Symposium on Experimental Rhinology and Immunology of the Nose (SERIN), Ghent, Belgium,October 23-25, 2018
Available from: 2018-10-15 Created: 2018-10-15 Last updated: 2019-07-10Bibliographically approved
Saber, A., Gont, C., Dash-Wagh, S., Kirkegaard, M., Strand, S. P. & Ulfendahl, M. (2010). Assessment of in Vitro and in Vivo Transfection Efficiency of the Biodegradable Polymer Chitosan in the Inner Ear. The Journal of International Advanced Otology, 6(3), 307-315
Open this publication in new window or tab >>Assessment of in Vitro and in Vivo Transfection Efficiency of the Biodegradable Polymer Chitosan in the Inner Ear
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2010 (English)In: The Journal of International Advanced Otology, ISSN 1308-7649, Vol. 6, no 3, p. 307-315Article in journal (Refereed) Published
Abstract [en]

Background: Sensorineural hearing loss is a significant problem worldwide and a condition that is not completely cured by currently available therapy. Gene therapy of the inner ear offers an exciting alternative and it has been suggested that this therapeutic modality could be used in treatment aiming at preventing, reversing or managing cochlear disorders. Because of their desired properties as an alternative to the viral vectors, non-viral vectors have been extensively explored for gene delivery. One example is chitosan, a biodegradable cationic polymer.

Objective: To evaluate the in vitro and in vivo transfection efficiency of chitosan as a non-viral gene carrier for gene delivery to cells of the inner ear.

Materials and Methods: Organotypic cultures of the hearing organ, the organ of Corti, were prepared from postnatal day 2 rats, and exposed to chitosan carrying plasmid DNA (pDNA) encoding for green fluorescent protein (GFP) for 24-48 hours. The in vivo transfection efficiency was tested at two time points, at one day or seven days after infusing chitosan/pDNA polyplexes through osmotic pumps into the cochlea of adult guinea pigs (n=41). The tissue was then processed for anti-GFP immunostaining (in vitro and in vivo) and RT-PCR (in vivo).

Results: The in vitro assessment showed prominent GPF transfection after 24-48 hours, while the in vivo GFP transfection in the inner ear was inconsistent and did not show good correlation with the in vitro transfection. Conclusion: It can be concluded that the using chitosan as a carrier for the in vivo transfection, is associated with varying and in consistent degree of transfection.

Place, publisher, year, edition, pages
European Academy of Otology and Neurotology and the Politzer Society, 2010
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75078 (URN)000285986200002 ()2-s2.0-78650762031 (Scopus ID)
Funder
Swedish Research Council
Note

Funding Agencies:

European Commission

Tysta Skolan Foundation

Petrus and Augusta Hedlund Foundation

Swedish Council for Working Life and Social Research through the FAS Center 

Hawler Medical University-Iraq (HMU)

Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-07Bibliographically approved
Saber, A. (2010). Round window membrane and delivery of biologically active agents into the cochlea. (Doctoral dissertation). Universitetsservice US-AB
Open this publication in new window or tab >>Round window membrane and delivery of biologically active agents into the cochlea
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Establishing efficient methods for local administration of drugs to the inner ear has great clinical relevance for the management of inner ear disorders. However, the administration route remains a critical issue. The most feasible approach for a non invasive drug delivery to the inner ear is application of medication to the middle ear cavity on the promise that it will diffuse through the thin round window membrane (RWM) separating the inner ear from the middle ear cavity. Gene therapy represents a promising future in otology and offers an exciting therapeutic alternative as it could be used in prevention or management of cochlear disorders. Also for a gene therapy approach, RWM application seems most feasible administration route. Exploring and characterizing the RWM route of administration is thus a fundamentally important area of research for the development of future treatment of inner ear disorders. The objectives of the thesis were to evaluate the efficacy of two drug and gene delivering vehicles to the inner ear, sodium hyaluronate (HYA) and chitosans, which can be applied to the cochlea. Ultimate aim is to establish an efficient drug delivery system and gene transfection for the inner ear. HYA and chitosans loaded with the ototoxic drug neomycin as tracer for drug release have been instilled into the middle ear of the guinea pigs. Effects on RWM and cochlear hair cells were evaluated after a single instillation of HYA (day 7 and 28), chitosans and saline solution (day 7). The hearing organ was analysed for hair cell loss and the thickness and ultrastructural properties of the RWM were analysed by light and transmission electron microscopy. The in vitro transfection efficiency of chitosan was tested by exposing organotypic cultures of the organ of Corti, prepared from postnatal day 2 rats, to chitosan carrying plasmid DNA (pDNA). The in vivo transfection efficiency was tested at one day or seven days after infusing chitosan/pDNA polyplexes with the use of osmotic pumps into the cochlea of adult guinea pigs. Tissue analysis was made by immunohistochemsitry and RT-PCR. HYA and chitosans, especially glycosylated derivative, are safe vehicles that can be used for drug transport into the inner ear through the RWM. Both vehicles successfully released the loaded neomycin, which exerted toxic effects on cochlear hair cells in a degree depending on the concentrations used. The vehicles per se had no noxious effect on the cochlear hair cells but they provoked a comparable effect on the thickness and morphology of the RWM. The thickness of the RWM returned to normal 4 weeks after exposure to HYA. Chitosan as a carrier for inner ear transfection, was associated with inconsistent transfection in vitro and in vivo . The importance of the RWM as a portal for local therapy of inner ear disorders is highlighted in this thesis by focusing on efficiency and effects of the vehicles, applied to the RWM for delivering biologically active agents into the cochlea. The difficulties and variability associated with applying substances to the RWM were explored. The results of this thesis add new knowledge concerning mechanisms of passage of biologically active agents through the RWM and may help us to better understand the role of RWM in the local cochlear therapy and problems of local treatment of inner ear diseases.

Place, publisher, year, edition, pages
Universitetsservice US-AB, 2010. p. 60
Keywords
Round window membrane, sodium hyaluronate, chitosan, local cochlear drug delivery and gene delivery
National Category
Medical and Health Sciences Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75083 (URN)978-91-7457-067-0 (ISBN)
Public defence
2010-10-27, 09:10 (English)
Opponent
Supervisors
Available from: 2019-08-06 Created: 2019-07-11 Last updated: 2019-08-07Bibliographically approved
Saber, A., Strand, S. & Ulfendahl, M. (2010). Use of the Biodegradable Polymer Chitosan as a Vehicle for Applying Drugs to the Inner Ear. In: : . Paper presented at 33rd Annual MidWinter Research Meeting of the Association for Research in Otolaryngology, Anaheim, California, USA, February 6-10, 2010.
Open this publication in new window or tab >>Use of the Biodegradable Polymer Chitosan as a Vehicle for Applying Drugs to the Inner Ear
2010 (English)Conference paper, Poster (with or without abstract) (Refereed)
Abstract [en]

Development of efficient local delivery systems for the auditory organ has an important role in clinical practice for the management of inner ear disorders using pharmacological means. Chitosan, a biodegradable polymer, is a good drug carrier with bioadhesive properties. The aim of this study was to investigate the feasibility of using chitosan to deliver drugs to the inner ear across the round window membrane (RWM). Three structurally different chitosans loaded with a tracer drug, neomycin, were injected into the middle ear cavity of albino guinea pigs (n=35). After 7 days the effect of chitosans and neomycin was compared among the treatment groups. The hearing organ was analysed for hair cell loss and the RWM evaluated in term of thickness. All tested chitosan formulations successfully released the loaded neomycin which then diffused across the RWM, and exerted ototoxic effect on the cochlear hair cells in a degree depending on the concentrations used. Chitosans per se had no noxious effect on the cochlear hair cells. It is concluded that the chitosans, and especially glycosylated derivative, are safe and effective carriers for inner ear therapy.

National Category
Otorhinolaryngology Pharmaceutical Sciences
Identifiers
urn:nbn:se:oru:diva-69322 (URN)
Conference
33rd Annual MidWinter Research Meeting of the Association for Research in Otolaryngology, Anaheim, California, USA, February 6-10, 2010
Available from: 2018-10-05 Created: 2018-10-05 Last updated: 2019-07-10Bibliographically approved
Saber, A., Strand, S. P. & Ulfendahl, M. (2010). Use of the biodegradable polymer chitosan as a vehicle for applying drugs to the inner ear. European Journal of Pharmaceutical Sciences, 39(1-3), 110-115
Open this publication in new window or tab >>Use of the biodegradable polymer chitosan as a vehicle for applying drugs to the inner ear
2010 (English)In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 39, no 1-3, p. 110-115Article in journal (Refereed) Published
Abstract [en]

Development of efficient local delivery systems for the auditory organ has an important role in clinical practice for the management of inner ear disorders using pharmacological means. Chitosan, a biodegradable polymer, is a good drug carrier with bioadhesive properties. The aim of this study was to investigate the feasibility of using chitosan to deliver drugs to the inner ear across the round window membrane (RWM). Three structurally different chitosans loaded with a tracer drug, neomycin, were injected into the middle ear cavity of albino guinea pigs (n=35). After 7 days the effect of chitosans and neomycin was compared among the treatment groups. The hearing organ was analysed for hair cell loss and the RWM evaluated in term of thickness. All tested chitosan formulations successfully released the loaded neomycin which then diffused across the RWM, and exerted ototoxic effect on the cochlear hair cells in a degree depending on the concentrations used. Chitosans per se had no noxious effect on the cochlear hair cells. It is concluded that the chitosans, and especially glycosylated derivative, are safe and effective carriers for inner ear therapy.

Place, publisher, year, edition, pages
Elsevier, 2010
Keywords
Round window membrane, Chitosan, Hair cell, Drug delivery
National Category
Pharmaceutical Sciences
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75076 (URN)10.1016/j.ejps.2009.11.003 (DOI)000274672300016 ()19931387 (PubMedID)2-s2.0-73749087296 (Scopus ID)
Funder
Forte, Swedish Research Council for Health, Working Life and WelfareSwedish Research Council
Note

Research funders:

European Commission FP6, EUROHEAR (LSHG-CT-20054-512063)

Tysta Skolan Foundation

Petrus and Augusta Hedlund Foundation

Hawler Medical University-Iraq (HMU)

Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-07Bibliographically approved
Barzinjy, V. B., Dabbagh, R. & Saber, A. (2009). Fungal and Other Microorganisms Involved in Otomycosis in Hawler Area. Middle East Journal of Internal Medicine, 2(1), 7-10
Open this publication in new window or tab >>Fungal and Other Microorganisms Involved in Otomycosis in Hawler Area
2009 (English)In: Middle East Journal of Internal Medicine, ISSN 1837-9052, Vol. 2, no 1, p. 7-10Article in journal (Refereed) Published
Abstract [en]

Background: Patients with clinical manifestations suggestive of otitis externa.

Objective: To study the prevalence of mycotic infection in these patients.

Methods: Ear swabs were taken from these patients and both direct examination and culture were done.

Results: The most common pathogenic fungal isolates were Candida species (8.4%) and Aspergillus niger (4.2%). Conclusion: Besides Aspergillus and Candida species isolated in these patients, many bacteria were also seen. Dry climate, wearing head clothes, presence of middle ear infection, wearing hearing aids and swimming were predisposing factors for this infection.

Place, publisher, year, edition, pages
Australia: Medi World International, 2009
Keywords
External otitis. Otomycosis
National Category
Medical and Health Sciences Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Infectious Diseases
Identifiers
urn:nbn:se:oru:diva-75084 (URN)
Available from: 2019-07-11 Created: 2019-07-11 Last updated: 2019-08-07Bibliographically approved
Saber, A., Laurell, G., Bramer, T., Edsman, K., Engmér, C. & Ulfendahl, M. (2009). Middle Ear Application of a Sodium Hyaluronate Gel Loaded with Neomycin in a Guinea Pig Model. Ear and Hearing, 30(1), 81-89
Open this publication in new window or tab >>Middle Ear Application of a Sodium Hyaluronate Gel Loaded with Neomycin in a Guinea Pig Model
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2009 (English)In: Ear and Hearing, ISSN 0196-0202, E-ISSN 1538-4667, Vol. 30, no 1, p. 81-89Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Establishing methods for topical administration of drugs to the inner ear have great clinical relevance and potential even in a relatively short perspective. To evaluate the efficacy of sodium hyaluronate (HYA) as a vehicle for drugs that could be used for treatment of inner ear disorders.

METHODS: The cochlear hair cell loss and round window membrane (RWM) morphology were investigated after topical application of neomycin and HYA into the middle ear. Sixty-five albino guinea pigs were used and divided into groups depending on the type of the treatment. Neomycin was chosen as tracer for drug release and pharmacodynamic effect. HYA loaded with 3 different concentrations of neomycin was injected to the middle ear cavity of guinea pigs. Phalloidin stained surface preparations of the organ of Corti were used to estimate hair cell loss induced by neomycin. The thickness of the midportion of the RWM was measured and compared with that of controls using light and electron microscopy. All animal procedures were pe rformed in accordance with the ethical standards of Karolinska Institutet.

RESULT: Neomycin induced a considerable hair cell loss in guinea pigs receiving a middle ear injection of HYA loaded with the drug, demonstrating that neomycin was released from the gel and delivered to the inner ear. The resulting hair cell loss showed a clear dose-dependence. Only small differences in hair cell loss were noted between animals receiving neomycin solution and animals exposed to neomycin in HYA suggesting that the vehicle neither facilitated nor hindered drug transport between the middle ear cavity and the inner ear. One week after topical application, the thickness of the RWM had increased and was dependent upon the concentration of neomycin administered to the middle ear. At 4 weeks the thickness of the RWM had returned to normal.

CONCLUSION: HYA is a safe vehicle for drugs aimed to pass into the inner ear through the RWM. Neomycin was released from HYA and transported into the inner ear as evidenced by hair cell loss.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2009
National Category
Otorhinolaryngology
Research subject
Oto-Rhino-Laryngology; Biology
Identifiers
urn:nbn:se:oru:diva-75077 (URN)10.1097/AUD.0b013e31818ff98e (DOI)000262312000010 ()19125030 (PubMedID)2-s2.0-64849084054 (Scopus ID)
Funder
Swedish Research Council
Note

Funding Agencies:

European Commission

Tysta Skolan

Pettus and Augusta Hedlund Foundation

AFA Insurance

Hawler Medical University-Iraq

Available from: 2019-07-10 Created: 2019-07-10 Last updated: 2019-08-07Bibliographically approved
Saber, A., Laurell, G., Tobias, B., Katarina, E., Cecilia, E. & Mats, U. (2008). Middle Ear Application of Hyaluronic Acid Gel Loaded with Neomycin in a Guinea Pig Model. In: : . Paper presented at 31st Annual MidWinter Meeting of the Association for Research in Otolaryngology, Phoenix, AZ, USA, February 16-21, 2008.
Open this publication in new window or tab >>Middle Ear Application of Hyaluronic Acid Gel Loaded with Neomycin in a Guinea Pig Model
Show others...
2008 (English)Conference paper, Poster (with or without abstract) (Refereed)
Abstract [en]

Background: One alternative for controlled drug delivery to the inner ear is application of medication to the middle ear cavity on the promise that it will diffuse through the round window membrane (RWM) into the inner ear.

Objective: to evaluate the efficacy of hyaluronic acid gel (HYA) as a vehicle for drugs that are aimed to treat inner ear disorders.

Methods: in this study the cochlear hair cell loss and RWM morphology were investigated after topical application. Neomycin was chosen as tracer for drug release and the ototoxic effect was evaluated. HYA, (0.5%) loaded with 3 different concentrations of neomycin, was injected to the middle ear cavity of guinea pigs. the ototoxic effect of neomycin in HYA gel was compared to that of neomycin solution applied to the middle ear cavity. Phalloidin stained surface preparation of the organ of Corti was used to estimate hair cell loss induced by neomycin. the thickness of the midportion of the RWM was measured and compared with that of controls using light and electrom microscope.

Result: Neomycin induced a considerable hair cell loss in guinea pigs receiving middle ear injection of HYA loaded with the drug. One week after topical application the thickness of the RWM was dependent upon the concentration of neomycin administered to the middle ear. At 4 weeks the thickness of the RWM had returned to normal.

Conclusion: HYA is a safe vehicle for drugs aimed to pass into the inner ear through the RWM. Neomycin was released from HYA and transported into the inner ear as evidenced by hair cell loss. Key words: Round window membrane (RWM), hyaluronic acid gel, HYA, hair cell loss, thickness.

National Category
Otorhinolaryngology Pharmaceutical Sciences
Identifiers
urn:nbn:se:oru:diva-69319 (URN)
Conference
31st Annual MidWinter Meeting of the Association for Research in Otolaryngology, Phoenix, AZ, USA, February 16-21, 2008
Available from: 2018-10-05 Created: 2018-10-05 Last updated: 2019-07-10Bibliographically approved
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