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Article ; Online: High Throughput siRNA Screening Using Reverse Transfection.

von Schantz, Carina / Saarela, Jani

Methods in molecular biology (Clifton, N.J.)

2016 Volume 1470, Page(s) 25–37

Abstract: RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we ... ...

Abstract	RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis.
Language	English
Publishing date	2016
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-6337-9_3
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Genome-wide siRNA screening reveals several host receptors for the binding of human gut commensal Bifidobacterium bifidum.

Kainulainen, Veera / von Schantz-Fant, Carina / Kovanen, Ruusu-Maria / Potdar, Swapnil / Laamanen, Karoliina / Saarela, Jani / Satokari, Reetta

NPJ biofilms and microbiomes

2022 Volume 8, Issue 1, Page(s) 50

Abstract: Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, the ... ...

Abstract	Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, the protective mechanisms of bifidobacteria on intestinal epithelium at molecular level are poorly understood. In this study, we developed a high-throughput in vitro screening assay to explore binding receptors of intestinal epithelial cells for Bifidobacterium bifidum. Short interfering RNAs (siRNA) were used to silence expression of each gene in the Caco-2 cell line one by one. The screen yielded four cell surface proteins, SERPINB3, LGICZ1, PKD1 and PAQR6, which were identified as potential receptors as the siRNA knock-down of their expression decreased adhesion of B. bifidum to the cell line repeatedly during the three rounds of siRNA screening. Furthermore, blocking of these host cell proteins by specific antibodies decreased the binding of B. bifidum significantly to Caco-2 and HT29 cell lines. All these molecules are located on the surface of epithelial cells and three out of four, SERPINB3, PKD1 and PAQR6, are involved in the regulation of cellular processes related to proliferation, differentiation and apoptosis as well as inflammation and immunity. Our results provide leads to the first steps in the mechanistic cascade of B. bifidum-host interactions leading to regulatory effects in the epithelium and may partly explain how this commensal bacterium is able to promote intestinal homeostasis.
MeSH term(s)	Bifidobacterium/genetics ; Bifidobacterium/metabolism ; Bifidobacterium bifidum/genetics ; Caco-2 Cells ; HT29 Cells ; Humans ; Infant ; RNA, Small Interfering/genetics ; RNA, Small Interfering/metabolism
Chemical Substances	RNA, Small Interfering
Language	English
Publishing date	2022-06-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2817021-0
ISSN	2055-5008 ; 2055-5008
ISSN (online)	2055-5008
ISSN	2055-5008
DOI	10.1038/s41522-022-00312-0
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Article ; Online: Genome-wide siRNA screening reveals several host receptors for the binding of human gut commensal Bifidobacterium bifidum

Veera Kainulainen / Carina von Schantz-Fant / Ruusu-Maria Kovanen / Swapnil Potdar / Karoliina Laamanen / Jani Saarela / Reetta Satokari

npj Biofilms and Microbiomes, Vol 8, Iss 1, Pp 1-

2022 Volume 9

Abstract: Abstract Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, ... ...

Abstract	Abstract Bifidobacterium spp. are abundant gut commensals, especially in breast-fed infants. Bifidobacteria are associated with many health-promoting effects including maintenance of epithelial barrier and integrity as well as immunomodulation. However, the protective mechanisms of bifidobacteria on intestinal epithelium at molecular level are poorly understood. In this study, we developed a high-throughput in vitro screening assay to explore binding receptors of intestinal epithelial cells for Bifidobacterium bifidum. Short interfering RNAs (siRNA) were used to silence expression of each gene in the Caco-2 cell line one by one. The screen yielded four cell surface proteins, SERPINB3, LGICZ1, PKD1 and PAQR6, which were identified as potential receptors as the siRNA knock-down of their expression decreased adhesion of B. bifidum to the cell line repeatedly during the three rounds of siRNA screening. Furthermore, blocking of these host cell proteins by specific antibodies decreased the binding of B. bifidum significantly to Caco-2 and HT29 cell lines. All these molecules are located on the surface of epithelial cells and three out of four, SERPINB3, PKD1 and PAQR6, are involved in the regulation of cellular processes related to proliferation, differentiation and apoptosis as well as inflammation and immunity. Our results provide leads to the first steps in the mechanistic cascade of B. bifidum-host interactions leading to regulatory effects in the epithelium and may partly explain how this commensal bacterium is able to promote intestinal homeostasis.
Keywords	Microbial ecology ; QR100-130
Subject code	570
Language	English
Publishing date	2022-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
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Article ; Online: Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells.

Denisova, Oxana V / Merisaari, Joni / Kaur, Amanpreet / Yetukuri, Laxman / Jumppanen, Mikael / von Schantz-Fant, Carina / Ohlmeyer, Michael / Wennerberg, Krister / Aittokallio, Tero / Taipale, Mikko / Westermarck, Jukka

Scientific reports

2022 Volume 12, Issue 1, Page(s) 13796

Abstract: Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially ... ...

Abstract	Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.
MeSH term(s)	Antineoplastic Agents ; Glioblastoma/drug therapy ; Humans ; Protein Kinase Inhibitors/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Protein Phosphatase 2 ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Staurosporine/pharmacology
Chemical Substances	Antineoplastic Agents ; Protein Kinase Inhibitors ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Protein Phosphatase 2 (EC 3.1.3.16) ; Staurosporine (H88EPA0A3N)
Language	English
Publishing date	2022-08-13
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-18118-7
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Development of actionable targets of multi-kinase inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

Oxana V. Denisova / Joni Merisaari / Amanpreet Kaur / Laxman Yetukuri / Mikael Jumppanen / Carina von Schantz-Fant / Michael Ohlmeyer / Krister Wennerberg / Tero Aittokallio / Mikko Taipale / Jukka Westermarck

Scientific Reports, Vol 12, Iss 1, Pp 1-

2022 Volume 9

Abstract: Abstract Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby ... ...

Abstract	Abstract Therapeutic resistance to kinase inhibitors constitutes a major unresolved clinical challenge in cancer and especially in glioblastoma. Multi-kinase inhibitors may be used for simultaneous targeting of multiple target kinases and thereby potentially overcome kinase inhibitor resistance. However, in most cases the identification of the target kinases mediating therapeutic effects of multi-kinase inhibitors has been challenging. To tackle this important problem, we developed an actionable targets of multi-kinase inhibitors (AToMI) strategy and used it for characterization of glioblastoma target kinases of staurosporine derivatives displaying synergy with protein phosphatase 2A (PP2A) reactivation. AToMI consists of interchangeable modules combining drug-kinase interaction assay, siRNA high-throughput screening, bioinformatics analysis, and validation screening with more selective target kinase inhibitors. As a result, AToMI analysis revealed AKT and mitochondrial pyruvate dehydrogenase kinase PDK1 and PDK4 as kinase targets of staurosporine derivatives UCN-01, CEP-701, and K252a that synergized with PP2A activation across heterogeneous glioblastoma cells. Based on these proof-of-principle results, we propose that the application and further development of AToMI for clinically applicable multi-kinase inhibitors could provide significant benefits in overcoming the challenge of lack of knowledge of the target specificity of multi-kinase inhibitors.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2022-08-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses.

Uusi-Rauva, Kristiina / Blom, Tea / von Schantz-Fant, Carina / Blom, Tomas / Jalanko, Anu / Kyttälä, Aija

International journal of molecular sciences

2017 Volume 18, Issue 5

Abstract: Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying ... ...

Abstract	Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5) disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs) harbouring the most common
MeSH term(s)	Cell Differentiation ; Cell Lineage ; Cells, Cultured ; Humans ; Induced Pluripotent Stem Cells/cytology ; Induced Pluripotent Stem Cells/metabolism ; Lysosomal Membrane Proteins ; Membrane Proteins/genetics ; Mutation ; Neuronal Ceroid-Lipofuscinoses/genetics ; Neuronal Ceroid-Lipofuscinoses/pathology ; Phenotype
Chemical Substances	CLN5 protein, human ; Lysosomal Membrane Proteins ; Membrane Proteins
Language	English
Publishing date	2017-05-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms18050955
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Article ; Online: The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

Pietiainen, Vilja / Saarela, Jani / von Schantz, Carina / Turunen, Laura / Ostling, Paivi / Wennerberg, Krister

Combinatorial chemistry & high throughput screening

2014 Volume 17, Issue 4, Page(s) 377–386

Abstract: The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and ...

Abstract	The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.
MeSH term(s)	Biological Specimen Banks ; Cooperative Behavior ; Drug Discovery/methods ; Drug Discovery/organization & administration ; Europe ; Finland ; Genomics/methods ; Genomics/organization & administration ; High-Throughput Screening Assays ; Humans ; Microscopy ; Molecular Medicine/methods ; Molecular Medicine/organization & administration ; Precision Medicine/methods ; RNA Interference ; RNA, Small Interfering ; Translational Medical Research/organization & administration ; Workforce
Chemical Substances	RNA, Small Interfering
Language	English
Publishing date	2014-02-19
Publishing country	United Arab Emirates
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2064785-2
ISSN	1875-5402 ; 1386-2073
ISSN (online)	1875-5402
ISSN	1386-2073
DOI	10.2174/1386207317666140323195927
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Article ; Online: Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses

Kristiina Uusi-Rauva / Tea Blom / Carina von Schantz-Fant / Tomas Blom / Anu Jalanko / Aija Kyttälä

International Journal of Molecular Sciences, Vol 18, Iss 5, p

2017 Volume 955

Abstract	Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5) disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs) harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X), were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.
Keywords	CLN5 ; iPS cells ; NCL ; sphingolipid ; lysosomal storage disease ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Language	English
Publishing date	2017-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Screening out irrelevant cell-based models of disease.

Horvath, Peter / Aulner, Nathalie / Bickle, Marc / Davies, Anthony M / Nery, Elaine Del / Ebner, Daniel / Montoya, Maria C / Östling, Päivi / Pietiäinen, Vilja / Price, Leo S / Shorte, Spencer L / Turcatti, Gerardo / von Schantz, Carina / Carragher, Neil O

Nature reviews. Drug discovery

2016 Volume 15, Issue 11, Page(s) 751–769

Abstract: The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative ... ...

Abstract	The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.
MeSH term(s)	Animals ; Cell Culture Techniques/methods ; Cell Line, Transformed ; Drug Discovery/methods ; Drug Evaluation, Preclinical/methods ; High-Throughput Screening Assays/methods ; Humans ; Induced Pluripotent Stem Cells/drug effects ; Induced Pluripotent Stem Cells/physiology ; Models, Biological ; Pharmaceutical Preparations/administration & dosage
Chemical Substances	Pharmaceutical Preparations
Language	English
Publishing date	2016
Publishing country	England
Document type	Review
ZDB-ID	2062954-0
ISSN	1474-1784 ; 1474-1776
ISSN (online)	1474-1784
ISSN	1474-1776
DOI	10.1038/nrd.2016.175
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Zs.A 5564: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: A novel aspartylglucosaminuria mutation affects translocation of aspartylglucosaminidase.

Saarela, Jani / von Schantz, Carina / Peltonen, Leena / Jalanko, Anu

Human mutation

2004 Volume 24, Issue 4, Page(s) 350–351

Abstract: The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) ... ...

Abstract	The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient.
MeSH term(s)	Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Aspartylglucosaminuria ; Aspartylglucosylaminase/genetics ; Aspartylglucosylaminase/physiology ; COS Cells/enzymology ; Cell Line/enzymology ; Cercopithecus aethiops ; Cricetinae ; DNA Mutational Analysis ; Endoplasmic Reticulum/enzymology ; Finland/epidemiology ; Heterozygote ; Humans ; Hydrophobic and Hydrophilic Interactions ; Lysosomal Storage Diseases/enzymology ; Lysosomal Storage Diseases/epidemiology ; Lysosomal Storage Diseases/genetics ; Lysosomes/enzymology ; Male ; Mesocricetus ; Molecular Sequence Data ; Mutation, Missense ; Point Mutation ; Protein Sorting Signals/genetics ; Protein Transport/genetics ; Recombinant Fusion Proteins/metabolism ; Structure-Activity Relationship ; Transfection
Chemical Substances	Protein Sorting Signals ; Recombinant Fusion Proteins ; Aspartylglucosylaminase (EC 3.5.1.26)
Language	English
Publishing date	2004-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1126646-6
ISSN	1098-1004 ; 1059-7794
ISSN (online)	1098-1004
ISSN	1059-7794
DOI	10.1002/humu.9276
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