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Article ; Online: Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration.

Ramaswami, Gokul / Yuva-Aydemir, Yeliz / Akerberg, Brynn / Matthews, Bryan / Williams, Jenna / Golczer, Gabriel / Huang, Jiaqi / Al Abdullatif, Ali / Huh, Dann / Burkly, Linda C / Engle, Sandra J / Grossman, Iris / Sehgal, Alfica / Sigova, Alla A / Fremeau, Robert T / Liu, Yuting / Bumcrot, David

Scientific reports

2024 Volume 14, Issue 1, Page(s) 2153

Abstract: Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic ... ...

Abstract	Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.
MeSH term(s)	Humans ; Microglia/metabolism ; Induced Pluripotent Stem Cells ; Pluripotent Stem Cells ; Transcription Factors/metabolism ; Neurodegenerative Diseases/genetics ; Neurodegenerative Diseases/metabolism
Chemical Substances	Transcription Factors
Language	English
Publishing date	2024-01-25
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-024-52311-0
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Article ; Online: Author Correction: Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration.

Scientific reports

2024 Volume 14, Issue 1, Page(s) 9346

Language	English
Publishing date	2024-04-23
Publishing country	England
Document type	Published Erratum
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-024-60234-z
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Article ; Online: Methods to Identify and Characterize siRNAs Targeting Serpin A1 In Vitro and In Vivo Using RNA Interference.

Milstein, Stuart / Qian, Kun / Zhang, Linying / Sehgal, Alfica

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

2017 Volume 1639, Page(s) 115–126

Abstract: RNAi is a powerful tool that can be used to probe gene function as well as for therapeutic intervention. We describe a workflow and methods to identify screen and select potent and specific siRNAs in vitro and in vivo using qPCR-based methods as well as ... ...

Abstract	RNAi is a powerful tool that can be used to probe gene function as well as for therapeutic intervention. We describe a workflow and methods to identify screen and select potent and specific siRNAs in vitro and in vivo using qPCR-based methods as well as an AAT activity assay. We apply these techniques to a set of siRNAs targeting rat AAT, and use this set to exemplify the cell-based and in vivo data that can be generated using these methods.
MeSH term(s)	Animals ; Cell Line ; DNA, Complementary/genetics ; Female ; Gene Knockdown Techniques ; Humans ; Mice ; Molecular Biology/methods ; RNA Interference ; RNA, Small Interfering/metabolism ; Rats ; Reproducibility of Results ; Transfection ; alpha 1-Antitrypsin/genetics ; alpha 1-Antitrypsin/metabolism
Chemical Substances	DNA, Complementary ; RNA, Small Interfering ; alpha 1-Antitrypsin
Language	English
Publishing date	2017-07-20
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-7163-3_11
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Article ; Online: Thrombin generation in plasma of patients with haemophilia A and B with inhibitors: Effects of bypassing agents and antithrombin reduction.

Livnat, Tami / Sehgal, Alfica / Qian, Kun / Van Nguyen, Huy / Madigan, Kate / Sorensen, Benny / Kenet, Gili

Blood cells, molecules & diseases

2020 Volume 82, Page(s) 102416

Abstract: Antithrombin (AT) reduction has been shown to improve thrombin generation (TG) in haemophilia with or without inhibitors. As treatment with bypassing agents (BPAs) may be required in patients with breakthrough bleeding while receiving AT-lowering therapy, ...

Abstract	Antithrombin (AT) reduction has been shown to improve thrombin generation (TG) in haemophilia with or without inhibitors. As treatment with bypassing agents (BPAs) may be required in patients with breakthrough bleeding while receiving AT-lowering therapy, we assessed TG in platelet-poor plasma samples from haemophilia patients in the presence of BPA (recombinant activated factor VII [rFVIIa; 1.25 or 2.5 μg mL
MeSH term(s)	Adolescent ; Adult ; Antithrombins/blood ; Blood Coagulation Factor Inhibitors/blood ; Child ; Child, Preschool ; Hemophilia A/blood ; Hemophilia B/blood ; Humans ; Infant ; Male ; Middle Aged ; Prospective Studies ; Thrombin/metabolism
Chemical Substances	Antithrombins ; Blood Coagulation Factor Inhibitors ; Thrombin (EC 3.4.21.5)
Language	English
Publishing date	2020-02-11
Publishing country	United States
Document type	Clinical Trial ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1237083-6
ISSN	1096-0961 ; 1079-9796
ISSN (online)	1096-0961
ISSN	1079-9796
DOI	10.1016/j.bcmd.2020.102416
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Article ; Online: Progesterone receptor membrane component 1 (PGRMC1) binds and stabilizes cytochromes P450 through a heme-independent mechanism.

McGuire, Meredith R / Mukhopadhyay, Debaditya / Myers, Stephanie L / Mosher, Eric P / Brookheart, Rita T / Kammers, Kai / Sehgal, Alfica / Selen, Ebru S / Wolfgang, Michael J / Bumpus, Namandjé N / Espenshade, Peter J

The Journal of biological chemistry

2021 Volume 297, Issue 5, Page(s) 101316

Abstract: Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, ...

Abstract	Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.
MeSH term(s)	Amino Acid Substitution ; Animals ; Cytochrome P-450 Enzyme System/genetics ; Cytochrome P-450 Enzyme System/metabolism ; Enzyme Stability ; HeLa Cells ; Heme/genetics ; Heme/metabolism ; Humans ; Membrane Proteins/genetics ; Membrane Proteins/metabolism ; Mice ; Mice, Knockout ; Mutation, Missense ; Receptors, Progesterone/genetics ; Receptors, Progesterone/metabolism
Chemical Substances	Membrane Proteins ; PGRMC1 protein, human ; PGRMC1 protein, mouse ; Receptors, Progesterone ; Heme (42VZT0U6YR) ; Cytochrome P-450 Enzyme System (9035-51-2)
Language	English
Publishing date	2021-10-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2021.101316
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Article ; Online: Opportunities and challenges for innovative and equitable healthcare.

Ecker, David J / Aiello, Clarice D / Arron, Joseph R / Bennett, C Frank / Bernard, Amy / Breakefield, Xandra O / Broderick, Timothy J / Callier, Shawneequa L / Canton, Barry / Chen, Janice S / Fishburn, C Simone / Garrett, Banning / Hecht, Sidney M / Janowitz, Tobias / Kliegman, Melinda / Krainer, Adrian / Louis, Chrystal U / Lowe, Christopher / Sehgal, Alfica /

Tozan, Yesim / Tracey, Kevin J / Urnov, Fyodor / Wattendorf, Daniel / Williams, Thomas W / Zhao, Xuanhe / Hayden, Michael R

Nature reviews. Drug discovery

2024 Volume 23, Issue 5, Page(s) 321–322

MeSH term(s)	Humans ; Delivery of Health Care ; Health Equity ; Healthcare Disparities ; COVID-19/epidemiology
Language	English
Publishing date	2024-02-26
Publishing country	England
Document type	Journal Article ; News
ZDB-ID	2062954-0
ISSN	1474-1784 ; 1474-1776
ISSN (online)	1474-1784
ISSN	1474-1776
DOI	10.1038/d41573-024-00032-4
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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: Liver as a target for oligonucleotide therapeutics.

Sehgal, Alfica / Vaishnaw, Akshay / Fitzgerald, Kevin

Journal of hepatology

2013 Volume 59, Issue 6, Page(s) 1354–1359

Abstract: Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be ... ...

Abstract	Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to overcome. To date,hepatic delivery of oligonucleotides has been the area with greatest progress, and thus we have focused on liver-targeted therapeutics that have shown promise at the preclinical and/or clinical level.The liver is the largest internal organ in the body, playing a central role in metabolism, detoxification, synthesis, and secretion of major plasma proteins (carrier proteins, coagulation factors,complement components, hormones, and apolipoproteins),and iron homeostasis. It is therefore not surprising that a large number of disease targets reside in the liver where they are susceptible to modulation by oligonucleotide therapies.
MeSH term(s)	Angiopoietin-like Proteins ; Angiopoietins/physiology ; Cholesterol, LDL/blood ; Humans ; Liver Diseases/drug therapy ; Oligonucleotides/therapeutic use ; Oligonucleotides, Antisense/therapeutic use
Chemical Substances	ANGPTL3 protein, human ; Angiopoietin-like Proteins ; Angiopoietins ; Cholesterol, LDL ; Oligonucleotides ; Oligonucleotides, Antisense
Language	English
Publishing date	2013-12
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	605953-3
ISSN	1600-0641 ; 0168-8278
ISSN (online)	1600-0641
ISSN	0168-8278
DOI	10.1016/j.jhep.2013.05.045
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Article ; Online: Discovery and Targeting of the Signaling Controls of

Schwartz, Brian E / Rajagopal, Vaishnavi / Smith, Cynthia / Cohick, Evan / Whissell, Gavin / Gamboa, Mario / Pai, Rutuja / Sigova, Alla / Grossman, Iris / Bumcrot, David / Sasidharan, Kavitha / Romeo, Stefano / Sehgal, Alfica / Pingitore, Piero

Cells

2020 Volume 9, Issue 10

Abstract: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and ... ...

Abstract	Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (
MeSH term(s)	Animals ; Humans ; Male ; Mice ; Non-alcoholic Fatty Liver Disease/genetics ; Phospholipases A2, Calcium-Independent/metabolism ; Signal Transduction ; Transfection
Chemical Substances	PNPLA3 protein, mouse (EC 3.1.1.3) ; Phospholipases A2, Calcium-Independent (EC 3.1.1.4)
Language	English
Publishing date	2020-10-07
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells9102247
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Article: Oxygen-dependent, alternative promoter controls translation of tco1âº in fission yeast

Sehgal, Alfica / Hughes, Bridget T / Espenshade, Peter J

Nucleic acids research. 2008 Apr., v. 36, no. 6

2008

Abstract: Eukaryotic cells respond to changes in environmental oxygen supply by increasing transcription and subsequent translation of gene products required for adaptation to low oxygen. In fission yeast, the ortholog of mammalian sterol regulatory element ... ...

Abstract	Eukaryotic cells respond to changes in environmental oxygen supply by increasing transcription and subsequent translation of gene products required for adaptation to low oxygen. In fission yeast, the ortholog of mammalian sterol regulatory element binding protein (SREBP), called Sre1, activates low-oxygen gene expression and is essential for anaerobic growth. Previous studies in multiple organisms indicate that SREBP transcription factors function as positive regulators of gene expression by increasing transcription. Here, we describe a unique mechanism by which activation of Sre1-dependent transcription downregulates protein expression under low oxygen. Paradoxically, Sre1 inhibits expression of tco1âº gene product by activating its transcription. Under low oxygen, Sre1 directs transcription of tco1âº from an alternate, upstream promoter and inhibits expression of the normoxic tco1âº transcript. The resulting low-oxygen transcript contains an additional 751 nt in the 5' untranslated region that is predicted to form a stable, complex secondary structure. Interestingly, polysome profile experiments revealed that this new longer transcript is translationally silent, leading to a decrease in Tco1 protein expression under low oxygen. Together, these results describe a new mechanism for oxygen-dependent control of gene expression and provide an example of negative regulation of protein expression by an SREBP homolog.
Keywords	5' untranslated regions ; Schizosaccharomyces pombe ; binding proteins ; eukaryotic cells ; gene expression ; gene expression regulation ; genes ; mammals ; oxygen ; protein synthesis ; sterols ; transcription (genetics) ; transcription factors ; translation (genetics)
Language	English
Dates of publication	2008-04
Size	p. 2024-2031.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkn027
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Article ; Online: Oxygen-dependent, alternative promoter controls translation of tco1+ in fission yeast.

Sehgal, Alfica / Hughes, Bridget T / Espenshade, Peter J

Nucleic acids research

2008 Volume 36, Issue 6, Page(s) 2024–2031

Abstract	Eukaryotic cells respond to changes in environmental oxygen supply by increasing transcription and subsequent translation of gene products required for adaptation to low oxygen. In fission yeast, the ortholog of mammalian sterol regulatory element binding protein (SREBP), called Sre1, activates low-oxygen gene expression and is essential for anaerobic growth. Previous studies in multiple organisms indicate that SREBP transcription factors function as positive regulators of gene expression by increasing transcription. Here, we describe a unique mechanism by which activation of Sre1-dependent transcription downregulates protein expression under low oxygen. Paradoxically, Sre1 inhibits expression of tco1(+) gene product by activating its transcription. Under low oxygen, Sre1 directs transcription of tco1(+) from an alternate, upstream promoter and inhibits expression of the normoxic tco1(+) transcript. The resulting low-oxygen transcript contains an additional 751 nt in the 5' untranslated region that is predicted to form a stable, complex secondary structure. Interestingly, polysome profile experiments revealed that this new longer transcript is translationally silent, leading to a decrease in Tco1 protein expression under low oxygen. Together, these results describe a new mechanism for oxygen-dependent control of gene expression and provide an example of negative regulation of protein expression by an SREBP homolog.
MeSH term(s)	5' Untranslated Regions/chemistry ; Gene Expression Regulation, Fungal ; Nucleic Acid Conformation ; Oxygen/metabolism ; Promoter Regions, Genetic ; Protein Biosynthesis ; Schizosaccharomyces/genetics ; Schizosaccharomyces/metabolism ; Schizosaccharomyces pombe Proteins/biosynthesis ; Schizosaccharomyces pombe Proteins/genetics ; Schizosaccharomyces pombe Proteins/metabolism ; Sterol Regulatory Element Binding Proteins/metabolism
Chemical Substances	5' Untranslated Regions ; Schizosaccharomyces pombe Proteins ; Sre1 protein, S pombe ; Sterol Regulatory Element Binding Proteins ; Oxygen (S88TT14065)
Language	English
Publishing date	2008-02-14
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkn027
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