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Analyses of CSF reveal decreased levels of four proteins in ALS patients, but no changes upon analgesia in patients with neuropathic pain
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
University of Cluj Napoca, Cluj Napoca, Romania.
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-220139OAI: oai:DiVA.org:uu-220139DiVA: diva2:704633
Available from: 2014-03-12 Created: 2014-03-11 Last updated: 2014-04-29
In thesis
1. Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
Open this publication in new window or tab >>Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed.

In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences.

In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA).

In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls.

In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS.

In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 980
Keyword
proximity ligation assay, proximity barcoding assay, sensitive, in situ, multiplex, profiling, amyloid-beta, MARK, tau, CSF, biomarker, protein complexes, Alzheimer’s disease, amyotrophic lateral sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-220070 (URN)978-91-554-8901-4 (ISBN)
Public defence
2014-04-25, B21, Biomedicinskt centrum, Husargatan 3, SE-75123, Uppsala, 13:00 (English)
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Supervisors
Available from: 2014-04-03 Created: 2014-03-10 Last updated: 2014-04-29

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Anaesthesiology and Intensive CareMolecular toolsScience for Life Laboratory, SciLifeLabCancer Pharmacology and Computational MedicineDepartment of Engineering SciencesBiochemial structure and functionDepartment of Chemistry - BMC
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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