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Compaction of rolling circle amplification products increases signal strength and integrity
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction.
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-217748OAI: oai:DiVA.org:uu-217748DiVA: diva2:693538
Available from: 2014-02-04 Created: 2014-02-04 Last updated: 2014-03-21Bibliographically approved
In thesis
1. Making Visible the Proximity Between Proteins
Open this publication in new window or tab >>Making Visible the Proximity Between Proteins
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.

In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment.  

In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.

The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.

The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 973
Keyword
Proximity ligation assay, In situ PLA, rolling circle amplification, protein interaction, protein-protein interaction, in situ, single cell, single molecule, protein complex, antibody, cancer, tissue section, microscopy, image analysis, system biology, multiplex, dynamic range, methods development, systems biology
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology Biochemistry and Molecular Biology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-217772 (URN)978-91-554-8878-9 (ISBN)
Public defence
2014-03-28, B8, Biomedicinskt centrum, Husargatan 3, SE-75123 Uppsala, 09:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 259796EU, FP7, Seventh Framework Programme, 278568
Available from: 2014-03-06 Created: 2014-02-04 Last updated: 2014-04-29Bibliographically approved

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Clausson, Carl-MagnusSöderberg, OlaArngården, LindaIshaq, OmerWählby, CarolinaNilsson, MatsKrzywkowski, Tomasz

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Clausson, Carl-MagnusSöderberg, OlaArngården, LindaIshaq, OmerWählby, CarolinaNilsson, MatsKrzywkowski, Tomasz
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Science for Life Laboratory, SciLifeLabDepartment of Immunology, Genetics and PathologyDivision of Visual Information and Interaction
Medical Biotechnology

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