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Enhanced in situ proximity ligation assays via Unfolding PLA probes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Söderberg)ORCID iD: 0000-0002-1053-5856
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Söderberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Söderberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Landegren)
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-248874OAI: oai:DiVA.org:uu-248874DiVA: diva2:801276
Funder
Swedish Foundation for Strategic Research EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 264737
Available from: 2015-04-08 Created: 2015-04-08 Last updated: 2017-05-04
In thesis
1. Improvements and Applications of in situ Proximity Ligation Assays
Open this publication in new window or tab >>Improvements and Applications of in situ Proximity Ligation Assays
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cells building up the human body is in constant communication with each other. This communication is done through large complex networks of signaling pathways for inter- and intracellular signal transduction. The signaling activity regulates many important processes, for example cell death, proliferation and differentiation. Information within the signaling networks is communicated over the cell membrane, through the cytoplasm and entering the nucleus by protein activities such as protein-protein interactions (PPIs) and post translation modifications (PTMs). The cells adapts to their own environment, responding to multiple stimuli from their surroundings. This in combination with memory of previous responses, difference in cell cycles stages and sometimes altered genetic background generates heterogeneous cell populations in which every cell is slightly different from its neighbor. This calls for methods to study the activity of endogenous proteins in individual cells within a population.

In situ proximity ligation assay (in situ PLA) was originally developed to visualize interaction between endogenous proteins in fixed cells and tissue and can also be applied to detect PTMs. This thesis describe the application of in situ PLA to study PPIs in signaling pathways and the work to further develop and improve techniques for proximity dependent detection. 

In paper I in situ PLA is used to study cross talk between the Hippo and the TGFβ signaling pathways. The study shows the complex formation by the transcription co-factors of the Hippo pathway, Yap and Taz, and the main effectors of the TGFβ pathway Smad2/3. Furthermore the density dependent localization of the interaction is described.

Paper II presents a new version of the in situ PLA probes for simultaneous detection of multiple complexes. Visualization of various complexes involving EGFR, Her2 and Her3 is presented as a proof of concept.

The efficiency of in situ PLA is limited by several factors, one being the design of PLA probes and oligonucleotide systems. Even upon proximal binding of the probes there is a risk of formation of non-circular ligation products, which cannot be amplified and detected. In Paper III two new PLA probes are presented aiming to reduce the formation of non-circular ligation product and hence increase the detection efficiency of in situ PLA.

Paper IV presents a new method for detection of protein complexes and phosphorylation; proxHCR. ProxHCR combines signal amplification by enzyme free hybridization chain reaction (HCR) with the requirement of proximal binding of two affinity probes. As a proof of principle the method is used to detect multiple complexes and protein phosphorylation in fixed cells and tissue.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1099
National Category
Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biomedical Laboratory Science/Technology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-248876 (URN)978-91-554-9233-5 (ISBN)
Public defence
2015-05-29, Biomedicinskt centrum (BMC), B/B42, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research
Available from: 2015-05-07 Created: 2015-04-08 Last updated: 2015-07-07
2. Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis
Open this publication in new window or tab >>Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine.  Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment.  In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection.

In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA.

In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA).  We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer.

In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold.

In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 83 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1336
Keyword
protein detection, proximity ligation assays, proximity extension assay, rolling circle amplification, ELISA, flow cytometry, fluorescence microscopy
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-320380 (URN)978-91-554-9930-3 (ISBN)
Public defence
2017-06-14, B/B42, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2017-05-22 Created: 2017-04-25 Last updated: 2017-06-08

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