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dc.contributor.author
Jordi, Christian Alain
dc.contributor.supervisor
Reddy, Sai
dc.contributor.supervisor
Tay, Savas
dc.contributor.supervisor
Treutlein, Barbara
dc.date.accessioned
2020-07-20T06:41:57Z
dc.date.available
2020-07-19T14:47:03Z
dc.date.available
2020-07-20T06:40:00Z
dc.date.available
2020-07-20T06:41:57Z
dc.date.issued
2020
dc.identifier.uri
http://hdl.handle.net/20.500.11850/426950
dc.identifier.doi
10.3929/ethz-b-000426950
dc.description.abstract
Single cell analysis is a booming research field in biology. Cells are the fundamental building blocks of biological systems, and are therefore key to their understanding. For example it is necessary to study how they communicate with each other; how they organize themselves into larger structures like tissues, or the immune system. Understanding these processes has interesting implications for the study of diseases like cancer or chronic inflammation that can start from the dysregulation of single cells. Due to their small size, single cells cannot be studied using traditional biological methods that rely on the analysis of whole cell populations. Therefore new technologies are necessary. Some of the most promising advances have been made in the field of single cell mRNA sequencing. Through the application of microfluidic devices, it is now possible to sequence the mRNA of thousands of single cells. In comparison to that, the tools to analyse single cell protein content are still comparably limited. Since proteins catalyse most biological reactions, it would be more desirable to study biological systems on the protein level. In this thesis we took advantage of a protein analysis technique called the Proximity Ligation Assay (PLA) to develop new tools for single cell protein analysis. PLA relies on antibodies conjugated to short DNA strands. If two antibodies bind their target protein simultaneously, the short DNA molecules can be joined during a ligation step. This procedure allows to convert the presence of a protein into a DNA signal, and makes it possible to take advantage of highly developed DNA analysis methods like quantitative PCR, and Next Generation Sequencing (NGS). We developed a highly sensitive digital PLA assay that was able to reproducibly measure the protein content of single mammalian cells. This assay was combined with state of the art RT-PCR workflows to measure proteins, and mRNA from the same cell simultaneously. This revealed a very low correlation between mRNA, and protein expression levels in single cells, which stands in contrast to observations on the population level. PLA also has another interesting property. Due to the double recognition of target molecules it is possible to detect whether two proteins are in spatial proximity to each other. Since most proteins perform their function in complexes, this is a potentially very rich source of information. Therefore, we developed a PLA protocol that uses NGS to read out protein signals (PLA-Seq); like this, we were able to simultaneously measure 11 proteins, and their interactions. The new assay was tested using bulk samples, and was able to clearly distinguish two cell lines from each other based on their PLA signals. Since we designed PLA-Seq to be compatible with state of the art, high-throughput single cell RNA-Seq workflows we expect that it will soon be feasible to integrate our PLA-Seq assay in similar fashion, to allow high-throughput measurements of protein expression levels and their interactions.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.rights.uri
http://rightsstatements.org/page/InC-NC/1.0/
dc.title
Development of Proximity Ligation Assay variants for single cell proteomics
en_US
dc.type
Doctoral Thesis
dc.rights.license
In Copyright - Non-Commercial Use Permitted
dc.date.published
2020-07-20
ethz.size
146 p.
en_US
ethz.code.ddc
DDC - DDC::5 - Science::570 - Life sciences
en_US
ethz.identifier.diss
26148
en_US
ethz.publication.place
Zurich
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02060 - Dep. Biosysteme / Dep. of Biosystems Science and Eng.::03952 - Reddy, Sai / Reddy, Sai
en_US
ethz.date.deposited
2020-07-19T14:47:16Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2020-07-20T06:40:12Z
ethz.rosetta.lastUpdated
2021-02-15T15:31:42Z
ethz.rosetta.versionExported
true
ethz.COinS
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