Quantitative quadruplexed single cell c-Jun/ AP-1 analysis and its application in COPD monitoring by a non-invasive means using live cells from sputum
File(s)
Author(s)
Lu, Shao-Ju, Ryan
Type
Thesis
Abstract
In this thesis, I demonstrate the establishment of four c-Jun related assays using microfluidic
antibody capture (MAC) chips which are capable of quantifying the copy numbers of total c-
Jun, two phosphorylated c-Jun at two specific sites and its active heterodimer complex with
single cell sensitivity either in immortalized cancer cell lines or clinical samples of Chronic
obstructive pulmonary disease (COPD).
A bimultiplexed c-Jun assay has also been developed, where a total c-Jun assay is multiplexed alongside either a phosphorylated c-Jun assay or a heterodimer assay. The cellular heterogeneity in the correlation between total c-Jun and two phosphorylated forms and c-Jun heterodimer across six cell lines was explored. In addition, in drug perturbation experiments the bimultiplexed c-Jun assay was capable of reflecting the stimulation of pS63-c-Jun/ c-Jun pathway in response to drugs. In addition, the investigation of c-Jun expression heterogeneity with the effect of cell cycle was deciphered using a fluorescent transfection system. The results suggested that cell cycle could be one of the causes of c-Jun and pS63-c-Jun expression variation.
I demonstrate that the bimultiplexed c-Jun assay can be applied to clinical samples either from excised lung tissue or induced sputum for COPD monitoring. The readouts from the assay are independently capable of distinguishing epithelial cells extracted from the lungs of patients diagnosed with COPD, from those from healthy patients. Moreover, I show that the minimum number of cells required to independently achieve a 99% confidence in diagnosis of COPD with each of these markers is 1 for total c-Jun and 2 for phospho c-Jun.
Lastly, I describe the establishment of an integrated c-Jun assay in a quadruplexed format which provides four c-Jun related readouts simultaneously. The proof-of-concept experiments using this quadruplexed c-Jun assays may provide a detailed insight in c-Jun single-cell signaling and cellular heterogeneity.
antibody capture (MAC) chips which are capable of quantifying the copy numbers of total c-
Jun, two phosphorylated c-Jun at two specific sites and its active heterodimer complex with
single cell sensitivity either in immortalized cancer cell lines or clinical samples of Chronic
obstructive pulmonary disease (COPD).
A bimultiplexed c-Jun assay has also been developed, where a total c-Jun assay is multiplexed alongside either a phosphorylated c-Jun assay or a heterodimer assay. The cellular heterogeneity in the correlation between total c-Jun and two phosphorylated forms and c-Jun heterodimer across six cell lines was explored. In addition, in drug perturbation experiments the bimultiplexed c-Jun assay was capable of reflecting the stimulation of pS63-c-Jun/ c-Jun pathway in response to drugs. In addition, the investigation of c-Jun expression heterogeneity with the effect of cell cycle was deciphered using a fluorescent transfection system. The results suggested that cell cycle could be one of the causes of c-Jun and pS63-c-Jun expression variation.
I demonstrate that the bimultiplexed c-Jun assay can be applied to clinical samples either from excised lung tissue or induced sputum for COPD monitoring. The readouts from the assay are independently capable of distinguishing epithelial cells extracted from the lungs of patients diagnosed with COPD, from those from healthy patients. Moreover, I show that the minimum number of cells required to independently achieve a 99% confidence in diagnosis of COPD with each of these markers is 1 for total c-Jun and 2 for phospho c-Jun.
Lastly, I describe the establishment of an integrated c-Jun assay in a quadruplexed format which provides four c-Jun related readouts simultaneously. The proof-of-concept experiments using this quadruplexed c-Jun assays may provide a detailed insight in c-Jun single-cell signaling and cellular heterogeneity.
Version
Open Access
Date Issued
2018-04
Date Awarded
2019-08
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Keith, Willison Willison
Tate, Ed
Klug, David
Sponsor
Ministry of Education Republic of China (Taiwan)
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)