Approches protéomiques pour l’analyse des exosomes de liquides biologiques pour la recherche de biomarqueurs

Abstract : A biomarker is a molecule (or a cluster of molecule) which will reflect the occurrence of a pathological state, giving us the ability to detect a disease, to predict its severity or to assess drug efficiency. Biological fluids are the golden standards for biomarker research in human as they are routinely collected for patients’ follow-up and are less invasive than biopsies. During my PhD, I focused on exosomes that can be found in these biological fluids. Exosomes are nanovesicles with a diameter ranging between 30 and 100 nanometers. Exosomes are secreted by all cell types and harbor cytoplasmic and membranous proteins specific of their cells of origins. One of the major interest of exosomes enriched from biological fluids is that they represent a valuable source of biomarkers. They can be considered as a « liquid biopsy ». Their analysis could complete classical diagnosis and follow-up tools. In this project, we applied high resolution, high throughput proteomic techniques for exosomes analysis. We firstly focused on protein profiles in urinary exosomes in the context of two urinary tract diseases: cystinuria and kidney cancer. Cystinuria is an inherited autosomal recessive disease that is characterized by the formation of cystine stones in the kidneys. To date, there are no markers to predict the evolution toward end stage renal disease. We developed a method to prepare exosomes in order to reproducibly analyze their protein profiles. We applied this method to eight cystinuria patients and compared their profiles to those of ten healthy subjects. A panel of 38 differentially expressed proteins in patients were found and validated by western blots. We also applied this method to patients with clear cell renal cell carcinoma, for which invasive biopsies are necessary for clear diagnosis. We analyzed urinary exosomes form eight patients before and after nephrectomy. We were able to highlight 25 overexpressed proteins in patients’ exosomes. Eventually, the last part of my thesis was dedicated to the analysis of exosomes enriched from bronchoalveolar lavage fluid collected in cystic fibrosis patients, a disease that affects mostly the lungs. Bronchoalveolar lavage fluid exosomes analysis could give a new insight on the mechanisms of this disease. We compared protein profiles in exosomes from four cystic fibrosis patients and six asthmatic patients. The whole point of this work is to show that proteomic analysis of exosomes isolated from biological fluids could become a golden standard for the discovery of diagnosis or prognosis biomarkers.
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Matthieu Bourderioux. Approches protéomiques pour l’analyse des exosomes de liquides biologiques pour la recherche de biomarqueurs. Physiologie [q-bio.TO]. Université Sorbonne Paris Cité, 2015. Français. ⟨NNT : 2015USPCB102⟩. ⟨tel-01535959⟩

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