Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (p. 158-169).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 91-100).

published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Physiology / Master / Master of Philosophy

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Σκοπός της συγκεκριμένης ερευνητικής εργασίας ήταν η αποσαφήνιση του ρόλου της Κυκλοσπορίνης στην πρόκληση νεφρικής ίνωσης, διαμέσου του συστήματος ΕΤ-1/ΝΟ. Για το λόγο αυτό πραγματοποιήθηκαν in vitro πειράματα, στα οποία χρησιμοποιήθηκαν ΗΚ-2 κύτταρα και διερευνήθηκε η επίδραση της κυκλοσπορίνης στην βιωσιμότητα των κυττάρων, στην παραγωγή ΝΟ, στην συσσώρευση των mRNA των et-1, etr-a, etr-b, enos και inos καθώς και τη συσσώρευση των αντίστοιχων πρωτεϊνών τους. Προέκυψε ότι η κυκλοσπορίνη τροποποιεί την έκφραση των γονιδίων et-1 και των υποδοχέων της etr-a και etr-b, των συνθετασών του ΝΟ, enos και inos, τη συσσώρευση των αντίστοιχων πρωτεϊνών, καθώς και του παραγόμενου NO. Επίσης, η αλβουμίνη δεν επηρεάζει τη συσσώρευση της ακτίνης, παρουσιάζει ωστόσο κυτταροτοξική δράση. / The aim of this research was the elucidation of the role of Cyclosporine in the induction of renal fibrosis, through the activation of the ET-1/NO system. We performed in vitro experiments using HK-2 cells (human proximal tubular epithelial cells) and we studied the impact of Cyclosporine on cell viability production of NO, expression of et-1, etr-a, etr-b, enos and inos and the accumulation of the subsequent proteins, as well as actin. Cyclosporine was found to alter the expression of et-1, etr-a, etr-b and enos and the accumulation of the subsequent proteins. Moreover, it affects the amount of the synthesized and secreted NO, while having no effect on the accumulation of actin.

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Body fluids have gained widespread importance for proteomics based biomarker discovery as they have proved to reveal many candidate biomarkers for a variety of physiological diseases. Urine is a particularly favourable body fluid when profiling diseases associated with proximal tissues (kidney, urinary tract, etc.). The collection of urine is also non-invasive compared to other fluids such as blood, plasma and amniotic or cerebral spinal fluids. The main objective of this work was to determine an optimal protocol for profiling of the urine proteome via peptide mass sequencing. Subsequently, the urine proteome was characterized as a potential source for protein biomarker(s) related to kidney obstruction. Of particular relevance to a quantitative investigation, it was found that the conventional urinary proteome workflow inadvertently introduces a sampling bias. Demonstrated here is the fact that the sediment proteins of urine, which are typically discarded prior to analysis, contain important protein constituents which were previously reported in the literature as candidate biomarkers. A solution-based intact protein separation workflow was demonstrated for the analysis of urinary proteins. The mass-based separation platform, namely gel-eluted liquid fractionation entrapment electrophoresis (GELFrEE), incorporates the use of sodium dodecyl sulphate (SDS). As a known signal suppressant in mass spectrometry (MS), the MS tolerance of SDS in a proteome workflow was determined. Simple and effective protocols for the isolation of proteins from SDS-containing solutions are presented. Also presented is a comparison between GELFrEE and the conventional „GeLC? protocol for SDS-based protein separation, with similar numbers of proteins identified by the two platforms. Lastly, a novel strategy for isotope labelling of proteins at the intact level is presented. This intact labelling of proteins is therefore compatible with intact proteome prefractionation, as seen with GELFrEE-MS2 separation. This quantitative workflow was applied to biomarker profiling from urine samples obtained for a model (rodent) kidney obstruction. / A PhD thesis discussing optimal approaches for urine proteomics, a new strategy for separation and labelling of intact proteins for the purpose of protein quantitation and some possible biomarkers for kidney obstructions in a rat model.