טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentGinsberg Darrell Joseph
SubjectA Novel Method for Detecting Nuclear Localization of
Transcription Factors in Blood Leukocytes
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Peretz Lavie
Dr. Lena Lavie
Full Thesis textFull thesis text - English Version


Abstract

Obstructive Sleep Apnea (OSA) is a highly prevalent disorder that is independently associated with an increased risk of cardiovascular morbidity and mortality.  The hallmark of OSA, chronic intermittent hypoxia (CIH), is thought be the major cause of the increased oxidative stress and inflammatory status of OSA patients that promotes an increased risk of cardiovascular consequences. The redox-sensitive transcription factor Nuclear Factor kB (NFκB), the master regulator of the immune response, is directed to the nucleus subsequent to its activation. Thus, nuclear localization of NFκB is correlated with its activation. The purpose of this study was to validate a novel flow cytometry-based method that analyzes isolated nuclei from leukocytes for the determination of the relative change in the nuclear localization of transcription factors, specifically NFκB. This study details the development and validation of a flow cytometry based method. Dose and time kinetic experiments with activators and inhibitors of NFκB demonstrated the methods’ sensitivity and specificity. In addition, as a secondary validation of NFκB activation, the NFκB-directed cytokine Interleukin-8 (IL-8) was measured by flow cytometry in human polymorphonuclear cells (PMNs). Subsequent to method validation, nuclear translocation of NFκB in human PMNs isolated from healthy non-OSA human controls subjected to a 6 hour treatment of intermittent or sustained hypoxia using a computer controlled hypoxia system is measured. The results show a significantly increased NFκB nuclear localization in PMNs exposed to a 6 hour treatment of intermittent or sustained hypoxia. However, there was no significant difference between the PMNs subjected to intermittent and sustained hypoxia. These results further support the evidence that intermittent hypoxia, induces an increased inflammatory state which can lead to negative cardiovascular consequences. In conclusion, this validated flow cytometry method is a practical and cost effective tool to measure the relative nuclear localization of transcription factors as a marker of their activation. It is suggested that this method be employed to measure the activation of other transcription factors possibly associated with OSA, simultaneously, using in-vitro models of intermittent hypoxia as well as in patients with OSA.