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Potential Signaling Partners of Sry-Related HMG Box-C (SOXC)

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Potential Signaling Partners of Sry-Related HMG Box-C (SOXC) Potential Signaling Partners of Sry-Related HMG Box-C (SOXC) Transcription Factors in Renal Development A Thesis Submitted to the Graduate Faculty in Partial Fulfilment of the Requirements for the Degree of Masters of Science Department of Biomedical Sciences Faculty of Veterinary Medicine University of Prince Edward Island Junzhuo Huang Charlottetown, PEI June, 2013 © 2013. Junzhuo Huang "!! CONDITIONS FOR THE USE OF THE THESIS The author has agreed that the Library, University of Prince Edward Island, may make this thesis freely available for inspection. Moreover, the author has agreed that permission for extensive copying of this thesis for scholarly purposes may be granted by the professor or professors who supervised the thesis work recorded herein or, in their absence, by the Chair of the Department or the Dean of the Faculty in which the thesis work was done. It is understood that due recognition will be given to the author of this thesis and to the University of Prince Edward Island in any use of the material in this thesis. Copying or publication or any other use of the thesis for financial gain without approval by the University of Prince Edward Island and the author’s written permission is prohibited. Requests for permission to copy or to make any other use of material in this thesis in whole or in part should be addressed to: Chair of the Department of Biomedical Sciences Faculty of Veterinary Medicine University of Prince Edward Island Charlottetown, P. E. I. Canada C1A 4P3 ""!! PERMISSION TO USE GRADUATE THESIS Title of Thesis: “Potential Signaling Partners of the Sry-Related HMG Box-C (SOXC) Transcription Factors in Renal Development” Name of Author: Junzhuo Huang Department: Biomedical Sciences Degree: Master of Science Year: 2013 Name of Supervisor(s): Drs. Sunny Hartwig and Glenda Wright In presenting this thesis in partial fulfilment of the requirements for a graduate degree from the University of Prince Edward Island, the author has agreed that the Robertson Library, University of Prince Edward Island, may make this thesis freely available for inspection and gives permission to add an electronic version of the thesis to the Digital Repository at the University of Prince Edward Island. Moreover the author further agrees that permission for extensive copying of this thesis for scholarly purposes may be granted by the professor or professors who supervised the author’s thesis work, or, in their absence, by the Chair of the Department or the Dean of the Faculty in which the author’s thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without the author’s written permission. It is also understood that due recognition shall be given to the author and to the University of Prince Edward Island in any scholarly use which may be made of any material in the author’s thesis. Signature: ___________________________ Address: Departmert of Biomedical Sciences Atlantic Veterinary College University of Prince Edward Island University Avenue, 550 Charlottetown, PE C1A 4P3 CANADA. Date: _______________________________ """! ! University of Prince Edward Island Faculty of Veterinary Medicine Charlottetown CERTIFICATION OF THESIS WORK We, the undersigned, certify that Junzhuo Huang, candidate for the degree of Master of Science, has presented his thesis with the following title: Potential Signaling Partners of the Sry-Related HMG Box-C (SOXC) Transcription Factors in Renal Development that the thesis is acceptable in form and content, and that a satisfactory knowledge of the field covered by the thesis was demonstrated by the candidate through an oral examination held on June 10, 2013. Examiners: Dr. Luis Bate: _______________________________________ Dr. Sunny Hartwig: _______________________________________ Dr. Sandra McConkey: _______________________________________ Dr. Collins Kamunde: _______________________________________ Dr. Karen Samis: _______________________________________ Date: _________________ "#! ! TABLE OF CONTENTS Title page i Conditions of use ii Permission of use iii Certification of thesis work iv Table of contents v Abstract vii Acknowledgements viii Dedication ix List of figures x List of tables xi List of abbreviations xii Gene and protein nomenclature xv 1.! INTRODUCTION!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!#! 1.1! Kidneys!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%! 1.1.1!Kidney structure and functions!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%! 1.2! Kidney development!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!&! 1.2.1!Mouse metanephric kidney development!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!&! 1.2.2!Pronephros!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!'! 1.2.3!Mesonephros!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!'! 1.2.4!Metanephros!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!(! 1.2.5!Ureteric bud branching!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%&! 1.3! Wilms’ Tumor Suppressor 1(WT1)!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%)! 1.3.1!Kidney diseases!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%)! 1.3.2!Wilms’ Tumor!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%'! 1.3.3!WT1!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!%(! 1.4! SOX transcription factors!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!*%! 1.4.1!SOX family!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!*%! 1.4.2!SOXC subfamily: SOX4, SOX11 and SOX12!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!*&! 1.5! Protein partners of SOXC transcription factors!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!*+! 1.5.1!SOX protein partners!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!*+! 1.5.2!SOXC protein partners!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!*(! 1.6! Research objectives!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,-! 1.6.1!Rationale!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,-! 1.6.2!Hypotheses!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,%! 2.! MATERIALS AND METHODS!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!$%! 2.1! Tissue isolation!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,*! #!! 2.2! RNA isolation and reverse transcription!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,*! 2.3! Primer design and Reverse Transcription (RT)-PCR!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,.! 2.4! Real time PCR!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!,(! 2.5! Analysis!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!+%! 3.! RESULTS!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!&$! 3.1! List of potential SOXC protein partners!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!+,! 3.2! RT-PCR!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!+.! 3.3! Realtime PCR!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!)-! 4.! DISCUSSION!"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!'(! 4.1! The shortlist of SOXC candidate partners!$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$!)(!
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