WWW.DISSERTATION.XLIBX.INFO
FREE ELECTRONIC LIBRARY - Dissertations, online materials
 
<< HOME
CONTACTS



Pages:   || 2 | 3 | 4 | 5 |   ...   | 22 |

«by BLAKE R. ZELICKSON VICTOR M. DARLEY-USMAR, COMMITTEE CHAIR JOHN C. CHATHAM YABING CHEN LOUIS J. DELL’ITALIA AIMEE LANDAR C. ROGER WHITE A ...»

-- [ Page 1 ] --

ASSESSMENT OF MITOCHONDRIAL STRESSORS ON CELLULAR

BIOENERGETICS

by

BLAKE R. ZELICKSON

VICTOR M. DARLEY-USMAR, COMMITTEE CHAIR

JOHN C. CHATHAM

YABING CHEN

LOUIS J. DELL’ITALIA

AIMEE LANDAR

C. ROGER WHITE

A DISSERTATION

Submitted to the graduate faculty of The University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Doctor of Philosophy

BIRMINGHAM, ALABAMA

2011

ASSESSMENT OF MITOCHONDRIAL STRESSORS ON

CELLULAR BIOENERGETICS

BLAKE R. ZELICKSON

MOLECULAR AND CELLULAR PATHOLOGY

ABSTRACT

The mitochondrion plays a central role in the maintenance of bioenergetic func- tion through the production of ATP and essential metabolites. The development of mi- tochondrial bioenergetic defects is a hallmark of important pathologies such as cardi- ovascular and liver diseases. It is well established that a decrease in mitochondrial func- tion, typically of 20-40%, is associated with the progression of these pathologies. Causal relationships have been more difficult to establish because of the challenge of assessing mitochondrial function in a cellular setting. Specifically, it is known that mitochondria function at less than their maximal respiratory capacity and the remainder, known as re- serve or spare capacity, is thought to be utilized for increased work or combating oxida- tive stress. The following questions have been addressed in this dissertation: 1) Does re- serve capacity change under conditions of diminished oxygen availability?, 2) Do the variations in mitochondrial phenotype caused by differences in the mitochondrial DNA sequence amongst different populations modify the response to a pathological stress?, and 3) Does the metabolism of alcohol in the liver interact with the mitochondria to change reserve capacity and the response to nitric oxide and hypoxia? In testing these concepts, we have used in vivo models of alcoholic liver disease and cardiac volume overload and an ex vivo model of vascular ischemia/reperfusion. Taken together, our da- ii    ta support the concept that mitochondrial bioenergetics are a key determinant of the re- sponse of a wide variety of cells to pathological stressors.

Keywords: Mitochondrial Function, Extracellular Flux, 4-Hydroxynonenal, Nitric Oxide, Hypoxia, Reserve Capacity iii 

–  –  –

The completion of this dissertation and the research within would not have been possible without the help of several people throughout my tenure in graduate school.

First and foremost, I would like to thank my mentor, Victor Darley-Usmar. In the four and a half years I spent in your lab, you helped me to grow not only as a scientist, but also as a person. You always took the time to figure out the best way to push me to succeed, and I am truly grateful for all of the support, encouragement, patience, and even the “bollockings” you have given me over the years. I can only hope to one day be as good of a mentor to someone else as you have been to me.

To my “lab family,” both past and present, thank you so much for everything that you’ve done for me over the years. To Michelle Johnson, my “lab mom,” your proficiency in all things science is always impressive and beyond compare. You always seem to know how to do every possible kind of assay, as well as the secrets of how to make them work. But more importantly, you were always willing to take the time out of your absurdly busy day to share your expertise with me. To Gloria Benavides, my “lab wife,” I have thoroughly enjoyed working and traveling with you over the past few years. We made an excellent team, and I can’t think of anyone with whom I would have rather spent all those countless hours running up to eight Seahorse experiments per day. You helped me to become more patient, understanding, and an all-around better person, and for that I

–  –  –

in-crime for the past four and a half years. You taught me all the basics of bench-work and were always willing to discuss my data and give me advice, even when it meant that you would have to stay late as a result. We have been through a lot together, and I will definitely miss all of our chats and life-coaching sessions. To Sam Giordano, my “little lab sister,” I enjoyed acting as your pseudo-mentor while you were rotating through our lab, and maybe even a little bit afterwards as well. To Balu Chacko, I have really enjoyed all of our scientific discussions. You know so much about so many different things, and always seem to have a new and interesting perspective about what different results could mean. To Brad Hill, I loved working with you during your short time at UAB. I learned so much about how to be successful in science from you, and your never-ending energy and enthusiasm continue to amaze me. To Joo Yeun Oh, the Korean ninja, you were a constant source of entertainment in the lab. In addition, you are probably one of the most selfless and giving people I have ever met, and I will always be in awe of all that you do. To Mi Jung Chang, never in my entire life have I witnessed anyone do as much work as you managed to do on a daily basis. I will miss working with you and your help with my Korean, even if I only know two phrases. To Sarah Owusu, I really enjoyed working with you for the year that you were here. We have so much in common, and it was really great to have a partner in the avoidance of all things unkosher.





Thanks also to Colin Reily, Philip Kramer, Elena Ulasova, Yaozu Ye, Brian Dranka, and Jessica Perez for helping to make it so that I never dreaded going to work, even when I knew it would be a really long day. I have enjoyed working with you all.

–  –  –

You have more enthusiasm for science than I have ever seen before, which inspires me to be more enthusiastic and interested as well. I have always been impressed by how you manage to juggle being a clinician and a researcher, and I hope to be able to achieve even a percentage of the success that you have in your career. I would also like to thank several members of the Dell’Italia lab for all of their support throughout all of our collaborations. To Doug Gladden, thanks for always being willing to isolate myocytes whenever they were needed, even if you sometimes decided that they didn’t need to be counted.

Working with you has been interesting and fun to say the least, and I will miss all of our random conversations and your unique perspective on things. To Eddie Bradley, rarely have I seen such impressive skills as I have witnessed observing you perform various surgeries or procedures on mice and rats. To Pam Powell, thanks for always being willing to perform immunohistochemistry for me, even when I came to you with ideas that could have potentially kept you busy for months at a time.

To Aimee Landar, thank you for always being willing to share your expertise with me whenever I needed it. I don’t know anyone who can troubleshoot a Western blot as well as you can. Thank you also to the members of the Landar lab, both past and present, for all of their support over the years. To Stephanie Wall, I have enjoyed interacting with you whenever I got the chance. Trying to get you to be just a little bit less nice was always a great source of entertainment for me. To Fen Zhou, thank you so much for ordering antibodies for me so quickly when I decided last minute that I needed to probe for just one more thing. Thanks also to Anne Diers and Praveen Vayalil, my fellow investigators into bioenergetics in hypoxia.

–  –  –

the years. Between scheduling meetings, getting reimbursements, figuring out what exactly the training grant would pay for, and helping me track down Victor, you made my time in graduate school go so much more smoothly than it ever could have without you.

To Nicole Newton, you were always an incredibly valuable source of information.

Thank you so much for always having answers to my one million questions, particularly as I was preparing to graduate. To Rakesh Patel, thank you for all of your advice over the years. You always knew what my best course of action was, and probably helped me avoid a lot of grief in the process. To my committee, John Chatham, Yabing Chen, Lou Dell’Italia, Aimee Landar, and Roger White, thank you so much for your ideas regarding my research and for always pushing me to look for additional explanations for my data.

Last, but certainly not least, I would like to thank my family for everything that they have done for me throughout my entire life. To my parents, thank you so much for always supporting and believing in me, even when it was not deserved. You are the smartest and hardest working people I know, and I can only hope to live up to the example that you have set for me. To my grandparents, thank you for all the faith you have had in me. You make me strive to succeed in everything that I do in the hopes that I can one day deserve how proud you always are of me. To my brothers, aunts, uncles, and cousins, thank you as well for your encouragement over the years. I could never have made it this far without your constant support.

“Truly great friends are hard to find, difficult to leave, and impossible to forget.”

–  –  –

Abstract

DEDICATION

ACKNOWLEDGEMENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER 1 INTRODUCTION

Role of the Mitochondrion in Physiology

Bioenergetics

Mitochondrial ROS Production

Importance of mtDNA Haplotype

Role of the Mitochondrion in Pathology

Bioenergetic Dysfunction

Diseases Associated with Mitochondrial Dysfunction

Alcoholic Liver Disease

Ischemia/Reperfusion in the Vasculature

Cardiac Volume Overload

Summary

–  –  –

Introduction

Role of Nitric Oxide in Alcohol-Induced Hepatotoxicity

Chronic Alcohol Consumption Causes Mitochondrial Dysfuntcion

Materials and Methods

Materials

Alcohol Feeding

Hepatocyte Preparations

Mitochondrial Enzyme Activity Assays

Immunoblot Analysis

Mitochondrial Bioenergetics

Hypoxia Exposure

Immunohistochemistry

Statistics

Results

Hepatocyte Density for Measurement of Mitochondrial Bioenergetics

Alcohol Induces Characteristics of Liver Toxicity in Hepatocytes

Hepatocytes Exhibit Bioenergetic Defect in Response to Alcohol Exposure

Chronic Alcohol Increases Hepatocyte Sensitivity to Nitric Oxide

Alcohol Exacerbates Bioenergetic Alterations Induced by Hypoxia and Nitric Oxide

Alcohol-Induced Hypoxia in the Liver in iNOS-Dependent...............59 Discussion

3 HYPOXIA/REOXYGENATION ALTERS THE RESPONSE OF

CELLULAR BIOENERGETICS TO 4-HYDROXYNONENAL

IN ENDOTHELIAL CELLS

Introduction

–  –  –

Materials and Methods

Materials

Cell Culture

Measurement of Oxygen Consumption in Endothelial Cells..............77 Mitochondrial Function Assay

Exposure to Hypoxia and Reoxygenation

Immunoblot Analysis

Statistics

Results

Oxygen Dependence of Endothelial Cell Mitochondrial Function

Reactive Lipid-Induced Alterations to Endothelial Cell Bioenergetics

Oxygen-Dependence of Endothelial Cell Bioenergetic Alterations Induced by HNE

Exacerbation of Reoxygenation-Induced Loss of Mitochondrial Function by HNE

Discussion

4 MITOCHONDRIAL HAPLOTYPE DETERMINES SUSCEPTIBILITY

TO CARDIOMYOCYTE DYSFUNCTION RESULTING FROM

CARDIAC VOLUME OVERLOAD

Introduction

Effects of Acute Volume Overload on the Cardiomyocyte Mitochondrion

Elucidating Role of mtDNA Haplotype on Susceptibility to Volume Overload

Materials and Methods

Reagents

Generation of Mitochondria:Nuclear Exchange (MNX) Mice

Aortocaval Fistula Surgery

Cardiomyocyte Isolation

xi    Mitochondrial Enzyme Activities

Immunoblot Analysis

Cellular Bioenergetics

Echocardiography and Hemodynamic Measurements

Immunohistochemistry

Transmission Electron Microscopy

Statistics

Results

Comparison of C57BL/6 and C3H/HeN Mitochondrial Phenotypes

Bioenergetic Response of C57BL/6 and C3H/HeN Mice to Volume Overload

Morphometry and Left Ventricular Function of Mice after Volume Overload

Volume Overload-Induced Alterations in Bioenergetic Function

Alterations in Desmin Organization in Cardiomyocytes after Volume Overload

Effect of Volume Overload on Mitochondrial Morphology..............136 Discussion

5 DISCUSSION

Introduction

Reserve Capacity and the Cellular Response to Pathological Stresses

Alcohol-Induces Loss of Mitochondrial Function

Role of Nitric Oxide in Alcohol-Dependent Hepatotoxicity.............149 Alcohol Consumption Alters Hepatocellular Response to Hypoxia

Hypoxic Signaling as a Mediator of Alcohol-Induced Bioenergetic Alterations

Effect of Hypoxia/Reoxygenation on Bioenergetic Function

Endothelial Cell Mitochondrial Function is Altered in Hypoxia

Reoxygenation Induces Progressive Loss of

–  –  –

Susceptibility to Bioenergetic Alterations is Mediated by mtDNA Haplotype

Bioenergetic Responses to Volume Overload in C57BL/6 and C3H/HeN Mice

Effect of mtDNA on Changes in Morphometry and Function by Volume Overload

Volume Overload-Induced Alterations in Mitochondrial Function in MNX Mice

Loss of Desmin Mediates Bioenergetic Alterations Induced by Volume Overload

Volume Overload-Induced Changes in Cardiomyocyte Morphology



Pages:   || 2 | 3 | 4 | 5 |   ...   | 22 |


Similar works:

«OREGON STATE UN VERS TY LIBRARIES 1111 1111 11 11111 1111 12 0003896558 Tillamook Prehistory and Its Relation to the Northwest Coast Culture Area by Thomas M. Newman /-iPARTMENT OF ANTHROPfottLIBRARY, TILLAMOOK PREHISTORY AND ITS RELATION TO THE NORTHWEST COAST CULTURE AREA by THOMAS M. NEWMAN A THESIS Presented to the Department of Anthropology and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1959...»

«Loughborough University Institutional Repository Manufacturing systems interoperability in dynamic change environments This item was submitted to Loughborough University's Institutional Repository by the/an author.Additional Information: • A Doctoral Thesis. Submitted in partial fullment of the requirements for the award of Doctor of Philosophy of Loughborough University. https://dspace.lboro.ac.uk/2134/13174 Metadata Record: c Neil Hastilow Publisher: Please cite the published version. This...»

«EXPERIMENTAL CHARACTERIZATION AND MULTIDISCIPLINARY CONCEPTUAL DESIGN OPTIMIZATION OF A BENDABLE LOAD STIFFENED UNMANNED AIR VEHICLE WING By VIJAY NARAYAN JAGDALE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1 © 2010 Vijay Narayan Jagdale 2 To my idol and inspiring father, Late Narayan L Jagdale; my caring mother, Kaushalya N. Jagdale; my loving wife,...»

«c 2012 Vijay Raman TRAFFIC-AWARE CHANNEL ALLOCATION AND ROUTING IN MULTICHANNEL, MULTI-RADIO WIRELESS NETWORKS BY VIJAY RAMAN DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Electrical and Computer Engineering in the Graduate College of the University of Illinois at Urbana-Champaign, 2012 Urbana, Illinois Doctoral Committee: Professor Nitin Vaidya, Chair Assistant Professor Matthew Caesar Assistant Professor Sayan Mitra Professor...»

«Making Room: The Geography of Rooming House Regulation in Toronto. by Lisa Marie Freeman A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Geography University of Toronto © Copyright by Lisa Marie Freeman 2013 Making Room: The Geography of Rooming House Regulation in Toronto Lisa Marie Freeman Doctor of Philosophy Department of Geography University of Toronto 2013 Abstract This dissertation addresses the contemporary moment of the...»

«kwb.45-variantology_inhalt_neu:kwb.31-variantology_end_5.0 19.02.10 14:12 Seite 215 david link Scrambling T-R-U-T-H Rotating Letters as a Material Form of Thought “Know that the secrets of God and the objects of His science, the subtle realities and the dense realities, the things of above and the things from below, belong to two categories: there are numbers and there are letters. The secrets of the letters are in the numbers, and the epiphanies of the numbers are in the letters. The numbers...»

«Ideas and considerations for detailed design and naming for Ōtautahi North Western Cluster of Schools A Ngāi Tūāhuriri Perspective An Example of Modern Māori Learning Environments and associated Cultural Identifiers Te Whare Maahunui Tuahiwi Marae Home of Ngāi Tūāhuriri Mana Whenua 1 Wednesday, 14 October 2015 Table of Contents Whāinga / Aim Kaupapa rapunga whakaaro / Philosophy Mana Whenua / Te Ngāi Tūāhuriri Te Ngāi Tūāhuriri/ Mana Whenua Ngāi Tahu Whānui and Te Rūnanga o...»





 
<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.