Development of Microfluidic Platforms for Muscle Strength and Aging Investigations in the Model Organism C. elegans by Md Mizanur Rahman, M.S. A Dissertation In Chemical Engineering Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved Dr. Siva A. Vanapalli Chair of Committee Dr. Jerzy Blawzdziewicz Dr. Mark W. Vaughn Dr. Carla Lacerda Dr. Alex Trindade Mark Sheridan Dean of the Graduate School December, 2016 Copyright 2016, Md Mizanur Rahman Texas Tech University, Md Mizanur Rahman, December 2016 ACKNOWLEDGMENTS I would like to thank my PhD advisor, Dr. Siva A. Vanapalli for his continued support and mentorship over last five years. I would also like to thank Dr. Monica Driscoll, professor of department of molecular biology and biochemistry at Rutgers University and Dr. Nate Szewczyk, associate professor of faculty of medicine & health sciences at university of Nottingham, UK for challenging me with research questions. I acknowledge the great contribution of Dr. Jerzy Blawzdziewicz, professor of department of mechanical engineering for helping me to learn the research strategies. I would also like to thank my parents, my sisters Poly & Lina, and my brother Mamun for their constant encouragement and support throughout my life. I’m thankful to my wife Dilruba for her continued support and sacrifice. Also, thanks to all my friends who encouraged me during the depressing days of my failures in early days of my PhD. ii Texas Tech University, Md Mizanur Rahman, December 2016 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................... ii ABSTRACT ......................................................................................................... vii LIST OF TABLES ............................................................................................... ix LIST OF FIGURES .............................................................................................. x CHAPTER 1 INTRODUCTION: GENERAL OVERVIEW ........................... 1 1.1 Introduction ................................................................................................. 1 1.2 Human mortality and age-related pathologies ............................................ 2 1.3 Theories and mechanism of aging............................................................... 6 1.4 Importance of muscle maintenance for healthy aging ................................ 8 1.5 Caenorhabditis elegans-The celebrated animal model ............................. 10 1.6 C. elegans: a powerful model organism for aging study .......................... 12 1.7 Microfluidics for worm aging & healthspan studies ................................. 14 1.8 References ................................................................................................. 17 CHAPTER 2 LITERATURE REVIEW: AGING, LIFESPAN AND HEALTHSPAN .................................................................................................. 25 2.1 Introduction ............................................................................................... 25 2.2 Functional measures of aging.................................................................... 26 2.2.1 Locomotion ...................................................................................... 27 2.2.1.1 Crawling ................................................................................. 27 2.2.1.2 Swimming .............................................................................. 30 2.2.2 Pharyngeal pumping......................................................................... 34 2.2.3 Reproductive health ......................................................................... 36 2.2.4 Stress response ................................................................................. 38 2.2.4.1 Oxidative Stress ..................................................................... 39 2.2.4.2 Thermal Stress ....................................................................... 40 2.2.5 Electrophysiology ............................................................................ 41 2.2.6 Neuronal morphology and neuro-muscular integrity ....................... 44 2.3 Cellular and molecular markers of aging .................................................. 46 2.3.1 Sarcomere disintegration .................................................................. 47 2.3.2 Mitochondrial fragmentation ........................................................... 49 2.3.3 Lipofuscin accumulation .................................................................. 51 2.4 Key studies on healthspan of C. elegans ................................................... 54 iii Texas Tech University, Md Mizanur Rahman, December 2016 2.4.1 Definition of healthspan ................................................................... 54 2.4.2 Role of muscle health on aging ........................................................ 60 2.5 Micropillar-based force detection-A tool for measuring muscle forces .. 64 2.5.1 Integrated fiber optic device as force sensor .................................... 64 2.5.2 Deformable pillar based device as force sensor ............................... 66 2.5.2.1 SU-8 force sensing pillar arrays ............................................. 66 2.5.2.2 Micropillar-based (PDMS) force measurement system ......... 67 2.6 Problem statement ..................................................................................... 71 2.7 References ................................................................................................. 73 CHAPTER 3 NEMAFLEX: A MICROFLUIDIC TOOL FOR MEASUREMENT OF MAXIMUM MUSCULAR STRENGTH OF C. ELEGANS .................... 81 3.1 Introduction ............................................................................................... 81 3.2 Materials and methods .............................................................................. 82 3.3 Results ....................................................................................................... 85 3.3.1 Basic principle of NemaFlex and strenght analysis ......................... 85 3.3.2 Validity of the force-deflection expression ...................................... 88 3.3.3 Experimental error in the measurement of forces ............................ 89 3.3.4 Influence of arena geometry and animal behavior on C. elegans' strength ................................................................................................................... 92 3.3.5 Design considerations for the micropillar arena .............................. 94 3.3.6 NemaFlex quantitates maximum muscular strength in C. elegans .. 95 3.3.7 Influence of body size on muscular strength in C. elegans.............. 98 3.3.8 Force balance of the pillar forces produced by active bending of C. elegans body ........................................................................................... 101 3.4 Discussion ............................................................................................... 102 3.5 References ............................................................................................... 104 CHAPTER 4 IMAGE-BASED SOFTWARE FOR MEASURING MUSCLE STRENGTH OF C. ELEGANS IN A MICROPILLAR ARRAY ................. 106 4.1 Introduction ............................................................................................. 106 4.2 Image processing algorithm .................................................................... 108 4.2.1 Imaging .......................................................................................... 108 4.2.2 Image pre-processing ..................................................................... 109 4.2.3 Pillar measurements ....................................................................... 110 4.2.4 Verification of pillar tracking algorithm ........................................ 113 4.3 Capability of the software: Parameters measured ................................... 115 4.4 Discussion ............................................................................................... 119 4.5 References ............................................................................................... 120 iv Texas Tech University, Md Mizanur Rahman, December 2016 CHAPTER 5 NEMALIFE: A SIMPLE MICROFLUIDIC DEVICE FOR RECORDING LIFESPAN AND HEALTHSPAN IN C. ELEGANS .......... 121 5.1 Introduction ............................................................................................. 121 5.2 Materials and methods ............................................................................ 126 5.3 Results ..................................................................................................... 129 5.3.1 Removal of progeny and retention of adults .................................. 130 5.3.2 Development of feeding protocol for lifespan study in microfluidic lifespan device ......................................................................................... 132 5.3.3 Influence of arena geometry on animal locomotion and lifespan .. 136 5.3.4 Mutant and RNAi screens in NemaLifes device ............................ 139 5.3.5 Simple control of food delivery in longitudinal dietary restriction assay in Nemalife .................................................................................................. 141 5.4 Characterization of healthspan measures in NemaLife device ............... 144 5.4.1 Pharyngeal pumping of aging