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Caenorhabditis Elegans Canterbury Christ Church University’s repository of research outputs http://create.canterbury.ac.uk Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. Stastna, J. (2016) Natural Variation in lifespan and stress responses in Caenorhabditis elegans. Ph.D. thesis, Canterbury Christ Church University. Contact: [email protected] Natural Variation in Lifespan and Stress Responses in Caenorhabditis elegans by Jana J Stastna Canterbury Christ Church University Thesis submitted for the Degree of Doctor of Philosophy 2016 I II Acknowledgements This work would not have been possible without the help of a great number of people. First and foremost, I would like to thank my supervisor, Simon Harvey for his extreme patience, helpful guidance and stimulating discussions and in assisting me with my PhD project and for allowing me to develop into a real scientist! Thanks to Jan Kammenga and L. Basten Snoek from Wageningen University, Netherlands for the opportunity to work with brand new panel of 4- parental recombinant inbred lines as well as N2/CB4856 RILS and nearly isogenic lines and CB4856/N2 RILs. It has been a real privilege to work alongside the team from Wageningen and I am very grateful to Basten for his assistance with the 4-parental RIL mapping. Thanks to Alex Vujakovic for his invaluable assistance with the gruelling experiments without any pay. I am grateful to Marie McConnell and Yiru Wang for useful discussions and all the help I have received over the years. I would like to thank to all the academic and support staff for their support, advice and encouragement. I would also like to thank Lee Byrne and Peter Vujakovic on my supervisory panel as well as Katie Fowler and Alison Woollard for agreeing to sit on my examination panel. Thanks to Canterbury Christ Church University for funding of my PhD. Very special thanks to my partner Matthew Springham for the initial encouragement to pursue a career in science and for all the support and patience through out the years. Finally, I would like to thank to my family, especially my mum for her long distance support and encouragement, and to all my friends who had to put up with loads of worm talk over the years. III IV Table of Contents: Acknowledgements………………………………………………………….………………………………………………III Table of Contents……………………………..…………………………………………….…………………………………V List of Abbreviations…………………………………….…………….…………………………………………………….IX List of Figures…………………………………………………………………….……….………………………………………X List of Tables………………………………………….………………………………………………..………………………..XII Abstract………………………………………..……………………………………………………………………..……………XIII 1. CHAPTER ONE Introduction 1.1. Ageing and lifespan in human population………………………..……..…….…….……….. 1.2. Stress and dietary restriction……………………………………………….…….……..………….. 1.3. Mechanism(s) of DR………………………………………………………….…….…..………..……… 1.4. DR not working in model organisms………………………………….…….……....…………… 1.5. DR not working in non-model organisms………………………….…….……..….……….. 1.6. Timing of food restriction…………………………………………………….…….……..……….. 1.7. Genetic background………………………………………………………….…….……...…………..15 1.8. Summary of DR…………………………………………………………………….…….………..……..17 1.9. Ageing and lifespan in model organisms……………….………….…….…………..………18 1.10. Model organisms……………………………………………………………….…….……..…………..20 1.11. Caenorhabditis elegans as a model organism………..…………….…….…………..……24 1.11.1. History of C. elegans…………………………………………………….…….…...………….25 1.11.2. Ecology of C. elegans………………………………………………….…….………..……….27 1.12. Quantitative genetics……………………………………………………..…………….…….……...31 1.13. Ageing pathways…………………………………………………….…….…………….…….…….…34 1.14. Aims of the Thesis………………………………………….……………………….……..………….. V 2. CHAPTER TWO Natural variation in the effects of dietary restriction on lifespan in Caenorhabditis elegans 2.1. Introduction……………………………………………………………………………….……...……………… 2.2. Methods………………………………..……………………………………………………….……...………….. 2.2.1. Wors…………………………….………………………………..……………………….…….…………. 2.2.2. Lifespa assays……………………………………………………………………..……….…….……… 2.2.3. Materal hatchig…………………………………………….……………………….…….…..…….. 2.2.4. Data aalysis………………………………………………………………..………….…….……………. 2.3. Results………………………………………………………………………………..….……….…….…………… 2.4. Discussion……………………………………………………………..………………….…….……..…………… 3. CHAPTER THREE QTL Mapping in Multi-Parent Recombinant Inbred Lines (RILs) 3.1. Introduction………………………………………………………………………………….…….…..……….… 3.2. Methods…………………………………………………………………………………….…….…..…………… 3.2.1. Wors………………………………………………………..……………………….…….……………….. 3.2.2. Lifespan assays…………………………………………………………………….……...…..…………. 3.2.2.1. Worm lifespan assays………………………………………...………..……….…….…… 3.2.2.2. Statistical aalysis………………………………………..…….………….…….…..……... 3.2.3. Heat stress resistance………………………………………………..….……….…….………………67 3.2.4. Oxidative stress resistance………………………………….…….……..……….…..……………. 3.2.5. Cold stress resistance……………………………………………..………………….…….………….69 3.3. Results………………………………………………………………………..………….…….……………………. 3.3.1. Lifespan and DR………………………………………………..……………………….……..…………. 3.3.2. Survival in response to stress…………………………..……………………….…….…………… 3.4. Discussion……………………………………………………………………………..…………….…….……….. 4. CHAPTER FOUR QTL mapping in multi-parent RILs 4.1. Introduction…………………….…………………………………………….…….…………………………..…. 4.2. Methods……………………………….…………………………………………………..……….…….…………. 4.3. Results…………………………………………………………………………………….…….……..…………….. VI 4.3.1. Lifespan under normal conditions…………………………………..………….…….…………. 4.3.2. Lifespan under DR and Effect of DR…………………………………..…….….…….…………. 4.3.3. Stress response……………………………………………………………………..……...…………….. 4.4. Discussion………………………………………………………………………………….….…...…………….. 5. CHAPTER FIVE Cold Stress Candidate genes 5.1. Introduction…………………………………………………………………………….….….……..………… 5.2. Methods……………………………………………………………………………..…….….…….…………... 5.2.1. Worms………………………………………………………………..…………….…….…….………….. 5.2.2. Cold stress assay……………………………………………..……………….…….…….…………… 5.2.3. QTL mapping…………………………………………………..……………..…….…………………… 5.3. Results……………………………………………………………………..…………….………….……...……. 5.4. Discussion…………………………………………………………..…………..……….…….…….………….. 6. CHAPTER SIX Various Types of Dietary Restriction 6.1. Introduction…………………………….…………………………………..…………….…….….…………. 6.2. Methods…………………………………………………………………………..……….…….…….……….. 6.2.1. Worms…………………………………………………………………..………….…….……….………. 6.2.2. Total Starvation Assay…………..…………….………………………..……….……..….………. 6.2.3. DR assays on plates……………………….…….…………………….…….………...….…………. 6.2.4. Data analysis…………………………………….…………..…………………..….…….….…..……. 6.3. Results……………………………………………………………………………….…….…..……..….………. 6.3.1. Total starvation in liquid culture…………………………………….…….…..…..….………. 6.3.2. Various DR treatments…………………………………………………….……...…..….………… 6.4. Discussion…………………………………………………………………..……….………….……..…………. 7. General Discussion and Conclusions 7.1. Geeral discussio…………………………………………………………..…..……….…….…………… 7.2. Principal findings…………………………………………………………..…………….…….…………….. 7.3. Future directions……………………………………………………….…….……..……………………….. 7.4. Activities arisig fro this thesis………………………………..……..……………….…….………. VII 7.4.1. Publications……………………………………..……………………………….…….….….…….……. 7.4.2. Manuscripts in preparation………………………………..………….………….…….………… 7.4.3. Presentation………………………………………..……....……………….…….…………………….156 7.4.4. Research awards……………………………………………..…………….…….…….……….…….. 8. Appendices ……………………………………………………………….…………………….………….…….…….158 9. Bibliography………………………………………………………….……….……….….….…………………..……. VIII List of Abbreviations QTL Quantitative Trait Loci RILs Recombinant Inbred Lines ILs Isogenic Lines DR Dietary Restriction mTOR Mechanistic Target of Rapamycin PKA Protein Kinase A Ras A membrane-associated guanine nucleotide-binding protein N2 Collected from Bristol, most commonly used strain of C. elegans BO Collected from France, Bergerac strain is highly unstable LOD Logarithm Of the Odds SNP Single Nucleotide Polymorphism AFLP Amplified Fragment Length Polymorphisms eQTL Expression Quantitative Trait Loci IX List of Figures Figure 1.1 Insulin/insulin-like growth factor signalling (ILS) pathway in C. elegans……….……...36 Figure 2.1 Maternal hatching in N2/CB4856 RILs…………………………………………………….…….…..… Figure 2.2 Allelic effect on maternal hatching…………………………………………………….…….….……… Figure 2.3 Lifespan and the effect of dietary restriction in wild isolates………….…….…………….. Figure 2.4 Histograms of N2/CB ILs and Wild isolates………………………………….………….…………… Figure 2.5 Maternal hatching rate of the wild-isolates on DR and NGM…………..…….……………. Figure 3.1 Principal component analysis plot (PCA)……………………………………………….….…………. Figure 3.2 Lifespan under normal conditions and dietary restriction in the RILs…………….……. Figure 3.3 Lifespan on ad libitum and DR treatment………………………….………………………………… Figure 3.4 Block effect in RILs………………………………………………………….…………………………………… Figure 3.5 R-Simulation of lifespan differences…………………………………..…….……………….………… Figure 3.6 Variation in RILs for heat shock, cold shock and oxidative stress………………..……….. Figure 4.1 QTL
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