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OXIDATIVE STRESS in ALGAE: METHOD DEVELOPMENT and EFFECTS of TEMPERATURE on ANTIOXIDANT NUCLEAR SIGNALING COMPOUNDS Md Noman Siddiqui Purdue University

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OXIDATIVE STRESS in ALGAE: METHOD DEVELOPMENT and EFFECTS of TEMPERATURE on ANTIOXIDANT NUCLEAR SIGNALING COMPOUNDS Md Noman Siddiqui Purdue University Purdue University Purdue e-Pubs Open Access Theses Theses and Dissertations Spring 2014 OXIDATIVE STRESS IN ALGAE: METHOD DEVELOPMENT AND EFFECTS OF TEMPERATURE ON ANTIOXIDANT NUCLEAR SIGNALING COMPOUNDS Md Noman Siddiqui Purdue University Follow this and additional works at: https://docs.lib.purdue.edu/open_access_theses Part of the Fresh Water Studies Commons Recommended Citation Siddiqui, Md Noman, "OXIDATIVE STRESS IN ALGAE: METHOD DEVELOPMENT AND EFFECTS OF TEMPERATURE ON ANTIOXIDANT NUCLEAR SIGNALING COMPOUNDS" (2014). Open Access Theses. 256. https://docs.lib.purdue.edu/open_access_theses/256 This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. Graduate School ETD Form 9 (Revised/) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By MD NOMAN SIDDIQUI Entitled OXIDATIVE STRESS IN ALGAE: METHOD DEVELOPMENT AND EFFECTS OF TEMPERATURE ON ANTIOXIADANT NUCLEAR SIGNALING COMPOUNDS Master of Science For the degree of Is approved by the final examining committee: PAUL BROWN REUBEN GOFORTH THOMAS HOOK To the best of my knowledge and as understood by the student in the 7KHVLV'LVVHUWDWLRQ$JUHHPHQW 3XEOLFDWLRQ'HOD\DQGCHUWLILFDWLRQDisclaimer (Graduate School Form ), this thesis/dissertation DGKHUHVWRWKHSURYLVLRQVRIPurdue University’s “Policy on Integrity in Research” and the use of copyrighted material. PAUL BROWN Approved by Major Professor(s): ____________________________________ ____________________________________ Approved by:ROBERT SWIHART 04/22/2014 Head of the 'HSDUWPHQWGraduate Program Date OXIDATIVE STRESS IN ALGAE: METHOD DEVELOPMENT AND EFFECTS OF TEMPERATURE ON ANTIOXIDANT NUCLEAR SIGNALING COMPOUNDS A Thesis Submitted to the Faculty of Purdue University by Md Noman Siddiqui In Partial Fulfillment of the Requirements for the Degree of Master of Science May 2014 Purdue University West Lafayette, Indiana ii Dedicated to My wife, Farhana Islam Son , Nashwan Yusha and to My Parents iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. Paul Brown for his scholastic guidance to complete my study and research as well, without his guidance it was not possible for me to complete the research. My sincere appreciation for his patience, caring, critical thoughts and to provide me all supports to form my research in a shape. I would like to thanks my committee members Dr. Thomas Höök and Dr. Reuben Goforth for their time, suggestions and guidance to make my research successful. Special thanks to Department of Forestry and Natural Resources, Purdue University to provide me the funds for my study and research. I would like special thanks to Amber Jannasch and Dr. Bruce Cooper of Bindley Bioscience Center to help me to analyze my sample. Specially, Amber for her patience, effort and time. Without her close monitoring and help it was not possible for me to complete the analysis of samples. I would like to thank Sam Guffey, our lab manager for his help all the time to conduct my research and the staff of business office to help me to collect my required chemicals and other supplies. I would like to thank my parents and younger sisters and brother for their mental support to complete my study abroad. iv At last I would like to thanks my wife, Farhana Islam and my son Nashwan Yusha. Their contribution is inexpressible in words. They were my support, hope and inspiration in my good and bad times. Md Noman Siddiqui v TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES……………………………………………………………………....ix LIST OF ABBREVIATIONS ............................................................................................ xi ABSTRACT ............................................................................................................ xiii CHAPTER 1. INTRODUCTION ................................................................................. 1 1. 1 Introduction ……………………………………………………………………..1 1. 2 Assay development ………………………………………..……………………7 1.2.1 Introduction……………………………………….……………………….7 1. 3 Materials and Methods……………………..………………..…………..………...9 1.3.1 Collection of standards……………………...………………………...…..9 1.3.2 Selection of extraction solvent………………………………………….....9 1.3.3 Preparation of stock solution……………………………………..……...10 1.3.4 Sample extraction………………………………………………………...11 1.3.5 Liquid Chromatography/APPI Mass Spectrometry Analysis……………12 1.3.6 Liquid Chromatography/ESI Mass Spectrometry Analysis……………...13 1.4 Statistical analysis………………………………………………………..………14 1.5 Results……………………………………………………………………………15 1.5.1 Comparison of APPI and ESI method…………………………………...16 1.5.2 Compounds detected only by APPI method…………………………..…19 1.5.3 Compounds detected by only HPLC ESI/MS method…………………...23 1.6 Discussion………………………………………………………………………..24 CHAPTER 2. ABIOTIC STRESS ON ALGAE ....................................................... 26 2.1 Introduction ……………………………………………………………………26 vi Page 2.2 Objective of the study……………………………………………………………27 2.3 Materials and methods ……………………………………………………28 2.3.1 Stock Culture of phytoplankton………………………………….28 2.3.2 Experimental culture….………………………………………….28 2.3.3 Sample collection……………………………………………...…29 2.3.4 Preparation of stock solution…………………………………….29 2.3.5 Sample extraction………………………………………………...29 2.3.6 Liquid Chromatography/APPI Mass Spectrometry Analysis……30 2.3.7 Liquid Chromatography/ESI Mass Spectrometry Analysis…...…30 2.3.8 Thiobarbituric acid reactive substances assay (MDA)………......30 2.3.8.1 Reagents for MDA methods…………………………......31 2.3.8.2 Procedure…………………………………………...……31 2.4 Statistical analysis………………………………………………..31 2.5 Results ……………….……………………………………….….32 2.5.1 Number of cells at different temperatures…………….....32 2.5.2 Oxidative stress…………………………………………..33 2.5.3 Results from APPI analysis……………………………...34 2.5.4 Results from ESI analysis………………………………..42 2.6 Discussion………………………………………………..44 CHAPTER 3. SUMMARY AND CONCLUSIONS .................................................. 48 CHAPTER 4. SUGGESTED ADDITIONAL WORKS............................................. 49 LIST OF REFERENCES ................................................................................................. 50 APPENDIX………………………………………………………………………………61 Table 1. MRM table for analytes collected in APPI mode………………….…..61 Table 2. MRM table for analytes collected in ESI mode…..…………………….63 Table 3: Amount (ng) of different compounds detected by APPI method in Isochrysis sp. at different weight …………………….………………………….65 Table 4: Amount (ng) of different compounds detected by ESI method in Isochrysis sp. at different weight ……………………………………..…………66 vii Page Table 5: Number of cells at different temperatures with time interval ………….67 Table 6: MDA values( nmol/cell) in Isochrysis at different temperatures over time ……………………………………………………………………………...68 Table 7: Amount ( picogram/cell) of different compounds detected by APPI method in Isochrysis sp. at different temperature with varying time…………....69 Table 8: Amount (picogram/cell) of different compounds detected by ESI method in Isochrysis sp. at different temperature with varying time…………………….71 viii LIST OF TABLES Table .............................................................................................................................. Page 1 Identified exogenous and endogenous nuclear ligands…………………………...….....4 2 MRM approaches that detected targeted compounds from Isochrysis samples………15 3 Concentration of compounds detected by APPI and ESI methods…………………....16 ix LIST OF FIGURES Figure ............................................................................................................................. Page 1.1 Carotenoids biosynthetic pathway in seaweeds. .......................................................... 5 1.2 Comparison of canthaxanthin of Isochrysis sp.by APPI and ESI method ................. 16 1.3 Comparison of zeaxanthin of Isochrysis sp.by APPI and ESI method ...................... 17 1.4 Comparison of diatoxanthin of Isochrysis sp.by APPI and ESI method……………17 1.5 Comparison of B cyclocitral of Isochrysis sp.by APPI and ESI method…………....18 1.6 Comparison of violaxanthin of Isochrysis sp.by APPI and ESI method……………18 1.7 Concentration of fucoxanthin of Isochrysis sp.by APPI method …………………...19 1.8 Concentration of diadinoxanthin of Isochrysis sp.by APPI method………………...20 1.9 Concentration of astaxanthin of Isochrysis sp.by APPI method…………………....20 1.10 Concentration of antheraxantin of Isochrysis sp.by APPI method………………...21 1.11 Concentration of retinal Isochrysis sp.by APPI method…………………………...22 1.12. Concentration of retinol of Isochrysis sp.by APPI method……………………….22 1.13 Concentration of Retinoic acid Isochrysis sp.by ESI method ……………………..23 1.14 Concentration of β cryptoxanthin of Isochrysis sp.by ESI method………………..24 2.1 Number of Isochrysis cells with changing time……………………………………..33 2.2 MDA values in Isochrysis at different temperatures over time …………...………..34 2.3 Concentration of fucoxanthin in Isochrysis sp. using APPI method …………….....35 2.4 Concentration of diadinoxanthin in Isochrysis sp. using APPI method …………....36 2.5 Concentration of diatoxanthin in Isochrysis sp. using APPI method ………….…...37 2.6 Concentration of antheraxanthin in Isochrysis sp. using APPI method ………….…38 2.7 Concentration of violaxanthin in Isochrysis sp. using APPI method.........................39
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