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Analysis of Cellular Responses to Microwave Irradiation in E. Coli and Change of Oxygen Level and Culture Medium in Human Cancer Cell Lines Using RNA-Seq Based

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Analysis of Cellular Responses to Microwave Irradiation in E. Coli and Change of Oxygen Level and Culture Medium in Human Cancer Cell Lines Using RNA-Seq Based Analysis of cellular responses to microwave irradiation in E. coli and change of oxygen level and culture medium in human cancer cell lines using RNA-seq based transcriptomic profiling Eunike Ilona Hilson Biotechnology Submitted in partial fulfillment of the requirements for the degree of Master of Science Faculty of Mathematics and Science, Brock University St Catharines, Ontario © 2021 Abstract RNA sequencing (RNA-seq) is one of the applications of next-generation sequencing (NGS) with differential gene expression (DGE) analysis at the transcriptomic level as its primary objective. Among the NGS technologies, the Illumina platforms are the current standard for RNA-seq analysis for their best cost efficiency and sequencing accuracy. In this study, we employed Illumina-based RNA-seq to examine the gene expression profile change in E. coli cells after exposure to microwave irradiation (MWI) and in cancer cell lines in response to different culture conditions using breast cancer cell lines (MCF7) and prostate cancer cell lines (PC3) as the models. Our results in examining the gene expression change in E. coli showed that the non- thermal effects of MWI led to E. coli cells entering the stationary phase with most of the downregulated genes involved in metabolic and biosynthesis pathways. MWI also upregulated the expression of genes important for the maintenance of membrane integrity and adhesion associated with bacterial motility. In comparison with other similar studies, our methodology allowed us to observe the impact of non-thermal effects of MWI at 2.45 GHz via simultaneous cooling. Our results in examining the transcriptomic profile of MCF7 and PC3 cells in response to oxygen level and culture medium change showed that gene expression in MCF7 is highly affected by oxygen level and culture medium changes when compared to PC3, especially in DMEM at 18% O2. DNA replication, cell-cycle, and viral carcinogenesis are the most affected pathways observed from different culture conditions in both cell lines. In PC3, only the legionellosis seems to be most impacted by culture medium changes at 5% O2, involving 8 differentially expressed genes (DEGs), important for cancer cell development. DGE analysis also I provides the transcriptomic profile of MCF7 and PC3, showing that different nutrient composition (between DMEM and Plasmax) and oxygen levels (5% O2 and 18% O2) changes the metabolism and various signaling pathways in both cell lines differently suggesting that the oxygen level and culture medium are important factors impacting the outcome of cell culture- based experiments in cell type-specific fashion. Keywords: RNA-seq, differential gene expression analysis, differentially expressed genes, transcriptomic profile, microwave irradiation, E. coli, culture conditions, cancer cell lines, MCF7, PC3. II Acknowledgment First, I would like to thank Brock University for all the support and wonderful experiences I have during my study in Canada. I am grateful to my supervisor, Dr. Ping Liang, for his support, guidance, and encouragement throughout my master's program and in completing my research, helping me understand the subject matter and broaden my skill in Biotechnology as well as Bioinformatics. I am thankful to my committee members, Dr. Jeff Stuart and Dr. Tony Yan, for their support and guidance to help me understand more about the research topics by allowing me to participate in their research projects. I also want to acknowledge all my colleagues in Dr. Ping Liang’s lab (Zakia Dahi, Jerry Tang, Robert Martin, Arsala Ali, Radesh Nattamai, Daniel Tang, Vinay Kumar Chundi, Marina Casavecchia, Bruce Racey, and Dr. Kai Hu), Fereshteh Moradi (Dr. Jeff Stuart’s Ph.D. student), and Frank Betancourt Montoya (Dr. Tony Yan’s Ph.D. student) for their help and support in completing this thesis. Finally, I would like to thank my family and friends in Canada and Indonesia for their continued support physically and spiritually to achieve my goals, especially Mr. and Mrs. Horvarth and family, Rebecca Joseph, Daislyn Vidal, Pau Pin, Felicia Marija, Katelyn Stachow, Central Church Community, Brock Power to Change (P2C), my parents, my sister and her husband, my husband, and his family, Adriana Nana, Mertha Prana, Dircia Cannisia C, and Dr. Dhira Satwika. May all the glory, honor, and praise for God alone, for he makes all things possible and beautiful in his time. III Table of contents Abstract …………………………………………………………………………………………...I Acknowledgment ………………………………………………………………………………..III List of Tables ……………………………………………………………………………...……..X List of Figures ………………………………………………………………………………….XII List of Abbreviations ………………………………………………………………………….XIV Chapter 1: General introduction on differential gene expression analysis by RNA sequencing …1 1.1. RNA-sequencing for differential gene expression analysis ………………………1 1.2. Overview of RNA-seq workflow for DGE analysis ……………………………..3 1.3. Research design: experimental design for RNA-seq based DGE analysis ….…...3 1.3.1. Expression variation ……………………………………………………….4 1.3.2. Level of replications ……………………………………………………....5 1.3.3. Sequencing read depth ………………………………………………….....6 1.3.4. Sequencing read length …………………………………………………....7 1.3.5. Library type: single-end or paired-end sequencing …………………….....8 1.3.6. Selection of RNA species ………………………………………………....9 1.3.7. Strand and non-strand-specific sequencing ……………………………...10 1.3.8. Reference availability: reference vs non-reference …………………...….13 1.4. DGE analysis …………………………………………………………………...14 1.4.1. Pre-processing ……………………………………………………………15 1.4.2. Read mapping ……………………………………………………………15 1.4.3. De novo transcriptome assembly ………………………………………...18 1.4.4. Quantification of reads abundance ……………………………………….20 IV 1.4.5. Normalization ……………………………………………………………22 1.4.6. Generating the raw DEGs list ……………………………………………24 1.4.7. Filtering the list of DEGs ………………………………………………...27 1.4.8. Enrichment analysis ……………………………………………………...28 1.5. Overall research objectives ……………………………………………………..30 Chapter 2: Analysis of cellular responses to microwave radiation in E. coli using RNA-seq based transcriptome profiling ...………………………………………………………………………...31 2.1. Introduction and related literature review …………………………………………..31 2.1.1. Microwave irradiation …………………………………………………….31 2.1.2. MWI disrupts the cellular membrane activity ……………………………32 2.1.3. MWI changes the enzymatic activity ……………………………………..34 2.1.4. Research objectives ……………………………………………………….35 2.2. Methods and materials ……………………………………………………………...36 2.2.1. Sample preparation ……………………………………………………….36 2.2.2. Library preparation and sequencing ………………………………………37 2.2.3. Assessment of RNA-seq data ……………………………………………..38 2.2.4. RNA-seq reads alignment ………………………………………………...38 2.2.5. DGE analysis ……………………………………………………………..38 2.2.6. DGE downstream analysis ………………………………………………..40 2.2.7. Identification of unannotated transcripts …………………………………40 2.2.8. Functional enrichment analysis …………………………………………..40 2.2.9. Analysis of co-expressed DEGs …………………………………………..41 2.2.10. Comparing the transcriptomic data with the previous proteomic data ….41 V 2.2.11. Computational analysis ………………………………………………….41 2.3. Results ………………………………………………………………………………42 2.3.1. Overview of the RNA-seq data: quality check and summary statistics …..42 2.3.2. Overview of the gene expression profile in response to MWI …………...44 2.3.3. List of DEGs common in 3 tools …………………………………………47 2.3.4. Functional enrichment analysis of DEGs using DAVID …………………48 2.3.6. Co-expressed analysis …………………………………………………….51 2.3.7. Comparing the transcriptomic data with proteomic data from the previous study ……………………………………………………………………………..55 2.4. Discussion …………………………………………………………………………….57 2.4.1. E. coli reacted to enhance membrane integrity and adhesion for survival in response to MWI ……………………….………………………………………..58 2.4.1.1. The increased activity of bacterial efflux pumps………………………..58 2.4.1.2. Disruption of membrane protein responsible for stabilizing membrane integrity ………………………………………………………………………….60 2.4.1.3. Increased activity of the PTS, controlling carbon uptake and metabolism ……………………………………………………………………………………62 2.4.1.4. E. coli adhesion increased by MWI …………………………………….63 2.4.2. E. coli shuts down most metabolism and biosynthesis to enter the stationary phase in response to MWI ……………………………………………………….64 2.4.2.1. E. coli enters stationary phase during MWI …………………………….65 2.4.2.2. The downregulated expression of genes coding for F1 sector of membrane-bound ATP synthase ………………………………………………...66 VI 2.4.2.3. The suppression of NAD+ biosynthesis ………………………………...67 2.4.2.4. The downregulated expression of genes coding for aminoacyl-tRNA synthetases ………………………………………………………………………69 2.4.2.5. The suppression of one carbon pool by folate ………………………….71 2.4.2.6. The suppression of iron transport ………………………………………74 2.4.3. The differences between transcriptomic and proteomic data from E. coli in response to MWI ………………………………………………………………...76 2.4.4. Concluding statements and future perspectives …………………………..77 Chapter 3: Assessment of different culture condition’s impact on cell’s physiology in culture by gene profiling…………………………………………………………………………………….79 3.1 Introduction and related literature review ……………………………………………..79 3.1.1. The development of cell culture technique ……………………………….79 3.1.2. Cell culture medium ………………….…………………………………...79 3.1.3. The impact of different culture medium components on cell culture …….80 3.1.4. The impact of oxygen on cell physiology in culture ……………………...83 3.1.4.1. The impact of hyperoxia in cell culture
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