Effect of chronic exposure to shisha in oral keratinocytes

Contents- CHAPTER 1 - General Introduction 1.1 Structure and function of Oral mucosa 1.2 Histology and function of Oral keratinocytes 1.3 Forms of tobacco usage in middle eastern countries 1.4 Effect of Shisha on oral keratinocytes 1.5 Genetic expression pattern and genomic anomalies of shisha on oral keratinocytes CHAPTER 2- Literature review 2.1 Shisha Constituents and Health effects 2.2 Oral mucosal diseases associated with chronic use of shisha 2.3 Molecular studies and shisha exposure 2.4 High throughput studies on shisha exposure 2.5 Biomarkers and shisha-associated oral cancer CHAPTER 3 - Development of an in vitro model of chronic shisha exposure using normal oral keratinocytes 3.1 Overview 3.2 Materials and Methods 3.2.1 Cell culture 3.2.2 Preparation of shisha extract 3.2.3 Adaptation of oral keratinocytes to shisha extract 3.2.4 Cellular proliferation assays 3.2.5 Colony formation assays 3.2.6 Cellular invasion assays 3.3 Results and discussion 3.3.1 Chronic exposure to shisha induces phenotypic changes in oral keratinocytes and increases the proliferative and invasive ability of oral cells 3.4 Discussion 3.5 Conclusions CHAPTER 4 - Genomic alterations in oral keratinocytes chronically exposed to shisha 4.1 Overview 4.2 Materials and methods 4.2.1 Cell culture 4.2.2 Whole exome sequence library preparation 4.2.3 Somatic variant analysis 4.2.4 RNA Seq library preparation 4.2.5 Whole transcriptome sequence analysis 4.2.6 Integration of whole exome and RNA-Seq dataset 4.2.7 Data analysis 4.2.8 Data availability 4.3 Results 4.3.1 Whole exome sequence analysis 4.3.2 Transcriptome analysis 4.3.3 Integrative analysis of whole exome and RNA-Seq 4.3.4 Pathway analysis using Ingenuity Pathway Analysis (IPA) 4.3.5 Comparison between OKF6/TERT1-Smoke with OKF6/TERT1-Shisha 4.4 Discussion 4.5 Conclusions CHAPTER 5 - Proteomic alterations in oral keratinocytes upon chronic exposure to shisha in a time dependent fashion 5.1 Overview 5.2 Materials and methods 5.2.1 Sample preparation for mass spectrometric analysis and In-solution trypsin digestion 5.2.2 Tandem mass tag (TMT)-based labeling and Basic pH reversed phase liquid chromatography (bRPLC) 5.2.3 LC-MSn analysis 5.2.4 Data analysis 5.2.5 Accessibility of proteomic data 5.2.6 Bioinformatics analysis 5.3 Results 5.3.1 Chronic exposure to shisha extract results in global proteomic changes in oral keratinocytes 5.3.2 Chronic exposure to shisha increases the proliferative and invasive ability of oral cells and decreases immune response in oral keratinocytes 5.3.3 Chronic exposure to shisha extract increases pathways associated with cell growth and invasion and decreases immune response in oral keratinocytes 5.4 Discussion 5.5 Conclusions CHAPTER 6 - Global phosphoproteomic changes observed in oral keratinocytes in response to chronic shisha exposure 6.1 Overview 6.2 Materials and methods 6.2.1 Cell culture and adaptation of normal oral keratinocytes to shisha extract 6.2.2 Colony formation assays 6.2.3. Sample preparation for mass spectrometry analysis - In-solution digestion and TMT labeling 6.2.4 Basic reversed-phase liquid chromatography (bRPLC) and phosphopeptide enrichment using IMAC 6.2.5 LC-MS/MS analysis 6.2.6 Data analysis 6.2.7 Proteomic data access 6.2.8 Bioinformatics analysis 6.3 Results and discussion 6.3.1 Chronic treatment with shisha extract induces cellular transformation in oral keratinocytes 6.3.2 Shisha exposure results in global proteomic and phosphoproteomic changes in oral keratinocytes 6.3.3 Chronic shisha exposure enriches for unique cellular processes in oral keratinocytes 6.3.4 Chronic shisha exposure alters signaling processes associated with mTORC1 and translation initiation in oral keratinocytes 6.4 Conclusions CHAPTER 7 - Secretome analysis of oral keratinocytes chronically exposed to shisha 7.1 Overview 7.2 Materials and methods 7.2.1 Collection and processing of secretome 7.2.2 Protein extraction, TMT labeling and bRPLC fractionation 7.2.3 LC-MSn analysis 7.2.4 Data analysis 7.2.5 Bioinformatics analysis 7.2.6 Immunoblot assays 7.2.7 Statistical analysis and Data availability 7.3 Results 7.3.1 Quantitative proteomic analysis of OKF6/TERT1-Shisha secretome 7.3.2 Bioinformatics analysis of dysregulated proteins in OKF6/TERT1-Shisha secretome 7.3.3 Identification of potential cancer biomarkers using the OKF6/TERT1-Shisha secretome data 7.3.4 Immunoblot validation of differentially secreted proteins identified in OKF6/TERT1-Shisha secretome 7.4 Discussion 7.5 Conclusions Chapter 8 8.1 Conclusive remarks Summary Recommended research for future Complete list of references