Non Commercial. You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact xxxxxxxxxxx@xxx.xx.xx providing details, and we will remove access to the work immediately and investigate your claim. The Role of Mitochondria in the Development of Insulin Resistance and Type 2 Diabetes Kenan Direk BSc MSc Thesis submitted for the degree of Doctor of Philosophy King's College London Supervised by Xx Xxxx Xxxxxx and Xx Xxxxx Xxxxxx 2013 Table of Contents Contribution of Collaborators 6 Acknowledgements 7 Abstract 8 List of Tables 9 List of Figures 12 Abbreviations 14 Publications 17 Chapter 1: Introduction 18 Type 2 Diabetes 19 Insulin and Glucose Action 22 Measures of Insulin Resistance and Beta Cell Function 24 Diagnostic Test Criteria for T2D 28 Prevalence and Incidence 31 Worldwide 31 England 31 Mitochondrial Function and T2D 33 Genetics of T2D 35 Genetic Architecture of Common Disease 35 Genome-Wide Association Studies 35 Monogenic Forms of Insulin Resistance 40 Novel Biological Insights of Insulin Secretion/Resistance From GWA Studies 40 Mitochondria and Diabetes 44 How do Mutations in the Mitochondrial Genome Cause Diabetes? 46 Contribution of Mitochondrial and Nuclear Genome Variants to T2D 46 Why Might NEM Genes be Important in T2D? 48 Research Aims of this Thesis 52 Chapter 2: Materials and Methods 53 Samples 54 TwinsUK Sample 54 Wellcome Trust Case Control Consortium 59 National Institute for Diabetes and Digestive and Kidney disease (NIDDK) 59 Genotyping 60 TwinsUK 60 WTCCC1 61 African American 62 Quantitative Genetic Theory 63 Genetic Variance 66 Genetic Model Specification 67 Ordinary Least Squares Regression 71 Logistic Regression 73 Allelic and Genotypic Tests of Association 74 Classical Twin Modelling 76 Assumptions and Limitations 76 Heritability Estimates 78 Variance Components 78 Linkage Disequilibrium 84 Chapter 3: The Relationship Between DXA-based and Anthropometric Measures of Visceral Fat and Morbidity in Women. 90 Abstract 91 Introduction 92 Materials and Methods 94 Subjects and Data Collection 94 CT 94 DXA 94 Anthropometry 95 T2D 95 Hypertension 95 Carotid Intima-Media Thickness 96 Liver Function Tests 96 Model Validation 96 Heritability Analysis 97 Morbidity Association with Adiposity 98 Results 100 Estimation of Visceral Fat (Validation Sample) 100 Heritability 108 Visceral Fat as a Risk Factor of Morbidity (Study Sample) 110 Discussion 118 Chapter 4: A Candidate Gene Study for the PARL/ABCC5 Gene Region as a Novel Type 2 Diabetes Susceptibility Locus 121 Abstract 122 Introduction 123 Materials and Methods 126 Glycaemic Traits 126 Fine-Mapping 126 Statistical Genetic Methods 127 Results 130 Val262Leu (rs3732581, PARL) as a Candidate Marker for Insulin Resistance and T2D 132 LD in the PARL/ABCC5 Gene Region 134 The PARL/ABCC5 Gene Region as a Candidate for Insulin Resistance and T2D 138 Gene Expression Quantitative Trait Locus (eQTL) Analysis 144 Subcutaneous Adipose ABCC5 Expression as an Intermediate Phenotype for Fasting Insulin, Visceral Fat Accumulation and T2D 147 Discussion 149 Chapter 5: Mitochondrial Genetic Pathways and Susceptibility to T2D 153 Abstract 154 Introduction 155 Identification of Nuclear-Encoded Mitochondrial Genes 155 Pathway-based Analyses 160 Hypothesis 166 Statistical Analysis of Pathways 167 Gene Set Enrichment 170 Bias 172 Materials and Methods 175 Subjects and Data 175 Definition of NEM Genes 175 NEM Gene Sets 175 NEM Gene Set Tissue Specificity 176 Genomic Regions 176 Gene Set Size 176 Extent of NEM Gene Set Overlap 177 Set-Based Analysis 180 Over-Representation Analysis 180 Results 181 Discussion 195 Study Strengths and Limitations 201 Concluding Chapter 203 Future work 209 Supplementary Tables 212 Bibliography 224 Contribution of Collaborators Chapter 3 Acquisition of computed tomography data: Xx Xxxxxx Xxxxxxx. Acquisition of all other data on the twin sample: Department of Twin Research at King’s College London. Variance inflation factor and residual analyses: Xx Xxxx Xxxxxx. Chapter 4 Gene expression data: Multiple Tissue Human Expression Resource. Linkage disequilibrium maps: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Malécot test of association: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Chapter 5
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Non Commercial. You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact xxxxxxxxxxx@xxx.xx.xx providing details, and we will remove access to the work immediately and investigate your claim. The Role of Mitochondria To Boldly Go: How do women in senior positions in the Development Church of Insulin Resistance and Type 2 Diabetes Kenan Direk BSc MSc England construe their leadership? Research Based Thesis submitted for the degree of Doctor of Philosophy King's College London Supervised by Xx Xxxx Xxxxxx Doctorate in Theology and Xx Ministry Xxxxx Xxxxxx Xxxx June 2013 Table Acknowledgements 5 Abstract 6 Preface WHAT IS LEADERSHIP? WOMEN IN THE CHURCH 8 What is leadership? 9 Women in the church 14 Chapter 1 A RESEARCH JOURNEY AN EXPLORATION OF HOW A GROUP OF WOMEN INCUMBENTS CONSTRUE THEIR MINISTRY (MFS) 19 A Research Journey 20 Two key events: 1992 and 2012 21 The main questions 23 Two studies 26 An exploration of Contents Contribution how a group of Collaborators 6 Acknowledgements 7 Abstract 8 List women incumbents construe their ministry 28 Key themes 28 Person, Process and Context 29 Now and Ideal 36 Rankings and Ratings 39 Conclusions from the MFS 42 Chapter 2 EMPIRICAL RESEARCH METHODOLOGY 46 What does it mean to construe? 47 Rationale 49 Personal Construct Psychology in theory 51 Personal Construct Psychology in practice 55 Choosing Elements 56 Eliciting Constructs 57 Laddering 58 The Ministry Focused Study (MFS) 60 - Repertory Grid Questionnaire 60 The Research Based Thesis (XXX) 66 - Self Characterisation 67 - Repertory Grid Interview 71 Closing comments on methodology 75 Chapter 3 RESULTS 77 Introduction Detailed Case Study: Xxxx Summaries of Tables 9 List each of Figures 12 Abbreviations the 14 Publications 17 Case Studies Collective data Summary of the case studies looked at as a group Helps and Hindrances The '3 roles' - Xxxxxxxxxx, Xxxx, Theological Educator 105 105 108 111 The experience of the person, process and context in leadership How the women experience the person in leadership 116 116 How the women experience the process in leadership 119 How the women experience the context in leadership 124 Conclusions: Key themes and issues from the empirical research 129 Chapter 1: Introduction 18 Type 2 Diabetes 19 Insulin and Glucose Action 22 Measures 4 CONCLUSION 133 Is Personal Construct Psychology a useful tool? 134 The two studies 138 An exploration of Insulin Resistance and Beta Cell Function 24 Diagnostic Test Criteria for T2D 28 Prevalence and Incidence 31 Worldwide 31 England 31 Mitochondrial Function and T2D 33 Genetics how a group of T2D 35 Genetic Architecture of Common Disease 35 Genome-Wide Association Studies 35 Monogenic Forms of Insulin Resistance 40 Novel Biological Insights of Insulin Secretion/Resistance From GWA Studies 40 Mitochondria and Diabetes 44 women incumbents construe their ministry How do Mutations women in senior positions in the Mitochondrial Genome Cause DiabetesChurch of 138 140 England construe their leadership? 46 Contribution - The Person of Mitochondrial the leader: Managing the Role 140 Being ‘yourself’ as leader - The Process of leadership: Having Agency Being a ‘relational' leader - The Context in which leadership is exercised: Engaging with the Structures Being a ‘woman’ leader Conclusions General Observations Three important questions - Can 'Servant Leadership' be a good model for women? - Can tradition be a resource for women? - Is simply having more women enough? To boldly go? 143 146 150 150 152 152 153 155 157 BIBLIOGRAPHY and Nuclear Genome Variants REFERENCES 159 APPENDICES Appendix A 168 The 11 Corollaries in PCP 169 Appendix B 170 MFS Grid Questionnaires 171 Appendix C Research Based Thesis (XXX) Initial letter to T2D 46 Why Might NEM Genes the women 184 185 Interview information gathering - Biographical information (blank) 186 - Helps and Hindrances sheet (blank) 187 - Elements and Triadic Elicitation 188 - Grid with Elements (blank) 189 Appendix D Xxxxx's results with full Grid Suite analysis 190 Acknowledgements With profound gratitude to all who have supported me in various ways during my DThMin studies: People in the Dioceses of Birmingham, Peterborough, Southwark and Rochester Women’s Continuing Ministerial Education Trust XX Xxxx Fund, Xxxxxxxx College, Cambridge My supervisors Xxxxxxx Xxxxxx, Xxxx'x College, London Xxxx Xxxx, University of Hertfordshire My family Xxxxx Xxxxxx Xxxx and Xxxx a woman who always inspires me The women who took part in the study who gave of themselves as well as their time Xxxxxxx Xxxxx Xxxxxxx Xxxxxxxxx Hannah Xxxxx Xxxxx Xxxx Xxxx Xxxxx Xxxxxx Xxxx Xxxxx Xxxxxxx Abstract This thesis contains the results of two empirical studies focusing on the ministry and leadership of women in the Church of England. Two key events, one in 1992 and the other in 2012, almost exactly twenty years apart, provide the framework and context in which these studies take place. The thesis helps to explain why so few women apply for senior positions in the Church of England. The first study, explores how women incumbents in a diocese make sense of their particular roles and ministries. The focus is initially on women incumbents, as the senior leaders for the church emerge from this group. This Ministry Focused Study (MFS) begins not only to address the issue itself but also acts as a pilot study. The second and substantive Research Based Thesis (XXX) uses these insights to investigate the primary question of the thesis: How do women in senior positions in the Church of England construe their leadership? Fourteen women (out of 15) who were in some of the most senior positions in the Church of England - Cathedral Deans, Archdeacons and Theological College Principals - took part in the research project. The central argument is that effective leadership for these women consists of three core dimensions: The Person of the leader - this includes the connections between person and role, especially being able to ‘be Important yourself’ as a leader, as well as managing the role in T2D? 48 Research Aims the public and private spheres. The Process of leadership - summarised as ‘agency enhanced by communion’. The emphasis here is having the ability to bring about change and make things happen; fundamental to this is being a relational leader who seeks to develop interactions based on integrity and trust. The Context in which leadership takes place - involves a deep understanding and engagement with the structures of the church, within which the leadership role is experienced and exercised. Both studies employ a method of interviewing called Repertory Grids, which derive from a theory of personality proposed by Xxxxxx Xxxxx known as Personal Construct Psychology (PCP). From the standpoint of those who are interviewed, it enables the discovery of some of the key issues and perspectives, as well as giving insight into both the relative importance of those issues and how those key perspectives interact with each other. PCP has proved useful in a variety of contexts, including education, social science (Xxxxx et al., 2000) and business research (Xxxxxxx and Xxxxxxx, 1980); being used with individuals, groups or organisations to explore a range of issues from beliefs, feelings and attitudes of individuals to understanding organisational transitions and corporate values (Fransella, 2003). Until now, however, it has not been employed in a major empirical study in theology and ministry. Although there has been much written about women in ministry, and to a lesser extent women in church leadership, over the last decade or so, relatively little of this Thesis 52 Chapter 2: Materials and Methods 53 Samples 54 TwinsUK Sample 54 Wellcome Trust Case Control Consortium 59 National Institute for Diabetes and Digestive and Kidney disease (NIDDK) 59 Genotyping 60 TwinsUK 60 WTCCC1 61 African American 62 Quantitative Genetic Theory 63 Genetic Variance 66 Genetic Model Specification 67 Ordinary Least Squares Regression 71 Logistic Regression 73 Allelic and Genotypic Tests of Association 74 Classical Twin Modelling 76 Assumptions and Limitations 76 Heritability Estimates 78 Variance Components 78 Linkage Disequilibrium 84 Chapter 3: The Relationship Between DXA-has been based and Anthropometric Measures of Visceral Fat and Morbidity in Womenon primary data such as that which these two research studies have been able to generate. 90 Abstract 91 Introduction 92 Materials and Methods 94 Subjects and Data Collection 94 CT 94 DXA 94 Anthropometry 95 T2D 95 Hypertension 95 Carotid Intima-Media Thickness 96 Liver Function Tests 96 Model Validation 96 Heritability Analysis 97 Morbidity Association with Adiposity 98 Results 100 Estimation of Visceral Fat (Validation Sample) 100 Heritability 108 Visceral Fat as This thesis comes at a Risk Factor of Morbidity (Study Sample) 110 Discussion 118 Chapter 4: A Candidate Gene Study for the PARL/ABCC5 Gene Region as a Novel Type 2 Diabetes Susceptibility Locus 121 Abstract 122 Introduction 123 Materials and Methods 126 Glycaemic Traits 126 Fine-Mapping 126 Statistical Genetic Methods 127 Results 130 Val262Leu (rs3732581, PARL) as a Candidate Marker for Insulin Resistance and T2D 132 LD significant point in the PARL/ABCC5 Gene Region 134 The PARL/ABCC5 Gene Region history of the Church of England1 as it continues to discover a Candidate for Insulin Resistance way forward following the rejection, by General Xxxxx in November 2012, of proposed legislation, which would have allowed women to become bishops. Within this context, the thesis explores how these women in senior positions make sense of their leadership roles, what enables them in their roles and T2D 138 Gene Expression Quantitative Trait Locus (eQTL) Analysis 144 Subcutaneous Adipose ABCC5 Expression asks whether women leaders in the church are able, ‘to boldly go...’ 1 Hereafter referred to as an Intermediate Phenotype for Fasting Insulin, Visceral Fat Accumulation and T2D 147 Discussion 149 Chapter 5: Mitochondrial Genetic Pathways and Susceptibility to T2D 153 Abstract 154 Introduction 155 Identification of Nuclear-Encoded Mitochondrial Genes 155 Pathway-based Analyses 160 Hypothesis 166 Statistical Analysis of Pathways 167 Gene Set Enrichment 170 Bias 172 Materials and Methods 175 Subjects and Data 175 Definition of NEM Genes 175 NEM Gene Sets 175 NEM Gene Set Tissue Specificity 176 Genomic Regions 176 Gene Set Size 176 Extent of NEM Gene Set Overlap 177 Set-Based Analysis 180 Over-Representation Analysis 180 Results 181 Discussion 195 Study Strengths and Limitations 201 Concluding Chapter 203 Future work 209 Supplementary Tables 212 Bibliography 224 Contribution of Collaborators Chapter 3 Acquisition of computed tomography data: Xx Xxxxxx Xxxxxxxthe 'church'. Acquisition of all other data on the twin sample: Department of Twin Research at King’s College London. Variance inflation factor and residual analyses: Xx Xxxx Xxxxxx. Chapter 4 Gene expression data: Multiple Tissue Human Expression Resource. Linkage disequilibrium maps: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Malécot test of association: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Chapter 5Preface WHAT IS LEADERSHIP?
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Jul. 2021 Engaging with austerity: austerity management, discourse and the regimes of Mitochondria in the Development of Insulin Resistance and Type 2 Diabetes Kenan Direk BSc MSc Thesis engagement Xxx xxx Xxx Xxxxxx submitted for the degree a PhD in Public Management and Organisation Studies King’s College, London February, 2020 Contents Tables, figures and abbreviations 7 List of Doctor tables 7 List of Philosophy King's College London Supervised by Xx Xxxx Xxxxxx and Xx Xxxxx Xxxxxx 2013 Table of Contents Contribution of Collaborators 6 Acknowledgements 7 Abstract figures 8 List of Tables abbreviations 9 List Acknowledgements 10 Abstract 12 Part 1 – Introduction 13 Chapter 1 : Introduction 13 Austerity and public administration 13 Studying austerity management 14 Engaging with austerity 16 Introducing the thesis 18 Part 2 – Literature reviews 22 Chapter 2 : Austerity management 22 Introduction 22 Tracking the austerity management literature 23 The content: savings 26 The situation: austerity 29 The practice: managing austerity 32 Agency: austerity managers 35 Conceptualising austerity management 37 Chapter 3 : Conceptualising austerity management: the regimes of Figures 12 Abbreviations 14 Publications 17 engagement 40 Introduction 40 Theorising situated action 41 Regimes of engagement 43 Two types of moral pluralism 49 Situations, uncertainty and crisis 52 Materiality and discourse 54 Conclusion 56 Chapter 4 : Discourse and the management of austerity 58 Introduction 58 The need for a discursive approach 59 Austerity discourse 61 Critical Discourse Analysis 64 Conclusion: a discursive pragmatism 69 Part 3 – Methods 72 Chapter 5 : Methods 72 Introduction 72 Theoretical and philosophical commitments 73 Research design 77 Gathering data 79 Analysing practice 83 Generating theory 89 Conclusion 91 Part 4 – Organisations under austerity 93 Chapter 6 : Introducing the practices 94 Attaining tenuous futures 94 Constructing possibility 97 Reconfiguring quality 100 Conducting and contesting financial tests 102 Reshaping engagements 104 Practices of austerity management 106 Chapter 7 : Dryas: case narrative 109 Introducing Dryas 109 Period 1: Introduction 18 Type 2 Diabetes 19 Insulin Cost Improvement and Glucose Action 22 Measures of Insulin Resistance and Beta Cell Function 24 Diagnostic Test Criteria for T2D 28 Prevalence and Incidence 31 Worldwide 31 England 31 Mitochondrial Function and T2D 33 Genetics of T2D 35 Genetic Architecture of Common Disease 35 Genome-Wide Association Studies 35 Monogenic Forms of Insulin Resistance 40 Novel Biological Insights of Insulin Secretion/Resistance From GWA Studies 40 Mitochondria and Diabetes 44 How do Mutations in the Mitochondrial Genome Cause Diabetes? 46 Contribution of Mitochondrial and Nuclear Genome Variants to T2D 46 Why Might NEM Genes be Important in T2D? 48 Research Aims of this Thesis 52 Chapter reframing 110 Period 2: Materials and Methods 53 Samples 54 TwinsUK Sample 54 Wellcome Trust Case Control Consortium 59 National Institute for Diabetes and Digestive and Kidney disease (NIDDK) 59 Genotyping 60 TwinsUK 60 WTCCC1 61 African American 62 Quantitative Genetic Theory 63 Genetic Variance 66 Genetic Model Specification 67 Ordinary Least Squares Regression 71 Logistic Regression 73 Allelic and Genotypic Tests of Association 74 Classical Twin Modelling 76 Assumptions and Limitations 76 Heritability Estimates 78 Variance Components 78 Linkage Disequilibrium 84 Chapter Scrutiny 120 Period 3: Bridging 124 Coda: The Relationship Between DXA-based tactical decision 128 Interpreting the case 129 Conclusion and Anthropometric Measures theoretical reflections 134 Chapter 8 : Thyme: case narrative 138 Introducing Thyme 138 Period 1: Financial recovery, financial strategy 139 Period 2: Making financial plans real 144 Period 3: Destabilising 149 Interpreting the case 153 Conclusion and theoretical reflections 158 Chapter 9 : Aloe: case narrative 162 Introducing Aloe 162 Period 1: Becoming real 163 Period 2: External shock and suspicion 169 Period 3: Internalising instability 173 Interpreting the case 176 Conclusion and theoretical reflections 180 Chapter 10 : Austerity management across sites 185 Introduction 185 Attaining tenuous futures 186 Constructing possibility 192 Reconfiguring quality 196 Conducting and contesting financial tests 199 Reshaping engagements 201 Conclusion: modelling austerity management 205 Part 5 – Theorising austerity management 211 Chapter 11 : Situating work 211 Introduction 211 Conceptualising the situation 212 Situations and healthcare austerity 215 Situating work 225 Conclusions and implications for organisations 230 Chapter 12 : ‘Looking again’: opening and closing one’s eyes 234 Introduction 234 Uncertainty and austerity 235 Uncertainty and the regimes of Visceral Fat engagement 236 Managing uncertainty under austerity 238 Theorising cycles of opening and Morbidity in Women. 90 Abstract 91 Introduction 92 Materials and Methods 94 Subjects and Data Collection 94 CT 94 DXA 94 Anthropometry 95 T2D 95 Hypertension 95 Carotid Intima-Media Thickness 96 Liver Function Tests 96 Model Validation 96 Heritability Analysis 97 Morbidity Association with Adiposity 98 Results 100 Estimation of Visceral Fat (Validation Sample) 100 Heritability 108 Visceral Fat as a Risk Factor of Morbidity (Study Sample) 110 Discussion 118 Chapter 4: A Candidate Gene Study for the PARL/ABCC5 Gene Region as a Novel Type 2 Diabetes Susceptibility Locus 121 Abstract 122 Introduction 123 Materials and Methods 126 Glycaemic Traits 126 Fine-Mapping 126 Statistical Genetic Methods 127 Results 130 Val262Leu (rs3732581, PARL) as a Candidate Marker for Insulin Resistance and T2D 132 LD in the PARL/ABCC5 Gene Region 134 The PARL/ABCC5 Gene Region as a Candidate for Insulin Resistance and T2D 138 Gene Expression Quantitative Trait Locus (eQTL) Analysis 144 Subcutaneous Adipose ABCC5 Expression as an Intermediate Phenotype for Fasting Insulin, Visceral Fat Accumulation and T2D 147 Discussion 149 Chapter 5: Mitochondrial Genetic Pathways and Susceptibility to T2D 153 Abstract 154 Introduction 155 Identification of Nuclear-Encoded Mitochondrial Genes 155 Pathway-based Analyses 160 Hypothesis 166 Statistical Analysis of Pathways 167 Gene Set Enrichment 170 Bias 172 Materials and Methods 175 Subjects and Data 175 Definition of NEM Genes 175 NEM Gene Sets 175 NEM Gene Set Tissue Specificity 176 Genomic Regions 176 Gene Set Size 176 Extent of NEM Gene Set Overlap 177 Set-Based Analysis 180 Over-Representation Analysis 180 Results 181 Discussion 195 Study Strengths and Limitations 201 Concluding Chapter 203 Future work 209 Supplementary Tables 212 Bibliography 224 Contribution of Collaborators Chapter 3 Acquisition of computed tomography data: Xx Xxxxxx Xxxxxxx. Acquisition of all other data on the twin sample: Department of Twin Research at Kingclosing one’s College London. Variance inflation factor and residual analyses: Xx Xxxx Xxxxxx. Chapter 4 Gene expression data: Multiple Tissue Human Expression Resource. Linkage disequilibrium maps: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Malécot test of association: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Chapter 5eyes 249 Conclusion 252
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The Role of Mitochondria in the Development of Insulin Resistance and Type 2 Diabetes Kenan Direk BSc MSc Thesis submitted for the degree of Doctor of Philosophy King's College London Supervised by Xx Xxxx Xxxxxx and Xx Xxxxx Xxxxxx 2013 Table of contents Contents Contribution Table of Collaborators 6 Acknowledgements contents 1 Abstract 5 Acknowledgments 7 Abstract 8 List of Tables 9 List of Figures 12 Abbreviations 14 Publications 17 Chapter 1: General Introduction 18 Type 2 Diabetes 19 Insulin and Glucose Action 22 Measures 8 Historical perspective 8 Innervation of Insulin Resistance and Beta Cell Function 24 Diagnostic Test Criteria the GI tract 8 Enteroendocrine cells 11 I cells 12 EC cells 13 Methods of studying endocrine cells 15 Mechanisms for T2D 28 Prevalence and Incidence 31 Worldwide 31 England 31 Mitochondrial Function and T2D 33 Genetics nutrient stimulation of T2D 35 Genetic Architecture endocrine cells 17 Mechanisms of Common Disease 35 Genome-Wide Association Studies 35 Monogenic Forms of Insulin Resistance 40 Novel Biological Insights of Insulin Secretion/Resistance From GWA Studies 40 Mitochondria and Diabetes 44 How do Mutations mechanotransduction in mammals 26 Mechanically evoked transmitter release 32 Mechanosensation in the Mitochondrial Genome Cause Diabetes? 46 Contribution intestinal epithelium 33 Role of Mitochondrial and Nuclear Genome Variants to T2D 46 Why Might NEM Genes be Important Voltage gated channels in T2D? 48 Research secretion 34 Calcium channels 39 Sodium channels 40 Voltage gated channels in enteroendocrine cells. 43 Transient receptor potential channels 44 TRP channels as sensory transducers 45 Aims of this Thesis 51 Abbreviations 52 Chapter 2: Materials and Methods 53 Samples 54 TwinsUK Sample Cell culture 54 Wellcome Trust Case Control Consortium Preparation of Primary Tissues 54 Crypt preparation 55 Coverslip coating 56 In Situ Hybridisation 56 RNA isolation, RT-PCR, q-RT-PCR, Gel Electrophoresis. 57 Primers 59 National Institute for Diabetes and Digestive and Kidney disease (NIDDK) 59 Genotyping 60 TwinsUK 60 WTCCC1 Drugs 61 African American 62 Quantitative Genetic Theory Solutions 63 Genetic Variance 66 Genetic Model Specification Electrophysiology 63 Intracellular Calcium Studies. 65 Statistics 67 Ordinary Least Squares Regression 71 Logistic Regression 73 Allelic and Genotypic Tests Mechanical Stimulation of Association 74 Classical Twin Modelling 76 Assumptions and Limitations 76 Heritability Estimates 78 Variance Components 78 Linkage Disequilibrium 84 cells 67 Chapter 3: The Relationship Between DXAVoltage gated ion channels in STC-1 and KRJ-based I cells 69 Results 69 PCR analysis of channel expression 69 KRJ-I cells 74 Electrophysiological and Anthropometric Measures Calcium studies on STC-1 cells 75 STC-1 cells express a large outward current 76 Sodium channels in STC-1 cells 85 Calcium channels in STC-1 cells 91 Electrophysiological studies of Visceral Fat and Morbidity in Women. 90 Abstract 91 Introduction 92 Materials and Methods 94 Subjects and Data Collection 94 CT 94 DXA 94 Anthropometry 95 T2D 95 Hypertension 95 Carotid IntimaKRJ-Media Thickness 96 Liver Function Tests 96 Model Validation 96 Heritability Analysis 97 Morbidity Association I cells 98 KRJ-I cells did not respond to Potassium Chloride 99 Two distinct outward currents were observed 99 Investigation with Adiposity 98 Results 100 Estimation of Visceral Fat (Validation Sample) 100 Heritability 108 Visceral Fat as a Risk Factor of Morbidity (Study Sample) 2-APB 101 Discussion 103 STC-1 cells 103 KRJ-I cells 110 Discussion 118 Chapter 4: A Candidate Gene Study for the PARL/ABCC5 Gene Region as a Novel Type 2 Diabetes Susceptibility Locus 121 Abstract 122 Introduction 123 Materials and Methods 126 Glycaemic Traits 126 Fine-Mapping 126 Statistical Genetic Methods 127 Results 130 Val262Leu (rs3732581, PARL) as a Candidate Marker for Insulin Resistance and T2D 132 LD The role of TRPA1 in the PARL/ABCC5 Gene Region 134 The PARL/ABCC5 Gene Region as a Candidate release mechanism of STC-1 cells 113 Results 113 TRPA1 expression in STC-1 cells 113 PCR analysis of channel expression 115 In situ hybridisation TRPA1 116 Fatty Acid activation of STC-1 cells 118 TRPA1 antagonist AP18 blocks fatty acid activation of STC-1 cells 122 A role for Insulin Resistance and T2D L-type calcium channels? 125 Denatonium activates STC-1 cells 127 Pharmacological investigation of indirect denatonium activation of STC-1 cells 130 Cold activation of STC-1 cells 131 Discussion 138 Gene Expression Quantitative Trait Locus (eQTL) Analysis 144 Subcutaneous Adipose ABCC5 Expression as an Intermediate Phenotype for Fasting Insulin, Visceral Fat Accumulation and T2D 147 Discussion 149 Chapter 5: Mitochondrial Genetic Pathways Primary cell extraction and Susceptibility EC cell mechanosensation 149 Results 149 Crypt extraction 149 KRJ-I cells respond to T2D hypotonic solutions 150 PCR identification of candidate osmo- and mechano-sensitive ion channel in KRJ-I cells 151 Calcium imaging with TRP agonists. 152 TRPA1 and TRPV1 152 Thymol 153 Direct mechanical stimulation of KRJ-I cells 156 Direct stimulation using piezo electric actuator elicits responses in cultured DRG neurons but not in KRJ-I cells 158 Discussion 160 Final summary 166 Supplementary Material 169 Abstract 154 Introduction 155 Identification The sensing of Nuclearluminal nutrients is a central function of the gut epithelium where enteroendocrine cells act as sensor cells to secrete hormones in response to the luminal contents. This thesis focuses on voltage gated ion channels and the transient receptor potential (TRP) superfamily in I cells and enterochromaffin cells using the model tumour cell lines STC-1 and KRJ-Encoded Mitochondrial Genes 155 PathwayI respectively. Electrophysiology and PCR was performed on STC-1and KRJ-based Analyses 160 Hypothesis 166 Statistical Analysis I model cell lines to elucidate the channels that may regulate hormone release. In KRJ-I cells two outward currents were observed. In STC-1 cells three types of Pathways 167 Gene Set Enrichment 170 Bias 172 Materials ionic currents were identified; a large outward voltage gated potassium current, a tetrodotoxin-sensitive voltage gated sodium current and Methods 175 Subjects a nifedipine-sensitive persistent calcium current. In intracellular calcium studies of STC-1 cells, the TRPA1 agonists cinnamaldehyde and Data 175 Definition AITC evoked increases in intracellular calcium concentration that could be inhibited by the TRPA1 antagonist AP18. Calcium responses evoked by fatty acids, the bitter tastant denatonium and cold temperatures below 25°C were also attenuated by the TRPA1 antagonist AP18. Studies to elucidate the mechanism of NEM Genes 175 NEM Gene Sets 175 NEM Gene Set Tissue Specificity 176 Genomic Regions 176 Gene Set Size 176 Extent action of NEM Gene Set Overlap 177 Setthese compounds were carried out using tools to block signalling pathways. As enterochromaffin cells are thought to be mechanically active, KRJ-Based Analysis 180 OverI cells were used as a model to study the potential molecular mechanism. PCR experiments showed the presence of the novel mechanosensitive channels. Three methodologies were developed to stimulate KRJ-Representation Analysis 180 Results 181 Discussion 195 Study Strengths I cells directly: intracellular calcium measurements with stimulation by either jets of saline or shear flow, and Limitations 201 Concluding Chapter 203 Future whole cell patch clamp with direct mechanical stimulation. No evidence for mechanical responsiveness was found. These studies provide valuable insight into these two model cell lines on which a large amount of work 209 Supplementary Tables 212 Bibliography 224 Contribution of Collaborators Chapter 3 Acquisition of computed tomography data: Xx Xxxxxx Xxxxxxxis being carried out. Acquisition of all other data Although cell models provide valuable insight into transduction mechanisms, studies on the twin sample: Department of Twin Research at King’s College London. Variance inflation factor and residual analyses: Xx Xxxx Xxxxxx. Chapter 4 Gene expression data: Multiple Tissue Human Expression Resource. Linkage disequilibrium maps: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Malécot test of association: Xx Xxxxxxx Xxx and Xx Xxxxxxx Xxxxxxxx. Chapter 5primary cell cultures remain paramount.
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