Thyroid Hormones: Methods and Protocols

This volume focuses on thyroid hormones and their transporters and receptors. Chapters focus on a wide range of methods and protocols that are important for advancing the elucidation of the role of thyroid hormones in our body. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Preface Contents Contributors Part I: An Overview Chapter 1: Thyroid Hormones and Co-workers: An Overview 1 The Hypothalamus-Pituitary-Thyroid Axis 2 Thyroid Gland and Thyroid Hormones 3 Genomic and Non-genomic Actions of Thyroid Hormones 3.1 Plasma Membrane Integrin ανβ3 3.2 TH Transporter Proteins 3.3 TH Nuclear Receptors (TRs) 3.4 Coactivators and Corepressors 4 Clinical Implications 4.1 Thyroid Cancer 4.2 Hypothyroidism 4.2.1 Clinical Presentation and Significance 4.3 Hyperthyroidism 4.4 Resistance to Thyroid Hormone 4.4.1 Mutations in TRα and TRβ Genes 4.4.2 Deiodination Defect 4.4.3 Relevance of mct8 in Health and Disease References Chapter 2: Thyroid Hormone Action by Genomic and Nongenomic Molecular Mechanisms 1 Nuclear Thyroid Hormone Receptors (TRs) 2 Transcriptional Regulation by TRs 2.1 Binding to TREs and Dimerization 2.2 Thyroid Hormone-Dependent Transcriptional Activation: Coactivators 2.3 Transcriptional Repression by Unliganded Receptors: Corepressors 2.4 Models of Thyroid Hormone-Dependent Transcriptional Activation 2.5 Thyroid Hormone-Dependent Transcriptional Repression 3 Non-genomic Extranuclear TR Actions 4 Non-genomic TR-Independent Thyroid Hormone Actions References Part II: Thiroid Hormones Chapter 3: A Sensitive Radioimmunoassay for T3 and T4 Determinations in Plasma and Tissues 1 Introduction 2 Materials 2.1 Testing Chromatographic Separation 2.2 Synthesis of High Specific Activity 125I-T3 and 125I-T4 2.3 Free Iodide Estimation 2.4 Purification of Tracers from Iodide by Paper Electrophoresis 2.5 T3 and T4 Extraction from Plasma Samples 2.6 Preparation of T3 and T4 Extraction Columns for Tissue Samples 2.7 T3 and T4 Extraction from Tissue Samples 2.8 Radioimmunoassays for T3 and T4 Determinations 3 Methods 3.1 Testing Chromatographic Separation 3.2 Synthesis of High Specific Activity 125I-T3 and 125I-T4 3.3 Free Iodide Estimation 3.4 Purification of Tracers from Iodide by Paper Electrophoresis 3.5 T3 and T4 Extraction from Plasma Samples 3.6 Preparation of T3 and T4 Extraction Columns for Tissue Samples 3.7 T3 and T4 Extraction from Tissue Samples 3.8 Radioimmunoassay for T3 Determination 3.9 Radioimmunoassay for T4 Determination 3.10 Radioimmunoassay Results 4 Notes References Chapter 4: Generation of a Mouse Model for the Study of Thyroid Hormones Regulatory Effect on the Immune System 1 Introduction 2 Materials 2.1 Animal Models 2.2 Cell Culture 2.3 Reagents, Solutions, and Preservation Media 2.4 Protein Extraction 2.5 Western Blotting 2.6 Immunohistochemistry 2.7 Cytokines and Glucose Quantification and Gene Expression Analysis 2.8 Transient Transfections and Luciferase Assays 2.9 ChIP Assays 3 Methods 3.1 Generation of Hypothyroid, Euthyroid, and Hyperthyroid Mice 3.2 Cell Culture 3.3 Generation of Endotoxemia in Animal Models 3.4 Sample Collection from Animal Models 3.5 Immunohistochemistry Analysis of Macrophages Immune Population 3.6 Protein Extraction 3.7 Western Blotting 3.8 Cytokine and Chemokine Quantification 3.9 Aspartate Aminotransferase (ATS) Activity 3.10 Real-Time Quantitative RT-PCR (qRT-PCR) to Gene Expression Analysis 3.11 Transient Transfections and Luciferase Assays 3.12 ChIP Assays 4 Notes References Chapter 5: Analysis of Apoptosis by Thyroid Hormone Induction 1 Introduction 2 Materials 2.1 Cell Culture and Treatments 2.2 Cell Extracts, Protein Quantification, and SDS/Polyacrylamide Gel (SDS/PAGE) 2.3 Immunoblotting and PARP Detection 2.4 PI Staining, Fluorescein Isothiocyanate-Conjugated Annexin V (Annexin V-FITC)/PI Double Staining, TMRM Assay and Flow Cyto... 3 Methods 3.1 Cell Culture and Treatments 3.2 Detection of PARP Cleavage by Western Blotting 3.3 Quantification of the Sub-G1 Hypodiploid Cell Population by PI Staining 3.4 Quantification of Apoptosis Based on the Redistribution of Phosphatidylserine to the Outer Leaflet of Plasma Membrane, by ... 3.5 Quantification of Loss of Mitochondrial Inner Membrane Potential by TMRM Staining 4 Notes References Chapter 6: Immunophenotyping of Leukocytes in Brain in Hypothyroid Mice 1 Introduction 2 Materials 2.1 Generation of Hypothyroid Mice 2.2 Generation of Cerebral Malaria Infections 2.3 Isolation and Preparation of Mouse Brain Cells 2.4 Cell Surface Staining 2.5 Equipment and Supplies 3 Methods 3.1 Generation of Hypothyroid Mice 3.2 Generation of Cerebral Malaria Infections 3.3 Isolation and Preparation of Mouse Brain Cells 3.4 Cell Surface Staining of Brain-Infiltrated Leukocytes 3.5 Samples Acquisition 3.5.1 Compensation Procedures 3.5.2 Gate Setting Strategies 4 Notes References Chapter 7: Immunophenotyping of Hematopoietic Cells in the Spleen in Hypothyroid Mice 1 Introduction 2 Materials 2.1 Generation of Hypothyroid Mice 2.2 Generation of Cerebral Malaria Infections 2.3 Isolation and Preparation of Mouse Splenic Hematopoietic Cells 2.4 Cell Surface Staining 2.5 Equipment and Supplies 3 Methods 3.1 Generation of Hypothyroid Mice 3.2 Generation of Cerebral Malaria Infections 3.3 Isolation and Preparation of Mouse Spleen Cells 3.4 Cell Surface Staining of Hematopoietic Spleen Cells 3.5 Samples Acquisition 3.5.1 Compensation Procedures 3.5.2 Gate Setting Strategies 4 Notes References Chapter 8: Mouse Embryonic Fibroblasts Reprogramming to Induced Pluripotent Stem Cells by T3 1 Introduction 2 Materials 2.1 Primary Cultures and Cells Lines 2.2 Cell Culture Media and Reagents 2.3 Viral Transduction, MEFs Infection and Reprogramming 2.4 Colony Quantification 2.5 Quantitative Real-Time PCR 2.6 Plastic Material 2.7 Instruments 3 Methods 3.1 Harvesting and Culturing MEFs 3.2 Culturing the Packaging Cell Line for Viral Production (Fugene 6 Method) and Infecting MEFs 3.3 MEFs Reprogramming 3.4 Colony Staining and Quantification 3.5 iPSC Colony Expansion 3.6 Quantitative Real-Time PCR 4 Notes References Chapter 9: DNA Methylation Analysis by Bisulfite Pyrosequencing of Mouse Embryonic Fibroblasts with Reprogramming Enhanced by ... 1 Introduction 2 Materials 2.1 Primer and Assay Design 2.2 DNA Extraction 2.3 Bisulfite Conversion 2.4 Alternative Conversion: Oxidative Bisulfite Conversion 2.5 DNA Amplification 2.6 DNA Amplification Gel Check 2.7 Pyrosequencing 3 Methods 3.1 Primer and Assay Design 3.1.1 Primer Design 3.1.2 Assay Design 3.2 Pyrosequencing Run Export 3.3 Pyrosequencing Assay Controls 3.4 DNA Extraction 3.5 Bisulfite Conversion 3.6 Alternative Conversion: Oxidative Bisulfite Conversion 3.7 DNA Amplification 3.8 DNA Amplification Gel Check 3.9 Pyrosequencing 4 Notes References Part III: Thyroid Hormones Receptors Chapter 10: Analysis of the Pattern of RNA Expression in the Skin of TR-Deficient Mice By RNA-seq 1 Introduction 2 Materials 2.1 Skin Tissue Preservation in RNAlater 2.2 RNA Extraction from Mice Skin Preserved in RNAlater Solution 2.3 RNA Quantification and Integrity Assessment 2.4 Library Preparation and Library Size Assessment 2.5 Transcriptome Sequencing 2.6 Bioinformatic Analyses 3 Methods 3.1 Tissue RNA Preservation in RNAlater 3.2 RNA Extraction from Skin Tissue Preserved in RNAlater 3.3 RNA Quality Assessment and Quantification 3.4 Library Preparation and Library Size Assessment 3.5 Transcriptome Sequencing 3.6 Bioinformatic Analyses 4 Notes References Chapter 11: Validation of miR-21, miR-31, miR-34A, and miR-203 Changes by Quantitative Polymerase Chain Reaction in Isolated K... 1 Introduction 2 Materials 2.1 Animals 2.2 Skin Digestion 2.3 Isolation of Keratinocytes from Skin Epidermis 2.4 Quantitative Real-Time PCR Assays from miRNA 2.4.1 Purification of Total RNA Including miRNA 2.4.2 RNA Quality and Concentration Determinations 2.4.3 Reverse Transcription 2.4.4 miRNA PCR Amplification 3 Methods 3.1 Skin Digestion 3.2 Isolation of Keratinocytes from Skin Epidermis 3.3 Quantitative Real-Time PCR Assays from miRNA 3.3.1 Purification of Total RNA Including miRNA 3.3.2 RNA Quality and Concentration Determinations 3.3.3 Reverse Transcription 3.3.4 PCR Amplification 3.3.5 Normalization and Analysis Methods 4 Notes References Part IV: Thyroid Hormone Transporter MCT8 Chapter 12: Magnetic Resonance Imaging Techniques for Investigating the MCT8-Deficient Brain in Murine Disease Models 1 Introduction 2 Materials 2.1 Time-of-Flight Magnetic Resonance Angiography (TOF-MRA) 2.2 Diffusion Tensor Imaging (DTI) 3 Methods 3.1 TOF-MRA 3.2 DTI 4 Notes References Part V: Zebrafish Model Chapter 13: Experimentally Manipulating the Thyroid Hormone Axis in Zebrafish 1 Introduction 2 Materials 3 Methods 3.1 Troubleshooting 4 Notes References Part VI: Clinical Studies Chapter 14: Review of Surgical Interventions in the Thyroid Gland: Recent Advances and Current Considerations 1 Introduction 2 Thyroid Diseases 3 Types of Surgery on the Thyroid Gland 4 Key Points of Thyroid Surgery 5 Complications and Postoperative Considerations in Thyroid Gland Surgery 6 Indications for Thyroid Surgery 7 Techniques and Recent Advances in Thyroid Surgery 8 Outcomes and Effectiveness in Thyroid Gland Surgery 9 Ongoing Research and Emerging Areas of Study 10 Conclusions References Chapter 15: Immunohistochemical Study Protocol in Thyroid Pathology 1 Introduction 1.1 Antibodies in Normal and Neoplastic Thyroid Parenchyma 1.2 Molecular Immunohistochemical Markers 2 Materials 2.1 Management of Surgical Specimens 2.2 Inmunohistochemical Solutions 2.3 Immunocytochemical Solutions 3 Methods 3.1 Immunohistochemical Study 3.1.1 Management of Surgical Specimens 3.1.2 Sample Processing 3.1.3 Immunohistochemistry Protocol 3.1.4 Mounting for Visualization 3.2 Immunocytochemical Study 3.2.1 Sample Processing from Fine Needle Aspirations of the Thyroid 3.2.2 Staining 4 Notes References Chapter 16: Thyroid Hormones Determination in Euthyroid and LT4-Treated Patients During COVID-19 Hospitalization 1 Introduction 1.1 Patients ́ Selection 1.2 Risk Factors Selected to Be Included in the Study 1.2.1 Age 1.2.2 Gender 1.2.3 Obesity 1.2.4 Diabetes Mellitus 1.2.5 Chronic Respiratory Disease Chronic Obstructive Pulmonary Disease (COPD) Asthma 1.2.6 Cardiovascular Disease Hypertension Cardiovascular Disease (CVD) 1.2.7 Immunocompromised States: Cancer 2 Materials 3 Methods 3.1 TSH Measurement 3.2 Free Thyroxine (FT4) Measurement 3.3 Free Triiodothyronine (FT3) Measurement 3.4 Results Interpretation 4 Notes References Index