Mitochondrial Medicine: Volume 2: Assessing Mitochondria (Methods in Molecular Biology, 2276)

Preface
Contents
Contributors
Chapter 1: Mitochondrial Dysfunction in Mitochondrial Medicine: Current Limitations, Pitfalls, and Tomorrow
	1 Introduction
	2 Methods to Assess Mitochondrial Dysfunctions
		2.1 Basis in Bioenergetic
		2.2 Analyses of Maximal ETC Activities
		2.3 Structural Analyses: BN-PAGE
		2.4 Functional Analyses: Respiration Rates
			2.4.1 Devices
				The O2K Oxygraph
				The Seahorse  XF
			2.4.2 Permeabilized Tissues and Cells
			2.4.3 Intact Cells
		2.5 Mitochondrial Membrane Potential
		2.6 Metabolites Dosages and Metabolomics
			2.6.1 Metabolite Measurements
			2.6.2 Metabolomics
	3 Discussion
	References
Chapter 2: Preparation of ``Functional´´ Mitochondria: A Challenging Business
	1 Introduction
	2 Isolated Mitochondria: Addressing Composition and Function
	3 First Step: The Inevitable
	4 Isolating ``Intact´´ Mitochondria: A Challenging Business
	5 Reproducibility and Quality Control: Guarantee for Successful Analysis
	6 Conclusion
	References
Chapter 3: Isolation and Quality Control of Functional Mitochondria
	1 Introduction
	2 Materials
		2.1 Mitochondria Fractionation
		2.2 Linear Saccharose Gradient
		2.3 Marker Enzyme Assays
			2.3.1 Succinate Dehydrogenase (SDH) Assay
			2.3.2 Acidic Phosphatase Assay
			2.3.3 Basic Phosphatase Assay
			2.3.4 Catalase Assay
			2.3.5 Glucose-6-Phospatase (G-6-Pase) Assay
			2.3.6 JC-1 Uptake Assay
	3 Methods
		3.1 Subcellular Fractionation
			3.1.1 Preparation of Linear Saccharose Gradient
			3.1.2 Homogenization
			3.1.3 Differential Centrifugation
			3.1.4 Isopycnic Density Gradient Centrifugation
		3.2 Protein Measurement
		3.3 Marker Enzyme Assays
			3.3.1 Mitochondria: Succinate Dehydrogenase (SDH) (See Ref. 7)
			3.3.2 Lysosomes: Acid Phosphatase (See Ref. 8)
			3.3.3 Plasma Membrane: Basic Phosphatase (See Ref. 8)
			3.3.4 Peroxisome: Catalase (See Ref. 9)
			3.3.5 Endoplasmic Reticulum: Glucose-6-Phophatase (See Ref. 10)
			3.3.6 Mitochondria (Functional Integrity): JC-1 Uptake Assay (See Ref. 11)
	4 Notes
	References
Chapter 4: Purification of Functional Platelet Mitochondria Using a Discontinuous Percoll Gradient
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Optional Solutions
		2.3 Percoll Gradient Preparation
		2.4 Blood Collection Preparation
	3 Methods
		3.1 Platelet Isolation
		3.2 Crude Mitochondrial Extraction
		3.3 Mitochondria Purification
		3.4 Mitochondrial Yield by Flow Cytometry
		3.5 Mitochondrial Yield by Bicinchoninic Acid Assay (BCA)
	4 Notes
	References
Chapter 5: Mechanical Permeabilization as a New Method for Assessment of Mitochondrial Function in Insect Tissues
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Substrates
		2.3 Uncoupler
		2.4 Inhibitors
	3 Methods
		3.1 Insects
		3.2 Sample Preparation
			3.2.1 A. aegypti Head Dissection (Fig. 1a-c)
			3.2.2 D. melanogaster Thorax Dissection (Fig. 1d-g)
		3.3 Mechanical Permeabilization and Respirometry Measurements Protocols
			3.3.1 A. aegypti Heads
			3.3.2 D. melanogaster Thorax
		3.4 Data Interpretation
		3.5 Protocols Validation
			3.5.1 A. aegypti Heads
			3.5.2 D. melanogaster Thorax
		3.6 Troubleshooting
	4 Notes
	References
Chapter 6: Analysis of Mitochondrial Retrograde Signaling in Yeast Model Systems
	1 Introduction
	2 Materials
		2.1 Cells and Growth Media
		2.2 Buffers, Reagents, and Labware
			2.2.1 Real-Time  PCR
			2.2.2 Protein Extraction
			2.2.3 SDS-Polyacrylamide Gel Electrophoresis
			2.2.4 Western Blotting
	3 Methods
		3.1 Analysis of RTG-Dependent Target Gene mRNAs
			3.1.1 Cell Growth and Low pH Shift
			3.1.2 RNA Isolation and Real-Time  PCR
		3.2 Analysis of CIT2p-GFP
			3.2.1 Cell Lysis
			3.2.2 Western  Blot
			3.2.3 Fluorescence Microscopy
		3.3 Analysis of Rtg3p Phosphorylation
			3.3.1 Total Yeast Protein Extraction
			3.3.2 Western Blotting
	4 Notes
	References
Chapter 7: Native Gel Electrophoresis and Immunoblotting to Analyze Electron Transport Chain Complexes
	1 Introduction
	2 Materials
		2.1 Preparation for Native Electrophoresis (See Note 1)
		2.2 Preparation for Western Blotting
	3 Methods
		3.1 Protocol Native Electrophoresis
		3.2 Sample Preparation
		3.3 Immunoblotting
	4 Notes
	References
Chapter 8: Measuring Mitochondrial Hydrogen Peroxide Levels and Glutathione Redox Equilibrium in Drosophila Neuron Subtypes Us...
	1 Introduction
		1.1 Redox Sensitive Green Fluorescent Proteins to Detect Glutathione Redox Equilibrium and H2O2 Levels
		1.2 Directing Cell-Specific Gene Expression in Drosophila
		1.3 Capturing and Measuring Oxidation Status of Mito-roGFP2-Grx1 and Mito-roGFP-Orp1 in Drosophila Dopaminergic Neuron Mitocho...
	2 Materials
		2.1 Drosophila Stocks
		2.2 Food Preparation
		2.3 Anesthetizing Flies
		2.4 Drosophila Brain Dissection and Immunolabeling
		2.5 Mounting the Brains on Microscope Slides
		2.6 Dopaminergic Neuron Cluster Image Capture
		2.7 Measuring roGFP Fluorescence Reporters
	3 Methods
		3.1 Standard Corn Meal Molasses Drosophila Food Preparation
		3.2 Drosophila Stocks and Maintenance
		3.3 Crossing Drosophila to Obtain Progeny Expressing Mito-roGFP2-Grx1 or Mito-roGFP2-Orp1 in Dopaminergic Cells
		3.4 Drosophila Brain Dissection
		3.5 Tyrosine Hydroxylase Immunolabeling
		3.6 Mounting Drosophila Brains on Microscope Slides
		3.7 Capturing Images of roGFP2s in Dopaminergic Neuron Mitochondria
		3.8 Image Processing
		3.9 Data Analysis
	4 Notes
	References
Chapter 9: Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Cells and Reagents
	3 Methods
		3.1 Optimization of Saponin Concentration and Cell Density
		3.2 Electron Flux Assay Mix Preparation
		3.3 Cell Suspension
		3.4 Step-by-Step Instructions
		3.5 Analysis of the Results
		3.6 Interpretation of the Results
	4 Notes
	References
Chapter 10: Whole-Cell and Mitochondrial dNTP Pool Quantification from Cells and Tissues
	1 Introduction
	2 Materials
		2.1 General Equipment
		2.2 Whole-Cell dNTP Isolation from Cells
		2.3 Whole-Cell dNTP Isolation from Tissues
		2.4 Mitochondrial dNTP Isolation
		2.5 Binding, Polymerization, and Detection
	3 Methods
		3.1 Whole-Cell dNTP Isolation from Cells
		3.2 Whole-Cell dNTP Isolation from Tissues
		3.3 Mitochondrial dNTP Isolation
		3.4 Affinity Capture of Oligonucleotides (Fig. 1 I)
		3.5 Preparation of Standard Series and Sample Dilutions
		3.6 Polymerase Reaction and Detection (Fig. 1 II-III)
		3.7 Data Analysis
	4 Notes
	References
Chapter 11: Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitocho...
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Fluorescence Imaging
	3 Methods
		3.1 Rat Heart Extraction
		3.2 Extraction of Cardiac Mitochondria
		3.3 Coverslip Preparation
		3.4 Experimental Procedure for Mitochondria Imaging
		3.5 Imaging of Mitochondria
		3.6 Treatment of Images with ``Fiji´´
		3.7 Mitochondria Follow-Up with ``TrackMate´´
		3.8 Python Scripts for Quantitative Analyses
	4 Notes
	References
Chapter 12: Investigation of Mitochondrial ADP-Ribosylation Via Immunofluorescence
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Seeding
		3.2 Treatment of Cells to Increase Mitochondrial ADP-Ribosylation
		3.3 Fixation and Permeabilization
		3.4 Blocking and Immunostaining
		3.5 DAPI Staining and Mounting
		3.6 Analysis/Quantification of the Immunofluorescence Signal
	4 Notes
	References
Chapter 13: Assessment of Mitochondrial Ca2+ Uptake
	1 Introduction
	2 Materials
		2.1 Chemical Fluorescent Indicator Components
		2.2 Components for the Use of Genetically Encoded Ca2+ Indicators
		2.3 Components for Mitoplast Isolation and Patch-Clamp Recordings
		2.4 Components for Measuring Cellular O2 Consumption Rates
	3 Methods
		3.1 Measuring Indirect Ca2+ Uptake Via Calcium Green
		3.2 Measuring Direct Mitochondrial Ca2+ Uptake Via Fura-2 or Rhod-2
		3.3 Transfection of Genetically Encoded Ca2+ Indicators
		3.4 Real-Time Recordings of [Ca2+]cyto and [Ca2+]mito
		3.5 Mitoplast Patch-Clamping Recording
		3.6 Assessing Ca2+-Dependent Changes in Mitochondrial Metabolism
	4 Notes
	References
Chapter 14: Assessment of Mitochondrial Membrane Potential and NADH Redox State in Acute Brain Slices
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents for NADH Determination
		2.3 Reagents for Mitochondrial Membrane Potential Determination
	3 Methods
		3.1 Acute Brain Slices Preparation Procedure
		3.2 NADH Determination Procedure
		3.3 Mitochondrial Membrane Potential Determination Procedure
	4 Notes
	References
Chapter 15: Evaluation of Mitochondria Content and Function in Live Cells by Multicolor Flow Cytometric Analysis
	1 Introduction
	2 Material
		2.1 Preparation of the Single-Cell Suspension
		2.2 Detection of Mitochondrial Mass, Activity, and ROS (See Notes 1 and 2)
		2.3 Surface Marker Staining
	3 Methods
		3.1 Tissue Harvest and Generation of the Single-Cell Suspension
		3.2 Quantification of Mitochondria Mass and Membrane Potential
		3.3 Surface Marker Staining
		3.4 Reactive Oxygen Species Detection
		3.5 Data Analysis
	4 Note
	References
Chapter 16: Analysis of Mitochondrial Dysfunction During Cell Death
	1 Introduction
	2 Materials
		2.1 Assessment of Cytochrome c Release from the Mitochondria of Apoptotic Cells
		2.2 Assessment of the Mitochondrial Membrane Potential Alterations in Apoptosis
		2.3 Assessment of Oxygen Consumption in Intact Apoptotic Cells
		2.4 Assessment of Mitochondrial Respiration in Apoptotic Cells with Digitonin-Permeabilized Plasma Membrane
		2.5 Assessment of Mitochondrial Production of Superoxide Radical
	3 Methods
		3.1 Evaluation of Cytochrome c Release from the Mitochondria of Apoptotic Cells
		3.2 Assessment of the Mitochondrial Membrane Potential
		3.3 Assessment of Oxygen Consumption in Intact Apoptotic Cells
		3.4 Assessment of Mitochondrial Respiration in Apoptotic Cells with Digitonin-Permeabilized Plasma Membrane
		3.5 Assessment of Mitochondrial Superoxide Radical Production
	4 Notes
	References
Chapter 17: Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes
	1 Introduction
	2 Materials (See Note 1)
		2.1 General Solutions for Running BN  Gels
		2.2 Compromise Buffer (CB) for Incubations and BN Gel Extraction
		2.3 Solutions for In-Gel Complex V Assay
	3 Methods
		3.1 Mitochondrial Incubation and Sample Extraction for BN Gel Analysis
		3.2 BN Gel Electrophoresis
		3.3 Cx-V In-Gel Assay
	4 Notes
	References
Chapter 18: Patch-Clamp Recording of the Activity of Ion Channels in the Inner Mitochondrial Membrane
	1 Introduction
	2 Materials
		2.1 Stock Solutions
		2.2 Mitochondria Isolation
		2.3 Mitoplast Preparation
		2.4 Patch-Clamp Recordings
		2.5 Single-Mitoplast  PCR
	3 Methods
		3.1 Mitochondria Isolation
		3.2 Mitoplast Preparation
		3.3 Patch-Clamp Recordings
		3.4 Single-Mitoplast  PCR
	4 Notes
	References
Chapter 19: Assessment of Mitochondrial Protein Glutathionylation as Signaling for CO Pathway
	1 Introduction
	2 Materials
		2.1 Mitochondria Isolation from Cell Culture
		2.2 Mitochondria Isolation from Brain Cortex
		2.3 CO Treatment
		2.4 Immunoprecipitation
		2.5 Immunoblotting
	3 Methods
		3.1 CO Treatment
		3.2 Mitochondria Isolation from Cell Culture
		3.3 Mitochondria Isolation from Brain Tissue
		3.4 Immunoprecipitation of Proteins in Isolated Mitochondria
		3.5 Immunoblotting
	4 Notes
	References
Chapter 20: 3D Optical Cryo-Imaging Method: A Novel Approach to Quantify Renal Mitochondrial Bioenergetics Dysfunction
	1 Introduction
		1.1 Mitochondrial Dysfunction
		1.2 Optical Metabolic Imaging
	2 Materials
		2.1 Optical Components
		2.2 Mechanical Components
		2.3 Sample Preparation Requirements
	3 Methods
		3.1 Black Mounting Medium (BMM) Preparation
		3.2 Kidney Sample Preparation
		3.3 Cryo-Imaging Procedure
			3.3.1 Mounting
			3.3.2 Image Acquisition
		3.4 Image Processing
		3.5 Data Interpretation
	4 Notes
	References
Chapter 21: Simultaneous Quantification of Mitochondrial ATP and ROS Production Using ATP Energy Clamp Methodology
	1 Introduction
		1.1 2DOG ATP Energy Clamp
		1.2 Use of the 2DOG ATP Energy Clamp to Quantify ATP Production in Isolated Mitochondria and Simultaneous Assessment of H2O2 P...
		1.3 Utilization in Recent Studies
	2 Materials
		2.1 Isolation of Mitochondria
		2.2 Assay Incubation
		2.3 Sample Preparation for NMR Spectroscopy
		2.4 NMR Spectroscopy
	3 Methods
		3.1 Isolation of Mitochondria from Tissue
		3.2 Further Purification of Isolated Mitochondria (See Note 6)
		3.3 Assay Incubation
		3.4 Fluorescent Assessment of H2O2 Production
		3.5 Processing the Well Contents for NMR-Based ATP Assay
		3.6 NMR Spectroscopy for Quantifying ATP Production
		3.7 Calculation of ATP Production Rates
	4 Notes
	References
Chapter 22: High-Throughput Image Analysis of Lipid-Droplet-Bound Mitochondria
	1 Introduction
		1.1 Background
		1.2 Training Images Used for WEKA Training  Set
		1.3 Mitochondria Morphological Parameters That Describe Mitochondrial Networking
		1.4 Segmentation of Cytosolic Mitochondria Versus Peridroplet Mitochondria Reveals Significant Differences in H2O2 Levels
			1.4.1 Trainable WEKA Segmentation Is Significantly More Effective at Mitochondrial Segmentation Compared to Conventional Thres...
			1.4.2 Validation of Machine-Learning Classifier
		1.5 Benefits of a Software Macro
	2 Materials
		2.1 Primary Brown Adipocytes Culture
			2.1.1 Primary Preadipocytes Isolated from BAT Growth Media
			2.1.2 Primary Preadipocytes Differentiation Media to  BA
			2.1.3 Adenoviral Transduction to Deliver H2O2 Reporters
		2.2 Microscopy
			2.2.1 Basic Requirements of a Confocal Microscopy System
			2.2.2 Basic Requirements of Images
			2.2.3 Fluorophores and Imaging Settings
		2.3 Analysis
			2.3.1 Training a WEKA Classifier
			2.3.2 Using a Macro
	3 Methods
		3.1 Primary Pre-adipocytes Transfer to an Imaging Plate
		3.2 Transduction of BA with Adenovirus Encoding H2O2 Reporters
		3.3 Microscopy
		3.4 Image Analysis (See Note 12)
			3.4.1 Brown Adipocyte (BA)  Analysis
			3.4.2 Using WEKA Classifiers
	4 Notes
	References
Chapter 23: Cell Energy Budget Platform for Multiparametric Assessment of Cell and Tissue Metabolism
	1 Introduction
	2 Materials
		2.1 Critical Equipment
		2.2 Cells and Reagents
		2.3 Solutions
	3 Methods
		3.1 T-ECA and L-ECA Assays
		3.2 OCR Assay
			3.2.1 Rates of Cellular Oxygen Consumption
			3.2.2 Conduction of Measurements Using Animal Tissue
		3.3 ATP Analysis
		3.4 Total Protein (Biomass) Analysis
		3.5 Data Processing and CEB Analysis
		3.6 Examples and Interpretation of CEB  Data
		3.7 Conclusions
	4 Notes
	References
Chapter 24: Fluorescence-Based Assay For Measuring OMA1 Activity
	1 Introduction
	2 Materials
		2.1 Fluorogenic Reporter Peptide (AFRATDHG): Serves as the Substrate for the OMA1 Assay
		2.2 Protein Sample/Unknown
		2.3 TPEN (Used as an OMA1 Inhibitor)
		2.4 OMA1 Buffer
		2.5 96-Well Microplate
		2.6 Plate Reader
	3 Methods
		3.1 Procedure
		3.2 Analysis
	4 Notes
	References
Chapter 25: Studying Mitochondrial Network Formation by In Vivo and In Vitro Reconstitution Assay
	1 Introduction
	2 Materials
		2.1 Observation of MNF in Cells
		2.2 In Vitro Reconstitution System
			2.2.1 Protein Purification
			2.2.2 Mitochondria Isolation
			2.2.3 In Vitro Reconstitution Assay
	3 Methods
		3.1 Cell Transfection and Observation
		3.2 In Vitro Reconstitution System
			3.2.1 Motor Protein Purification
			3.2.2 Mitochondrial Isolation
			3.2.3 Flow Chamber Assembly
			3.2.4 Preparation of Polymerized Microtubule Filaments
			3.2.5 Gliding Assays to Confirm the Activity of Purified Motor Proteins
			3.2.6 In Vitro Reconstitution of MNF
	4 Notes
	References
Chapter 26: Extraction of Functional Mitochondria Based on Membrane Stiffness
	1 Introduction
	2 Materials
	3 Methods
		3.1 Design and Fabrication of the Microfluidics-Based Cell Shredder
		3.2 Cell Culture
		3.3 Cell Disruptions and Mitochondria Extraction
			3.3.1 Optimization of Mitochondria Extraction by the Dounce Homogenizer
			3.3.2 Optimization of Mitochondria Extraction by the Microscale Cell Shredder
		3.4 Cell Disruption Efficiency Determined by Flow Cytometer
		3.5 Determination of Total Protein Yield (Bradford Assay)
		3.6 Characterization of Mitochondrial Membrane Potential
		3.7 Characterization of Mitochondrial Integrity (Citrate Synthase Assay)
	4 Notes
	References
Chapter 27: A Protocol for Untargeted Metabolomic Analysis: From Sample Preparation to Data Processing
	1 Introduction
	2 Materials
	3 Method
		3.1 LC/MS Protocol
		3.2 LC Method Setup
		3.3 MS Method Setup
		3.4 Acquisition Sequence Order
		3.5 Biofluid Sample Preparation and Metabolite Extraction for Positively Charged Polar Metabolites
		3.6 Data Analysis
	4 Notes
	Box 1 Definitions of Common Terms
	References
Chapter 28: A Method for Analysis of Nitrotyrosine-Containing Proteins by Immunoblotting Coupled with Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 2D Electrophoresis
		2.2 Immunoblotting
		2.3 Staining
		2.4 Chemiluminescence Detection
		2.5 In-Gel Digestion and nLC-MS/MS Analysis
	3 Methods
		3.1 Sample Preparation and Isoelectric Focusing
		3.2 SDS-PAGE
		3.3 Immunoblotting and Chemiluminescence Detection
		3.4 In-Gel Digestion
		3.5 Analysis of Tryptic Digests from Spots with nLC-MS/MS
	4 Notes
	References
Chapter 29: In Vivo Visualization and Quantification of Mitochondrial Morphology in C. elegans
	1 Introduction
	2 Materials
	3 Methods
		3.1 Culture of C. elegans
		3.2 Microscopy Slide Preparation
		3.3 Imaging of Mitochondria
		3.4 Automated Image Analysis (See Note 11)
		3.5 Mitochondrial Quantification
	4 Notes
	References
Chapter 30: Assessing Impact of Platinum Complexes on Mitochondrial Functions
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Primer
		2.3 Solutions
		2.4 Fluorescent Dyes
		2.5 Cell Culture Components
	3 Methods
		3.1 Synthesis of TPP+-PtII (a)
		3.2 Synthesis of TPP+-PtII (b)
		3.3 Synthesis of TPP+-PtIV (a)
		3.4 Synthesis of TPP+-PtIV (b)
		3.5 Mitochondrial Uptake
		3.6 Determination of Mitochondrial Superoxide (mtSOX)
		3.7 Mitochondrial Membrane Potential (JC-1 Assay)
		3.8 Mitochondrial Morphology
		3.9 mtDNA Damage
		3.10 Transcription of Mitochondrial Genes
		3.11 Mitochondrial Bioenergetics
			3.11.1 Oxygen Consumption Rate (OCR)
			3.11.2 Extracellular Acidification Rate (ECAR)
		3.12 Proteins Relevant to Mitochondrion-Mediated Apoptosis
	4 Notes
	References
Chapter 31: In Silico Modeling of the Mitochondrial Pumping Complexes with Markov State Models
	1 Introduction
	2 Ransac Model of the bc1 Complex
	3 Markov State Models
	4 States and Reactions
	5 Estimation of Rate Constants
	6 The Membrane Potential and Charge Transfer Reactions
	7 Thermodynamics and Interactions
	8 Simulations
	9 Protein Conformation
	References
Chapter 32: Monitoring the Mitochondrial Presequence Import Pathway In Living Mammalian Cells with a New Molecular Biosensor
	1 Introduction
	2 Materials
		2.1 Recombinant Vectors
			2.1.1 pCI-Neo Mammalian Expression Vectors Encoding Probes 1, 2, and 3
			2.1.2 Recombinant Lentiviral Vector Encoding Probe 1
		2.2 Materials and Reagents
			2.2.1 Equipment and Cell Culture Material
			2.2.2 Cell Culture Media, Buffers, Reagents
			2.2.3 Transfection Reagents
			2.2.4 Chemical Compounds
			2.2.5 Immunocytochemistry
	3 Methods
		3.1 Cell Culture: Seeding, Transfection or Lentiviral Transduction, Induction of the Probes
			3.1.1 HEK293T Cells
			3.1.2 Human Primary Fibroblasts
			3.1.3 Mouse Primary Cortical Neurons
		3.2 Mitochondrial Import Reporter Assay
			3.2.1 Induction of the Mitochondrial Import Probe
			3.2.2 RGFP Fluorescence Assay
			3.2.3 Luciferase Bioluminescence Reporter Assay (Rluc)
			3.2.4 Analysis of the Results
	4 Notes
	References
Index