Title Page
ABSTRACT
국문 초록
PREFACE
Contents
LIST OF ABBREVIATIONS 23
CHAPTER Ⅰ. INTRODUCTION 26
Ⅰ. Introduction 27
CHAPTER Ⅱ. LITERATURE REVIEW 29
2.1. FTIR microscopy 30
2.1.1. Electromagnetic waves and InfraRed 30
2.1.2. Radiation and matter interaction: molecular vibration 30
2.1.3. Infrared spectroscopy 34
2.1.4. Data Assessment: Qualitative/Quantitative Analysis 38
2.1.5. Concept and Application of Infrared Microscopy 41
2.1.6. Structure of Infrared Microscopy 43
2.1.7. Fourier transform infrared image 45
2.1.8. Infrared Spectrum Data Analysis and Application 49
2.1.9. The Future of Infrared Microscopy 55
2.2. Adipose Tissue 57
2.2.1. Adipocytes 57
2.2.2. Adipocyte differentiation 59
2.2.3. Structure and Physiology of White Adipose Tissue 60
2.2.4. Regulation of white fat formation of White Adipose Tissue 62
2.2.5. Structure and Physiology of Brown Adipose Tissue 64
2.2.6. Regulation of brown fat formation of Brown Adipose Tissue 65
2.2.7. Analysis of Characteristics in Adipocytes 66
CHAPTER Ⅲ. DENTIFICATION OF BIOCHEMICAL DIFFERENCES IN WHITE AND BROWN ADIPOCYTES USING FTIR SPECTROSCOPY 70
3.1. Introduction 71
3.2. Materials and Methods 72
3.2.1. Culture and Differentiation 72
3.2.2. Oil-Red-O Staining 73
3.2.3. Reverse Transcription Quantitative Polymerase Chain Reaction Analysis 73
3.2.4. Preparation and Collection of Infrared Spectroscopy Spectra 74
3.2.5. Statistical Analysis 74
3.3. Results 74
3.3.1. Differentiation of 3T3-L1 White Preadipocytes and Immortalized Brown Preadipocytes 74
3.3.2. FTIR Spectroscopy Spectra of Adipocyte Conditioned Media 75
3.3.3. FTIR Spectroscopy Spectra of Adipocytes 78
3.3.4. Discrimination of Adipocytes 80
3.4. Discussion 81
CHAPTER Ⅳ. IDENTIFICATION OF BROWNING IN HUMAN ADIPOCYTES BY PARTIAL LEAST SQUARES REGRESSION (PLSR), INFRARED SPECTRAL BIOMARKERS, AND PARTIAL LEAST SQUARES DISCRIMINANT ANALYSIS (PLS-DA) USING FTIR SPECTROSCOPY 87
4.1. Introduction 88
4.2. Materials and Methods 90
4.2.1. Human Adipose-Derived Stem Cell Culture and Differentiation 90
4.2.2. Oil-Red-O Staining 91
4.2.3. Quantitative Reverse Transcription PCR (qRT-PCR) Analysis 91
4.2.4. Preparation and Collection of FTIR Spectra 92
4.2.5. Partial Least-Squares Regression (PLSR) and Partial Least-Squares Discriminant Analysis (PLS-DA) Modeling 92
4.2.6. Infrared Spectral Biomarkers 93
4.2.7. Statistical analysis 93
4.3. Results 97
4.3.1. PLSR using FTIR Spectra of Established Human Adipocytes 97
4.3.2. PLSR using FTIR Spectra of Human Adipocytes and Human Adipocyte-Conditioned Media on a Slide Glass 99
4.3.3. Comparing PLSRs 102
4.3.4. Infrared Spectral Biomarkers on Human Beige Adipocytes 102
4.3.5. Infrared Spectral Biomarkers on Human Beige Adipocyte-Conditioned Media on a Slide Glass 105
4.3.6. PLS-DA, Classification of Adipocytes and Expression Distribution of Adipogenesis Genes in Adipocytes 105
4.4. Discussion 108
CHAPTER Ⅴ. GENERAL SUMMARY AND CONCLUSIONS 115
REFERENCES 120
APPENDICES 136
A. Applied Sciences. 2022; 12(6):3071 137
B. Photonics. 2023; 10(1):2 147
Table 2.1. Near InfraRed (NIR), Mid InfraRed (MIR), and Far InfraRed (FIR) 35
Table 2.2. The spectral interpretations of absorbed wavenumber 51
Table 2.3. Targets of PPARγ targets and their functions 63
Table 3.1. List of primers (sequence 5'→3') used for qPCR 73
Table 4.1. List of primers used for qRT-PCR 92
Table 4.2. Comparing PLSRs using FTIR spectra of human adipocyte 94
Table 4.3. Summarization of objective, methods, results, and discussion of this study 95
Table 4.4. Inclusion of important wavenumbers in the infrared region for each PLSR. 102
Table 4.5. Infrared spectral biomarkers for browning in human adipocytes in adipocyte/conditioned medium. 104
Figure 2.1. Electromagnetic wave 30
Figure 2.2. Dividing electromagnetic wave depending on energy, wavenumber, and wavelength 31
Figure 2.3. Interaction of radiation and matter 31
Figure 2.4. Mechanical model of a vibration diatomic molecule 32
Figure 2.5. Normal modes of vibration molecular 32
Figure 2.6. IR spectrum. (A) IR-spectrum of water, (B) Lambert and Beer's Law, (C) IR-spectrum of hexane 33
Figure 2.7. Principle layout of IR spectrometer 33
Figure 2.8. Procedure of IR spectrum. (A) Michelson interferometer, (B) the result of Fourier transformation, (C) transmission spectrum T(ν) =... 35
Figure 2.9. Major compartments of FTIR microscopy. (A) types of source, (B) types of beam splitter, (C) types of detector depending on wavelength range 36
Figure 2.10. Spectrum related to resolution 37
Figure 2.11. IR spectrum of chemical function groups 39
Figure 2.12. Absorbance and calibration in FTIR. (A) A = log(I₀ / I) = ε·c·l, (B) univariate calibration, (C) multivariate calibration 41
Figure 2.13. FTIR microscopy's application. (A) particle analysis, (B) contamination analysis, (C) polymer multilayer analysis, (D) tissue analysis 42
Figure 2.14. Defect analysis in polymer film using FTIR microscopy. (A) selecting defect area by light microscopy, (B) getting IR spectrum in suspecting... 43
Figure 2.15. Stand-alone FTIR microscopy appearance and layout 44
Figure 2.16. Combined FTIR microscopy appearance and layout 45
Figure 2.17. Principle of FTIR imaging. (A) to get IR image sequentially in stand-alone FTIR microscopy (middle) and simultaneously in combined FTIR... 46
Figure 2.18. FTIR band related to CH₃ anti-/symmetric stretching, CH₂ anti-/symmetric stretching 47
Figure 2.19. Chemical image by FTIR microscopy during incubation. (A) temporal variations in various biomolecule domains by infrared spectroscopy, (B)... 48
Figure 2.20. FTIR Image of plant tissue (wheat seed) 49
Figure 2.21. Spectral pre-process of FTIR microspectroscopic image 50
Figure 2.22. Various tools in metabolism 52
Figure 2.23. Publications related to FTIR(dark) and Raman(light). (A) by organ, (B) by year 53
Figure 2.24. FTIR diagram controlling temperature and humidity 54
Figure 2.25. How to find biomarkers by FTIR microscopy 56
Figure 2.26. Visual (left) and FTIR images of White Adipose Tissue on lipid hydrocarbon (middle) and amide I (right) basis 56
Figure 2.27. Detecting micronucleus in RBC by FTIR microscopy 57
Figure 2.28. Fat distribution influences risks associated with obesity in human 58
Figure 2.29. Molecular signature of adipocyte lineages 58
Figure 2.30. Differentiation into white, beige, or brown adipocytes 59
Figure 2.31. Transcription factors are induced during adipocyte differentiation 60
Figure 2.32. Distribution of white adipose tissue in the human body 61
Figure 2.33. A complex transcriptional cascade regulates adipogenesis 62
Figure 2.34. Regulation of adipogenesis by extracellular factors 64
Figure 2.35. 2-[¹⁸F]fluoro-2-deoxyglucose (FDG) uptake into Brown Adipose Tissue in summer and winter by FDG-PET scans 65
Figure 3.1. Characteristics of adipocytes assessed by Oil-Red-O and expression of adipocyte markers. (A) Expression of white adipocyte markers in white... 76
Figure 3.2. FTIR spectra of various adipocyte-conditioned media. (A) MWCM (red), WPCM (green) and WAM (blue), (B) MBCM (pink), BPCM (green) and... 77
Figure 3.3. The difference of (A) amide I/amide II and (B) amide I/amide I + amide II of various adipocyte conditioned media. ** p 〈0.01; n.s., not significance. 78
Figure 3.4. FTIR spectra from different types of adipocytes. (A) WPA (red), MWA (pink), (B) BPA (green), MBA (blue). 79
Figure 3.5. Analyses of FTIR spectra on different types of adipocytes. (A) CH₂ sym/asym, (B) amide I/II ratio, and (C) CH₂ asym/CH₃ asym. sym:... 80
Figure 3.6. Discrimination of adipocytes by principal component analysis. (A) Two-dimensional drawing of identification by PCA, (B) three-dimensional drawing... 81
Figure 3.7. Raw spectra of conditioned media [WAM (black), WPCM (red), MWCM (blue), BAM (green), BPCM (yellow), MBCM (orange)] and adipocytes... 83
Figure 3.8. Spectra used in PCA for discrimination of adipocytes 85
Figure 3.9. Cluster by dendrogram for discrimination of adipocytes 85
Figure 4.1. Characteristics of human adipocytes assessed via ORO, expression of human adipogenic/thermogenic markers, and PLSR using FTIR spectra of human... 98
Figure 4.2. Analysis of brown-like adipocyte characteristics of adipocytes in differentiated hBA. (A) The expression ratio of UCP1/FABP4 in... 99
Figure 4.3. Raw spectra of hBA, hPA and hWA on a slide glass. 100
Figure 4.4. Raw spectra of hBA-CM, hPA-CM, and hWA-CM on a slide glass. 100
Figure 4.5. PLSR using FTIR spectra of human adipocytes on a gelatin-coated slide glass and human adipocyte-conditioned media on a slide glass. (A) PLSR... 101
Figure 4.6. Infrared spectral biomarkers of hBA. (A) The differences between mean spectra of hBA and hWA. (B) PCA-LDA cluster vector for hBA. (C) U-test per wavenumber for hBA. (D) Fisher's score per wavenumber for hBA.... 103
Figure 4.7. Infrared spectral biomarkers of hBA-CM. (A) The differences between mean spectra of hBA-CM and hWA-CM. (B) PCA-LDA cluster vector for hBA-... 106
Figure 4.8. Classification of adipocytes and expression distribution of adipogenic genes in adipocytes determined using PLS-DA. (A) Classification of adipocytes using PLS-DA. (B) UCP1's expression distribution using PLS-DA.... 107
Figure 4.9. The 3D scatter plots of PCA-LDA, PCA, and PLS using FTIR spectra of human adipocytes. 109
Figure 4.10. The 3D scatter plots of PCA-LDA, PCA, and PLS using FTIR spectra of human adipocytes conditioned media. 109
Figure 4.11. Test set validation (A) Test set validation of PLSR for hBA. (B) Test set validation of PLSR for hBA on a slide glass. (C) Test set validation of... 110
Figure 4.12. ROC of PLS-DA used in expression distribution of adipogenic genes in adipocytes. 111
Figure 4.13. ROC of PLS-DA used in classification of adipocytes. 114