TABLE OF CONTENTS:
CHAPTER 1
INTRODUCTION………8
1. Thyroid gland generalities………....8
1.1 Thyroid gland, functional unit……….8
1.2 Thyroid hormone synthesis and secretion……….8
1.3 Regulatory cascades in human thyroid……….9
2. Thyroid tumors………13
2.1 Types of thyroid tumors………13
2.2 Thyroid carcinoma………...14
2.2.1 Epidemiology, mortality……….14
2.2.2 Papillary thyroid carcinoma……….16
3. Reactive oxygen species and H2O2………...22
3.1 H2O2 in the thyroid -physiological role………..23
3.2 Role of H2O2 in thyroid mutagenesis ……….25
4. Ionizing Radiation……….27
4.1 Generalities………27
4.2 Ionizing Radiation dose……….28
4.3 Ionizing Radiation induced damage……….31
4.3.1 DNA damage, response and repair………..……..32
5. Radiation and the thyroid………36
5.1 External radiation epidemiological studies……….36
5.2 External radiation – in vitro data………37
5.3 Iodine-131 (131I) and the thyroid………38
5.3.1 131I in thyroid therapeutics………39
5.3.2 Accidental exposure to 131I from nuclear fallouts……….39
5.3.3 131I and the thyroid - in vivo, in vitro data………..45
CHAPTER 2 AIM OF THE CURRENT WORK……….46
CHAPTER 3 MATERIALS AND METHODS……….48
Cell culture………..48
Cell treatments……….49
Western Blot………..50
Cell viability test………..51
DNA damage detection………..51
Cell proliferation assay………53
RNA extraction and purification………54
RNA quantification and quality measurements……….54
Reverse transcription………55
RT-quantitative Polymerase Chain Reaction………55
Next Generation Sequencing………..56
Data visualization………59
Statistical analysis………60
CHAPTER 4 RESULTS………..61
1. Characterization of the effect of TSH in human primary cultured thyrocytes at baseline……….61
Differentiation state, gene expression………61
Iodide uptake………66
Capacity to proliferate………67
DNA damage et baseline………..68
2. Characterization of the effect of TSH on thyrocytes DNA damage following exposure to 131I, γ radiation and H2O2……….71
Cell survival………71
Thyrocyte DNA damage following exposure to 131I, γ radiation and H2O2…….72
P53bp1 co-localized with γ H2AX foci in human thyrocytes confirming DNA double strand breaks……….75
Inhibitors of iodine uptake, NaClO4 and KI, partially alleviated TSH effect on 131I-induced DNA damage………...76
The effect of TSH on 131I-induced DNA damage was observed in PCCL3 cell line………77
Role of NIS in 131I-induced DNA damage in COS-7 /COS NIS-6 cell lines……….78
3. Characterization of the effect of TSH on thyrocytes gene expression profiles following exposure to 131I, γ radiation and H2O2 using Next Generation RNA sequencing………..79
4. Thyrocyte cellular fate following exposure to 131I, γ radiation and H2O2. Role of TSH………96
TSH did not influence DNA damage repair………..96
131I and γ radiation are persistent inhibitors of thyrocyte proliferation in contrast to H2O2, independent to TSH presence………..96
131I and γ radiation did not induce apoptosis but cell cycle arrest via upregulation of p21 in human thyrocytes, independent to TSH presence……99
CHAPTER 5 DISCUSSION, PERSPECTIVES……….100
CHAPTER 6 GENERAL CONSLUSION………111
BIBLIOGRAPHY………113 ANNEXES
