12 research outputs found
H. pylori์ EGFR ์ ํธ์ ๋ฌ์ ๋ฏธ์น๋ ์ํฅ ๋ฐ celecoxib์ ์์๋ฐ์ ์ต์ ๊ธฐ์ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ์ํ๊ณผ, 2012. 8. ๊น๋์.๋ฐฐ๊ฒฝ : Helicobacter pylori (H. pylori)์ ๊ฐ์ผ์ ์ธํฌ์ ์์กด ์กฐ์ ๋ฅ๋ ฅ์ ์ ํดํ์ฌ ์์์ ๋ฐ์์ํ์ ์ฆ๊ฐ์ํจ๋ค. H. pylori ๊ฐ์ผ์ ์ธํฌ๋ด EGFR์ ํ์ฑํ์ ๊ด๋ จ์ด ์์ผ๋ฉฐ, ํ๋ถ๋จ๊ณ์ phosphatidylinositol 3-OH kinase (PI3K)-Akt-Glycogen synthase kinase-3 (GSK3) ์ ๊ฐ์ ๊ฒฝ๋ก๋ฅผ ํ์ฑํ์์ผ ์ธํฌ์ ์์กด๊ณผ ์ด๋์ ์กฐ์ ํ๋ ๊ฒ์ผ๋ก ์๋ ค์ ธ ์๋ค. ๋ํ H. pylori๊ฐ์ผ์ COX-2์ ๊ณผ๋ฐํ์ ์ ๋ฐํ๋๋ฐ, ์ด์ ์ฐ๊ตฌ์์ COX-2 ์ต์ ์ ์ธ celecoxib๊ฐ Akt ์ ํธ์ ๋ฌ๊ฒฝ๋ก๋ฅผ ์ต์ ํ๋ค๊ณ ๋ฐํ ๋ฐ ์๋ค. EGFR๊ณผ COX-2๋ Akt ์ ํธ์ ๋ฌ๊ฒฝ๋ก๋ฅผ ๊ณต์ ํ๋ฉฐ ๊ธด๋ฐํ ์ํฅ์ ์ฃผ๊ณ ๋ฐ์ ๊ฐ๋ฅ์ฑ์ด ์๋ค. ์ด์ ๋ณธ ์ฐ๊ตฌ๋ H. pylori๊ฐ EGFR ์ ํธ์ ๋ฌ์ ๋ฏธ์น๋ ์ํฅ์ ํ์ธํ๊ณ celecoxib์ด ์ด๋ฅผ ์ต์ ํ๋์ง ํ์ธํ๊ณ ์ ํ์๋ค.
์ฐ๊ตฌ์ฌ๋ฃ ๋ฐ ๋ฐฉ๋ฒ : AGS ์์์ธํฌ์ฃผ๋ฅผ H. pylori (cagA+, vacA+) G27๊ณผ cagE ๋์ฐ๋ณ์ด๋ฅผ ๊ฐ์ง G27์ 24์๊ฐ๋์ ๊ฐ์ผ์ํจ ํ mRNA์ ๋จ๋ฐฑ์ง์ ๋ฐํ์ ํ์ธํ์๋ค. COX-2, EGFR, TGF-ร, Snail, Slug, E-cadherin์ mRNA๋ฐํ์ RT-PCR๋กํ์ธํ์๋ค. ๊ทธ๋ฆฌ๊ณ ๋ค์ํ ๋๋(0, 10, 20, 30 ยตmol/L)์ ceclecoxib๋ฅผ ์ฒ๋ฆฌํ์ฌ COX-2, EGFR, tAkt, pAkt์ pGSK3ร์ ๋จ๋ฐฑ์ง ๋ฐํ์ ํ์ธํ์๋ค.
๊ฒฐ๊ณผ : AGS ์์์ธํฌ์ฃผ์์ wild type์ H. pylori ๊ฐ์ผ์ COX-2, EGFR, TGF-ร, Snail, Slug์ mRNA ๋ฐํ์ ์ฆ๊ฐ์์ผฐ์ผ๋ฉฐ E-cadherin์ mRNA๋ฐํ์ ๊ฐ์์์ผฐ๋ค. ๋ํ wild type์ H. pylori ๊ฐ์ผ์ COX-2, EGFR, pAkt, pGSK3ร ์ ๋จ๋ฐฑ์ง ๋ฐํ์ ์ฆ๊ฐ์ํจ ๋ฐ๋ฉด, cagE ๋์ฐ๋ณ์ด๋ก ์ธํด IVํ ๋ถ๋น๊ณ์ ๊ฒฐํจ์ด ์๋ H. pylori์ ์ํ ๊ฐ์ผ์ EGFR์ ํ์ฑํ์ํค์ง ๋ชปํ์๋ค. Celecoxib์ H. pylori์ ์ํด ๊ณผ๋ฐํ๋๋ COX-2 (p=0.015), EGFR (p=0.025), pAkt (p=0.025)์ pGSK3ร (p= 0.029)๋ฅผ ์ต์ ์์ผฐ๋ค.
๊ฒฐ๋ก : AGS ์์์ธํฌ์ฃผ์์IVํ ๋ถ๋น๊ณ๊ฐ ์ ์์ธH. pylori์ ์ํ ๊ฐ์ผ์ EGFR ์ ํธ์ ๋ฌ์ฒด๊ณ๋ฅผ ํ์ฑํ์์ผฐ์ผ๋ฉฐ, celecoxib์ ์ด๋ฅผ ์ต์ ํ๋ ํจ๊ณผ๋ฅผ ๋ณด์๋ค.Background and aim: Helicobacter pylori (H. pylori) infection increases the risk of gastric cancer through disrupting the regulation of cell survival. H. pylori infection is associated with epithelial growth factor receptor (EGFR) activation. EGFR downstream targets, such as phosphatidyl inositol 3-OH kinase (PI3K)-Akt-glycogen synthase kinase-3 (GSK3) pathways, regulate cell survival and migration. H. pylori infection also induces cyclooxygenase-2 (COX-2) over-expression, and previous study suggested that selective COX-2 inhibitor, celecoxib, blocks Akt signaling pathways. COX-2 and EGFR may cross talk through Akt-signaling pathways. The aim of the present study was to evaluate the effect of H. pylori on EGFR signaling pathways and to find out whether celecoxib has inhibitory effect on the EGFR signaling pathway or not.
Methods: AGS gastric epithelial cell lines were co-cultured with the toxigenic H. pylori cagA+, vacA+ G27 and the cagE- mutant of G27. The expressions of COX-2, EGFR, TGF-ร, Snail, Slug and E-cadherin were measured by real-time PCR. In the next, western blot analyses of COX-2, EGFR, total Akt (tAkt), phosphorylated Akt (pAkt) and pGSK3ร were carried out at various concentrations (0, 10, 20, 30ฮผmol/L) of celecoxib treatment for 24 hours in H. pylori treated AGS cell lines.
Results: H. pylori infection significantly up-regulated the mRNA levels of COX-2, EGFR,TGF-ร, Snail, Slug and down-regulated E-cadherin in RT-PCR. AGS cell lines treated with cagE- mutants, which have a defective type IV secretion system did not show EGFR up-regulation. Celecoxib had inhibitory effects on H. pylori-induced over-expression of COX-2 (p=0.015), EGFR (p=0.025), pAkt (p=0.025) and pGSK3ร (p=0.029) in AGS cell lines in Western blot analysis.
Conclusion: Infection by H. pylori with intact type IV secretion system activates EGFR signal pathways in AGS cell lines and celecoxib has inhibitory effect on this pathway. These finding provide insights into the anti-gastric cancer effect of celecoxib.์ด๋ก I
๋ชฉ์ฐจ โ
ข
LIST OF TABLES โ
ฃ
LIST OF FIGURES โ
ค
๋ณธ๋ฌธ
์๋ก 1
์ฐ๊ตฌ์ฌ๋ฃ ๋ฐ ๋ฐฉ๋ฒ 2
์ฐ๊ตฌ๊ฒฐ๊ณผ.. 5
๊ณ ์ฐฐ 13
์ฐธ๊ณ ๋ฌธํ 17
๊ตญ๋ฌธ์ด๋ก 23Maste
์ค์ฌ๋ฆฝ ์ ๋ฆฌ๊ณผ์ ์์ pericentrin ์ญํ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ (๋ฐ์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ ์์ฐ๊ณผํ๋ํ ์๋ช
๊ณผํ๋ถ, 2017. 8. ์ด๊ฑด์.A centrosome is composed of a centriole surrounded by protein matrix, called pericentriolar materials (PCM). The microtubule organization is the prime function of the centrosome. Cilia formation is another important function of the centrosome in quiescent cells. Duplication and segregation of centrioles occur in tight link to cell cycle. A daughter centriole is assembled next to the mother centriole during S phase, and remained in an engaged state until the cell exits M phase. New daughter centrioles may be generated only after the mother and daughter centrioles in the previous cycle are separated. Therefore, centriole separation is considered a licensing step for centriole duplication. However, it is largely unknown how centriole engagement is maintained and disrupted during the cell cycle.
Pericentrin (PCNT) is a PCM protein which is important for maturation process of centrosome to become spindle poles during mitotic entry. PCNT is also involved in induction of centriole separation during mitotic exit. PCNT is specifically cleaved, which is considered an essential step for centriole separation during mitotic exit. The purpose of my research is to elucidate mechanistic aspects of PCNT functions in centriole engagement and separation during M phase.
In chapter 1, I report that PCNT has to be phosphorylated by PLK1 in order to be a suitable substrate of separase. The phospho-resistant mutants of PCNT are not cleaved by separase and eventually inhibit centriole separation. Furthermore, phospho-mimetic PCNT mutants rescue centriole separation even in the presence of BI2536. Based on these results, I propose that PLK1 phosphorylation is a priming step for separase-mediated cleavage of PCNT and eventually for centriole separation. PLK1 phosphorylation of PCNT provides an additional layer of regulatory mechanism to ensure the fidelity of centriole separation during mitotic exit.
In chapter 2, I generated PCNT knockout cell lines and analyzed the phenotypes in relation to PCM assembly and centriole association. Deletion of PCNT hardly affected interphase centrosomes but conferred defects in centrosome maturation in cells entering M phase. The centrioles in PCNTโdeleted cells were prematurely separated in early phase of mitosis and frequently amplified in M phase-arrested cells. Abnormal multi-nuclear cells repeatedly appeared in PCNT-deleted cells at interphase. My results confirmed that PCNT is critical for centriole association during M phase.BACKGROUND 1
1. Structure of centrosome 1
1.1 Discovery of centrosome 1
1.2 Centrioles 1
1.3 Pericentriolar material (PCM) 2
2. Functions of centrosome 6
2.1 Microtubule network formation in interphase 6
2.2 Spindle formation during mitosis 6
2.3 Primary cilia formation in quiescent cells 7
3. Centrosome cycle 12
3.1 Initiation of centriole duplication 12
3.2 Centriole elongation 13
3.3 Centrosome maturation 13
3.4 Centrosome separation 14
3.5 Bipolar spindle formation 14
3.6 Centriole disengagement 14
4. Licensing mechanism for centriole duplication: Centriole engagement and disengagement 19
PURPOSE 23
CHAPTER 1. PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit 24
ABSTRACT 25
INTRODUCTION 26
MATERIAL AND METHODS 29
Plasmids, siRNA, and cell culture 29
Antibodies 30
Immunoprecipitation and immunoblot analyses 31
Immunostaining analysis 33
Statistical analysis 34
RESULTS 35
BI2536 blocks both PCM disassembly and centriole separation 35
PLK1 phosphorylation is necessary for PCNT cleavage 36
PLK1 phosphorylates PCNT in vivo 38
PCNT phosphorylation is necessary for centriole separation 40
PCNT and CEP215 are essential for centriole association 41
Dual functions of PLK1 phosphorylation of PCNT 43
Discussion 60
CHAPTER 2. Phenotypic analyses of PCNT-deleted cells 64
ABSTRACT 65
INTRODUCTION 66
MATERIAL AND METHODS 68
Plasmids, siRNA, and cell culture 68
Generation of knockout cell lines with CRISPR/CAS9 system 69
Antibodies 69
Immunoblot analysis 70
Immunostaining analysis 71
Statistical analysis 72
RESULTS 73
Generation of PCNTTP53 double knockout cell lines 73
Interphase centrosomes in the PCNT-deleted cells 73
Spindle poles in the PCNT-deleted mitotic cells 74
Active role of PCNT for centriole association 77
DISCUSSION 94
PERSPECTIVE 98
REFERENCES 101
ABSTRACT IN KOREAN (๊ตญ๋ฌธ ์ด๋ก) 108Docto
๊ธ๋๋ ธ ์ ์๋ฅผ ์ด์ฉํ switchable-linker ๊ธฐ๋ฐ ๊ฒ์ถ๋ฒ์ ์์ง๊ณผ ์นจ์ ๋ฐ์์ ์ํฅ์ ๋ฏธ์น๋ ์์์ ๊ดํ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ๋์
์๋ช
๊ณผํ๋ํ ๋์๋ช
๊ณตํ๋ถ, 2018. 8. ์ต์์ง.Aggregation of gold nanoparticles (AuNPs) has been used in various fields for assorted purposes. In particular, detection of analytes through visual color changes induced by aggregation of AuNPs has been extensively studied in recent years. In our previous research, switchable-linker based detection system was introduced. In the system, sensing of specific targets is achieved based on the quantitative correlations between linkers, targets and particles. When the number of particles and linkers are ideally matched, a maximum aggregation occurs in suspension inducing aggregates settling. This linker range where aggregates precipitation dominantly occurs is called exhibiting visual color change (REVC). Since REVC plays a critical role in the system, it is important to gain an understanding of the factors that could affect in REVC formation. However, little research has been done about the basic mechanisms of REVC formation based on AuNPs aggregation and precipitation, especially focusing on the effects of particles size and surface area.
In this study, the effects of total surface area of AuNPs in REVC formation were identified by comparing 3 different cases. In the first case, 11 nm and 19 nm-sized particles were used in the previous study. Under the same plasmon absorbance, as the size of particle increased, the total surface area decreased. In this case, REVC appeared at lower linker concentrations with a narrower range. After the addition of target streptavidin, REVC shifted more in 19 nm particles which had smaller surface area. As for the second case, 12, 18, and 24 nm-sized particles were used. In this case, the number of particles was identically adjusted, thus as the particle size increased, the total surface area of AuNPs increased. In larger surface area, REVC appeared at higher linker concentrations in a broader range which correspondsss to the previous results. With the addition of target streptavidin, REVC shifted less as the surface area increased. Lastly, the surface area of AuNPs was identically adjusted by controlling the number of particles using 13, 21, 30, and 36 nm-sized particles. Under the same surface area, REVC was formed at a similar linker concentrations regardless of individual particle sizes and REVC shifting occurred identically under the addition of target streptavidin. Three cases indicated that as the surface area of AuNPs decreased, REVC worked more sensitively as a sensor.
As part of an effort to confirm the reaction time according to particles, the third case was intensively interpreted. When the surface area of AuNPs was identically controlled, it was observed that in 21 nm-sized particles, REVC was formed the slowest among others in observation time of 200 minutes. From DLS size measurements of aggregates and Stokes law, settling velocity of aggregates in REVC was calculated. At an early settling stage, aggregates of 30 and 36 nm-sized particles precipitated more rapidly. However, as time went by, settling speed of aggregates composed of 13 and 21 nm-sized particles reached beyond the speed of larger particles. At around 120 minutes, the settling velocity of aggregates composed of 13 nm-sized particles went over than that of 21 nm-sized particles. This phenomenon might be explained by the fact that smaller individual particles made clusters more rapidly in much bigger sizes, thus settling speed inversion seemed to be happened.
The results of this study suggest the importance of the surface area in REVC formation and the aggregates precipitation speed inversion was revealed by the actual calculations using Stokes law. These can be contributed to the further development of switchable-linker based detection system and can be used as clues in other aggregation-based applications.โ
. INTRODUCTION 10
โ
ก. MATERIALS AND METHODS 13
2.1. Materials 13
2.2. Instrumentation 13
2.3. Preparation of gold nanoparticles 14
2.4. Preparation of streptavidin-coated gold nanoparticles (stAuNP) 18
2.5. Adjustment of the total surface area of AuNPs 22
2.6. Schematic explanations of the switchable-linker based detection system 24
โ
ข. RESULTS AND DISCUSSION 28
3.1. Effects of AuNPs surface area on REVC formation under the different conditions 28
3.1.1. REVC formation under the same absorption spectra 28
3.1.2. REVC formation under the same number of particles 32
3.1.3. REVC formation under the same AuNPs surface area 35
3.2. Settling velocity of aggregates in REVC interpreted by the modified Stokes law 38
3.2.1. Aggregates sizes obtained by DLS and TEM measurements of 13, 21, 30, and 36 nm-sized particles 39
3.2.2. Settling velocity of aggregates in REVC using the modified Stokes law and occurrence of speed inversion phenomenon 47
โ
ฃ. CONCLUSION 53
โ
ค. REFERENCES 55
๊ตญ๋ฌธ์ด๋ก 59Maste
Cyclosporin A ์น๋ฃ์ ๋ถ์์ฉ์ธ ์น์ ๊ณผ์ฑ์ฅ์ ๋ํ azithromycin์ ์์ฉ๊ธฐ์ ์ ๋ํ ์ฐ๊ตฌ
Dept. of Medical Science/์์ฌ[ํ๊ธ]
๋ชฉ์ : Cyclosporin A (CsA)๋ 1970๋
๋ ์ด๋๋ก ์ฅ๊ธฐ์ด์์ด๋ ๊ณจ์์ด์ ์์ ์ ๋ฐ์ ํ์๋ค์๊ฒ ๋ณดํธ์ ์ผ๋ก ์ฒ๋ฐฉ๋๋ ๋ฉด์ญ์ต์ ์ ์ด๋ค. ๊ทธ๋ฌ๋ CsA๋ ํ๊ธฐ์ ์ธ ํจ๊ณผ๋ฅผ ๊ฐ๊ณ ์์์๋ ๋ถ๊ตฌํ๊ณ , ์ ๋
์ฑ, ๊ฐ๋
์ฑ ๋ฑ ๋ค์ํ ๋ถ์์ฉ์ ๋ํ๋ด๋ฉฐ, ์น์๊ณผ์ฑ์ฅ๋ ๊ทธ ์ค ํ๊ฐ์ง์ด๋ค. ์ด์ ์์ ์ ๋ฐ์ ํ, CsA๋ฅผ ๋ณต์ฉํ ํ์๋ค์์ ๋ํ๋๋ ์น์๊ณผ์ฑ์ฅ์ ํ๊ท 30%๋ก ๋์ ๋น์จ๋ก ๋ณด๊ณ ๋์๋ค. ์ต๊ทผ ์์์ฐ๊ตฌ๋ฅผ ํตํด์ CsA๋ก ์ธํ ์น์๊ณผ์ฑ์ฅ์ macrolide ๊ณ์ด์ ํญ์์ ์ธ azithromycin(AZI)์ ์ํด ํจ๊ณผ์ ์ผ๋ก ์ต์ ๋๋ค๋ ๊ฒฐ๊ณผ๊ฐ ๋ณด๊ณ ๋์๋ค. ๊ทธ๋ฌ๋ ํ์ฌ๊น์ง CsA์ ์ํ ์น์๊ณผ์ฑ์ฅ์ ์ ๋๊ธฐ์ ๊ณผ, AZI์ ์ํ ์ต์ ๊ธฐ์ ์ ๋ช
ํํ ๋ฐํ์ง์ง ์์๋ค. ๊ทธ๋ฌ๋ฏ๋ก, ๋ณธ ์ฐ๊ตฌ์์๋ CsA์ ์ํ ์น์๊ณผ์ฑ์ฅ ์ ๋ ๊ธฐ์ ๊ณผ AZI์ ์ํ ์ต์ ๊ธฐ์ ์ ๋ฐํ๊ณ ์ ํ๋ค.
๋ฐฉ๋ฒ: ์น์ ์ฌ์ ์์ธํฌ๋ ๊ณผ๊ฑฐ์ CsA๋ฅผ ๋ณต์ฉํ์ฌ ์น์๊ณผ์ฑ์ฅ์ ๋ณด์ธ ํ์์ CsA๋ฅผ ๋ณต์ฉํ ๊ฒฝํ์ด ์๋ ๊ฑด๊ฐํ ํ์์ ์น์์์ explant culture๋ก ๋ถ๋ฆฌํ์๋ค. ์น์ ์ฌ์ ์์ธํฌ์ CsA์ ์ํ ์ฆ์๋ฅ ๊ณผ AZI์ ์ํ ์ต์ ๋ฅ ์ MTT assay์ ์ํด ์ธก์ ํ์๊ณ , ์ฝ๋ฌผ์ ์ํ ์น์ ์ฌ์ ์์ธํฌ์ ํํํ์ ๋ณํ๋ฅผ ๊ดํ ํ๋ฏธ๊ฒฝ์ ์ด์ฉํ์ฌ ๊ด์ฐฐํ์๋ค. ๊ทธ๋ฆฌ๊ณ MMPs (matrix metalloproteinases)์ mRNA์ ๋ฐํ๊ณผ ํ์ฑ๋๋ ๊ฐ๊ฐ reverse transcription - polymerase chain reaction (RT-PCR)๊ณผ zymography๋ก ์กฐ์ฌํ์๋ค.
๊ฒฐ๊ณผ: CsA์ ์ํ ์ธํฌ์ ์ฆ์๋ฅ ์ ๊ฑด๊ฐํ ํ์๋ณด๋ค CsA๋ฅผ ๋ณต์ฉํ ๊ฒฝํ์ด ์๋ ํ์๋ก๋ถํฐ ๋ถ๋ฆฌํ ์น์ ์ฌ์ ์์ธํฌ์์ ๋๋ ทํ๊ฒ ์ฆ๊ฐ๋์๋ค. ๊ทธ๋ฌ๋ฏ๋ก ์ดํ์ ์ฐ๊ตฌ๋ CsA๋ฅผ ๋ณต์ฉํ ๊ฒฝํ์ด ์๋ ํ์์ ์น์ ์ฌ์ ์์ธํฌ๋ก ์งํํ์๋ค. 120์๊ฐ ๋์ CsA (10ng/ml)์ AZI๋ฅผ ํจ๊ป ์ฒ๋ฆฌํ ๊ฒฝ์ฐ, AZI์ ๋๋์ ๋น๋กํ์ฌ ์น์ ์ฌ์ ์์ธํฌ์ ์ฆ์๋ฅ ์ด ๊ฐ์ํ์๋ค. CsA์ ์ํ ํํํ์ ๋ณํ๋ ๊ด์ฐฐ๋์ง ์์์ง๋ง, CsA์ AZI์ ํจ๊ป ์ฒ๋ฆฌํ์ ๊ฒฝ์ฐ์๋ ํ์ํ ๋ชจ์์ผ๋ก ๋ณํ๋์๋ค. CsA๋ฅผ ์ฒ๋ฆฌํ ์น์ ์ฌ์ ์์ธํฌ๋ MMP-2์ mRNA ๋ฐํ์๊ณผ ํ์ฑ๋๊ฐ ์ต์ ๋์๋ค. ๊ทธ๋ฌ๋ AZI๊ณผ CsA๋ฅผ ํจ๊ป ์ฒ๋ฆฌํ์ ๊ฒฝ์ฐ์๋ MMP-2์ mRNA ๋ฐํ์์ CsA๋ง ๋จ๋
์ฒ๋ฆฌํ ๊ฒฝ์ฐ์ ๋น๊ตํ์ฌ ์ ์์ ์ผ๋ก ํ๋ณต๋์๊ณ , MMP-2์ MMP-9์ ํ์ฑ์ด ์ ๋๋์๋ค. Membrane-type (MT)1 - MMP์ MT3 - MMP๋ฅผ ํตํด latent MMP-2์ ํ์ฑ๋๋ฅผ ์ต์ ํ๋ค๊ณ ๋ณด๊ณ ๋ testican 1์ CsA์ ์ํด mRNA์ ๋ฐํ์ด ๋ง์ด ์ฆ๊ฐ๋์์ผ๋, AZI๊ณผ ํจ๊ป ์ฒ๋ฆฌํ ์น์ ์ฌ์ ์์ธํฌ์์๋ ๋์กฐ๊ตฐ๊ณผ ๋น์ทํ ์ ๋๋ก ๋ฐํ์์ด ๊ฐ์๋์๋ค.
๊ฒฐ๋ก : AZI์ ์ํ ์ธํฌ์ฆ์ ์ต์ ์ MMP-2์ ํ์ฑ๋์ ์ฆ๊ฐ๋ CsA์ ์ํด ์ ๋๋ ์น์๊ณผ์ฑ์ฅ์ ์ต์ ํ๋๋ฐ ์ค์ํ๋ค.
ํต์ฌ๋๋ ๋ง: cyclosporin A, azithromycin, ์น์๊ณผ์ฑ์ฅ, matrix metalloproteinase-2, testican 1
[์๋ฌธ]Cyclosporin A (CsA) is the most frequently used immunosuppressor in transplant surgery, but gingival overgrowth (GO) remains one of its adverse side effects. Many previous reports have demonstrated that GO can be effectively treated by azithromycin (AZI), a macrolide antibiotic of the azalide subclass that suppresses protein synthesis of both gram-positive and gram-negative aerobes. However, the mechanism by which AZI suppresses CsA-induced GO (CIGO) has not yet been elucidated. In the present study, we examined the inhibitory effect of AZI on CIGO and its mechanism of action, as well as the mechanism whereby CsA induces GO. Human gingival fibroblasts were isolated from the gingival tissues of healthy subjects and patients exhibiting CIGO. The cell proliferation was significantly increased by CsA exposure for 5 days in fibroblasts isolated from gingival tissue of patients taking CsA. In contrast, AZI significantly inhibited CsA-induced cell proliferation. Morphological alterations were observed in the gingival fibroblasts stimulated with CsA, AZI, or the combination of AZI and CsA. The mRNA level and activity of matrix metalloproteinase-2 (MMP-2) were inhibited by CsA, but were increased by the combination of AZI and CsA. The activities of the latent and active forms of MMP-2 were increased by AZI in a dose-dependent manner. Interestingly, both the latent and active forms of MMP-9 were secreted from gingival fibroblasts exposed to the combination of AZI and CsA. The increased mRNA expression level of testican 1 in CsA-treated gingival fibroblasts was inhibited by the combination of AZI and CsA in a dose-dependent manner. Testican 1 has been known to inhibit pro-MMP-2 activation by membrane-type 1 (MT1)-MMP or MT3-MMP. These results suggest that AZI alters the synthesis
degradation of the extracellular matrix (ECM) by regulating the expression of testican 1, which consequently mediates the activation of MMP-2. Therefore, the induction of MMP-2 by AZI treatment might play an important role in the inhibitory mechanism of AZI on CIGO.
Key words : cyclosporin A, azithromycin, gingival overgrowth,
matrix metalloproteinase-2, testican 1ope
์ฐจ์ธ๋ ์ํ์ฑ์ ์ด์ฉํ ์๋ด ์ธ๊ท ๊ณผ ์์ ๋ฐ๋ณ์ ๊ด๋ จ์ฑ์ ๊ดํ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ (๋ฐ์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ์๊ณผ๋ํ ์ํ๊ณผ ๋ถ์์ ์ ์ฒด์ ๊ณต, 2016. 2. ๊น๋์.Introduction: Little is known about the role of gastric microbiota except for Helicobacter pylori (HP) in human health and disease. We compared the differences of human gastric microbiota according to gastric cancer or control and HP infection status and assessed the role of bacteria other than HP.
Materials and methods: Gastric microbiota of 63 antral mucosa, 18 corpus mucosa samples were analyzed by barcoded 454-pyrosequencing of the 16S rRNA gene. Antral samples were divided into the four subgroups based on HP positivity in pyrosequencing and presence of cancer. The analysis was focused on bacteria other than HP, especially, nitrosating or nitrate reducing bacteria (NB). The changes of NB in antral mucosa of 16 subjects were followed-up.
Results: The number of NB other than HP (non-HP-NB) was two times higher in the cancer groups than control groups but it did not reach statistical significance. The number of non-HP-NB tends to increase over time, but this phenomenon was prevented by HP eradication in the HP-positive control group, but not in the HP-positive cancer group.
Discussion: We found out HP plays more important role than other bacteria in the gastric carcinogenesis.1. Introduction 1
2. Materials and Methods 3
3. Results 10
4. Discussion 30
5. References 36
๊ตญ๋ฌธ์ด๋ก 41Docto
PLK1์ ์ํ pericentrin ์ธ์ฐํ๊ฐ ์ค์ฌ๋ฆฝ ์ ๋ฆฌํ์์ ๋ฏธ์น๋ ์ํฅ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ์๋ช
๊ณผํ๋ถ, 2013. 8. ์ด๊ฑด์.์ค์ฌ์ฒด๋ ๋ง์ด ์๋ ๋ํ์ ์ธ ์ธํฌ์๊ธฐ๊ด์ผ๋ก์, ์ธํฌ๋ถ์ด๊ธฐ์ ๋ฐฉ์ถ๊ทน์ผ๋ก ์์ฉํ์ฌ ๋ฐฉ์ถ์ฌ๋ฅผ ํ์ฑํ๋ค. ์ผ์์ฒด ์ ์ด์์ ์ํด ๋ฐ์๋๋ ๋ค๊ทน ๋ฐฉ์ถ์ฒด๋ ์ผ์์ฒด๋ถ์์ ์ฑ ํน์ ์ธํฌ์ฌ๋ฉธ์ ์ผ์ผํจ๋ค. ๋ฐ๋ผ์, ์ ๊ตํ ์ค์ฌ์ฒด ์ ์กฐ์ ๊ธฐ์์ ์ธํฌ๊ฐ ์์ ์ ์ ์ ๋ฌผ์ง์ ์์ ์ ์ผ๋ก ๋ธ์ธํฌ์ ์ ๋ฌํด์ฃผ๋๋ฐ ์ค์ํ ์ญํ ์ ํ๋ค.
์ค์ฌ๋ฆฝ ์ ๋ฆฌํ์์ ์ค์ฌ์ฒด์ ์๋ฅผ ์กฐ์ ํ๋ ํต์ฌ ๊ธฐ์์ผ๋ก ๋ธ์ค์ฌ๋ฆฝ์ด ๋ชจ์ค์ฌ๋ฆฝ์์ ๋จ์ด์ง๋ ํ์์ ๋งํ๋ค. ๋ํ ์ต๊ทผ ์ฐ๊ตฌ๊ฒฐ๊ณผ์ ๋ฐ๋ฅด๋ฉด separase์ ์ํ pericentrin์ ์ ๋จ์ด ์ค์ฌ๋ฆฝ ์ ๋ฆฌํ์์ ์ผ์ผํค๋ ์ฃผ์ํ ์์ธ์ผ๋ก ๋ฐํ์ก๋ค. ๋ณธ ์ฐ๊ตฌ๋ PLK1์ ์ํด ์กฐ์ ๋๋ pericentrin ์ ๋จ ๊ธฐ์์ ๋ํด ์ฐ๊ตฌํ์๋ค. Pericentrin์ PLK1์ ์ํด 2259๋ฒ ๋ฐ 2267๋ฒ ์๋ฏธ๋
ธ์ฐ์ด ์ธ์ฐํ๋๋ฉฐ, ์ด ๋ ์๋ฏธ๋
ธ์ฐ์ด ์๋ผ๋์ผ๋ก ์นํ๋ ๋์ฐ๋ณ์ด์์๋ pericentrin์ ์ ๋จ ๋ฐ ์ค์ฌ๋ฆฝ ์ ๋ฆฌํ์์ด ์ค์ด๋ฆ์ ๊ด์ฐฐํ์๋ค. ์ด ๊ฒฐ๊ณผ๋ PLK1์ด ์ค์ฌ๋ฆฝ ์ ๋ฆฌํ์์ pericentrin์ ์ธ์ฐํ๋ฅผ ํตํด ์กฐ์ ํ ์ ์์์ ์ ์ํ๋ค.Centrosome is a non-membrane bound organelle which functions as a spindle pole body during mitosis. Abnormalities in centrosome number cause multipolar spindles which result in chromosomal instability or cell death. Therefore the precise regulation of centrosome number is important for maintaining genome stability. During mitotic exit, procentrioles are disengaged from the mother centriole during mitotic exit, which is considered a licensing step for centriole duplication. It was recently reported that pericentrin cleavage is necessary and sufficient for centriole disengagement during mitosis. Here, I report that the pericentrin cleavage is regulated by PLK1. PLK1 phosphorylates S2259 and S2267 residues of pericentrin. FLAG-PCNT S2259A, S2267A in which these two residues are substituted to alanine is not cleaved during mitosis. Finally, centriole disengagement was inhibited in the pericentrin-depleted cells rescued with the phospho-resistant FLAG-PCNT mutants at S2259 and S2267. Based on the results, I propose that PLK1 phosphorylation is prerequisite to pericentrin cleavage and eventually to centriole disengagement during mitotic exit.ABSTRACT 1
CONTENTS 3
LIST OF FIGURES 5
INTRODUCTION 7
MATERIALS AND METHODS 9
DNA constructs and transfection 9
RNA interference 9
Cell culture and stable cell lines 10
Cell cycle synchronization and drug treatments 10
Antibodies 11
Immunofluorescence microscopy 11
Immunoblot analysis 12
RESULTS 13
The PLK1 activity is required for pericentrin cleavage during mitotic exit. 13
Specific phosphorylation of pericentrin is critical for its cleavage. 14
Phosphorylation at S2259 and S2267 residues is important for the pericentrin cleavage. 15
Specific phosphorylation of pericentrin is prerequisite to centriole disengagement during mitotic exit. 18
DISCUSSION 46
REFERENCES 48
ABSTRACT IN KOREAN(๊ตญ๋ฌธ์ด๋ก) 52Maste
์๋งค์ฌ์ ์์์ ์์ค๋ถํฌ ์ถ์ ๋ฐฉ๋ฒ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ(์์ฌ) --์์ธ๋ํ๊ต ๋ํ์ :ํต๊ณํ๊ณผ,2007.Maste
Application of radon tracer to evaluate the interaction characteristics between groundwater and surface water with time variance around the groundwater heat pump system
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ์ง๊ตฌํ๊ฒฝ๊ณผํ๋ถ, 2015. 8. ์ด๊ฐ๊ทผ.The water exchange between groundwater and surface water has a significant impact on developing water resources. A geothermal energy system using indirect open loop ground source heat pump (GSHP) has been developed at Han River Environmental Research Center (HRERC) in Yangsu-ri, Yangpyeong-gun, Korea. For designing a high efficiency performance of the geothermal heat pump system in the study area, the characteristics of interaction between groundwater and surface water has to be figured out because the aquifer system of the study site is directly affected by Han Rivers. Therefore, radon-222 was used as an indicator because it has much higher concentration in groundwater than in surface water. In this study, 12 groundwater wells were used for monitoring radon concentration, hydrogeochemical parameters, and water level from May, 2014 to Apr., 2015. The statistical analysis and mixing analysis were also conducted for quantitative interpretation of the obtained radon and hydrogeochemical data. There are no significant correlations between the hydrogeochemical parameters and radon concentration. However, each group classified by the cluster analysis was reflected its own hydrogeochemical properties. The spatial distribution of radon concentration was affected by seasonal effect and fluctuation of Han River stage from surrounding dam activity. The results of mixing analysis represented the quantitative interpretation of radon data. This study demonstrated radon-222 can be used as an appropriate environmental tracer in examining the characteristics of interaction between groundwater and surface water in consideration of river discharge rate. These results help to provide useful background information on the development of GSHP in the riverside area.ABSTRACT โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโ
ฐ
TABLE OF CONTENTS โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ โ
ฒ
FIGURE LIST โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโ
ด
TABLE LIST โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ โ
ท
1. INTRODUCTION โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 1
1.1 Research Backgrounds โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 1
1.2 Objectives and Scope โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 4
2. LITERATURE REVIEW โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 5
2.1 Radon as a Tracer โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 5
3. STUDY AREA โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 7
3.1 Site Description โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 7
3.2 Geological Characteristics โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 9
3.3 Hydrological Characteristics โฆโฆโฆโฆโฆโฆโฆโฆโฆ 18
4. METHODS AND MATERIALS โฆโฆโฆโฆโฆโฆโฆโฆโฆ 21
4.1 Well Installation โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 21
4.2 Water Sampling and Hydrogeochemical Analysis 24
4.3 Monitoring of Groundwater Flow โฆโฆโฆโฆโฆโฆโฆ 25
4.4 Measurement of Radon Concentration โฆโฆโฆโฆโฆ 26
4.5 The Mass Balance Equation for Mixing Analysis 28
4.6 Statistical Analysis of Data โฆโฆโฆโฆโฆโฆโฆโฆโฆ 31
5. RESULTS AND DISCUSSION โฆโฆโฆโฆโฆโฆโฆโฆโฆ 33
5.1 Long Term Monitoring of Groundwater and Surface
Water โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 33
5.2 Statistical Application to Interpret
Hydrogeochemical Properties in Groundwater โฆโฆโฆ 45
5.3 Short Term Monitoring of Groundwater and
Surface Water โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 54
5.4 Mixing Analysis by Using Radon-222 โฆโฆโฆโฆ 58
6. SUMMARY AND CONCLUSTION โฆโฆโฆโฆโฆโฆโฆ 69
REFERENCE โฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆโฆ 72Maste
์์คํธ๋ก๊ฒ ์์ฉ์ฒด-์ํ์ Ufmylation์ด ์์คํธ๋ก๊ฒ ์ ํธ์ ๋ฌ๊ณผ์ ์ ๋ฏธ์น๋ ์ํฅ์ ๊ดํ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ์๋ช
๊ณผํ๋ถ, 2014. 2. ์ ์งํ.UFM1, ubiquitin-like protein, is post-translationally conjugated to cellular proteins. The ufmylation reaction is catalyzed by an UFM1-activating E1 enzyme (UBA5), an UFM1-conjugating E2 enzyme (UBC1), and UFM1 E3 ligases (UFL1), in a manner similar to ubiquitination.
Estrogen receptor ฮฑ(ERฮฑ) is a member of a large conserved superfamily of steroid hormone nuclear receptors, acting as ligand-regulated transcription factor. ERฮฑ regulates many physiological pathways in response to its ligand, E2 (17ฮฒ-estradiol). ERฮฑ is undergoing different types of post-translational modifications, such as phosphorylation, sumoylation, acetylation, and ubiquitination, which regulate its transcriptional activation and/or stability.
Here, ERฮฑ was found to be a target for modification by UFM1. I also identified the major UFM1 acceptor site is in the AF1 domain. Moreover, knock down of UBA5 by RNA interference reduced the ERฮฑ transcriptional activity and accelerated E2-induced proteolysis. These results indicate that UFM1 modification plays a critical role in positive regulation of ERฮฑ function in transcriptional activation by preventing its degradationAbstract 1
Introduction 2
Materials and Methods 6
Plasmids and antibodies
Cell culture and transfections
Assays for ufmylation and deufmylation
Ni-NTA-agarose pull-down analysis and immunoprecipitation
Luciferase assays
Results 9
ERฮฑ๏ is a substrate for UFM1 modification
Formation of poly-UFM1 chain via K69-linked isopeptide bonds
Identification of the UFM1 acceptor sites in ERฮฑ
Reversal of ERฮฑ ufmylation by UFSP2
Requirement of ERฮฑ ufmylation for E2-induced ERฮฑ transactivation
Effect of ERฮฑ ufmylation for E2-induced ERฮฑ๏ proteolysis
Inverse correlation between E2-induced ufmylation and ubiquitination
of ERฮฑ
Discussion 31
References 36
๊ตญ ๋ฌธ ์ด ๋ก 40Maste