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초파리 모델 시스템에서 파킨 단백질이 매개하는 감염과 상처 발생에 대한 생체 내 반응과 TSPO-VDAC 복합체의 상호작용
No full textParkin, an E3 ubuquitin ligase associated with Parkinson's disease (PD), has recently been implicated in mediating innate immunity. However, molecular details regarding parkin-mediated immune response remain to be elucidated. Here, we identified mitochondrial TSPO-VDAC complex to genetically interact with parkin in mediating responses against infection and wound in Drosophila. The loss-of-function mutation in parkin results in defective immune response against bacterial infection. Additionally, parkin mutant larvae showed hypersensitivity against wound regardless of bacterial infection. Interestingly, the combinatorial trans-heterozygotic mutations in parkin and TSPO, or parkin and VDAC showed similar lethal tendency with parkin homozygous mutants. Furthermore, knockdown of TSPO alone also resulted in defective responses to infection and wound analogously to parkin mutants. Taken together, we propose that parkin cooperates with TSPO-VDAC complex to mediate responses against infection and wound. ⓒ 2016 DGISTⅠ. INTRODUCTION 1 --
Ⅱ. MATERIALS AND METHOD 3 --
2.1. Fly strains 3 --
2.2. Prediction of transmembrane domain of dTSPO 3 --
2.3. Infection and injury experiment 3 --
2.4. Analysis of survival rate 4 --
2.5. Single colony isolation of remaining bacteria inside the body after infection 4 --
2.6. RNA extraction, cDNA synthesis, reverse transcription-PCR (RT-PCR) and quantitative PCR (qPCR) 5 --
Ⅲ. RESULT 6 --
3.1. Parkin loss-of-function mutation induces defects in immune defense and wound repair 6 --
3.2. Parkin and mitochondrial protein TSPO genetically interact in mediating immune response 8 --
3.3. Loss of TSPO function shows similar defects to parkin mutants in responses to infection and wound 10 --
3.4. Mitochondrial TSPO-VDAC complex is involved in parkin-mediated immune defense 12 --
IV. DISCUSSION 13 --
Figures 16 --
References 35 --
Summary in Korean 39파킨슨 씨 병 (Parkinson’s disease)의 주요 발병 원인 단백질로 알려진 Parkin은 최근 선천성 면역 (Innate immunity) 반응의 매개에 관여한다고 보고 되고 있다. 하지만 parkin 단백질이 매개하는 선천성 면역 반응의 자세한 분자 수준적 기전은 아직까지 분명히 밝혀진 바가 없다. 본 연구에서 우리는 미토콘드리아 막 (mitochondrial membrane)에 존재하는 TSPO-VDAC 복합체가 감염 (infection)과 상처 (wound)로 인하여 유도 되는 선천성 면역반응의 매개 수준에서 유전적으로 parkin과 함께 상호작용하여 관여한다는 것을 초파리 모델 시스템 (Drosophila model system)을 사용하여 밝혀 내었다. Parkin 유전자에 돌연변이 (mutation)이가 발생하여 기능을 상실하게 되면 박테리아 감염 (bacterial infection) 시 발생하는 면역 반응에 결함이 생긴다는 것을 알게 되었다. 게다가, 이 단백질의 돌연변이가 생긴 초파리 애벌래 (larvae)는 박테리아 감염이 일어나지 않고 상처만 생겨도 과민감성 (hypersensitivity)을 보여서 높은 치사율 (lethality)을 보이는 것을 관찰할 수 있었다. 흥미롭게도, 박테리아 감염 시 parkin과 TSPO 혹은 parkin과 VDAC 각각의 이형 접합적 돌연변이체 (heterozygous mutant)가 결합하여 만들어진 이중 이형 접합적 돌연변이체 (trans-heterozygotic mutations)가 parkin 단백질 단독의 동형 접합적 돌연변이체 (homozyogous mutant)와 유사한 치사율 경향성 (lethal tendency)을 보이는 것을 발견하였다. 이는 parkin과 TSPO 그리고 parkin과 VDAC이 서로 유전적으로 상호작용할 수 있음을 간접적으로 보여주는 결과이다. 더욱이, TSPO 유전자의 활성화를 낮춘 실험군 (knockdown of TSPO) 또한 감염과 상처에 대한 면역 반응에 결함이 parkin 단백질 단독의 동형 접합적 돌연변이체 실험군과 같은 경향성으로 나타나는 것을 확인하였다. 종합하면, 우리는 parkin이 TSPO-VDAC 복합체와 함께 외부 위험물질로 인한 감염과 상처에 방어를 위해 유도되는 면역 반응을 매개한다는 것을 새롭게 밝혀내었다. ⓒ 2016 DGISTMasterdCollectio
Mechanism of inhibition of camptothecin-induced apoptosis by dexamethasone in C6 glioma cells
No full text의학과/박사[한글]중추신경계 종양의 50%를 차지하며 성인에서 가장 호발하는 악성교종은 수술, 항암제 및 방사선치료의 다병합요법에도 높은 재발률로 매우 불량한 예후를 보인다. 이는 종양 세포 자체 및 외적인 요소 등의 다양한 내성 기전에 의한 것으로 이들 내성 기전에 대한 연구는 질병의 완치율을 높이기 위해 필수적이다. 최근 뇌교종세포에서 dexamethasone이 camptothecin의 세포고사(apoptosis) 유도 효과를 감소시키며, 동시에 세포생존신호인 Bcl-xL 상승을 동반함이 밝혀졌다. 임상에서 뇌종양 환자에서 부종을 감소시키기 위해 늘 쓰이고 있는 스테로이드가 세포내 anti-apoptotic signal인 Bcl-xL 발현을 증가시키는 것은 항암제나 방사선과 같이 세포고사를 유도하는 치료법의 암세포 사멸효과를 저하시키는 역할을 할 수 있다는 점에서 중요한 의미를 가진다고 할 수 있다. 따라서 본 논문은 뇌교종 세포에서 스테로이드 투여 후 Bcl-xL 발현 증가를 위한 세포내 신호전달 과정상 상위전사조절물질들에 대한 연구를 통해 dexamethasone에 의해 camptothecin 유도 세포고사가 억제되는 전체적인 신호전달 기전을 규명하고자 하였다.뇌교종세포주인 백서 C6 교종세포를 대상으로 실험을 진행하였으며, 세포고사 유도를 위해 camptothecin 2 μM을 배지에 첨가하였다. Camptothecin에 의해 유도되는 세포고사의 확인 및 정량적 분석을 위해서 caspase-3 특이 활성도를 측정하는 CaspACETM？(Promega, Madison, USA) system 과 DNA 분절 정도를 확인하는 Cell Death Detection ELISA Plus Kit？(Roche, Mannheim, Germany)를 이용하였다. Camptothecin에 의해 증가되었던 caspase-3 특이 활성도 및 DNA 분절 현상은 dexamethasone 전처치로 크게 감소하였다. Dexamethasone 투여 후 Bcl-xL의 시간대별 증가 양상을 재확인 하였으며, 세포고사 억제 효과도 이러한 Bcl-xL 발현의 시간에 따른 변화에 상응하는 양상이었다. Dexamethasone에 의한 세포고사의 유도 및 Bcl-xL의 상승은 glucocorticoid receptor antagonist인 RU486(Mifepristone)에 의해 역전되어, 이 기전의 초기 단계에 glucocorticoid receptor (GR)가 관여함을 알 수 있었다. 일부 보고에서 STAT5가 Bcl-xL 유전자의 주된 조절자 중의 하나로 밝히고 있으며, 본 실험에서 사용된 뇌교종세포에서 dexamethasone에 의한 Bcl-xL 상승에 STAT5가 상위조절인자로 역할을 하는지를 보기 위한 실험을 진행하였다. Dexamethasone 투여 후 인산화로 활성화된 STAT5 단백질은 그 발현이 증가하였으며, electrophoretic mobility shift assay (EMSA)상 dexamethasone의 투여는 STAT5의 DNA 결합능도 증가시켰다. 또한 STAT5 단백질을 dominant negative 형태로 발현 가능한 재조합 아데노바이러스인 AdStat5aΔ740을 뇌교종 세포주인 C6에 감염시킨 후 세포내에서 Bcl-xL 단백질의 발현양이 확연히 감소됨을 확인하였다. 따라서 뇌교종세포주인 C6에 dexamethasone의 처리로 인하여 증가되는 Bcl-xL 단백질의 발현에 있어 STAT5 단백질이 전사 수준에서 직접적으로 관여하고 있다는 사실을 확인할 수 있었다. 또한 dexamethasone 처리 후 뇌교종세포주에서 STAT5와 GR 사이에 어떤 형태의 관련성이 있는지를 보고자 시행된 co-immunoprecipitation상 dexmethasone 투여 후 초기 2시간 이내에 STAT5와 GR의 물리적 결합이 이루어짐을 확인할 수 있었다.결론적으로 C6 뇌교종세포주에서 dexamethasone이 투여되면 이는 GR에 ligand 로서 결합을 하게 되고, GR은 STAT5의 활성을 유도하고, 이와 물리적 결합을 하게 된다. 이 두 단백질 복합체는 핵 내로 이동하여 Bcl-xL의 프로모터의 STAT5 단백질이 결합할 수 있는 DNA 영역에 붙어서 전사를 조절하게 된다. STAT5의 하위신호전달 물질인 Bcl-xL 단백질이 전사조절단계에서 조절되어 그 양이 증가되고, 결국 이는 camptothecin에 의해 유도되는 세포고사를 억제하는 것으로 생각된다.따라서 임상에서 뇌종양 환자에서 흔히 쓰이고 있는 스테로이드 투여는 각종 항암제에 대해 세포사멸효과를 떨어뜨릴 수 있으므로 향후 이 기전 연구를 바탕으로 dexamethasone에 의해 생길 수 있는 가능한 내성요인을 극소화하는 방법을 모색해야 할 것이다.
[영문]Malignant glioma, which is the most common primary central nervous system tumor in adults, carries extremely poor prognosis despite aggressive treatment with surgery, irradiation, and/or chemotherapy. These tumor cells are notoriously resistant to apoptosis. Resistance to apoptosis can be achieved via the up-regulation and/or down-regulation of anti-apoptotic or pro-apoptotic Bcl-2 family members, respectively. Recently dexamethasone, a synthetic glucocorticoid, is reported to induce partial resistance to anticancer drugs in glioma cells by transcriptional activation of Bcl-xL gene. The notion that the steroid, used commonly to relieve edema associated with brain tumor, protects cells from camptothecin, directed this study to investigate the upstream regulators for Bcl-xL transactivation after dexamethasone treatment.For the induction of apoptosis in C6 glioma cells, 2 μM of camptothecin was added to the culture medium. Apoptotic cell death was confirmed by a colorimetric caspase-3 assay with CaspACETM？(Promega, Madison, USA), and DNA breakage by Cell Death Detection Elisa Kit？(Roche, Mannheim, Germany). Increased caspase-3 activity and DNA fragmentaion after camptothecin alone was markedly reduced in cells pre-treated with 1 μM of dexamethasone. This effect was abolished by glucocorticoid receptor (GR) antagonist RU486, indicating that the protection due to dexamethasone is mediated via this receptor. Bcl-xL, which is markedly up-regulated in a time-dependent fashion following exposure to dexamethasone. The extent of apoptosis inhibition by dexamethasone corresponded well with the change of Bcl-xL expression according to exposure time to dexamethasone. In order to study the role of STAT5 in this signaling cascade, we determined Bcl-xL expression with introduction of dominant-negative STAT5. C6 glioma cells infected with the dominant negative STAT5 adenovirus, AdStat5aΔ740, suppressed strongly expression of Bcl-xL gene, indicating that Bcl-xL expression was controlled in transcriptional level by STAT5. Upon dexamethasone treatment, phosphorylated STAT5 increased within 2 hour and gradually decreased from 4-6 hour on western blot. On electrophoretic mobility shift assay (EMSA) to investigate nuclear DNA binding activity of STAT5 protein, the binding activity increased gradually up to 4 hour and then decreased thereafter. Nuclear extract was immunoprecipitated with a GR receptor specific antiserum, and developed on immunoblot with a STAT5 specific antiserum. Untreated control cells showed minimal activity of phosphorylated STAT5, whereas cells treated with dexamethasone for 2-4 hour had increased phosphorylated STAT5 activity. In summary, dexamethasone binds to GR, which then activates STAT5. Activated GR complexes with STAT5, and helps it to direct the STAT5 DNA binding site, providing positive enhancement of STAT5 transactivation for Bcl-xL regulation. Ultimately this mechanism study will have a role to identify therapeutic drugs or methods to reverse the effect of inhibition of camptothecin-induced apoptosis by dexamethasone which is needed essentially in the treatment with brain tumor.ope
Influences of gamma-ray irradiation upon the resistance of hookworm and the hosts
No full text의학과/박사[한글]
Influences of Gamma-ray Irradiation upon the Resistance of Hookworm and the Hosts
Jae-Ho Cho, M.D.
Department of Parasitology
Yonsei University College of Medicine, Seoul, Korea
(Director: Professor Chin Thack Soh, M.D.
There have been many reports about the influence of irradiation upon the eggs and
larvae of nematodes.
Parthes (1904) first reported that growth and maturity of ascaris eggs were
inhibited by X-ray irradiation. Tyzzer and Honeij(1916) and Zaiman et al.(1655)
reported that Trichinella spiralis larvae attenuated by exposure to a suitable dose
of gamma-irradiation induced a certain degree of resistance to challenging
infection with normal larvae, and stimulated production of antibodies. Miller(1964,
1965 b, c) found that a single subcutaneous infection with 40-KR-irradiated larvae
showed a highly significant resistance to a subsequent challenge of normal larvae
and a sterilizing effect on the female worm of Ancylostoma caninum. Naiman(1944)
reported that X-ray irradiation of rats increased the severity of Trypanosoma
lewisi infection, and diminished the ablastic antibody production.
The present investigation was designed to study on the influence of
gamma-irradiation upon the following areas; infectivity and reproductivity of
hookworm, pathogenicity and productivity of antibodies in the host
Ancylostoma caninum eggs were obtained from a dog feces and cultured for 7 days
at 25∼30。C. The filariform larvae were inoculated subcutanequslyon the thigh
region of experimental animals. Parasites free dogs and rats were used for
experiments.
Serum collection: The dog serum collected after 15min centrifugation at 3,000rpm
with 2 to 3 ml of their blood which was placed at room temperature for 20 minutes.
The blood of rats was taken from their cut tails with the capillary tube, and the
sera were obtained after 5 minutes centrifugation at 11,000rpm by Adams Autocrit.
Total serum protein was measured by Micro-Kjeldahl method and the serum protein
fraction was checked using paper electrophoresis in which serum was run to the
Whatman No. 1 filter paper for 16 hours at 70 volt. The reading was done by Junior
spectrophotometer in 520 mμ light length. Irradiation: Gamma-ray irradiation was
performed with a "Gamma cell 220" Cobalt-60 irradiator. Each of the larval pools
and rats were exposed to the following dosages;
Pool 0: No irradiation Pool 1: 5,000 R
Pool 2: 50,000 R Pool 3: 100,000 R
Pool 4: 1,000,000 R
Control rat : No irradiation Rat Ⅰ: 100 R
Rat Ⅱ: 500 R Rat Ⅲ: 1,000 R
Egg productivity: E.P.G(Eggs per gram of feces) was counted 21 to 46 days after
the infection by Stoll's eggs counting method at five day interval. Infectivity:
The rats were sacrificed 3 to 30 days after skin infection of the irradiated
larvae, and the migrating larvae were collected by Baermann's larvae collection
method from lungs, liver and carcase of the experimental animals. In dogs, adult
worms were collected from in he small bowel of the host 5 weeks after the
infestation.
Pathogenicity: The routine pathological examinations of lungs, liver and skin of
the sacrificed rats were carried out.
The results are summarize as follow:
Infectivity and reproductivity on gamma-irradiated larvae of Ancylostoma caninum:
In rats the proper host for A.caninum, the migrating larvae were collected from
lung, liver and muscles of the sacrificed animals.
In the 5,000 R-group, 25.7% the larvae were recovered three days after infection
with 1,000 irradiated larvae, and 29.1% in 100,000 R-group and 14.9% in 1,000,000
R-group respectively, while in the control group showed 38.3%b recovery.
In the control group, 74 larvae were recovered 30 days after infection with 1,000
non-irradiated filariform larvae. None was seen in both 50,000R and 100,000R
groups.
Whereas in 1,000,000 R-group the larvae was not found at 15 days after infection.
In the experimental groups for the challenge infection of non-irradiated larvae,
247 larvae were recovered in 5,000 R-group, 165 in 50,000 R-group, 254 in 100,000
R-group and 348 in the control group respectively.
In dogs, the proper host to A.caninum, 25 adult worms were recovered in 5,000
R-group after infected with 100 irradiated filariform larvae, 41 adults in the
control group. However, the adult was not found at all in 100,000 R-group.
In 5,000 R-group 41 adults were recovered after the challenging infection on
control larvae, but 57 adults in 100,000 R-group and 76 adults in the control
group. A single infection of 5,000 to 50,000 R-irradiated larvae showed a higher
resistance to a subsequent challenge of normal larvae.
The E.P.G. of 5,000 R-group in the dog decreased compared with control group
during 36 days observation after infection of irradiated filariform larvae, and
none was seen in 100,000 R-irradiated group.
Serum γ-globulin of rats and dogs infected with gamma-irradiated larvae of
A.caninum:
In all groups of rats, the abnormal host to A.caninum, serum γ-globulin
increased remarkably from 10.2% to 22.5% in comparison with the normal control
(10.2% to 19.6%) and to 100,000 R-group (10.0% to 21.1%). In dogs, the normal host
to A.caninum, γ-globulin tended to be similar to the rat groups. On the contrary,
A/G ratio decreased in al infection group during 5 weeks after infection of
hookworm.
Pathogenicity of rats infected with gamma-irradiated larvae of A.caninum:
In the rats infected with 5,ooo R-irradiated larvae, some inflammatory process
and hemorrhages were found in the lung but tissues were similar to the normal rats.
The pathological changes of the skin were similar with the picture of the lungs.
Infectivity and serum γ-globulin on gamma-irradiated rats infected with
non-irradiated larvae of A.caninum:
In infectivity slightly increased in 1,00 R-group in comparison to the normal
control rats. Serum γ-globulin decreased slightly in 100 R-group three weeks after
infection.
Whereas 1,000 R-irradiated rats which were infected with the normal larvae were
died within two weeks after infection.
The above results indicate that:
1. The infectivity of the gamma-ray irradiated filariform larvae of A.caninum was
inhibited at the dosage of 50,000R
2. The reproductivity of the gamma-ray irradiated filariform larvae of A.caninum
decreased at dosage of 5,000R
3. The pathogenicity of the gamma-ray irradiated filariform larvae wa milder than
contry group. The more the dosage of gamma-ray irradiation increased, the milder
pathological changes were occured.
4. The γ-globulin in serum of the host enhanced remarkably in the group of
50,000 R irradiated filariform larvae, and then the A/G ratio was decreased
according to the increase of γ-globulin.
5. In the irradiated host the discovery of the migrating larvae increased
slightly at the dosage of 1,000R. on the other hand the γ-globulin decreased
slightly in 100 R-irradiated host.
The above results indicate that gamma-ray irradiation decreases the infectivity
and reproductivity of the parasite. On the other hand, the irradiation upon the
host decreases the resistance of the host against parasite infection.
[영문]
There have been many reports about the influence of irradiation upon the eggs and larvae of nematodes.
Parthes (1904) first reported that growth and maturity of ascaris eggs were inhibited by X-ray irradiation. Tyzzer and Honeij(1916) and Zaiman et al.(1655) reported that Trichinella spiralis larvae attenuated by exposure to a suitable dose
of gamma-irradiation induced a certain degree of resistance to challenging infection with normal larvae, and stimulated production of antibodies. Miller(1964, 1965 b, c) found that a single subcutaneous infection with 40-KR-irradiated larvae
showed a highly significant resistance to a subsequent challenge of normal larvae and a sterilizing effect on the female worm of Ancylostoma caninum. Naiman(1944) reported that X-ray irradiation of rats increased the severity of Trypanosoma lewisi infection, and diminished the ablastic antibody production.
The present investigation was designed to study on the influence of gamma-irradiation upon the following areas; infectivity and reproductivity of hookworm, pathogenicity and productivity of antibodies in the host
Ancylostoma caninum eggs were obtained from a dog feces and cultured for 7 days at 25∼30。C. The filariform larvae were inoculated subcutanequslyon the thigh region of experimental animals. Parasites free dogs and rats were used for
experiments.
Serum collection: The dog serum collected after 15min centrifugation at 3,000rpm with 2 to 3 ml of their blood which was placed at room temperature for 20 minutes.
The blood of rats was taken from their cut tails with the capillary tube, and the sera were obtained after 5 minutes centrifugation at 11,000rpm by Adams Autocrit.
Total serum protein was measured by Micro-Kjeldahl method and the serum protein fraction was checked using paper electrophoresis in which serum was run to the Whatman No. 1 filter paper for 16 hours at 70 volt. The reading was done by Junior
spectrophotometer in 520 mμ light length. Irradiation: Gamma-ray irradiation was performed with a "Gamma cell 220" Cobalt-60 irradiator. Each of the larval pools and rats were exposed to the following dosages;
Pool 0: No irradiation Pool 1: 5,000 R
Pool 2: 50,000 R Pool 3: 100,000 R
Pool 4: 1,000,000 R
Control rat : No irradiation Rat Ⅰ: 100 R
Rat Ⅱ: 500 R Rat Ⅲ: 1,000 R
Egg productivity: E.P.G(Eggs per gram of feces) was counted 21 to 46 days after the infection by Stoll's eggs counting method at five day interval. Infectivity: The rats were sacrificed 3 to 30 days after skin infection of the irradiated larvae, and the migrating larvae were collected by Baermann's larvae collection
method from lungs, liver and carcase of the experimental animals. In dogs, adult worms were collected from in he small bowel of the host 5 weeks after the infestation.
Pathogenicity: The routine pathological examinations of lungs, liver and skin of the sacrificed rats were carried out.
The results are summarize as follow:
Infectivity and reproductivity on gamma-irradiated larvae of Ancylostoma caninum:
In rats the proper host for A.caninum, the migrating larvae were collected from lung, liver and muscles of the sacrificed animals.
In the 5,000 R-group, 25.7% the larvae were recovered three days after infection with 1,000 irradiated larvae, and 29.1% in 100,000 R-group and 14.9% in 1,000,000 R-group respectively, while in the control group showed 38.3%b recovery.
In the control group, 74 larvae were recovered 30 days after infection with 1,000 non-irradiated filariform larvae. None was seen in both 50,000R and 100,000R groups.
Whereas in 1,000,000 R-group the larvae was not found at 15 days after infection.
In the experimental groups for the challenge infection of non-irradiated larvae, 247 larvae were recovered in 5,000 R-group, 165 in 50,000 R-group, 254 in 100,000 R-group and 348 in the control group respectively.
In dogs, the proper host to A.caninum, 25 adult worms were recovered in 5,000 R-group after infected with 100 irradiated filariform larvae, 41 adults in the control group. However, the adult was not found at all in 100,000 R-group.
In 5,000 R-group 41 adults were recovered after the challenging infection on control larvae, but 57 adults in 100,000 R-group and 76 adults in the control group. A single infection of 5,000 to 50,000 R-irradiated larvae showed a higher resistance to a subsequent challenge of normal larvae.
The E.P.G. of 5,000 R-group in the dog decreased compared with control group during 36 days observation after infection of irradiated filariform larvae, and none was seen in 100,000 R-irradiated group.
Serum γ-globulin of rats and dogs infected with gamma-irradiated larvae of A.caninum:
In all groups of rats, the abnormal host to A.caninum, serum γ-globulin increased remarkably from 10.2% to 22.5% in comparison with the normal control (10.2% to 19.6%) and to 100,000 R-group (10.0% to 21.1%). In dogs, the normal host
to A.caninum, γ-globulin tended to be similar to the rat groups. On the contrary, A/G ratio decreased in al infection group during 5 weeks after infection of hookworm.
Pathogenicity of rats infected with gamma-irradiated larvae of A.caninum:
In the rats infected with 5,ooo R-irradiated larvae, some inflammatory process and hemorrhages were found in the lung but tissues were similar to the normal rats.
The pathological changes of the skin were similar with the picture of the lungs.
Infectivity and serum γ-globulin on gamma-irradiated rats infected with non-irradiated larvae of A.caninum:
In infectivity slightly increased in 1,00 R-group in comparison to the normal control rats. Serum γ-globulin decreased slightly in 100 R-group three weeks after infection.
Whereas 1,000 R-irradiated rats which were infected with the normal larvae were died within two weeks after infection.
The above results indicate that:
1. The infectivity of the gamma-ray irradiated filariform larvae of A.caninum was inhibited at the dosage of 50,000R
2. The reproductivity of the gamma-ray irradiated filariform larvae of A.caninum decreased at dosage of 5,000R
3. The pathogenicity of the gamma-ray irradiated filariform larvae wa milder than contry group. The more the dosage of gamma-ray irradiation increased, the milder pathological changes were occured.
4. The γ-globulin in serum of the host enhanced remarkably in the group of 50,000 R irradiated filariform larvae, and then the A/G ratio was decreased according to the increase of γ-globulin.
5. In the irradiated host the discovery of the migrating larvae increased slightly at the dosage of 1,000R. on the other hand the γ-globulin decreased slightly in 100 R-irradiated host.
The above results indicate that gamma-ray irradiation decreases the infectivity and reproductivity of the parasite. On the other hand, the irradiation upon the host decreases the resistance of the host against parasite infection.restrictio
Collagen type II and TGF-β1 control specific gene expression of chondrocyte by MAP kinase activation
No full text의학과/박사[한글]
관절 연골 (articular cartilage)의 손상은 재생 능력이 매우 제한적이므로 여러 방법의 연골 재생이 시도 되고 있다. 본 논문은 연골 세포의 주요한 세포외 산물인 제 2형 교원질 (type II collagen)이 단순한 세포 산물이 아닌 동적인 작용으로 TGF-β1 (transforming growth factor-β1) 등의 성장 인자와 같이 연골세포의 탈분화 (dedifferentiation)를 방지하고 세포의 표현형 유지와 연골 세포의 특이 유전자 발현을 조절하며 이러한 조절이 Mitogen activated protein (MAP) kinase의 경로를 통함을 확인하고자 하였다. 이를 위해 단층 배양 및 alginate 삼차원 배양에서 세포 표현형의 변화를 관찰하였고, 세포외 교원질의 부착에 관여하는 integrin의 발현 변화를 면역형광 염색으로 관찰 하였으며, Western blot으로 integrin 및 MAP kinase의 발현 및 활성을 확인하였다. 또한 역전사-중합 효소 연쇄 반응으로 제 2형 교원질 및 aggrecan 유전자의 mRNA 발현을 확인하였다. 본 실험의 결과로 제 2형 교원질이 피복(coating)된 배양 조건의 관절 연골세포에서는 7일까지 integrin α2, FAK (focal adhesion kinase) 발현이 증가되었으며, 신호 전달 물질인 ERK의 인산화가 지속적으로 유지되었다. 이러한 세포 내 신호 전달 과정의 차이는 연골세포 내 세포 골격 (cytoskeleton)의 변화와 세포 모양에도 영향을 미치며, 관절 연골세포의 형태와 특이 유전자 발현은 intergin α2 β1-FAK 신호 전달 (signaling)이 중요한 역할을 하리라 사료된다. Alginate bead 배양 상에서 ERK의 활성은 TGF-β1과 제2형 교원질에 의해 각각 활성화되었으며 두 요소가 같이 처리된 군에서 ERK의 활성이 지속적으로 증가하였고, col2A1, aggrecan mRNA는 TGF- β1 뿐 아니라 제 2형 교원질에 의해 발현하였다. 최종적으로 본 연구 결과를 통해 연골세포와 제 2형 교원질의 상호 작용이 integrin α2β1-FAK 복합체(complex)를 통해 ERK1/2를 활성화 시키고 결국 col2A1 mRNA의 발현을 증가시킴을 알 수 있었다. 이를 종합하면, 이미 알려진 TGF-β1의 수용체를 통해 세포의 분화와 증식을 촉진 시키는 Ras-raf-ERK 기전과 더불어 세포 외 기질인 제 2형 교원질 역시 integrin-FAK를 거쳐 MAP kinase의 활성화로 연골 세포의 특이 유전자 발현과 그 형태 유지에 중요한 역할을 하리라 사료된다.
[영문]The regeneration potential of articular chondrocyte is limited. There has been little study about the role of extracellular matrix in cartilage regeneration. The main component of extracellular matrix of articular chondrocyte is collagen type II that is not only important extracellular matrix but also having dynamic effect on chondrocyte. In this study, Cell morphology and correlation between type II collagen and dedifferentiation of chondrocyte were observed. Western blot and immunofluorescence staining checked Integrin expression and MAP kinase. The aggrecan and type II collagen gene expression were checked by PT-PCR. In our results, integrin 2 and FAK expression were significantly increased at 7 days in cells grown in type II collagen coated plates. ERK was transiently activated at 1h, 10h, and 7 days in TCP attached cells whereas ERK activation was maintained from 1 h to 7 days in type II collagen attached cells. Also these different signaling pathways between TCP and type II collagen may influence on cytoskeletal reorganization and then cell morphology. The changes in morphology and gene expression may be regulated by integrin receptor (α2β1)-FAK signaling in articular chondrocytes. In alginate bead culture, ERK activation was increased and maintained by TGF-β1 and type II collagen treated chondrocytes until 4 day. In conclusion COL2A1 mRNA and MAP kinase were expressed and controlled by TGF-β1 and type II collagen.ope
The Direction for Foreign Investment in Ulsan City
No full text본 논문에서는 동북아시아의 경제협력, 울산경제의 특징, 울산과 중국간의 무역추이, 최근 외국인 투자추세를 분석하면서 울산시가 추진하고 있는 외국인 투자 유치를 통한 물류 및 서비스 중심의 산업구조로 전환하는 경제개발 전략의 한계를 지적하였다. 본 논문에서 제시하는 외국인 투자유치 방향은 제조업에 중심을 둔 거점형 해외투자유치를 적극이용하면서 동북아시아의 자원 중심적 동북아 지역 전진 기지화를 추진하는 것을 주장하였다. 따라서 울산시가 추진하고 있는 다양한 분야의 외국인 해외투자유치 보다는 한국의 대표적인 공업도시로서 제조업 중심의 비교우위가 있는 특정분야를 직접 육성하는 방향으로 울산의 장기 발전전략이 수립되어야 한다.This paper discusses the prospects for APEC, the trend of global foreign investment and the characteristics of Ulsan industries and then reviews the recent policy direction for foreign investment proposed by Ulsan city. This paper suggests that Ulsan city should develop the regional development plan based upon the Ulsan citys competitiveness on heavy-and chemical industries. This paper suggests that Ulsan citys plan should establish the foreign investment straegy aiming at the China and Russia markets under the APEC movement.This paper discusses the prospects for APEC, the trend of global foreign investment and the characteristics of Ulsan industries and then reviews the recent policy direction for foreign investment proposed by Ulsan city. This paper suggests that Ulsan city should develop the regional development plan based upon the Ulsan citys competitiveness on heavy-and chemical industries. This paper suggests that Ulsan citys plan should establish the foreign investment straegy aiming at the China and Russia markets under the APEC movement
(The) benefit of individualized custom bolus in the postmastectomy radiation therapy : numerical analysis wit
No full text의학과/석사[한글]
근치적 유방절제술 후 보조요법으로 시행되는 항암화학방사선 병합요법을 통해 국소진행성 유방암 환자들의 생존율이 크게 향상되어, 치료와 연관된 부작용을 최소화하기 위한 세심한 방사선치료기법의 중요성이 더욱 커지고 있다. 최근 방사선치료기법의 발달로 과거에 비해서 폐나 심장의 합병증 발생 빈도가 많이 줄었지만, 특히 방사선폐렴은 임상에서 흔히 볼 수 있는 합병증으로 이를 줄이기 위한 노력이 절실히 필요하다. 일반적으로 유방암의 수술 후 방사선조사시 흉벽의 두께가 한 환자에서 위치에 따라 많은 차이를 보이는데, 이런 문제점을 해결하기 위해 내유방림프절과 흉벽을 다른 에너지의 전자선으로 치료하기도 하고, 흉벽이 얇은 곳을 보상하는 조직보상체를 사용하기도 하며, 접사면 기법으로 치료하기도 한다. 본 연구에서는 폐나 심장의 정상 장기에 대한 합병증을 줄이고자 흉벽에 대한 전자선 치료시 매 환자의 흉벽 두께를 1cm² 간격으로 세밀히 측정하여 조사야 내 전체 흉벽 두께를 균일하도록 보상할 수 있는 개별화된 조직보상체를 제작하였다.
한편, 방사선치료는 최근 3차원 입체조형치료계획의 발달로 환자가 실제로 조사 받는 방사선의 체내 선량 분포를 3차원적으로 한 눈에 확인할 수 있게 되었으며, 나아가서 종양이나 정상 장기의 방사선 흡수 선량과 그 해당 체적의 상관관계를 보여주는 선량체적히스토그램을 얻어 이를 통해 정상 장기의 합병증 발생률까지도 수치적으로 예측하고 있다.
하지만, 아직 근치적 유방절제술 후 전자선을 이용한 수술 후 방사선 치료법을 3차원 입체조형치료계획으로 구현하여 정상조직합병증발생률을 분석한 보고는 거의 없는 상태이며, 또한 흉벽 두께 보상을 위한 개별 조직보상체의 효용성에 대한 연구 자료도 부족한 상태이다. 이에 본 연구는 이러한 개별화된 조직보상체의 유용성을 평가하기 위하여 3차원 입체조형치료계획을 시행하여 실제적인 선량분포를 확인하고 나아가서 정상 장기인 폐나 심장에 대한 합병증 발생률 측면에 있어서도 어느 정도의 효과를 얻을 수 있는지 분석하고자 하였다.
본 연구는 유방전절제술 후 방사선치료를 받는 10명의 환자를 대상으로 하였으며, 우측 유방암 환자가 3명, 좌측 유방암 환자가 7명이었다. 모든 환자는 먼저 치료계획용 컴퓨터단층촬영을 시행하였으며, 이를 바탕으로 1cm² 간격으로 흉벽 두께를 세밀히 측정하였다. 이후 주로 내유방림프절 근방인 가장 두꺼운 흉벽 부위를 기준으로 그 곳의 전방 흉막면에 80% 선량이 조사될 수 있는 방사선에너지를 설정하고, 이 부위를 기준으로 보다 얇은 곳을 포함한 전체 흉벽의 두께가 균일해지도록 하는 개별화된 조직보상체를 제작하
였으며, 제작된 조직보상체를 적용하여 다시 치료계획용 컴퓨터단층촬영을 시행하였다.
이후 각 환자의 두 영상자료를 이용하여 3차원 치료계획용프로그램으로 설계하였다. 매 환자에서 조직보상체 적용 전과 후로 등선량곡선 분포 및 선량체적히스토그램을 비교하였고, 정상조직합병증발생률의 변화 및 기타 선량통계값도 분석 비교하였다. 그 결과는 다음과 같다.
1. 조직보상체를 적용하였을 때 모든 예에서 처방선량의 80% 등선량곡선의 깊이가 흉벽두께와 거의 일치하였다. 조직보상체를 사용하지 않았을 때는 90% 이상의 등선량 곡선이 전방 흉막면을 지나 폐 실질 부위에 깊이 걸쳐 있는 경우가 많았으며, 특히 좌측 유방암의
경우에는 심장에도 불필요하게 높은 선량이 조사됨을 관찰할 수 있었다.
2. 선량체적히스토그램을 조직보상체 적용 전,후로 동측 폐, 반대측 폐 및 심장에 대하여 각각 비교하였는데, 모든 예에서 조직보상체를 사용하였을 때, 동측 폐의 선량체적히스토그램이 크게 향상된 소견을 보였으며, 심장의 경우 좌측 유방암 환자에서 특히 두드러진 향상을 보였다. 반대측 폐의 경우는 조직보상체 적용 전,후로 거의 변화가 없었다.
3. 동측 폐의 경우 조직보상체를 적용하지 않았을 때, 평균 NTCP 값이 80.2 ± 3.43% 이었으며, 조직보상체를 사용한 경우에는 평균 NTCP 값이 47.7 ± 4.61% 로 현저하게 감소하였다. 따라서 개별화된 조직보상체의 사용으로 24.5% - 40.5% 의 정상조직합병증발생률을 줄일 수 있다고 생각할 수 있었다. 각 환자의 NTCP값을 Paired t-test를 이용하여 그 차이를 분석해 본 결과 p-value 0.01 이하로 통계적으로 유의하였다.
4. 동측 폐와 심장에 대해서 평균 선량, V_(50)(처방선량 50%이상의 선량이 조사되는 체적의 백분율), V_(95)(처방선량 95%이상의 선량이 조사되는 체적의 백분율), 최대 선량, 최소 선량 등을 구하였다. 선량통계값에 있어서 조직보상체 적용 전,후에 두드러진 변화를 보인 것은 평균 선량, V_(50), V_(95) 이었다. 최대선량 및 최소 선량값은 별다른 차이를 보이지 않았다. 동측 폐의 평균선량값은 평균 2757 cGy에서 2327 cGy로 감소하였다(p < 0.01). 심장의 평균선량값은 893 cGy에서 729 cGy로 감소하였으며, V50 값은 조직보상체를 적용하지 않은 경우 동측 폐와 심장에서 각각 평균 54.5%와 16.8% 였는데, 적용한 경우 45.3%와 9.8% 로 크게 감소하였으며, V95 값은 동측 폐에서 평균 17.4%에서 11.0%로, 심장에서 6.1%에서 2.2%로 감소하였다. 우측 유방암을 가진 환자들의 심장에 대한 V_(50) 및 V_(95) 는 1예를 제외한 모든 예에서 조직보상체 적용 유무와 상관없이 2% 이내의 적은 값을 보였다.
결론적으로, 유방암 환자의 수술 후 방사선치료 시 개개 환자의 흉벽의 불균일성을 보상하는 조직보상체의 유용성을 3차원 입체조형치료계획을 통해서 확인하고자 한 본 연구에서 조직보상체를 적용하지 않은 경우에 비해 적용한 경우에 등선량곡선분포, 선량체적
히스토그램, Lyman-Kutcher 모델에 의한 정상조직합병증발생률 및 기타 선량통계값 등 모든 면에 있어서 우월성을 확인할 수 있었다. 따라서 이러한 개별화된 조직보상체의 확대 적용을 위한 과학적인 근거를 제시할 수 있었다. 향후로 임상에서 이러한 결과가 실질적인 합병증 발생률 감소와 잘 연계되는지 계속적인 추적관찰 및 연구가 필요할 것으로 생각된다
[영문]
Purpose : With the improved survival rate of patients with breast cancer by postoperative chemoradiotherapy, meticulous radiotherapy techniques to minimize side effects became more important. To reduce the dose to lung and heart in the case of chest wall irradiation using an oppositional electron beam, we used an individualized custom bolus which was precisely designed to compensate the difference of chest wall thickness. The benefits were evaluated by comparing the normal tissue complication probabilities (NTCPs) and dose statistics with boluses to those without boluses.
Methods and Materials : Boluses were made and the effects were evaluated for ten patients treated with the reverse hockey stick technique. Electron beam energy was determined in order to irradiate 80% of the prescription dose to the deepest lung-chest wall boarder, which was usually located at the internal mammary lymph node chain. An individualized custom bolus was made to compensate the chest wall thinner than the prescription depth by meticulously measuring the chest wall thickness at 1cm² interval on the planning CT images. Second planning CT was obtained overlying the individualized custom bolus to each patient's chest wall. 3-D treatment planning was performed using ADAC-Pinnacle³ for each patient with or without bolus. NTCPs based on "the Lyman-Kutcher" model were analyzed and the mean, maximum, minimum doses, V_(50) and V_(95) for heart and lung were computed.
Results : The average NTCPs in the ipsilateral lung were reduced from 80.2 ± 3.43% to 47.7 ± 4.61% when individualized custom boluses were used, which shows statistically meaningful reduction (p<0.01). The mean lung dose in the ipsilateral lung was also significantly reduced about 430 cGy from 2757 cGy to 2327 cGy
(p<0.01). V_(50) and V_(95) in the ipsilateral lung markedly decreased from the average 54.5% and 17.4% to the average 45.3% and 11.0%. V_(50) and V_(95) in the heart also decreased from the average 16.8% and 6.1% to the average 9.8% and 2.2%. The NTCP in the contralateral lung as well as the heart were 0% even in the case of no bolus due to the small effective radiation volumes, mean value 4.4% and 7.1%, respectively.
Conclusion : The use of an individualized custom bolus in the radiotherapy of postmastectomy chest wall reduced the NTCP of ipsilateral lung about 24.5% - 40.5%, which can increase the complication free cure probability of breast cancer patients.ope
Arthroscopic medical reefing for patella maltracking with tracking surveillance : 관절경적 내측 지대 보강술 및 외측 지대 이완술
No full text의학과/석사[한글]
슬개골 재발성 탈구 또는 아탈구에 대한 치료 방법은 다양한 술식이 소개되어왔다. 그러나 수술 후의 정복 된 위치 외에 슬개골의 기울기 또는 슬관절 운동시 슬개골 궤도에 대한 수술 중 평가는 정확히 이뤄지지 못하였다. 저자들은 본 연구를 통하여 슬개골 재발성 탈구 또는 아탈구에 대한 관절경적 수술 방법과 결과를 평가하고자 하였다.1991년 1월부터 1996년 12월까지 슬개골 재발성 탈구 또는 아탈구에 대해 관절경적 수술을 시행한 34 명의 환자 36례를 대상으로 성별, 연령, 원인, 슬관절 내의 동반 병변을 조사하였고 방사선학적으로 슬개고위증(patella alta), 외반 슬, 대퇴 외과 발육 부전 등을 관찰하였다. 외측 및 내측으로 과도한 운동성을 보인 슬개골인 4례(11%)는 관절경적내측 보강술을 시행하였다. 습관성 탈구로 슬관절 외측 구축이 있던 1471(39%)는 관절경적 외측 이완술
과 내측지대 보강술로 슬개골의 위치, 기울기 그리고 슬관절 운동시 슬개골의 궤도를 정상화 시켰다 슬개골의 아탈구와 비정상적 기울기를 보인 1271(33%)는 외측 이완술 및 내측 보강술을 시행하였다. 관절경적 술식으로 정확한 궤도를 얻지 못하고 Q각이 20도 이상인 6례(17%)의 경우 Elmslie-Trillat 술식을 추가하였다.
34명(3671)의 대상 환자의 성별분포는 남자 9명 여자 25명이었다. 관절경적 수술 중 1177(31%)에서 동반된 골연골편 유리체가 관찰되었고,32례(89%)에서 연골연화증이 슬개골 또는 대퇴 외과에서 관찰되었으며 5례(14%)는 술전 또는 교정하는 관절경 술식 중 슬개
내측 추벽(medialpatella plica)이 관찰되었다. 5례의 내측 반월상 연골 파열, 2례의 외측반월상 연골 손상, 2례의 외측 원판형 연골, 1례의 전방 십자 인대내의결절종이 동반되어 있었으며 이러한 관절내 병변은 슬개골 탈구 또는 아탈구에 대한 술식과 동시에 치료하였다.
Elmslie-Trillat 술식을 시행한 6례를 제외한 환자는 술후 3-4일에 슬개골 보조기 착용 후 관절 운동 및 대퇴 사두근 강화 운동을 허용하였다. 통증 소실 여부에 따라 체중 부하를 시작하여 술 후 4-5개월에 운동(sports)을 허락하였다.
술 후 평균 48.1개월(25-93개월) 추시 관찰상 3271(89%)에서 관절 운동범위가 정상이며 통증없이 운동이 가능하였다. 운동 범위가 신전 0도에서 골곡 120도로 제한된 환자가 3711(8%)였으며, 90도 이내의 관절 운동제한을 보인 환자가 1례 있었다. 이는 관절경적 유착제거술로 치료하였다. 1례는 추시 도중 다른 외상에 의해 재탈구 소견을 보여 관절경적 수술을 다시 시행하였다.
슬관절 재발성 탈구 또는 아탈구에 대한 관절경적 치료는 슬개골의 위치를 직접 관절경으로 확인하며, 위치와 기울기, 궤도를 정확히 교정함으로 과교정을 막을 수 있다. 또한 관절 내부의 동반된 병변에 대한동시 치료가 가능하고 합병증이 적은 장점이 있다. 여성 환자의 경우 관절경 술식이 미용적으로 우수하다.관절경적 술식은 술 후 재활시기의 통증이 적고, 우수한 결과를 가져오는 술식으로 사료된다.
[영문]
Many surgical treatment for patella dislocation and subluxation have been introduced but there were no delicate description about the importance of patella tracking and tilting during arthroscopic operation. The purpose of this study is to review the results and benefit of arthroscopic medial reefing and lateral release operation with tracking and tilting surveillance. We review 36 cases patella subluxation and dislocation. Patients were examined clinically as well as radiologically. The average length of follow up was 48 months. 14 cases of recurrent dislocation with lateral tightness were treated by medial reefing and lateral release. 4 cases of hypermobil patella were treated by medial imbrication.
12 cases of subluxation or patella tilting were treated by lateral release with medial reefing. Despite arthroscopic operation ,there were 6 cases of maltracking with increased Q-angle which were treated by additional Elmslie-Trillat operation
with arthroscopic tracking surveillance. We found 32 cases associated chondromalasia, 11 cases of osteochondral fragment, 5 cases medial patella plica syndrome on preoperation or during operation and other intraarticular problem. Which were treated by arthroscopically. The function were evaluated by Crosby and
Insall criteria. 32 cases revealed excellent result,3 cases were good and 1 case was fair There were no iatrogenic medial patella plica syndrome or tilting. One case of recurrent dislocation and one case of limitation of motion less than 90 degrees were treated by arthroscopic methods. The arthroscopic operation with
suveillance of tracking and tilting during range of motion exercise bring consistent excellent result, low mobidity, cosmetic advantage.ope
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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