효소 활성 측정과 생물 영상화를 위한 반응기반 탐침의 개발

학위논문 (박사)-- 서울대학교 대학원 자연과학대학 화학부, 2017. 8. 홍종인.The detection of enzyme activity provides many clues to the nature of living systems and the interaction between biomolecules. Enzyme reaction triggers chemical transformation of substrate to regulate its function relating to many biological events. The most general approach for enzyme activity assay is a reaction-based strategy using the chemical transformation of substrates. Numerous probes have been developed based on the enzymatic reaction. However, only a few strategies to control a signal have been developed, and most of them have not been applied practically. Herein, several probes based on two basic strategies are proposed. The chemical transformation of a probe by enzymatic reaction triggers intramolecular cyclization to form fluorescent N-methylisoindole, which is a basic strategy to detect the enzyme activity. Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), which exhibited little change in the kinetic parameters, but a stronger emission than non-reducing conditions. Probe 1 also showed stronger intensity upon treatment with sulfatase under neutral conditions than acidic conditions, but it still has limitations in the selectivity of a specific sulfatase. Nevertheless, the fluorescent signal of the released N-methylisoindole provides a new assay for measuring sulfatase activity that could be applied for high-throughput screening (HTS). Bacterial arylsulfatases are crucial to biosynthesis in many microorganisms, as they cleave aromatic sulfate esters for use as a sulfur source. They are associated with pathogenesis and are applied in many areas, such as industry and agriculture. The hydrolysis of probe 1 by sulfatases induced fluorescence intensity enhancement and the generation of colored precipitates through the polymerization of N-methylisoindole, which enabled the monitoring of bacterial arylsulfatase activity and the discrimination of periplasmic sulfatases from cytosolic sulfatases through liquid- and solid-phase colony-based assays. For imaging analysis, the fluorescence of N-methylisoindole, the product of probe 1 from the cleavage upon sulfatase activity emitted a signal at the shorter wavelength. To overcome this limitation, fluorescence resonance energy transfer (FRET), aggregation-induced emission (AIE), and the excimer formation process were introduced. The FRET-based probe showed no fluorescence changes upon the treatment of sulfatase because the distance between N-methylisoindole and naphthalimide, an FRET acceptor, was 6.6 Å, which was out of range of the Förster distance. The AIE-based probe exhibited decreasing fluorescence and color changes, meaning that N-methylisoindole generation and polymerization did not intensify the AIE process. The excimer formation-based probe showed decreasing excimer emission, but increasing monomer and N-methylisoindole emissions. It also generated colored precipitates. When the probe was applied to bacteria species, the tendency of the emission was different from that in the results of the test in the homogenous condition, but precipitates were still formed. Thus, the excimer formation-based probe was only applicable to staining periplasmic sulfatase-expressing bacteria. The next strategy to design a probe was based on intramolecular charge transfer (ICT). The basic scaffold was 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) unit conjugated to a modified electron-donating group. Leucine aminopeptidases (LAPs) are widely distributed in organisms, from bacteria to humans, and play crucial roles in cell maintenance and growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this paper, we report a small-molecule probe, DCDHF conjugated to a leucine residue, which exhibits colorimetric and fluorogenic changes according to LAP activity. Human steroid sulfatase (STS) plays a pivotal role in the regulation of biologically active steroid to bind to the estrogen receptor (ER), which is related to hormone-dependent diseases. DCDHF conjugated with phenyl sulfate ester exhibits pH-dependent emission, which was appropriate for discriminating STS, whose optimal pH is 7other arylsulfatases usually exhibit the maximal activity at pH 5. DCDHF conjugated with phenyl sulfate interposing a self-immolative spacer between them is a ratiometric probe that could detect and monitor sulfatase activity in a complex cellular context.I. Introduction 1 1. Enzyme activity assays 2 2. Bioimaging 9 3. Fluorescent probe for detecting enzyme activity 16 4. Design strategy of fluorescent probe 21 II. Probes inducing signal changes by intramolecular cyclization upon the treatment of enzyme 34 1. Sulfatase activated probe generating N-methyl isoindole in buffer containing reducing agents 35 1.1. Introduction 35 1.2. Results and Discussion 37 1.3. Experiments 43 2. Detection of bacterial sulfatase activity and discrimination of periplasmic sulfatase activity through liquid- and solid-phase colony-based assays 59 2.1. Introduction 59 2.2. Results and Discussion 63 2.3. Experiments 74 3. Efforts for improvement of probe 1 using diverse signal generation strategies 86 3.1. Introduction 86 3.2. Results and Discussion 89 3.3. Experiments 97 III. Probes inducing fluorescence changes by intramolecular charge transfer process upon the treatment of enzyme 116 1. Small-molecule probe using dual signals to monitor leucine aminopeptidase activity 125 1.1. Introduction 117 1.2. Results and Discussion 118 1.3. Experiments 124 2. Selectvie detection of Steroide sulfatase (STS) and ratiometric detection of sulfatase activity in living cells 131 1.1. Introduction 131 1.2. Results and Discussion 134 1.3. Experiments 134 References 155 국문 초록 (Abstract in Korean) 173Docto

Comparison between Fluid Intake and Output Measurement Methods of the Patients Hospitalized in Medical Units

Purpose: The purpose of this study was to compare the fluid intake and output (I&O) measurement methods in order to figure out more effective and easier method for medical patients. Methods: 71 hospitalized patients participated in the study. In “liquid only (LO)” method, all amount of water was summed up including any liquid types of food and IV fluids. In “whole food(WF) intake,” all liquid and solid food intake and IV fluids were added up. Results: The average amount of fluid intake was 2105.29 ml for LO method and 2523.54 ml for WF method. The average amount of fluid output was 2148.98 ml. The intra-class correlations (ICC) between the intake and output measures by the two different methods was 0.803 and 0.826, respectively. The correlation between the differences of intake/output and body weight change in two different methods was r=.347 (p=.003), and r=.376 (p=.001), respectively. Conclusion: The results of this study indicate that both LO and WF method may be useful in monitoring patients’ fluid balance. Given the comparability of using LO over WF, it is suggested that measuring just liquid only intake as the indicator of patient’s intake is applicable in clinical setting.ope

MVRDV 건축의 datascape 공간조직방법 연구 : Villa VPRO, 1997와 Media Galaxy(Eyebeam Institute), 2001를 중심으로

학위논문(석사)--서울대학교 대학원 :건축학과,2003.Maste

글루탐산 분해효소(glutamate dehydrogenase)의 구조, 알로스테릭 조절, 및 작용기전

Dept. of Medical Technology/박사[영문] [한글] 글루탐산 분해효소 (GDH)의 구조 및 분자적 특성을 알아 보기 위하여 다음과 같은 연구 들을 수행하였다. 우선 조미료로 많이 이용되고 있으며 Chinese restaurant syndrome을 유발시키는 물질로 잘 알려져 있는 글루탐산나트륨 (MSG)이 뇌조직 내의 GDH에 미치는 영 향을 조사하였다. 장기간 MSG 복용에 의한 뇌에 미치는 영향을 알아보기 위해, 1주된 alb ino 쥐에게 1년간 식수에 MSG를 첨가하여 먹인 그룹과 먹이지 않은 두 그룹으로 나뉘어 실험해 보았다. 쥐의 몸무게와 뇌의 무게, 총 DNA와 RNA양 그리고 단백질 양에는 두 그룹 간의 유의한 차이를 보이지 않았으나 뇌의 생 추출물속의 글루탐산 농도를 비교한 결과, MSG를 먹인 그룹에서는 대조군에 비해 2배정도 높게 나타났다. 또한, 글루탐산 분해효소( GDH)의 mRNA 양은 두 그룹간의 유의한 차이를 보이지 않았으나, western blot 검사를 실 시한 결과 효소의 양은 MSG를 먹인 그룹이 현저해 감소한 것으로 보아, GDH 발현에 있어 후전사 조절에 영향을 미치거나 GDH의 분해율을 증가시키는 것으로 보여진다. 이 결과로, 장기간 MSG를 섭취하게 되면 쥐 뇌 속에 존재하는 GDH 활성을 감소시킴으로 글루탐산의 분해가 저하됨을 알 수 있었다. 다음으로, GDH의 구조적, 기능적 조절형태를 심도 있게 연구하기 위해 1557-base-pair의 인간 GDH 유전자를 합성하여 대장균주를 이용해 활성을 지닌 효소로 발현시켰다. 이렇게 발현된 효소는 사람과 소의 조직에서 추출한 효소와 여러 가지 생화학적 측면에서 유사 한 양상을 보였다. 자리지정 돌연변이방법을 이용해 이 효소의 Lys130 위치를 다른 아미 노산으로 바꾼 돌연체를 이용해 실험해 본 결과, Lys130 위치는 GDH의 기질이나 다른 조 효소가 결합하는 곳이 아닌 촉매작용을 일으키는데 관여하는 중요한 곳임을 알게 되었다. 다음으로, 인간 GDH의 알로스테릭 활성제인 ADP와 조효소인 NAD^+ 결합부위를 규명하기 위해, 각각 이에 해당되는 Tyr187과 Glu279부위에 대해 돌연변이를 일으켜 아미노산의 크 기와 소수성, 곁가지의 이온화 정도에 따라 다양한 돌연체들을 만들어 실험해 보았다. GD H의 자연형은 ADP와 반응시키면 3배정도 활성도가 증가된 반면 ADP 결합부위의 각 돌연체 효소들(Tyr187 돌연체)의 활성은 유의한 차를 보이지 않았다. 정지상태의 역학치수를 구 한 결과 Tyr187 돌연체 효소들은, 2-oxoglutarate와 NADH에 대한 K_m 값에는 유의한 차이 를 보이지 않았으나 V_(max) 값에는 자연형에 비해 4배 정도 감소된 결과를 얻었다. Glu2 79 돌연체 효소들은 NAD^+ 와 글루탐산에 대해 V_(max) 값은 22 ~ 28배 정도 감소한 반면 K_m 값은 NAD^+에 대해서만 20 ~ 25배 정도 감소하였다. ADP와 NAD^+ 결합부위를 규명하 기 위해 [α-^(32)P]8-azidoadenosine 5′-diphosphate (8N₃ADP)와 [^(32)P]nicotinamid e 2-azidoadenosine dinucleotide (2N₃NAD^+)를 이용하여 광흡착표지를 실시한 결과 GDH 의 자연형에 대한 [α-^(32)P]8N₃ADP와 [^(32)P]2N₃NAD^+의 광흡착 포화도에 대한 K_d 값은 25 μM 과 55 μM로 나타났다. 이때, 자연형의 경우에는 ADP와 NAD^+를 첨가하면 8N ₃ADP와 2N₃NAD^+의 광흡착이 현저히 감소되었으나, Tyr187와 Glu279 돌연체들은 각각 8 N₃ADP 및 2N₃NAD^+와 반응하지 않았다. 즉, 인간 GDH 유전자의 돌연변이와 광흡착표지 법을 통해 GDH의 Tyr187과 Glu279 위치는 각각 ADP와 NAD^+의 염기가 결합하는 부위임을 증명할 수 있었다. 마지막으로, GDH 결핍으로 신경퇴행성 질환이 생긴 환자에게 부족한 GDH를 단백질 차원 에서 보충하기위한 새로운 방법을 시도하였다. 이를 위해서 인간 GDH 유전자에 인간 면역 결핍 바이러스에 존재하는 TAT 단백질의 전이 부위를 융합 시켜 TAT-GDH 융합 단백질을 발현시켰다. 이렇게 얻어진 TAT-GDH 융합 단백질을 변성 시켜 신경세포 배양액에 분주한 결과 신경세포 내로 잘 침투하였고, 일단 세포 내로 들어가게 되면 변성된 단백질이 다시 제 형태를 갖추게 되어 정상적인 효소 활성을 가지게 되었다. 즉, TAT 잔기를 GDH에 첨 부하여도 GDH 활성에는 별 영향을 미치지 않으며, 세포 내 독성도 나타내지 않음을 알게 되었다. 이러한 결과는 GDH 결핍으로 인해 야기된 여러 질환을 앓고있는 환자에게 GDH를 직접 전달할 수 있는 새로운 방법을 제시해 주고 있다고 사료된다. -------------------- 핵심어: 글루탐산 분해효소, 글루탐산나트륨, 돌연변이, ADP 결합부위, NAD^+ 결합부위, 촉매작용부위, 광흡착표지법, TAT 융합 단백질, 단백질 치료, 퇴행성 신경질환, 유전자 공학, 단백질 공학 Structure, Allosteric Regulation, and Reaction Mechanism of Glutamate Dehydrogenase Glutamate in the form of its sodium salt (MSG; monosodium glutamate) is a widely used food additive and reported to be a cause of the Chinese restaurant syndrome. To investigate a long-term effect of MSG ingestion on the brain, one-week-old albino rats were kept for 1 year in equal groups with or without MSG in their drinking water. The concentrations of the glutamate in the brain crude extracts of the MSG treated group were 2-fold higher than those of the control group. There were no significant change in body weight, brain weight, and contents of protein, total RNA and DNA in the two groups of rats. The concentration of the enzyme on the western blot analysis was significantly decreased in the MSG treated group, whereas the level of GDH mRNA remained unchanged, suggesting a post-transcriptional control of the expression of GDH or an increased rate of degradation of enzyme protein. These results indicate that the prolonged MSG feeding reduces the activity of GDH and subsequently decreases the catabolism of glutamate in rat brain. To gain a deeper insight into the structural and regulatory basis of GDH, a 1557-base-pair gene that encodes human GDH has been synthesized and expressed in Escherichia coli as a soluble protein. The recombinant enzymes were indistinguishable in its biochemical properties from those isolated from human and bovine tissues. The results from the site-directed mutagenesis of Lys130 site indicate that Lys130 plays an important role in the catalysis of GDH and that Lys130 is an essential residue required for the catalysis of GDH but not for the substrate or coenzyme binding. To identify ADP (allosteric activator) and NAD^+ (coenzyme) binding sites within human GDH, a series of cassette mutations at Tyr187 and Glu279 positions were constructed for ADP and NAD^+ binding, respectively. The wild type GDH was activated up to 3-fold by ADP, whereas no significant activation by ADP was observed with the Tyr187 mutant GDH regardless of their size, hydrophobicity, and ionization of the side chains. Studies of the steady-state velocity of Tyr187 mutant enzymes revealed essentially unchanged apparent K_m values for 2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective apparent V_(max) values. The Glu279 mutant proteins showed a 22 ~ 28-fold decrease in the respective apparent V_(max) values and 20 ~ 25-fold increase in K_m values for NAD^+ with essentially unchanged K_m values for glutamate. The identification of the ADP and NAD^+ binding sites was further performed using photoaffinity labeling with [α-^(32)P]8-azidoadenosine 5′-diphosphate (8N₃ADP) and [^(32)P]nicotinamide 2-azidoadenosine dinucleotide (2N₃NAD^+). Saturation of photoinsertion with [α-^(32)P]8N₃ADP and [^(32)P]2N₃NAD^+ occurred apparent K_d values near 25 μM and 55 μM, respectively, for wild type GDH. The photoinsertions of 8N₃ADP and 2N₃NAD^+ were significantly decreased by ADP and NAD^+, respectively. Unlike wild type GDH, none of the mutant enzymes at Tyr187 or Glu279 was able to interact with 8N₃ADP and 2N₃NAD^+, respectively. The results with cassette mutagenesis and photoaffinity labeling indicate that the Tyr187 and Glu279 are required for efficient base-binding of ADP and NAD^+ to GDH, respectively. In an effort to replenish the GDH activity in the patients with the GDH-deficient neurodegenerative disorders, human GDH was transduced into PC12 cells by fusing with a gene fragment encoding the protein transduction domain of human immunodeficiency virus TAT protein to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH showed no cytotoxicity as determined by the ability to inhibit protein synthesis. These results may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.ope

홍천 지역어의 음운론적 연구

학위논문(석사)--서울대학교 대학원 :국어국문학과 국어학전공,2001.Maste

High-resolution heat capacity study of(1 - x)Pb(Yb½Nb½)O₃-xPbTiO₃

Maste

새로운 형광 단백질 탐침 : 분자 삼각체

Thesis(master`s)--서울대학교 대학원 :화학부 유기화학전공,2005.Maste

韓國産 향나무 녹병균(Gymnosporangium)의 形態的 特徵, 人工接種, 分子生物學을 利用한 同定, 奇主特異性 및 系統分類에 관한 硏究

Thesis (master`s)--서울대학교 대학원 :산림자원학과,2003.Maste

Photoaffinity labeling을 이용한 뇌조직 glutamate dehydrogenase isoproteins의 ADP와 NAD+ 결합부위 분석

The Graduate School Yonsei University/석사[한글] 본 연구에서는 소 뇌조직으로부터 분리 정제한 2가지 glutamate dehydngenase isoproteins (GDH Ⅰ 및 GDH Ⅱ)의 ADP 결합부위의 아미노산 서열을 규명하기 위해 [α**32P] 8N-azidoadenosine 5-diphosphate (8N^^3 ADP)로 photolabeling 을 하였다. Photolysis 를 시키지 않았을 때, 8N^^3 ADP와 유사하게 GDHⅠ 과 GDHⅡ 의 활성을 증가시켰다. Trypsin을 처리한 후, photolabel을 포함하고 있는 peptide를 immobilized aluminum affinity chromatography와 reversed-phase HPLC를 이용하여 분리하였으며, GDHⅠ과 GDHⅡ의 결과는 각각 E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-Y-D과 E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-Y-D-I-N 으로 나왔다. 한편 1mM ADP의 존재하에서 photolysis를 시킬 경우, peptide의 photolabeling이 90%정도 감소하였으나 다른 nucleotides를 사용한 경우에는 ADP처럼 효과적으로 photoinsertion 의 양을 감소시키지 못하였다. 이러한 결과로 미루어 ADP 결합부위에 대한 photoprobedml 특이성을 입증하게 되었고, 본 실험에서 사용한 photoprobe인 photoprobe인 [α**32 P] 8N^^3 ADP 로 확인한 peptide가 뇌조직 GDH isoproteinsdml ADP 결합 domain에 위치함에 알 수 있어다. 또한 [(32)**P] nicotinamide 2-azidoadenosine diphosphate (2N^^3 NAD**+) 을 photoaffinity probe로 사용하여 역시 소 뇌조직에서 분리한 GDHⅠ과 GDHⅡ의 NAD**+ 결합부위에 대하여도 조사하였다. photolysis를 시키지 않았을 때, 2N^^3 NAD**+는 NAD**+와 유사하게 GDH isoproteinsdml 기질로서 작용하였다. Probedml photoinsertion 은 GTP나 Glutarate 의해서는 증가하고 NAD**+나 ADP에 의해서는 감소하였다. Trypsin 처리 후 immobilized boronate affinity chromatography와 reversed-phase HPLC 로 photolabel-containing peptides를 분리하였다. 그 결과는 GDH isoproteins 둘 다 동일하게 C-I-A-V-G-X-S-D-G-S-I-W-N-P-D-G-I-D-P-K 로 나왔다. 이것은 아미노산 서열이 잘 밝혀진 소 간조직 GDH의 Cys**270 에서 Lys**289 의 위치와 일치함을 알게 되었다. 여기서, 'X'는 단백질 sequencing 과정에서 미확인 아미노산으로 분석된 것인데 기존에 알려진 GDH 의 아미노산 서열과 비교해 볼 때, photolabeled glutamate 임을 알 수 있었다. NAD**+ 존재하에서 photolysis를 시킨 경우, peptide의 photolabeling 이 감소하는 것으로 보아 photoprobe의 NAD**+ 결합부위내의 특이성을 입증할 수 있었고, 사용한 photoprobe 가 뇌조직 GDH isoproteins의 NAD**+ 결합 domain에 위치함을 알 수 있었다. [영문] Photoffinity labeling with [α**32P]8-azidhosphate (8N^^3 ADP) was used to identify the ADP binding site with two types of glutamate dehyrogenase isoproteins (GDH Ⅰ and GDH Ⅱ) isolated from bovine brain, 8N^^3 ADP, without photolysis, mimicked the activator properties of ADP on GDH Ⅰ and GDH Ⅱ sctivities, although maximal activity with 8N^^3 ADP was about 75% of maximal ADP - stimulated activity. Saturation of photoinsertion with [α**32 P] 8N^^3 ADP occurred at around 40-50μm photoprobe with apparent K^^4 values near 25 μm and 40μm for GDHⅠ and GDHⅡ, respectively. Photoinsertion of [α**32 P] 8N^^3 ADP was decreased best by ADP in comparison to other nucleotides. With the combination of immobilized alumminum affinity chromatograph and reversed-phase high performance liquid chromatography (HPLC), photolabel-containing peptides generated by tryptic digestion were isolated This identified a portion of the adenine ring binding domain of GDH isoproteins as region containing the sequence, E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-D and E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-Y-D-I-N for GDHⅠ and GDHⅡ, respectively. Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP. These results demonstrate selectivity of the photoprobe for ADP binding site and suggest that the peptide identified using the photoprobe islocaed in the ADP binding domain of the brain GDH ispproteins. Another photoaffinity probe, [(32)**P] nicotinamide 2 - azidoadenosine dinucleoteins (2N^^3 NAD**+), was also used to identify the NAD**+ binding site within ghnamate dehydrogenase isoproteins(GDHⅠ and GDHⅡ) isolated from bvine brain. In the absence of photolysis, 2N^^3 NAD**+ was a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the persence of saturating amounts of photoprobe, about 50% inhibition of the GDH activites was oberved. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD**+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, C-I-A-V-G-X-S-D-G-S-I-W-N-P-D-G-I-D-P-K for both GDH isoproteins, corresponding to Cys**270 through Lys**289 of the amino acid sequence of well known bovine liver GDH. The symbol X indicate a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designed as a photolabeled glutamate since the sequences inchuding the glutamate residue in question have a complete identity with those of NAD**+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD**+ binding site and suggest that the peptide identified using the photoprobe is located is located in the NAD**+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu**275 as the site of photoinsertionrestrictio

Development of a nursing intervention protocol based on nursing diagnosis of critical medical-surgical patients.

간호학과/석사[한글] 21세기 간호는 독자적인 실무를 통한 전문직 간호를 요구한다. 간호진단과 간호중재를 연계한 간호중재 프로토콜은 표준적인 간호활동을 제시하고 간호문제의 발견에서 진단, 수행하는 모든 활동을 독자적으로 수행하기 위한 도구로 사용될 수 있다. 따라서 본 연구의 목적은 내외과 중환자에게 필요한 간호진단을 파악하고 이에 따른 간호중재 및 활동을 규명함으로서, 내외과 중환자 간호수행에 지침이 되는 간호중재 프로토콜을 개발하는 것이다. 연구방법은 서울 소재 Y대학 부속 병원 중환자실 5곳에 근무 중인 간호사 중 중환자실 3년 이상의 간호경력을 가진 55명의 간호사를 대상으로, North American Nursing Diagnosis Association(NANDA)의 간호진단을 기본으로 한 설문지를 통해 성인 내외과 중환자에게 필요한 간호진단을 내용타당도 검증을 거쳐 선정하였다. 선정된 간호진단에 따라 Nursing Intervention Classification(NIC)에서 제시한 간호중재를 기초로, 전문가 집단이 모두 합의한 간호중재와 우선중재를 간호중재로 선정하였다. 전문가 집단은 55명의 간호사 중 중환자 간호경력 5년 이상 된 간호사와 간호대학 교수 3인으로 구성하였다. 선정된 간호중재에 대한 간호활동목록은 NIC에서 제시한 간호활동 목록을 한글수정작업을 거쳐 예비 간호중재 프로토콜로 작성하고 이에 대한 전문가 집단의 내용타당도 검증단계를 거친 후 최종 내외과 중환자 간호중재 프로토콜을 개발하였다. 본 연구의 결과는 다음과 같다. 1. 내외과 중환자에게 필요한 간호진단은 CVI(Index of Content Validation) .90이상의 점수를 받은 감염위험성, 기도 개방 유지불능, 기도 흡인 위험성, 비효율적 호흡 양상, 통증, 조직관류변화, 가스교환 장애, 체액 부족, 피부 손상 위험성, 호흡기능 부족 등 10개가 선정되었다. 2. 10개의 간호진단에 따른 간호중재는 NIC에서 제시한 간호중재 중 전문가가 모두 합의한 32개의 간호중재와 45개의 우선중재를 합해 총 67개의 간호중재를 선정하였다. 3. 간호활동은 예비간호중재 프로토콜로 전문가집단의 내용 타당도 검증을 실시한 결과 CVI를 통해 .83이상의 점수를 받은 979개의 간호활동으로 선정하였다. .83이상의 간호활동이 없는 피부손상 위험성의 진단에 대한 운동증진이라는 중재는 프로토콜에서 삭제하였다. 4. 그 결과, 10개의 간호진단, 66개의 간호중재, 979개의 간호활동으로 구성된 최종 내외과 중환자 간호중재 프로토콜을 개발하였다. 이러한 내외과 중환자 간호중재 프로토콜은 간호사의 의사결정을 도와 일관성있고 과학적인 간호를 제공할 수 있으며, 지속적인 개발을 통해 간호진단과 중재의 전산화 및 전문직으로서 위상을 높이는 데 기여할 수 있다고 본다. [영문] Professional nurses in the 21st century use research to promote independent practice and to link nursing diagnoses and nursing interventions. In this study a nursing intervention protocol for critical medical-surgical patients which can be used for instruction in nursing activities was to developed. Understanding of priority nursing diagnoses and identification of related nursing interventions and activities is an important link for nursing. The participants were 55 nurses with more than 3 years experience in one of five ICUs in Y medical center in Seoul. Through a survey based on the nursing diagnoses of the North American Nursing Diagnosis Association, content validity of the nursing diagnoses for critical medical-surgical patients was verified. For these nursing diagnoses and according to the nursing interventions suggested by Nursing Intervention Classification(NIC), priority nursing interventions were established and agreed upon by a team of experts. The experts were nurses with careers in ICU of 5 years or more and 3 professors of nursing. Following translation and updating of the NIC nursing activity list, a nursing activity list for the established nursing interventions was developed as the preliminary nursing intervention protocol. After verification for content validity by the expert group, a final nursing intervention protocol for critical medical-surgical patients was developed. A summary of the result of this study is presented below. 1. Nursing diagnoses for these patients were established as those which had a CVI (Index of Content Validation) of .90 or higher. Ten diagnoses met the criteria, Risk for infection, Ineffective airway clearance, Risk for aspiration, Ineffective breathing pattern, Pain, Altered tissue perfusion, Impaired gas exchange, Fluid volume deficit, Risk for impaired skin integrity, and Inability to sustain spontaneous ventilation, 2. From the nursing interventions suggested by NIC for the 10 diagnoses 32 were selected by all of the experts. As well as these interventions, 45 priority interventions were selected for a total of 67 interventions. 3. Using the preliminary nursing intervention protocol from the expert group, nursing activities were selected through examination of content validity. A total of 979 activities having a CVI of .83 or higher were selected. The intervention, "Exercise promotion" for the nursing diagnosis "Risk for impaired skin integrity" was deleted as none of the nursing activities met the CVI criteria. 4. The final nursing intervention protocol developed for critical medical-surgical patients consisted of 10 nursing diagnoses, 66 nursing interventions, and 979 nursing activities. Such nursing intervention protocols can help nurses make decisions and provide constant/scientific care. Through continuous development, they will also provide a computerized system of nursing diagnoses and interventions and improve the personal capability of nurses to be professional.ope