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dc.contributor.advisor이원재-
dc.contributor.author이경아-
dc.creator이경아-
dc.date.accessioned2016-08-26T04:08:04Z-
dc.date.available2016-08-26T04:08:04Z-
dc.date.issued2011-
dc.identifier.otherOAK-000000066953-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/209474-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000066953-
dc.description.abstractAll metazoan guts are in constant contact with microbes ranging from beneficial resident symbiotic commensal microorganisms to life-threatening opportunistic pathogens. Accordingly, mucosal epithelia must mount a conflicting strategy to draw the maximum benefits from normal microflora by efficiently combating pathogens while tolerating symbionts. However, the molecular mechanism of such immune system remains elusive. In the present study, we demonstrated that dual oxidase (DUOX)-dependent reactive oxygen species (ROS) play an important role in gut-microbe homeostasis in Drosophila. Genetic evidences demonstrated that the Gaq/PLCb controlled Ca2+ referred to as “DUOX-activity” pathway modulates DUOX enzymatic activity whereas the p38 MAP kinase-controlled ATF2 activation referred to as “DUOX-induction pathway” induces DUOX gene expression. In an infectious condition where microbial burdens are intense, both pathways are fully activated to produce maximum amounts of ROS to control the local microbes. However, in a routine condition where commensal bacteria are dominant, only DUOX-activity pathway is basally activated to antagonize occasionally introduced microbes. In this condition, DUOX-induction pathway is silenced despite of omni-presence of peptidoglycan, an agonist for p38 MAP kinase activation. We showed that this is due to the cross-regulation between two pathways wherein PLCb mediated MKP3 acts as a negative regulatory molecule leading to p38 dephosphorylation, thereby maintaining the DUOX-expression pathway in an off-state. Thus, the coordinated regulation of DUOX enables the host to achieve gut-microbe homeostasis by efficiently combating infection while tolerating commensal microbes.;장의 점막 표피세포는 다양한 종류의 미생물과 직접적으로 접촉하면서 항상성을 유지하고 있다. 때문에 점막 표피세포는 여러 가지 균을 차별적으로 인지하고 구분하여 그에 따라 서로 다른 면역기제를 적용해야 하는 세밀한 조절을 필요로 한다. 본 연구에서는 이러한 장의 항상성 유지에 중요한 역할을 하고 있는 DUOX유래의 ROS가 어떠한 경로를 통하여 조절되는지에 대하여 연구하였다. 먼저 평상시 음식과 함께 들어오는 섭식균을 포함한 공생균들의 정상적인 군집 유지에 Phospholipase C-bβ(PLCβ) 신호전달경로가 DUOX의 활성을 조절함으로써 매우 중요한 역할을 담당한다는 사실을 증명하였다. 장내 점막세포는 Gaq를 통한 PLCβ의 신속한 활성화로 칼슘의 생성을 유도하여 DUOX의 활성경로를 조절하고 평소의 정상의 미생물 군집 형성에 기여한다. 또한 감염균의 대량 감염이 동반되는 상황에서는 p38을 통한 DUOX 발현 조절경로를 가짐으로써 ROS가 충분히 생성될 수 있도록 하였다. 이 같은 DUOX의 발현조절 경로는 평소에는 억제되어 있다가 감염시에만 활성화 되는데 이는 과도한 ROS의 분비를 막는 것으로 DUOX의 활성조절 경로에 의하여 조절된다. 그러므로 본 연구에서는 장내 미생물에 따라 선택적으로 작용하는 DUOX의 복잡한 활성화-비활성화의 세밀한 조절이 숙주와 장내 미생물간의 항상성 유지에 필수적임을 규명하였다.-
dc.description.tableofcontentsINTRODUCTION 1 AIM OF REASERCH 18 MATERIALS & METHODS 19 Construct and Fly Strains 19 ROS Measurement 19 Calcium Measurement 20 IP3 Measurement 20 FRET Analysis 20 Survival Experiment 21 Cell and fly lines 21 Preparation of soluble microbial extracts 22 Immunostaining 22 Survival experiments 23 Real-time PCR analysis 23 Electrophoretic mobility-shift assay 23 ROS measurement 23 Lentivirus vector-mediated gene silencing in Caco-2 cells 24 Statistic analysis 24 PART I. Regulation of DUOX by the Gαq - Phospholipase Cβ - Ca2+ Pathway in Drosophila Gut Immunity 25 Abstract 26 Introduction 26 Result 28 I-1. Natural Drosophila-Yeast Interactions Are Controlled by DUOX-Dependent Gut Immunity 28 I-2. Yeast Induces DUOX-Dependent ROS in Drosophila S2 Cells 34 I-3. Yeast-Induced DUOX EnzymatiCβActivity Is Dependent on Inositol 1,4,5-Tri sphos phate Generation and Subsequent Ca2+ Mobilization through Gαq and PLCβ Activation 35 I-4. In Vivo Gut-Yeast Interaction Mobilizes Ca2+ for ROS Generation through DynamiCβActivation of Gαq and PLCβ 39 I-5. The Gαq-PLCβ-DUOX Pathway Is Required for Routine Control of Nutritional Microbes and Host Survival 48 Discussion 59 PART II. Coordination of multiple dual oxidase-regulatory pathway in responses to commensal and infectious microbe in Drosophila gut 63 Abstract 64 Introduction 64 Result 66 II-1. Microbial components induce Duox transcription 66 II-2. MKK3, p38 and ATF2 in Duox induction 70 II-3. PG receptor PGRP-LC and IMD in PG-dependent Duox induction 75 II-4. PLCβ in Duox induction and expression 78 II-5. PLCβ suppresses by inducing calcineurin B and MKP3 80 II-6. Physiological function of Duox expression pathway regulation 84 II-7. Conservation of DUOX-regulatory pathways 91 II-8. Existence of PLCβ-controlled DUOX-activity pathway and p38- controlled DUOX-Expression pathway in human intestinal Caco2 cells 97 Discussion 98 DISCUSSION 102 REFERENCES 109 논문 개요 116 ACKNOWLEDGEMANT 117-
dc.formatapplication/pdf-
dc.format.extent3842449 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleRegulatory mechanism of Dual oxidase In Drosophila Gut immunity-
dc.typeDoctoral Thesis-
dc.format.pageviii, 117 p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 생명·약학부생명과학전공-
dc.date.awarded2011. 2-
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