Microglial STAT3-mediated neuron-microglia interactions in major depressive disorder

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과, 2019. 2. 예상규.미세아교세포는 중추신경계에 존재하는 신경아교세포계의 한 종류로 뇌와 척수의 신경기능을 보조하거나 보호하는 기능을 한다. 특히, 신경-미세아교세포의 상호작용은 신경-면역계를 정상적으로 유지하는 대표적인 기전 중 하나로 알려져 있으며, 미아교세포의 활성화 상태를 조절하는 것은 신경-면역계의 균형을 유지하는데 매우 중요하다. 신경-면역계의 불균형은 곧 알츠하이머병, 파킨슨병, 주요우울장애 등 여러 신경정신질환의 원인이 되기도 한다. 하지만 신경-미세아교세포의 상호작용이 이러한 신경정신질환에서 어떤 기능을 하는지는 알려져 있지 않다. 신호변환 및 전사활성인자 3 (STAT3)는 일반적으로 면역세포에서 다양한 사이토카인과 성장인자들에 의해 발현되고, 세포 활성화를 일으켜 염증성반응을 일으키는 역할을 유전자이다. 이 연구에서는 미세아교세포-특이적 STAT3 결손이 주요우울장애같은 신경정신질환에서 신경-미세아교세포의 상호작용을 통해 신경면역계의 균형을 유지하고 증상을 완화시킬 것이라는 가설을 세워 연구를 진행했다. 먼저, 미세아교세포-특이적 STAT3 결손 (STAT3fl/flLysM-Cre+/-) 마우스 모델 제작하여 미세아교세포의 STAT3 유전자 활성을 억제시켰고, 장기간의 스트레스로 우울증을 유도한 후 행동분석실험인 강제 수영, 꼬리 매달리기, 자당 선호 및 오픈 필드 테스트를 수행하여 항우울 유사 행동을 보임을 확인했다. 흥미로운 점은 STAT3가 결손된 미세아교세포가 주변의 신경세포와 상호작용을 하여 신경세포로부터 대식세포 콜로니-자극인자 (M-CSF)의 분비를 증가시켰다는 것이다. 상호작용을 한 신경세포는 M-CSF를 자가분비하여 항우울 신호전달경로로 알려진 ERK1/2, Akt/GSK3β가 활성화 되었고, 뇌-유래 신경성장인자 (BDNF) 발현이 증가되었다. ERK1/2, Akt/GSK3β 의 활성화와 BDNF 단백질의 신경세포에서의 기능은 신경전달물질의 방출을 증가시키는 것으로 잘 알려져 있으며, 이는 곧 마우스 모델에서 보여준 항우울 유사 행동을 설명할 수 있는 근거가 된다. 결론적으로, 미세아교세포 STAT3의 기능 장애는 M-CSF에 의해 매개된 신경세포 활성을 통해 우울증 관련 행동을 조절하며, 미세아교세포 특이적인 STAT3 기능 억제는 기존의 우울증 치료가 갖고있는 한계점을 극복 할 수 있는 새로운 치료 전략이 될 수 있음을 시사한다.Neuron-microglia interactions play a crucial role in maintaining the neuroimmune system. The balance of neuroimmune system has emerged as an important process in the pathophysiology of depression. However, how neuron-microglia interactions contribute to major depressive disorders has been poorly understood. Herein, I demonstrated that microglia-derived neuronal changes induced antidepressive-like behavior by using microglia-specific signal transducer and activator of transcription 3 (STAT3) knockout (STAT3fl/flLysM-Cre+/-) mice. I found that microglia-specific STAT3 knockout mice showed antidepressive-like behavior in the forced swim, tail suspension, sucrose preference and open field tests. Surprisingly, the secretion of macrophage colony-stimulating factor (M-CSF) was increased from neuronal cells in the brains of STAT3fl/flLysM-Cre+/- mice. Moreover, the phosphorylation of antidepressant-targeting mediators and brain-derived neurotrophic factor (BDNF) expression were increased in the brains of STAT3fl/flLysM-Cre+/- mice as well as in neuronal cells in response to M-CSF stimulation. Collectively, microglial STAT3 regulates depression-related behaviors via M-CSF-mediated neuronal activity, suggesting that inhibition of microglial STAT3 might be a new therapeutic strategy for depression.ABSTRACT 1 CONTENTS 3 LIST OF TABLES AND FIGURES 4 LIST OF ABBREVIATIONS 7 INTRODUCTION 8 MATERIALS AND METHODS 13 RESULTS 26 FIGURES 36 DISCUSSION 89 REFERENCES 98 ABSTRACT IN KOREAN 110Docto

Design, Synthesis and Application of Chiral Diamine-based Building Blocks via Diaza-Cope Rearrangement (DCR)

학위논문 (박사)-- 서울대학교 대학원 : 화학부(유기화학전공), 2014. 2. 김병문.Much interest has been concentrated in the vininal diamine chemistry since many metal-based catalyst and organocatalysts as well as biologically active molecules containing the diamine structure have been synthesized. Diaza-Cope rearrangement (DCR) is a versatile tool for providing a variety of symmetrically-substituted as well as non-symmetrically-substituted chiral vicinal diamines. As the DCR route was introduced, it has rendered the structural tuning and thus electronic diversification of diamino functionality at ease so that many chemists could discover and develop new catalysts and applications owing to the simplicity and effectiveness of the rearrangement. However, use of the DCR for the construction of chiral building blocks for physiologically active molecules or peptidomimetic structures has not been as actively pursued as for catalysts. The DCR allows for stereospecific synthesis of chiral vicinal diamines through the rearrangement of diimines prepared from 1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane (hpen) and aldehydes. With a broad perspective, the chiral nonsymmetrical 1,2-disubstituted vicinal diamines could serve as suitable intermediates for the preparation of many physiologically active compounds. Using the hpen as a starting material, a number of useful chiral molecules containing diamine functionality can be synthesized. Piperazine-2-carboxylic acid has been utilized as an amino acid surrogate in many biologically active compounds. In Chapter 1, the development of an efficient route for enantiopure trans-3-arylpiperazine-2-carboxylic acid derivatives is described though the DCR process. A complete transfer of stereochemical integrity was observed for the transformation. Piperazine ring formation from the chiral 1,2-ethylenediamine derivatives using diphenylvinylsulfonium triflate, followed by oxidation using Ru(III)Cl3•H2O in the presence of NaIO4 provided the desired enantiopure trans-3-arylpiperazine-2-carboxylic acid derivatives. The rearrangement also allows us to prepare γ,δ-diamino acids, which may be useful for the synthesis of Tamiflu-type antiviral agents as well as various biologically active compounds. In Chapter II, we report the one-pot reaction for the synthesis of γ,δ-diimino esters with two adjacent chiral centers in enantiomerically pure form through DCR of diimines formed from (R,R)-hpen and aldehydes. DFT computation provides interesting understanding into the stereospecific rearrangement reaction. The crystal structure of the product diimine formed from the reaction of (R,R)-hpen and 2,6-dichlorobenzaldehyde shows that the reaction gives the product in S,S configuration. Preparation of vicinal quaternary carbon centers containing vicinal diamines is synthetically challenging task. In Chapter III, we show that the synthesis of a twinned alanine derivative is efficiently accomplished through the DCR method. Reaction between (R,R)-hpen and methyl pyruvate gives the diaza-Cope rearrangement product with good yield and excellent stereospecificity. The product containing two chiral quaternary carbon centers on vicinal position is characterized by high performance liquid chromatography (HPLC) and X-ray crystallography. DFT computation helps the understanding of why the diaza-Cope rearrangement takes place readily with methyl pyruvate but not with other ketones like acetone and substituted acetophenones. The structural prevalence of chiral vicinal diamines as versatile chiral building blocks has led to an intense demand of useful tools to simply and inexpensively determine the enantiomeric excess (ee) of the diamine molecules. In Chapter IV, we find that use of sugar molecules such as ribose and arabinose as chiral derivatizing agents is quite efficient in providing good analytical tool to determine the enantiopurities of C2-symmetric vicinal diamines. In this protocol, sugars or diamines do not need to be protected or modified in advance.CONTENTS Abstract 5 List of Figures 8 List of Schemes 9 List of Tables 10 Background 11 Chapter I. Synthesis of Chiral trans-3-Arylpiperazine-2- carboxylic Acid Derivatives 1. Introduction 24 2. Result and Discussion 26 3. Conclusion 33 4. Experimental 33 Chapter II. Stereospecific Synthesis of γ,δ-Diamino Esters 1. Introduction 48 2. Result and Discussion 49 3. Conclusion 55 4. Experimental 56 Chapter III. Stereospecific Synthesis of a Twinned Alanine Ester 1. Introduction 67 2. Result and Discussion 69 3. Conclusion 76 4. Experimental 77 Chapter IV. Sugar as Chiral Derivatizing Agent for the Determination of the Enantiopurity of Vicinal Diamines 1. Introduction 81 2. Result and Discussion 82 3. Conclusion 86 4. Experimental 86 References 90 Appendix A (NMR spectra) 104 Appendix B (HPLC spectra) 142 Appendix C (Computational data) 167 Appendix D (Crystal data) 172 국문초록 204Docto

Anterior clinoid mucocele coexisting with sphenoid sinus mucocele

An anterior clinoid mucocele, known to be extremely rare, can lead to visual complications due to its proximity to the optic nerve. We report a patient who developed visual disturbance due to an anterior clinoid mucocele. Interestingly, the anterior clinoid mucocele coexisted with a sphenoid sinus mucocele. When an anterior clinoid mucocele coexists with a sphenoid sinus mucocele, more deliberate diagnostic and therapeutic approaches must be considered according to our first experienceope

플라즈마 디웨팅을 이용한 금속 나노분말 array 제조에 관한 연구

학위논문 (박사)-- 서울대학교 대학원 : 재료공학부, 2013. 2. 이정중.A new method of producing metal nanoparticle arrays was developed with the help of plasma-induced dewetting. Various metal films (Cu, Ag, Au, Si, Ni, Co, Ti) were successfully turned into nanoparticle arrays at low operating temperatures. The mechanism of the plasma-induced dewetting was also discussed. TEM and SEM images showed that the dewetting proceeded through heterogeneous hole nucleation mechanism. The nanoparticles produced by plasma-induced dewetting were more uniformly distributed on the substrate than those produced by thermal annealing. It was revealed that low process temperature of the plasma treatment prevented coarsening of the nanoparticles, and uniformly distributed holes on the film surface, resulting in uniform nanoparticle arrays, were detected during plasma treatment. According to AES (auger electron spectroscopy), oxidation of the nanoparticles occurred less during plasma treatment because of the low operating temperature. Even at the low temperature, ion bombardment which transfers high energy to the substrate surface atoms made dewetting possible. The uniformity of nanoparticle arrays was controlled by varying plasma parameters. Plasma density and electron temperature were controlled by varying working pressure and applied RF power. Sheath voltage was also controlled directly using substrate bias. It was found that uniform nanoparticle arrays were produced when hole generation was increased by using high ion bombardment energy. When a low amount of energy was transferred to the substrate, small numbers of holes were generated on the film surface, resulting in non-uniformly distributed nanoparticles.1. Introduction 2. Research background and theory 2.1 Dewetting of thin film 2.1.1 Liquid thin film dewetting 2.1.2 Solid-state dewetting of thin film 2.1.2.1 Hole formation 2.1.2.2 Hole growth 2.2 Inductively coupled plasma 3. Experimental procedure 3.1 Film deposition and post-treatment 3.2 Analysis methods 4. Result and Discussions 4.1 Formation of Cu nanoparticle arrays by plasma-induced dewetting 4.2 The hole nucleation mechanism of the plasma-induced dewetting 4.2.1 Surface morphology analysis with SEM 4.2.2 TEM analysis 4.2.3 AFM analysis 4.2.4 SLP analysis 4.3 Uniformity control of nanoparticle arrays 4.3.1 Process temperature effect 4.3.2 Ion bombardment energy effect 4.4 Various metal nanoparticle arrays produced by plasma-induced dewetting 4.4.1 Low melting temperature metals 4.4.2 High melting temperature metals 5. ConclusionDocto

다공성 인산 칼슘 세라믹스의 제조 및 특성

학위논문(석사)--서울대학교 대학원 :재료공학부,2001.Maste

Genetic variation and transcriptome by developmental morphology and position of strobilus of Pinus densiflora f. multicaulis Uyeki

학위논문(박사)--서울대학교 대학원 :농업생명과학대학 산림과학부(산림환경학전공),2019. 8. 강규석.Pinus densiflora f. multicaulis Uyeki is one of the unique varieties of pine trees and is distributed mainly in Korea and Japan. Unlike the pine tree, the trunk is not developed straightly, but several branches are separated from the beginning of the ground. Thus, various types of tree appear for each individual, and they have excellent value. P. densiflora f. multicaulis are rarely distributed in nature and propagate by combining them for commercial or research purposes. In addition, Pinus species have been reported to have bisexual strobilus mixed male and female organ, and the development of bisexual strobilus has been observed more frequently in other species than in other species. and recently some transcriptome analysis has been conducted in two overseas species. Also, studies on the reasons for the development of bisexual strobili in the pine trees and the factors determining the reproductive organs have been continuously carried out. In Korea, the genome sequencing project of Pinus densiflora is in the beginning stage, and genetic research on P. densiflora f. multicaulis is also in the basic stage. In this study, we selected P. densiflora f. multicaulis Uyeki that have a low height and a lot of strobili, and analyzed the characteristics of the strobili and mature cones of P. densiflora f. multicaulis by the morphological, transcriptomical and genetical variances differences. As a result of the analysis of cones and seeds, the on P. densiflora f. multicaulis showed a developmental pattern of sex transformation consistent with the results of the other species of pine trees. As a result of morphological analysis of the cones and seeds developed at the apical and lateral sides, it was found that side cones and seeds from the side cones had lower germination times and growth than the cones and seeds developed at the apical them. However, it was confirmed that the seeds obtained from the side cones were close to the germination rate of the seeds obtained from the apical cones. As a result of analysis of transcriptome, the LEA gene family, cyplp029 gene, WRKY transcription factor ZmWRKY17, and dhn_ESK2 and so on were expressed in male strobili. In the beginning of biosexual strobili, we identified PtLTP1 to control ethylene, PIN1 to transport Auxin and NPF7 which transports nitrogen respectively. In female strobili, gibberellin, which is always expressed specifically in female tissues, the precursors of hormones such as CYPs and gibberellin metabolism genes, which are involved in the biosynthesis of gibberellin and brassinosteroid, in addition, in female strobili of side part, LINE-1 retrotransposable element was identified. As a result of the SNPs comparative analysis of normal population only developed apical cones and abnormal population developed side cones, only 16,666 SNPs were found in the two groups. Homo / homo SNPs 92, all of which matched within the population and differ between groups, were selected. Mapping to the sequence and RNA-seq data of the reference genome at the selected SNPs site revealed the transposon PpRT1 of Pinus pinaster. The function of the sequence of the selected SNPs should be clarified through further studies. From the results of this study, it was found that the same cone as the apical of branch is also developed in the lateral of branches. And genes that are presumed to be expressed by the flower developmental structure of P. densiflora f. multicaulis can be selected. In addition, we could find genetic differences according to the location of female strobili. Future studies such as the functional study of the genes found in this study and the development of the markers according to the results of SNPs molecular markers will be necessary.반송(Pinus densiflora f. multicaulis Uyeki)은 소나무의 독특한 품종 중 하나로써 한국과 일본에 주로 분포한다. 소나무와 달리 줄기가 곧게 발달하지 않고 지상부에서부터 세 개 이상 다수의 수간이 갈라져 나온다. 그리하여 개체 별로 다양한 수형이 나타나 관상적 가치가 뛰어나다. 반송은 자연에 집단분포하는 경우가 극히 드물며 상업적 또는 연구적인 목적으로 접목하여 증식해왔다. 더불어 소나무속 수종들은 암구화와 수구화가 혼합되어 있는 양성구화가 발생한다고 보고되어 왔으며, 반송에서는 양성구화의 발달이 다른 수종보다 많은 것이 관찰되어왔다. 최근에 들어서야 해외에서 소나무종의 두 가지 수종에서 전사체 분석이 진행되었다. 그리고 소나무속에서 양성구화가 발생하는 이유나 생식기관을 결정하는 요인에 대한 연구가 지속적으로 이루어지고 있다. 국내에서는 소나무(Pinus densiflora)의 유전체 해독 프로젝트가 시작하는 단계에 있으며, 반송에 대한 유전적인 연구 또한 기초단계에 있다. 본 연구에서는 소나무 속에서 수고가 낮고 구화가 많이 발달하는 반송을 선정하여 반송의 구화 및 성숙구과의 특성을 형태학적, 전사학적 그리고 유전학적인 차이를 비교 분석하였다. 구과와 종자 분석 결과, 반송이 기존 소나무 속의 타수종 연구 결과와 동일한 성전환하는 양성구화의 발달 형태를 보였다. 그리고 정단부와 측면부에서 발달한 구과 및 종자를 형태학적으로 비교 분석한 결과, 측면부에서 발생한 구과와 측면부 구과에서 생산된 종자가 정단부에서 발달한 구과 및 종자와 비교하여 발아하는 시기와 생장이 저조하다는 것이 밝혀졌다. 그러나 시간이 지나면 측면부에서 얻은 종자가 정단부에서 얻은 종자의 발아율에 가까워지는 것을 확인할 수 있었다. 구화들의 전사체 분석 결과, 수구화에는 LEA gene family와 cyplp029 gene, WRKY transcription factor ZmWRKY17, dhn_ESK2 등이 발현하였으며, 양성구화의 시작단계에서는 에틸렌(ethylene)을 조절하는 PtLTP1, 옥신(Auxin)을 수송하는 PIN1, 질소를 수송하는 NPF7.3을 동정하였다. 그리고 암구화에서는 조직에 상시 특이적으로 발현하는 지베렐린과 브라시노스테로이드 생합성에 관여하는 CYPs, gibberellin metabolism genes 등 호르몬의 전구체들이 주로 동정되었다. 특히, 측면부에서 발달하는 암구화에서는 LINE-1 retrotransposable element를 동정하였다. 당년지 정단에서만 구과가 발달하는 집단(AC, apical cone)과 정단 및 측면에서 구과가 발달하는 집단(SC, side cone)의 비교 분석 결과 16,666개의 SNP가 두 집단간에 차이가 있음을 발견하였으며, 그 중 집단 내에서 모두 일치하지만 집단간에 차이가 나는 homo/homo SNPs 92개를 선별하였다. 선별한 SNPs 위치의 reference genome의 서열과 RNA-seq 데이터에 mapping한 결과 Pinus pinaster의 transposon PpRT1을 발견할 수 있었다. 선발한 SNP에 대한 위치의 서열이 어떤 기능을 하고 있는지는 아직 추가 연구를 통하여 밝여나가야 할 것이다. 본 연구결과를 통하여 당년지 측면에서도 정단과 동일한 암구화가 발달한다는 것을 발견할 수 있었으며 반송의 화기 구조에 따라 발현하는 것으로 추정하는 유전자들을 선별할 수 있었다. 또한 암구화의 발생 위치에 따른 유전적 차이를 발견 할 수 있었다. 향후 본 연구에서 발견한 유전자들의 기능 연구와 SNPs 분자표지 분석 결과에 따른 표지 개발 등 후속 연구가 필요할 것이다.제1장 반송의 구화 발달 및 구과 분석 1 제1절 서론 1 제2절 연구사 3 제3절 재료 및 방법 9 3.1 반송의 구화 채취 9 3.2 구화 실체현미경 관찰 9 3.3 반송의 정단부 발달구과 및 측면부 발달구과 채취 10 3.4 구과 형태의 측정 및 분석 10 3.5 종자의 발아테스트 11 제4절 결과 12 4.1 구화의 구조 관찰 12 4.1.1 정단부와 측면부 구화의 형태적 비교 12 4.1.2 구화의 성숙과 수분 13 4.1.3 한 당년지 내에서 발달하는 구화의 조합 17 4.2 구과 및 종자 특성 비교 19 제5절 결론 및 고찰 29 제2장 반송 구화의 전사체 분석 32 제1절 서론 32 제2절 연구사 34 제3절 재료 및 방법 37 3.1 반송의 구화 채취 및 RNA 추출 37 3.2 Sequencing과 Quality control 37 3.3 de novo assembly and Annotation 37 3.4 Annotation of transcripts 38 제4절 결과 39 4.1 De novo assembly 39 4.2 Gene expression profiling 40 4.3 Grouping of subclusters by expression pattern 45 4.3.1 Annotation by gene data base and blastn searching 47 4.3.2 Expression pattern Analysis of Group 1 48 4.3.3 Expression pattern Analysis of Group 2 54 4.3.4 Expression pattern Analysis of Group 3 60 4.3.5 Expression pattern Analysis of Group 4 67 4.3.6 Expression pattern Analysis of Group 6 71 제5절 결론 및 고찰 75 제3장 반송 암구화 발달 위치에 따른 유전체 변이 78 제1절 서론 78 제2절 연구사 79 제3절 재료 및 방법 81 3.1 반송 샘플링과 DNA 추출 그리고 sequencing 81 3.2 SNP discovery 81 제4절 결과 83 4.1 Reads output of genomicDNA of the 10 samples 83 4.2 SNPs discovery and filtering 83 4.3 SNPs selection 83 4.4 SNPs mapping on transcripts data 83 제5절 결론 및 고찰 87 제4장 종합결론 89 제5장 향후 연구 방향 91 참고문헌 92 Abstract 105 Supplemental data 108Docto

OX40 리간드를 발현하는 지지 세포 기반 NK 세포 증폭 기술 개발

DoctorNatural killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. To increase NK cells in numbers and improve their antitumor activity for clinical applications, ex vivo cultivation is a effective method. Recently, a remarkable activation and expansion of NK cells was achieved using K562 cells genetically engineered to express cytokines and co-stimulatory factors and additional novel co-stimulatory factors for NK cell activation and expansion are continuously being sought. In addition, the mechanism of NK cell expansion through the interaction between genetically engineered feeder cells expressing co-stimulatory factors and NK cells has not been elucidated. To address this problem, we developed genetically engineered K562 feeder cells expressing OX40 ligand (K562-OX40L). Using this new feeder cells, we systematically studied the roles of OX40 ligand for NK cell expansion. First, we demonstrated that NK cell purity, expansion rate were significantly augmented cultured with K562-OX40L compared to conventional K562. Second, we identified transient exposure to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Finally, we demonstrated that homotypic interaction between NK cells through the OX40-OX40L axis is necessary for NK cell expansion

Dynamic micropatterning of cells on nanostructured surfaces using a cell-friendly photoresist

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Cyclooxygenase inhibitors induce apoptosis in sinonasal cancer cells by increased expression of nonsteroidal anti-inflammatory drug-activated gene

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) has recently been shown to be induced by nonsteroidal anti-inflammatory drugs (NSAIDs) and to have proapoptotic and antitumorigenic activities. Although sulindac sulfide induced apoptosis in sinonasal cancer cells, the relationship between NAG-1 and NSAIDs has not been determined. In this study, we investigated the induction of apoptosis in sinonasal cancer cells treated by various NSAIDs and the role of NAG-1 expression in this induction. The effect of NSAIDs on normal human nasal epithelial (NHNE) cells was also examined to evaluate their safety on normal cells. Finally, the in vivo anti-tumorigenic activity of NSAIDs in mice was investigated. In AMC-HN5 human sinonasal carcinoma cells, indomethacin was the most potent NAG-1 inducer and caused NAG-1 expression in a time- and dose-dependent manner. The induction of NAG-1 expression preceded the induction of apoptosis. Conditioned medium from NAG-1-overexpressing Drosophila cells inhibited proliferation of sinonasal cancer cells and induced apoptosis. In addition, in NAG-1 small interfering RNA-transfected cells, apoptosis induced by indomethacin was suppressed. In contrast, NAG-1 expression and apoptosis were not induced by NSAIDs or conditioned medium in NHNE cells. Furthermore, indomethacin induced a dose-dependent in vivo increase in the expression of NAG-1 mRNA in the mice tumors and the volume of xenograft tumors of AMC-HN5 cells in indomethacin-treated nude mice was reduced compared to that in control mice. In conclusion, indomethacin exerts proapoptotic and antitumorigenic effects in sinonasal cancer cells through the induction of NAG-1 and can be considered a safe and effective chemopreventive agent against sinonasal cancerope

Dynamic micropatterning of cells on nanostructured surfaces using a cell-friendly photoresist

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