기계 학습에 기반한 쥐들의 자발적 물체 인식 중의 행동 분석

학위논문(석사) -- 서울대학교대학원 : 자연과학대학 뇌인지과학과, 2024. 2. 이인아.자발적 물체 인식 패러다임은 간단하고 자연적이라는 특성을 가져 해마를 연구하기 위한 방법으로 널리 사용되어 왔다. 그러나 기존 연구에서는 탐색 행동의 정량화 기준이 너무 단순하거나 주관적이었다. 예를 들면, 쥐의 코가 물체로부터 일정 거리 내에 머무는 시간을 측정하는 방식이 주로 사용되어 왔다. 쥐가 물체 근처에서 보이는 다양한 행동들을 고려할 때, 이러한 측정 방법은 중요한 행동 특성들을 간과할 수 있다. 이 문제를 해결하기 위해 우리는 자세 추정 소프트웨어인 DeepLabCut을 사용하여 이전의 연구를 재분석했다. 우리는 24마리의 Long-Evans 쥐가 서로 다른 4가지 조건에서 물체 인식을 수행하는 동영상을 분석했다. 그 중 2가지는 물체 정체성 기억을 평가했고 나머지 2가지는 물체-장소 연합 기억을 평가했다. DeepLabCut은 쥐의 몸 부위들과 물체에 표시된 16개의 점들로 훈련되었고, 사용자 정의된 Matlab 프로그램을 사용하여 11가지 행동 유형을 분류했다. 전통적인 거리에 기반한 측정 방 식과 비교했을 때, DeepLabCut에 의해 정의된 '냄새 맡기'는 '새로운 물체 인식' 과제들에서 더 높은 식별 지수를 제공했다. '앞발을 올리고 둘러보기'는 새로운 물체에 대해 더 오래 발생했지만, 이 차이는 새로움의 정도가 클 때만 나타났다. 다른 행동들은 새로운 물체를 탐색하는 행동의 지표로 작용할 수 없었다. 우리의 연구 결과는 쥐가 보이는 물체 탐색 행동들을 다양하게 분류하고, 자발적 물체 인식 패러다임에서 어떤 행동이 중요한 표지로 간주되어야 하는지에 대한 통찰을 제공한다.The spontaneous object recognition (SOR) paradigm has been widely used as a simple and naturalistic method to investigate the hippocampus. However, in traditional studies, the quantification of the exploratory behavior has been too unsophisticated or subjective, such as measuring the duration a rats snout stays within a certain distance from an object. Given the various behaviors of the rats near the objects, such measurement might have overlooked some crucial behavioral characteristics. To address this issue, we used a pose-estimation software, DeepLabCut (DLC) to reanalyze our previous study. We analyzed the videos of 24 male Long-Evans rats performing 4 different object recognition tasks. Two of them assessed the object identity memory and the others assessed the object-place associative memory. DLC was trained with 16 points marked on the body parts of the rats and the objects, and a customized Matlab program was used to categorize 11 behavioral types. Compared to the distance-based measurements, sniffing defined by DLC yielded a higher discrimination index in the novel object recognition tasks. Step-on scanning occurred longer for the novel object only when the delay was longer. The other behaviors could not serve as indicators of novelty detection. Our findings propose an enhanced approach to quantify novelty-induced behavior of rats and provide insights into the types of behavior that can be considered as critical markers during the SOR.Chapter 1. Introduction 1 1.1 The spontaneous object recognition paradigm 1.2 Behavioral analysis with machine learning Chapter 2. Methods 4 2.1 Subjects 2.2 Surgery 2.3 Apparatus 2.4 Behavioral testing 2.5 Body part tracking (DeepLabCut) 2.6 Behavior classification 2.7 Statistical Analyses Chapter 3. Results 13 3.1 Rats exhibit various behaviors during the SOR 3.2 Object identity memory with a short-term delay 3.3 Object identity memory with a long-term delay 3.4 Object-place associative memory with 2 objects 3.5 Object-place associative memory with 3 objects Chapter 4. Discussion 25 4.1 The criteria for exploring behavior in the SOR 4.2 The relevance of fasciola cinereum 4.3 Further studies Bibliography 28 Abstract in Korean 31석

3차원 식물모델을 이용한 LED 식물공장 재배 상추의 수광량 및 광합성 속도의 예측

학위논문(석사)--서울대학교 대학원 :농업생명과학대학 식물생산과학부(원예과학전공),2019. 8. 손정익.In plant factories, light use efficiency (LUE) should be improved to reduce electrical cost. To evaluate LUE, light interception should be estimated under different lighting conditions. The objective of this study was to estimate the light interception, photosynthetic rate, and LUE of lettuces grown under LEDs. 3D-scanned plant models and ray-tracing simulation were used to estimate the light interception. Canopy photosynthetic rate was estimated by modified Farquhar-von, Caemmerer-Berry (FvCB) model based on simulation result. To analyze the accuracy, measured light intensities and canopy photosynthetic rates in a growth chamber with LEDs were compared with simulated values. Under several scenarios, changes in light interception under different light environments were analyzed. Light intensities and canopy photosynthetic rates obtained by simulation showed good agreements with measured ones. Canopy light distribution was affected by planting distance, but whole light interception was almost similar. The canopy light interception was gradually increased with decreasing lighting distance, but rather decreased at too intact lighting due to heterogenetic light distribution. With high floor reflectance, canopy light interception was more increased at larger planting distance. It was confirmed that this method could quantify the light environments and photosynthetic rate at various electrical light conditions and is useful tool to estimate LUE in plant factories.식물공장에서 전기 에너지 비용을 줄이기 위해서는 광 이용 효율을 높이는 것이 요구되며, 광 이용 효율을 평가하기 위해서는 다양한 인공광 조건에 대한 작물 수광의 예측이 필요하다. 본 연구의 목적은 시뮬레이션 방법을 통해 인공광 환경 하에서 작물의 수광과 광합성 속도 및 광 이용 효율을 예측하는 것이다. 작물의 수광량 예측을 위하여 3차원 스캐너를 통해 구축된 식물 모델과 광 추적 시뮬레이션이 이용되었다. 작물 군락의 총 광합성은 수정된 Farquhar-von, Caemmerer-Berry (FvCB) 엽 광합성 모델과 시뮬레이션 결과를 바탕으로 추정되었다. 본 방법론의 정확성에 대한 검증은 실제 생장 챔버에서 측정된 광도와 광합성 속도를 시뮬레이션을 통해 얻어진 결과와 비교함으로써 이루어졌다. 또한 시나리오 분석을 통해 다양한 인공광 환경에서 작물 군락의 수광 변화를 분석하였다. 시뮬레이션을 통해 도출된 광도의 분포와 광합성 속도를 측정값과 비교한 결과 높은 정확성을 보이는 것이 확인되었다. 서로 다른 재식간격에서 군락 광 분포는 다르게 나타났지만 총 수광량은 유사하였다. 예측된 광합성 속도를 기반으로 광 이용 효율을 분석한 결과, 상추 군락의 재식 간격에 따른 광 이용 효율은 유사하였고 낮은 광도에서 약 30% 낮은 광 이용 효율을 보였다. 시나리오 분석 결과 광원과 군락 간의 거리가 멀어질수록 총 수광량은 점차적으로 감소하는 경향을 보였으나, 그 거리가 지나치게 가까울 경우 불균등한 광 분포로 인하여 오히려 수광량이 감소하였다. 재배상 표면에 높은 반사율을 적용하였을 경우에는 재식 간격이 클수록 총 수광량이 증가하였다. 본 연구에서 제시한 방법을 활용하여 식물공장의 광환경과 광합성 속도를 정량화하였고 광이용 효율을 추정할 수 있음이 확인되었다.INTRODUCTION 1 LITERATURE REVIEW 4 MATERIALS AND METHODS 7 RESULTS 19 DISCUSSION 32 CONCLUSION 38 LITERATURE CITED 39 ABSTRACT IN KOREAN 45 APPENDICE 47Maste

Interleukin-18 suppresses adiponectin expression in 3T3-L1 adipocytes via a novel signal transduction pathway involving ERK1/2-dependent NFATc4 phosphorylation.

An inverse correlation between the pro-inflammatory cytokine interleukin-18 and the anti-atherogenic adipokine adiponectin has been reported in the chronic pathological conditions obesity, insulin resistance, coronary artery disease, and metabolic syndrome. We investigated whether this relationship is coincidental or has a causal basis. Here we show that interleukin-18 (IL-18) suppresses adiponectin transcription, mRNA expression, and secretion by 3T3-L1 adipocytes. IL-18 suppresses adiponectin promoter-reporter activity, an effect reversed by deletion or mutation of the NFATc4 core DNA-binding site. IL-18 induces NFATc4 phosphorylation (Ser(676)), nuclear translocation, and in vivo DNA binding. IL-18 induces ERK1/2 phosphorylation and enzyme activity, and pretreatment with the MEK inhibitor U0126, ERK1/2 inhibitor PD98059, or small interference RNA targeted to ERK1/2 attenuates ERK1/2 activation and NFATc4 phosphorylation. Finally, inhibition of ERK1/2 or NFATc4 knockdown reverses IL-18-mediated adiponectin suppression. In contrast to its inhibitory effects on adiponectin expression, IL-18 potently stimulates PAI-1 secretion. These data demonstrate for the first time that IL-18 selectively suppresses adiponectin expression via ERK1/2-dependent NFATc4 activation and suggest that the inverse relationship observed between IL-18 and adiponectin in various chronic pathological conditions is causally related. Thus, targeting IL-18 expression may enhance adiponectin expression and mitigate disease progression.ope

Serpina3c Regulates Adipogenesis by Modulating Insulin Growth Factor 1 and Integrin Signaling

Preadipocyte differentiation can be induced upon a hormonal treatment, and various factors secreted by the cells may contribute to adipogenesis. In this study, RNA-seq revealed Serpina3c as a critical factor regulating the signaling network during adipogenesis. Serpina3c is a secretory protein and is highly expressed in fat tissues. Knockdown of Serpina3c decreased adipogenesis by attenuating the mitotic clonal expansion of 3T3-L1 cells. These cells exhibited decreases in integrin α5, which abolished the phosphorylation of integrin β3. We found that Serpina3c inhibits a serine protease that regulates integrin α5 degradation. Knockdown of Serpina3c disrupted integrin-mediated insulin growth factor 1 (IGF-1) signaling and ERK activation. Serpina3c-mediated regulation of integrin-IGF-1 signaling is also associated with AKT activation, which affects the nuclear translocation of GSK3β. Altogether, our results indicate that Serpina3c secreted from differentiating adipocytes inhibits serine proteases to modulate integrin/IGF-1-mediated ERK and AKT signaling and thus is a critical factor contributing to adipogenesis.ope

How to Begin Molecular Research of Metabolic Diseases

Advancing higher education is an important goal within the scientific and medical communities. The Korean Endocrine Society has worked with medical researchers who hope to conduct molecular research in addition to their clinical education. Based on concepts developed at a 2016 educational workshop, this article summarizes the requirement for a strong foundation in the performance of molecular research. Specifically, recent articles in metabolic research are highlighted to provide examples of commonly used techniques in this field of study.ope

Role of cdk2 in the sequential phosphorylation/activation of C/EBPβ during adipocyte differentiation

Upon induction of differentiation, growth-arrested (G1 phase) 3T3-L1 preadipocytes express CCAAT/enhancer binding protein-β (C/EBPβ), initiating a transcriptional cascade. C/EBPβ immediately undergoes a priming phosphorylation (on Thr188) by MAPK/ERK. However, the acquisition of DNA binding and transactivation capacity of C/EBPβ is delayed until further phosphorylation (on Ser184 or Thr179) by GSK3β occurs. Phosphorylation by glycogen synthase kinase-3β (GSK3β) induces S phase entry and thereby mitotic clonal expansion (MCE), a requirement for terminal differentiation. Because MAPK activity is down-regulated before S phase is completed, we sought to identify the kinase that maintains C/EBPβ in the primed phosphorylated state throughout S phase and MCE. We show here that cdk2/cyclinA, whose expression is activated at the onset of S phase, functions in this capacity. Ex vivo and in vitro experiments show that cdk2/cyclinA catalyzes this delayed priming phosphorylation. Mass spectrometric analysis revealed that cdk2/cyclinA phosphorylates C/EBPβ on Thr188 and is required for phosphorylation (on Ser184 or Thr179) of C/EBPβ by GSK3β and maintenance of DNA binding activity. Suppression of cdk2 activity by RNA interference or pharmacologic inhibitor disrupts subsequent events in the differentiation program. Thus, MAPK and cdk2/cyclinA act sequentially to maintain Thr188 of C/EBPβ in the primed phosphorylated state during MCE and thereby progression of terminal differentiation.ope

Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation

AMP-activated protein kinase (AMPK) plays a key role in controlling energy metabolism in response to physiological and nutritional status. Although AMPK activation has been proposed as a promising molecular target for treating obesity and its related comorbidities, the use of pharmacological AMPK activators has been met with contradictory therapeutic challenges. Here we show a regulatory mechanism for AMPK through its ubiquitination and degradation by the E3 ubiquitin ligase makorin ring finger protein 1 (MKRN1). MKRN1 depletion promotes glucose consumption and suppresses lipid accumulation due to AMPK stabilisation and activation. Accordingly, MKRN1-null mice show chronic AMPK activation in both liver and adipose tissue, resulting in significant suppression of diet-induced metabolic syndrome. We demonstrate also its therapeutic effect by administering shRNA targeting MKRN1 into obese mice that reverses non-alcoholic fatty liver disease. We suggest that ubiquitin-dependent AMPK degradation represents a target therapeutic strategy for metabolic disorders.ope

Monoacylglycerol O-acyltransferase 1 (MGAT1) localizes to the ER and lipid droplets promoting triacylglycerol synthesis

Monoacylglycerol acyltransferase 1 (MGAT) is a microsomal enzyme that catalyzes the synthesis of diacylglycerol (DAG) and triacylglycerol (TAG). However, the subcellular localization and catalytic function domain of this enzyme is poorly understood. In this report, we identified that murine MGAT1 localizes to the endoplasmic reticulum (ER) under normal conditions, whereas MGAT1 co-localize to the lipid droplets (LD) under conditions of enriching fatty acids, contributing to TAG synthesis and LD expansion. For the enzyme activity, both the N-terminal transmembrane domain and catalytic HPHG motif are required. We also show that the transmembrane domain of MGAT1 consists of two hydrophobic regions in the N-terminus, and the consensus sequence FLXLXXXn, a putative neutral lipid-binding domain, exists in the first transmembrane domain. Finally, MGAT1 interacts with DGAT2, which serves to synergistically increase the TAG biosynthesis and LD expansion, leading to enhancement of lipid accumulation in the liver and fat.ope

High-dose Resveratrol Inhibits Insulin Signaling Pathway in 3T3-L1 Adipocytes

Background: Insulin resistance is a major factor in the development of metabolic syndrome and is associated with central obesity and glucose intolerance. Resveratrol, a polyphenol found in fruits, has been shown to improve metabolic conditions. Although it has been widely studied how resveratrol affects metabolism, little is known about how resveratrol regulates lipogenesis with insulin signaling in 3T3-L1 adipocytes. Methods: We treated differentiated 3T3-L1 adipocytes with resveratrol to observe whether resveratrol is effective at reducing lipid accumulation. Results: Resveratrol treatment after mitotic clonal expansion resulted in decreased lipid accumulation accompanied by reduced fatty acid synthase expression. Decreased glucose uptake was observed with inhibited GLUT4 translocation in cells treated with 100 μM resveratrol, suggesting that high doses of resveratrol block insulin signaling in adipocytes. Insulin-stimulated Akt phosphorylation is also dose-dependently reduced with resveratrol treatment. Interestingly, Akt phosphorylation is upregulated when cells are treated with long-term low doses of resveratrol, suggesting that only low doses of resveratrol improve metabolic conditions. Conclusion: High doses of resveratrol block the insulin signaling pathway, thereby reducing glucose uptake and lipid accumulation in vitro. The results also provide information about in vivo administration dosages and may explain the discrepancy between in vitro and in vivo effects of resveratrol.ope

Temporal profiling of the adipocyte proteome during differentiation using a five-plex SILAC based strategy

The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine-containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g., adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis, while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion.ope