Apobec2 plays a critical role in self-renewal of mouse ESCs and reprogramming of mouse iPSCs

학위논문 (석사)-- 서울대학교 대학원 : 융합과학기술대학원 분자의학 및 바이오제약학과, 2018. 8. 김효수.Apolipoprotein B mRNA-editing enzyme catalytic subunit 2 (APOBEC2) is known that highly expressed in heart and muscle tissues and plays an important role in regulating and maintaining muscle development in mammals. Besides regulation of muscle development, Apobec2 expression and function in other cells and tissues are still unknown – especially in pluripotent stem cells. Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the pre-implantation blastocyst. ESCs can be propagated stably in an undifferentiated state in vitro (self-renewal) and, under the appropriate culture conditions, can be induced to differentiate into a variety of cell types (pluripotency). Proper telomere length is essential for self-renewal and pluripotency of ESCs. Also, telomerase plays a critical role in reprogramming and self-renewal of induced pluripotent stem cells (iPSCs). However, the mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. Here, we reports that Apobec2 is specifically expressed in mouse ESCs and iPSCs, and regulates self-renewal of mouse ESCs as well as reprogramming of mouse iPSCs by regulating expression of Tert mRNA. Knock down of Apobec2 reduced colonization and proliferation of mouse ESCs. Furthermore, sphere-forming assay showed decreased self-renewal of mouse ESCs by Apobec2 knock down. During reprogramming of mouse iPSCs, the expression level of Apobec2 mRNA was increased in time dependent manner. Next, we performed knock down and overexpression of Apobec2 in prior to mouse iPSC reprogramming, and found that Apobec2 positively regulated reprogramming of mouse iPSCs. In this study, our results suggest that Apobec2 plays an important role in modulating self-renewal of mouse ESCs as well as iPSC reprogramming.Contents Abstract Contents 1. Introduction ……………………………………… 4 2. Materials and Methods ………………………… 7 3. Result ……………………………………………… 12 4. Discussion ………………………………………… 20 5. References ………………………………………… 23 6. Figures ……………………………………………… 28 국문 초록Maste

Brain Plasticity and Neurorestoration by Environmental Enrichment

In the adult mammalian brain, neural-lineage cells are continuously generated in the subventricular zone (SVZ) and dentate gyrus of the hippocampus. These cells in vivo arising from the adult SVZ may be regulated by environmental enrichment (EE). EE is a method of raising animals in a huge cage containing novel objects, running wheels and social interaction with a complex combination of physical, cognitive, and social stimulations. EE can affect neural plasticity via overexpression of growth factors such as brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and synaptic activity-regulating genes. EE also have advanced effects on brain functions including the enhancement of motor and cognitive functions in normal and pathological states. Additionally, behavioral changes by EE are related with molecular changes including neurogenesis, gliogenesis, angiogenesis, axonal sprouting, and dendritic branching in the adult brain. In this review, we focus on brain plasticity and neurorestoration associated with molecular changes of neurotrophic growth factors such as BDNF, VEGF, IGF-1, FGF-2 and synaptic activity-regulating genes that occurs in interaction to EE.ope

In Vivo Expression of Reprogramming Factors Increases Hippocampal Neurogenesis and Synaptic Plasticity in Chronic Hypoxic-Ischemic Brain Injury

Neurogenesis and synaptic plasticity can be stimulated in vivo in the brain. In this study, we hypothesized that in vivo expression of reprogramming factors such as Klf4, Sox2, Oct4, and c-Myc would facilitate endogenous neurogenesis and functional recovery. CD-1® mice were induced at 1 week of age by unilaterally carotid artery ligation and exposure to hypoxia. At 6 weeks of age, mice were injected GFP only or both four reprogramming factors and GFP into lateral ventricle. Passive avoidance task and open field test were performed to evaluate neurobehavioral function. Neurogenesis and synaptic activity in the hippocampus were evaluated using immunohistochemistry, qRT-PCR, and/or western blot analyses. Whereas BrdU+GFAP+ cells in the subgranular zone of the hippocampus were not significantly different, the numbers of BrdU+βIII-tubulin+ and BrdU+NeuN+ cells were significantly higher in treatment group than control group. Expressions of synaptophysin and PSD-95 were also higher in treatment group than control group. Importantly, passive avoidance task and open field test showed improvement in long-term memory and decreased anxiety in treatment group. In conclusion, in vivo expression of reprogramming factors improved behavioral functions in chronic hypoxic-ischemic brain injury. The mechanisms underlying these repair processes included endogenous neurogenesis and synaptic plasticity in the hippocampus.ope

Astroglial Activation by an Enriched Environment after Transplantation of Mesenchymal Stem Cells Enhances Angiogenesis after Hypoxic-Ischemic Brain Injury

Transplantation of mesenchymal stem cells (MSCs) has paracrine effects; however, the effects are known to be largely limited. Here we investigated the combination effects of cell transplantation and enriched environment (EE) in a model of hypoxic-ischemic brain injury. Brain damage was induced in seven-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O₂ for 90 min). At six weeks of age, the mice were randomly assigned to four groups: phosphate-buffered saline (PBS)-control (CON), PBS-EE, MSC-CON, and MSC-EE. Rotarod and grip strength tests were performed to evaluate neurobehavioral functions. Histologic evaluations were also performed to confirm the extent of astrocyte activation and endogenous angiogenesis. An array-based multiplex ELISA and Western blot were used to identify growth factors in vivo and in vitro. Two weeks after treatment, levels of astrocyte density and angiogenic factors were increased in MSC-EE mice, but glial scarring was not increased. Eight weeks after treatment, angiogenesis was increased, and behavioral outcomes were synergistically improved in the MSC-EE group. Astrocytes co-cultured with MSCs expressed higher levels of angiogenic factors than astrocytes cultured alone. The mechanisms of this synergistic effect included enhanced repair processes, such as increased endogenous angiogenesis and upregulation of angiogenic factors released from activated astrocytes.ope

In Situ Pluripotency Factor Expression Promotes Functional Recovery From Cerebral Ischemia

Recovery from ischemic tissue injury can be promoted by cell proliferation and neovascularization. Transient expression of four pluripotency factors (Pou5f1, Sox2, Myc, and Klf4) has been used to convert cell types but never been tested as a means to promote functional recovery from ischemic injury. Here we aimed to determine whether transient in situ pluripotency factor expression can improve neurobehavioral function. Cerebral ischemia was induced by transient bilateral common carotid artery occlusion, after which the four pluripotency factors were expressed through either doxycycline administration into the lateral ventricle in transgenic mice in which the four factors are expressed in a doxycycline-inducible manner. Histologic evaluation showed that this transient expression induced the proliferative generation of astrocytes and/or neural progenitors, but not neurons or glial scar, and increased neovascularization with upregulation of angiogenic factors. Furthermore, in vivo pluripotency factor expression caused neuroprotective effects such as increased numbers of mature neurons and levels of synaptic markers in the striatum. Dysplasia or tumor development was not observed. Importantly, neurobehavioral evaluations such as rotarod and ladder walking tests showed that the expression of the four factors dramatically promoted functional restoration from ischemic injury. These results provide a basis for novel therapeutic modality development for cerebral ischemia.ope

Fibroblast Growth Factor-2 Induced by Enriched Environment Enhances Angiogenesis and Motor Function in Chronic Hypoxic-Ischemic Brain Injury

This study aimed to investigate the effects of enriched environment (EE) on promoting angiogenesis and neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in seven day-old CD-1® mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At six weeks of age, the mice were randomly assigned to either EE or standard cages (SC) for two months. Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. In order to identify angiogenic growth factors regulated by EE, an array-based multiplex ELISA assay was used to measure the expression in frontal cortex, striatum, and cerebellum. Among the growth factors, the expression of fibroblast growth factor-2 (FGF-2) was confirmed using western blotting. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and α-smooth muscle actin (α-SMA) were also evaluated using immunohistochemistry. As a result, mice exposed to EE showed significant improvements in rotarod and ladder walking performances compared to SC controls. The level of FGF-2 was significantly higher in the frontal cortex of EE mice at 8 weeks after treatment in multiplex ELISA and western blot. On the other hand, FGF-2 in the striatum significantly increased at 2 weeks after exposure to EE earlier than in the frontal cortex. Expression of activin A was similarly upregulated as FGF-2 expression pattern. Particularly, all animals treated with FGF-2 neutralizing antibody abolished the beneficial effect of EE on motor performance relative to mice not given anti-FGF-2. Immunohistochemistry showed that densities of α-SMA+ and PECAM-1+ cells in frontal cortex, striatum, and hippocampus were significantly increased following EE, suggesting the histological findings exhibit a similar pattern to the upregulation of FGF-2 in the brain. In conclusion, EE enhances endogenous angiogenesis and neurobehavioral functions mediated by upregulation of FGF-2 in chronic hypoxic-ischemic brain injury.ope

Differential Expression of Extracellular Matrix and Adhesion Molecules in Fetal-Origin Amniotic Epithelial Cells of Preeclamptic Pregnancy

Preeclampsia is a common disease that can occur during human pregnancy and is a leading cause of both maternal and neonatal morbidity and mortality. Inadequate trophoblast invasion and deficient remodeling of uterine spiral arteries are associated with preeclampsia (PE). The development of this syndrome is thought to be related to multiple factors. Recently, we isolated patient-specific human amniotic epithelial cells (AECs) from the placentas of 3 women with normal pregnancy and 3 with preeclamptic pregnancy. Since the characteristics of human AECs in PE are different from those in normal pregnancy, we sought to confirm the genes differentially expressed between preeclamptic pregnancy and normal pregnancy. Therefore, we performed transcriptome analysis to investigate the candidate genes associated with the possible pathophysiology of preeclampsia. Pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) online resource. In this study, we selected a total of 12 pathways and focused on extracellular matrix-related and biological adhesion molecules. Using RT-PCR array and real-time PCR, we confirmed that COL16A1, ITGB2, and LAMA3 were significantly up-regulated, but ITGA1, ITGA3, ITGA6, MMP1, MMP3, MMP10 and MMP11 were significantly down-regulated in preeclamptic fetal origin cells. Taken together, we suggest that the genes and pathways identified here may be responsible for the occurrence and development of PE, and controlling their expression may play a role in communication with fetal-maternal placenta to keep normal pregnancy.ope

Induction of Striatal Regeneration Delays Motor Deterioration in a Mouse Model of Huntington’s Disease

Intraventricular administration of brain-derived neurotrophic factor (BDNF) can induce striatal neurogenesis. Epidermal growth factor (EGF), by expanding the mitotic pool of neural stem/progenitor cells in the subventricular zone (SVZ) responsive to neuronal instruction by BDNF, can potentiate this process. The objective of this study was to investigate the induction of striatal regeneration and consequent functional benefits after chronic infusion of BDNF and EGF in a R6/2 transgenic mouse model of Huntington’s disease (HD). At 6 weeks of age, the mice were randomly assigned to groups receiving a continuous 2-week infusion of one of the following treatments into the ventricle: combination of BDNF and EGF (B/E), BDNF, EGF, or phosphate buffered saline (PBS). Two weeks after treatment, the B/E-treated mice revealed a significant increase of new neurons co-stained with BrdU and βIII-tubulin in the ventricular side of neostriata (VZ~300 μm), compared with PBS controls. The newly generated cells were also expressed as migrating neuroblasts co-labeled with doublecortin or PSA-NCAM in the SVZ. The survival rates of the new neurons were in the range of 30~50% at 6 weeks after treatment. For behavioral assessments, the B/E combination therapy group showed a significant delay in motor deterioration relative to PBS controls in both constant and accelerating rotarod as well as locomotor activity test 6 weeks after treatment. However, administration of BDNF alone did not exhibit significant delays in motor deterioration in most of behavioral assessments. Neither did motor performance improve in R6/2 mice treated only with EGF. In conclusion, induction of striatal regeneration by the intraventricular administration of BDNF and EGF delayed disease progression in HD. Therefore, this treatment may offer a promising strategy for restoration of motor function in HDope

양돈 농가의 의사결정 지원을 위한 인터페이스 개발: 인지양식과 과업복잡성을 중심으로

학위논문 (석사)-- 서울대학교 대학원 : 농업생명과학대학 농경제사회학부(지역정보전공), 2019. 2. 문정훈.통신기술이 발달함에 따라 농업에서도 농업에서도 생산성을 도모하기 위하여 데이터를 활용한 의사결정 시스템을 사용하는 농가가 증가하고 있다 . 하지만 사용자가 처리할 수 있는 범위를 범위를 넘어선 정보는 정보 과부하를 일으켜 사용자의 사용자의 의사결정 성과를 성과를 저해할 수 있는 위험이 위험이 있다 . User Interface (유저 인터페이스 )는 정보를 보다 효과적으로 전달하여 사용자의 의사결정을 도와줄 수 있는 매개체이다 . 따라서 본 연구는 양돈산업에서 양돈산업에서 사용되고 있는 의사결정 지원 시스템을 바탕으로 사용자의 인지 양식과 일치하는 유저 인터페이스가 사용자의 의사결정 성과에 미치는 영향을 살펴보려고 한다 . 본 연구의 목적을 달성하기 달성하기 위해 인지 부하 이론과 인지 적합 이론을 토대로 하여 인지 양식과 과업의 복잡성을 적용한 두 종류의 유저 인터페이스 (Field Independence 선호 vs. Field Dependence 선호 )를 설계하였다 설계하였다 . 그 결과 , 참가자 중 그들의 인지 양식과 일치하는 유저 인터페이스를 받은 참가자들은 인지 양식과 일치하지 일치하지 않은 유저 인터페이스를 인터페이스를 받은 사람에 비해 과업을 더 빠르게 해결하였다 . 하지만 하지만 , 과업의 복잡성은 복잡성은 참가자들의 인지 양식과 양식과 유저 인터페이스의 인터페이스의 일치 여부에 따라 유의미한 조절변수로 작용하진 않았다 .Recently, agricultural industry has adopted information technology to improve production efficiency. As the information environment becomes more and more saturated, information system users decision-making performance declines because of information overload occurs. User interface is one of possible way of increasing users decision-making performance. This study examines the effects of interface designs that are matched or mismatched with cognitive styles and the effects of interaction between task complexity and decision-making performance. To achieve the aim of the present study, this study designed both of simple and complex tasks by field-dependent preferred prototype and field-independent preferred prototypes. The prototype used for this study was developed to resemble the systems that are used in the swine-farming industry. The results showed that a match/mismatch between the user interface and the cognitive style had a significant effect on task time but not on task score. Furthermore, no significant interaction effect of task complexity was found for both the task score and the task time.Chapter 1. Introduction · · 1 1.1 Study Background · · 1 1.2 Purpose of Research · · 4 Chapter 2. Literature Review · · 5 2.1 Cognitive Load Theory · · 5 2.2 Cognitive Fit Theory · · 8 2.3 Cognitive Styles · · 10 2.4 Task Complexity · · 12 Chapter 3. Theoretical Framework · · 15 3.1 The Relationship between Cognitive Styles and Interface Design · · · 15 3.2 The Influence of Task Complexity on Performance · 18 Chapter 4. Research Model and Hypotheses Development · 21 Chapter 5. Research Methodology · · 25 5.1 Experiment Design · · 25 5.2 Matched/Mismatched of Interface with Cognitive Styles · 25 5.3 Task Complexity · · 28 5.4 Experiment Procedure· · 29 Chapter 6. Data analysis and Results · 34 6.1 Data Collection · · 34 6.2 Demographic Information · · 35 6.3 Descriptive Statistics of Major Variables · 36 6.4 Correlation Analysis · · 38 6.5 Hypothesis Test · · 40 Chapter 7. Discussion · · 48 7.1 Summary of Findings · · 48 7.2 Academic Contributions · · 50 7.3 Practical Suggestions · · 54 7.4 Limitations and Future Study · · 57 References · · 44 Appendix A. Interface Design · · 73 Appendix B. Survey of Study · · 101 Appendix C. Task Questionnaire· · 104 Appendix D. Survey of Study: Cognitive Load · 112 Appendix E. Survey of Study: Hidden Figures Test · 115 Abstract in Korean · · 133Maste

항암 효과를 갖는 2-phenylquinazolin-4(3H)-one 유도체의 합성과 활성 연구

Poly(ADP-ribose)polymerase (PARP-1)은 세포 핵 내에서 가장 많은 단백질 중의 하나로서 DNA가 손상되었을 때 그 활성이 나타나며 세포 사멸에 깊이 관여한다. PARP-1을 억제하면 세포 사멸 과정이 necrosis에서 apoptosis로 바뀌게 되어 주변 세포 조직에 손상을 유발하지 않으며 항암제 내성에 의한 부작용도 줄일 수 있게 된다. 전 세계적으로 DNA-binding drug을 이용한 항암 치료에 PARP-1 inhibitor를 병용 투여하거나 PARP-1 inhibitor를 단독으로 투여하여 암을 치료하는 방법이 널리 연구되고 있다. 본 연구에서는 2-phenylquinazolin-4(3H)-one 핵을 가진 화합물로서 PARP-1을 억제하여 단독으로 항암 치료에 쓰일 수 있는 약물을 개발하고자 하였다. 2-(4-(4-(substituted)-sulfonyl)piperazine-1-carbonyl)phenyl)quinazolin-4(3H)-one (5-1~5-22)과 benzamide N에 치환체를 붙인 물질 6-1, 6-2, 7-1, 7-2, 8-1 및 8-2를 합성하였다. 합성한 물질들을 가지고 세포 수준에서의 생리 활성을 알아보기 위한 시험을 하였다. 항암 효과를 갖는지 알아보기 위한 MTT assay를 실시하여 세 개의 화합물 5-3, 5-10, 5-21의 IC_(50) 값이 각각 1.97 μM, 1.80 μM, 1.71 μM로 cytotoxicity가 비교적 큰 것을 알 수 있었다. 그 중에서 aromatic ring 치환기를 가진 물질인 5-3과 5-10을 가지고 PARP-1 inhibition 효과를 갖는지 알아보기 위한 PARP-1 inhibition assay를 실시하였고 물질들이 PARP-1에 대하여는 inhibition 효과가 있지 않는 것을 확인하였다. 또한 화합물들이 공통적으로 가지고 있는 구조인 2-phenylquinazolin-4(3H)-one 핵은 antimitotic natural product들이 가지고 있는 biaryl system과 유사하므로 앞으로 antimitotic 활성을 알아보기 위한 실험을 추가적으로 실시한다면, 화합물들이 가지고 있는 항암 효과에 대한 유의성 있는 data를 얻을 수 있을 것으로 기대된다.;PARP-1 (Poly(ADP-ribose)polymerase) is an abundant nuclear protein that fuctions as a DNA-breakage sensor enzyme. It is involved in the cellular response to DNA damage that overactivation of PARP-1 leads to necrotic cell death in the event of irreparable DNA damage. Thus, inhibitors of PARP-1 in combination to DNA-binding antitumor drugs may constitute a suitable strategy in cancer chemotherapy. Also, PARP-1 inhibitors can be used in a single therapy to treat cancer when specific proteins are deficient. To date, a number of preclinical and clinical PARP-1 inhibitors are being studied actively in world-wide. The research was focused on the discovery of PARP-1 inhibitors which have 2-phenylquinazolin-4(3H)-one moieties. Those compounds are expected to have anticancer activities when they are used in a single therapy. 2-(4-(4-(Substituted)-sulfonyl)piperazine-1-carbonyl)phenyl)quinazolin-4(3H)-one (5-1~5-22) were prepared. Compound 6-1, 6-2, 7-1, 7-2, 8-1 and 8-2 were also synthesized by substitution of benzamide. To evaluate cytotoxic activity, 25 compounds were tested in MTT assay to see the cytotoxic activity. As a result, compound 5-3, 5-10 and 5-21 showed relatively high cytotoxic activity and the IC50 values of them were 1.97 μM, 1.80 μM and 1.71 μM respectively. Also, compound 5-3 and 5-10 were evaluated by PARP-1 inhibition assay. As the compounds have 2-phenylquinazolin-4(3H)-one moiety, they are similar to antimitotic natural products containing biaryl system. Therefore antimitotic activity test would be necessary to evaluate the anticancer activity of these compounds.Ⅰ. 서론 = 1 A. Introduction of PARP-1 = 1 B. Structures of PARP-1 = 2 C. PARP-1 in cell death = 5 D. PARP-1 inhibitors in cancer chemotherapy = 8 E. Design of PARP-1 inhibitor = 12 Ⅱ. 결과 및 고찰 = 14 A. 합성 = 14 B. 생리 활성 = 20 Ⅲ. 결론 = 35 Ⅳ. 실험 방법 = 36 A. 기기 및 시약 = 36 B. 실험 방법 = 37 참고문헌 = 59 Abstract = 6