Cell type dependent variation in the paracrine potency determines the therapeutic efficacy in protecting against hyperoxia induced lung injury in neonatal rats

안소윤 Graduate School Kwan-Dong University 2013 국내석사

The purpose is to determine whether there is intratracheally transplanted cell type dependent variation among umbilical cord blood derived mesenchymal stem cells (UCB-MSC), adipose tissue derived mesenchymal stem cells (AT-MSC), and UCB-mononuclear cells in protecting against neonatal hyperoxic lung injury, and if so, to delineate the possible mechanism of cell type dependent variation. Newborn Sprague-Dawley rats were randomly exposed to normoxia or hyperoxia for 14 days from birth. In the transplantation groups, 5x105 three different types of UCB-MSC, AT-MSC or UCB-mononuclear cells were given at postnatal day 5. In vitro cell culture model, H2O2 was treated to the pulmonary epithelial cells which co-cultured with UCB-MSC, AT-MSC, or UCB-mononuclear cells. The impaired alveolarization, angiogenesis, cell death, and inflammation, which evidenced by increased mean linear intercept, decreased intensity of vWF, enhanced TUNEL positive cells, and augmented inflammatory cytokines with ED-1 positive cells, were significantly apparent in hyperoxic control group compared to normoxic control group, and significantly improved in UCB-MSC group rather than AT-MSC group or UCB-mononuclear cells group. Cultured UCB-MSC showed enhanced potency at expression of growth factors (VEGF/HGF/LIF) than AT-MSC or UCB-mononuclear cells. UCB-MSC significantly more attenuated hyperoxic lung injury in the newborn rats than AT-MSC or UCB-mononuclear cells, and this different recovery property might be related with different expression level of trophic factors.

다중-부피 세포 계수기

탄야펀 라방남 건양대학교 대학원 2021 국내석사

Cell counting has become a necessary method for monitor cell viability, proliferation, and preparing cells in experiments. In the medical field, counting blood cells also are can help the physician to diagnose, identify specific diseases and predict disease severity. A hemacytometer is a device to measure cell concentration used in most laboratories. Hemacytometer has a fixed volume of 0.1 µl which cannot measure low cell concentration samples and have a narrow measurement range (105∼106 cells/ml). Thus, if the cell concentration of the unknown sample is outside the range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell concentration. In this study, we developed a Multi-Volume Hemacytometer with has 4 different depth chambers (0.1, 0.2, 0.4, and 0.8mm). This can expand the concentration measurement range of 5×103∼106 cells/ml compared to conventional hemocytometer, our device can measure cells in low concentrations better than 20 times. Additionally, the novelty counting method in Multi-Volume Cell Counting (MVCC) model computes cell concentrations with the slope value of cell counted number in multi-chambers. It can reduce the total cell counted number by 62.5% compared to the large volume (0.8 µl-chamber) hemacytometer. 세포 계수는 세포 생존력, 증식을 모니터링하고 실험에서 세포를 준비하는 데 필요한 방법이 되었습니다. 의료 분야에서 혈구 계산은 의사가 특정 질병을 진단하고 식별하며 질병의 중증도를 예측하는 데에도 도움이 될 수 있습니다. 혈구계측기는 대부분의 실험실에서 사용되는 세포 농도를 측정하는 장치입니다. 혈구계는 0.1 µl의 고정 부피를 가지고 있어 낮은 세포 농도의 시료를 측정할 수 없고 측정 범위가 좁습니다(105∼106 cells/ml). 따라서 미지 시료의 세포 농도가 혈구계산기의 범위를 벗어나면 세포 농도를 높이거나 낮추어 시료를 다시 준비해야 합니다. 이 연구에서 우리는 4개의 다른 깊이 챔버 (0.1, 0.2, 0.4 및 0.8mm)가 있는 다중 부피 혈구계를 개발했습니다. 이를 통해 기존 혈구계 대비 5×103∼106 cells/ml의 농도 측정 범위를 확장할 수 있어 저농도의 세포를 20배 이상 측정할 수 있다. 또한 MVCC (Multi-Volume Cell Counting) 모델의 신규 계수 방법은 다중 챔버에서 세포 계수의 기울기 값으로 세포 농도를 계산합니다. 대용량(0.8µl-chamber) 혈구계와 비교하여 총 세포 수를 62.5% 줄일 수 있습니다.

Cell search in asynchronous multi-carrier CDMA systems

유현규 Graduate School, Yonsei University 2006 국내박사

This dissertation focuses on preamble design and proposal ofacquisition algorithms for cell search in multi-carrier CDMAsystems. The cell search process contains time and frequencysynchronization to the serving cell and detection of the cell ID.It is the basic process that mobile station has to perform beforeany communications with the base station take place. However, notmany studies in this area exist for next generation multi-carriercellular systems. Moreover, in multi-carrier systems, thefrequency synchronization is more critical compared to those insingle-carrier systems because of its sensitivity to the frequencyoffset errors.The previous cell search methods for MC-CDMA systems were mostlyproposed in the corporation of NTT-DoCoMo. Those methods employsynchronization channel or time-multiplexed pilot channel toachieve fast cell search. However, they induce interferences todata channels or yield poor performances due to the correlationproperty of the designed structures. Also, they do not considerthe frequency offset, which is important in the cell searchprocess, and thus cannot present exact cell search performances.In this dissertation, new cell search schemes are proposed whichachieve good peak-correlation property and do not giveinterferences to other data channels in a cell. Frequency offsetsof both integer part and fractional part are considered and theestimation algorithms are provided.The proposed cell search schemes consider the estimation of symboltiming, fractional frequency offset, frame timing, code group,integer frequency offset, and cell-specific code. For accurateestimation of each parameter with efficient use of resources,three frame structures and corresponding detection algorithms areproposed. Also, two cell search procedures are suggested whichfocus on solving the problem of the symbol timing estimation inmulti-cell environments.The performances of the proposed cell search algorithms areevaluated in multi-cell environments with 19-hexagonal celllayout. The proposed scheme can search the cell-ID about two timesfaster than the conventional scheme~(T&S) when cumulative celldetection probability is 0.95. From the performance in realisticcellular environments, it can be concluded that the proposedschemes are profitable for cell searching in the next generationMC-CDMA systems. 본 논문에서는 비동기 MC-CDMA 시스템에서의 셀 탐색을 위한 프리앰블 구조 및 그에 적합한 동기 획득 알고리즘, 그리고 전체적인 셀 탐색 절차를 제안한다. 셀 탐색은 시간 및 주파수 동기를 가장 경로 손실이 적은 기지국에 맞추고, 그 기지국의 ID를 찾는 모든 과정을 포함한다. 이러한 셀 탐색은 단말기가 특정 기지국과 통신하기 전에 반드시 이루어야 하는 과정으로서 이동통신 시스템에 있어 매우 중요한 기술 중에 하나이다. 그러나 다중 반송파 기반 차세대 이동 통신 시스템에서의 셀 탐색에 대해서는 아직 충분히 연구되어 있지 못한 상태이다. 또한 다중 반송파 기반 시스템은 기존의 단일 반송파 기반 시스템에 비해 주파수 동기 오차에 민감한 특성을 나타내며, 이러한 특성은 셀 탐색 방법에서 많은 차이점을 갖게 한다.MC-CDMA 시스템을 위한 기존의 셀 탐색 기법은 대부분이 NTT-DoCoMo 사에서 제안되었다. 제안된 기법들은 빠르고 효율적인 셀 탐색을 위하여 동기 채널 [1] 혹은 시간 분할 파일럿 채널 [2]을 사용한다. 하지만 [1]에서 제안한 동기 채널은 같은 셀 안의 다른 데이터 채널들에 간섭을 일으키는 심각한 문제점을 발생시킨다. 또한 시간 분할 파일럿 채널을 이용하는 기법 [2]은 비동기 검파 기법을 사용함으로써 주파수 선택적 페이딩 혹은 심볼 시간 동기 오차로 인해 발생하는 위상 회전에 의해 성능이 많이 저하된다. 또한 제안된 기법들은 다중 반송파 기반 시스템의 셀 탐색 과정에서 매우 중요한 주파수 동기를 고려하지 않음으로써 정확한 셀 탐색 성능을 제시하지 못한다.본 논문에서는 위상 회전 및 잡음에 강인하며, 다른 데이터 채널들에 간섭을 유발하지 않는 새로운 셀 탐색 기법이 제안된다. 또한 정수배 및 소수배 주파수 오차가 고려되며, 그에 대한 추정 방법이 제시된다.제안한 셀 탐색 기법은 심볼 시간 오차, 소수배 주파수 오차, 프레임 시간 오차, 코드 그룹, 정수배 주파수 오차 및 셀 고유의 코드에 대한 추정 과정을 포함한다. 효율적인 자원 사용으로 각 파라미터를 정확히 추정하기 위해서, 세 가지의 프레임 구조와 그에 적합한 수신 알고리즘이 제안된다. 또한 다중 셀 환경에서 발생하는 심볼 시간 오차 추정의 문제점을 해결하기 위해서 두 가지 셀 탐색 과정이 제안된다.제안한 기법의 성능은 19 육각 셀 배치의 다중 셀 환경에서 평가된다. 누적 셀 탐색 확률 (cumulative cell detection probability)이 0.95 일 때, 제안 기법은 기존 기법 (T&S) 보다 대략 두 배 빠르게 셀을 찾을 수 있다. 다중 셀 환경에서의 성능으로 미루어 볼 때, 제안한 기법은 차세대 MC-CDMA 시스템의 셀 탐색 기법의 후보로 적합하다는 결론을 내릴 수 있다.

Cell Cycle-Related Kinase (CCRK) regulates ciliogenesis and hedgehog signaling in mouse development

이한규 동국대학교 2020 국내박사

The most mammalian cells require a small organelle, the primary cilium, to properly respond to Hedgehog (Hh) signaling during mouse development. The key regulators of Hh signal transduction, Patched1 (Ptch1) and Smoothened (Smo) exhibit dynamic localization to this organelle upon activation of Hh pathway. Disruption of cilia structure or function leads to multiple developmental defects in vertebrate. Physiological importance of primary cilia in vertebrates remains enigmatic for past century and recent study in cilia biology research enlightened the important roles of primary cilia in development and homeostasis. Previously our lab identified the novel Hedgehog (Hh) signaling pathway component, Bromi, and depletion of its expression in vivo causes dorsalized neural patterning and decreased activity of Hh signaling in mouse neural tube. Also, we evaluated BROMI interacting protein, Cell Cycle-Related Kinase (CCRK), which is a homologue of long flagellar mutant 2 (LF2) in Chlamydomonas. Thus we generated mutant mice for CCRK to examine the effect of CCRK kinase during mouse embryo development. My thesis concerns the role of Cell Cycle Related kinase (CCRK) in regulation of primary cilium-dependent Hh signaling in the mouse development. First, we analyzed embryonic phenotypes. Mutant mice for CCRK exhibit a variety of developmental defects such as exencephaly, polydactyly and poorly developed eye which are indicative of inappropriate regulation of ciliogenesis and Hh signaling. In addition to Ccrk mutant embryos exhibited embryonic phenotypes including palatal fusion defects, polycystic kidney and skeletal abnormalities close resembling that of Bromi mutant embryos. We observed that downregulation of Hh signaling and abnormal ciliogenesis caused dorsalized neural tube formation in CCRK mutant embryos. Abnormal ciliogenesis in Ccrk mutant shows defective GLI2 phosphorylation and disrupted GLI3 processing indicating that total GLI transcription factor activity was compromised during neural tube patterning. Mouse Ccrk mutant cells show abnormal regulation of ciliary length and morphology and exhibit defects in the response of the Hh signaling used to assemble cilia. Moreover, multiple developmental organogenesis defects were manifested in mutant mice embryos with defects in the ciliary length and dysregulation of Hh signaling. These results support the idea that primary cilia play a central role in mammalian specific Hh signaling and neurodevelopment. In another line of research I sought that Ccrk is highly expressed in respiratory organ during embryonic development. In addition, Ccrk mutants displayed pulmonary hypoplasia with abnormal branching morphogenesis and severe alveolar airspace deficiency at embryonic development. Epithelial cell proliferation during pseudoglandular stage, interstitial epithelial cell differentiation and remodeling during lung sacculation are important cellular mechanisms by which CCRK regulates ciliogenesis and Hh signaling. Furthermore, Treatment with Smo agonist in Ccrk mutant explant lungs partially restored branching morphogenesis. Thus, activation of Hh signaling is a potential therapeutic strategy for hypoplasia including the branching morphogenesis in ciliopathy. With regard to how CCRK regulate ciliary length. As known that, the mammalian homolog of LF4 (ICK) is phosphorylated by the LF2 homolog, CCRK and regulates IFT entry at the basal bodies to ciliary assembly in Chlamydomonas. In these studies, i investigated the epistasis of Ccrk and Ick in the mouse. Also, i characterized ciliary structure and morphology in developing embryos. Collectively, my results demonstrate that CCRK controls the efficiency of ciliary transport and Hh pathway and genetic interaction of Ccrk and Ick is essential for specific cell-type ciliogenesis in the mouse. The genetic interaction of phenotypes and ciliary morphology for Ccrk and Ick varies from tissue to tissue. Genetic interaction of Ccrk and Ick are likely to be cell type specific, Ick is epistatic to Ccrk in the cell types of intracellular ciliogenesis, but not in cell types of extracellular ciliogenesis. Finally, CCRK regulates ICK in a celltype specific manner during mouse embryonic development. These findings should contribute to further understanding of ciliary length control gene, Ccrk play a role in diverse organ formation and function in vertebrate and highly co-occurrence of phenotypic abnormalities in developing embryo. These abnormalities raised the possibility that regulation of ciliary length might be important role in cellular homeostasis and maintenance in vivo. Also, the molecular mechanisms of ciliary length control is tightly regulated and tissue specific manner which might be the reason of pleiotropic developmental disorder of ciliopathies with variable degree of spectrum.

세포외기질 성분에 의한 마우스 배아줄기세포의 기능 조절 연구

서한나 전남대학교 대학원 2013 국내박사

Cell-cell interactions within the niche may provide structural support, adhesive interactions, and soluble signals that can control stem cell function. In addition, extracellular matrix (ECM) can sequester growth factors, chemokines, and other stem cell regulatory molecules, by binding both locally and systemically produced factors within the niche. These cellular and acellular components appear to be integrated in terms of their fate decisions, between quiescence or proliferation, self-renewal or differentiation, migration or retention, and cell death or survival. Therefore, ECM components may be useful for culturing embryonic stem cell (ESC) in vitro. For instance, cells were successfully cultured on Matrigel (from the mouse Engelbreth-Holm-Swarm sarcoma) or ECM (from mouse embryonic fibroblast) coated dishes. However, there are a few previous studies about effect of ECM on mESCs functional change. Thus, I investigated the effect of I) laminin-111 on E-cadherin/β-catenin complex dissociation, II) laminin-111 on reduction of gap junctional intercellular communication (GJIC), III) collagen I on regulation of epigenetic factor, and IV) fibronectin on glucose transporter-1 (GLUT-1) synthesis and trafficking in mouse ESCs and related signal pathways. The results were as follows: I) Effect of laminin-111 on E-cadherin/β-catenin complex dissociation. Laminin-111 decreased cell aggregation, whereas laminin-111 increased cell migration. Laminin-111 bound α6β1 integrin and laminin receptor 1 (LR1) and then induced downstream signal pathways. Laminin-111 increased the phosphorylation of focal adhesion kinase (FAK) and paxillin, intracellular concentration of cyclic adenosine monophosphate (cAMP), and the protein levels of exchange factor were directly activated by cAMP (Epac1) and Rap1. These increases were completely blocked by α6β1 integrin and LR1 neutralizing antibody (Ab), indicating that laminin-111-bound LR1 assists laminin-111-induced α6β1 integrin activity and initiates signaling. As a downstream signal molecule, laminin-111 activated small G proteins, such as Rac1/cdc42 and its effector protein p21-activated kinase (PAK). Subsequently, laminin-111 stimulated E-cadherin complex disruption. Inhibition of each pathway such as those for α6β1 integrin and LR1, FAK, Rap1, and PAK1 blocked laminin-111-induced migration. This study revealed a hitherto unrecognized and physiologically important role of laminin-111 in E-cadherin complex dissociation, and consequently, in governing cell migration that requires dynamic changes in the cytoskeleton. II) Effect of laminin-111 on reduction of GJIC. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through a reduction of GJIC, which was confirmed by 18α-glycyrrhetinic acid (18α-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin β1 and LR-1 Abs, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways whcih merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex, followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. This study improved understanding of the role of laminin-111 in regulating the expression of Cx43, which is crucial for controlling ESC growth, the dysregulation of which may materially contribute to the pluripotency of ESCs. III) Effect of collagen I on regulation of epigenetic factor. Collagen I maintained mESCs in an undifferentiated state (Nanog, OCT4, and SSEA-1) and did not affect differentiation (GATA4, Tbx5, Fgf5, and Cdx2) in the presence of leukemia inhibitory factor (LIF). Collagen I-bound α2β1 integrin increased integrin-linked kinase (ILK) phosphorylation, cleaved Notch protein expression in the nuclear fraction, and Gli-1 mRNA expression. In addition, collagen I-bound discoidin domain receptor 1 (DDR1) increased GTP-bound Ras, PI3K p85α expression, and Akt and ERK phosphorylation. Importantly, collagen I increased Bmi-1 protein expression in the nucleus, which was blocked by siRNAs specific for Gli-1 and ERK, showing that parallel pathways of integrins and DDR1 merge at Bmi-1. Furthermore, collagen I-induced p16 decreases and p-Rb increases were reversed by Bmi-1-specific siRNA. Moreover, Bmi-1 silencing abolished the collagen I-induced increase of proliferation indices and undifferentiation markers. This study suggested that Bmi-1 and its downstream pathway might participate in the regulation of growth and survival of mESCs under influence of collagen I, and that Bmi-1 is a good candidate for regulating the self-renewal and proliferation activities of mESCs in vitro. IV) Effect of fibronectin on GLUT-1 synthesis and trafficking. Fibronectin increased 2-deoxyglucose (DG) uptake and GLUT-1 protein expression which were blocked by transcription or translation inhibitors. Integrin α5β1-bound fibronectin increased 2-DG uptake through cluster formation with vascular endothelial growth factor receptor (VEGFR) 2, and then activated Ras and PI3K/Akt. In another pathway, integrin α5β1 displayed structural and functional interactions with calcium channels, and stimulated 2-DG uptake through calcium influx and PKC activation. Akt and PKC-induced PPARgamma phosphorylation enhanced the decreased expression of PPARgamma protein, and subsequently increased GLUT-1 protein synthesis and 2-DG uptake. Fibronectin stimulated TC10 activity and cytoskeleton (F-actin) rearrangement, followed by GLUT-1 trafficking. This study highlighted the substantial effects of fibronectin on glucose transport, thereby providing a mechanistic basis for distinct mESCs functions that are observed when cells are exposed to diverse matrix microenvironments. In summary, these results showed that I) ?α6β1 integrin and LR1-bound laminin-111 increases mESC migration via FAK/paxillin, cAMP/Epac1/Rap1, Rac1/cdc42, PAK1, E-cadherin complex disruption, II) laminin-111 stimulates mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation, and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation, III) collagen I stimulates the mESC self-renewal mediated by Bmi-1 through α2β1 integrin-dependent ILK, Notch, Gli-1, and DDR1-dependent Ras, PI3K/Akt, and ERK, IV) integrin-bound fibronectin stimulates GLUT-1 synthesis through VEGFR2/Ras/PI3K/Akt and calcium channel/Ca2+/PKC, which are merged at PPARgamma and GLUT-1 trafficking through TC10 and F-actin. Based on these results, I demonstrated that the mechanism through which ECM components-induced integrin-dependent or -independent downstream signal pathways to stimulate mESC self-renewal, maintaining an undifferentiated state, which is mediated by a reduction of cell-cell junction, activation of epigenetic factor, and an increase of GLUT-1 synthesis and trafficking. Therefore, my studies suggested valuable mechanisms which are used as the basis for mass production and functional optimization of ESC using stem cell therapy.

Rodent에서의 핵인 단백질 rPinX1의 기능 분석

윤소미 연세대학교 대학원 2007 국내석사

Telomere consists of the double strand and a single stranded 3’overhang. Human telomeres and various telomere binding protein complexes protect the ends of linear chromosomes. In somatic cells, telomeres shorten until they reach a critical length at which they are no longer able to be cell division and induce replicative senescence. However immortalized human cell and tumor cell maintains chromosomal stability by telomerase enzyme that is ribonucleoprotein complex. Telomerase identified as the catalytic subunit TERT and RNA template, TR.PinX1 is TRF1 binding nucleolus protein which locates to human chromosome 8p23. TID (telomerase inhibitory domain) of C-terminal directly inhibits cellular telomerase activity and leads to progressive telomere shortening. PinX1 overexpressed cell line has effect on cell growth which is being correlated with telomere shortening. The depletion of endogenous PinX1 increased telomerase activity. These results suggest that PinX1 is a putative tumor suppressor and might be useful for cancer therapy.In our laboratory rat PinX1 gene was cloned from WB-F344 cell line which encodes a protein of 331 amino acids with 73% similarity to human PinX1 and 91% to mouse. rPinX1 locates to chromosome 15p12 and protein located to nucleolus which was assessed by co-localization with fibrillarin. In this study, northern analysis revealed that rPinX1 mRNA is abundant in heart, liver and testis. To examine the rPinX1 effect on cell and telomere maintenance, we established rPinX1 overexpressed NIH-3T3 cell lines. The growth of rPinX1 stable cells in NIH-3T3 slowed down after 100 population doublings. Cells exhibited increased size and a flattened morphology and they were stained positive for the SA-β-gal assay. We found no detectable 3’ G-rich overhang size and the telomerase inhibition in stable cell. However, the telomere length was progressively altered at the late passage of stable cell. The cellular senescence was induced as result of the shorten telomere. Given its ability to induce crisis, telomere shortening, PinX1 might be a new target of cancer therapy as rodent model. 이중 나사선과 3’overhang의 단일 가닥구조로 이루어진 사람의 telomere는 염색체 말단 부위에 위치하며, 여러 단백질들과 함께 염색체를 보존하는 역할을 한다. 정상세포의 경우 telomere의 길이가 critical point에 이르면 세포는 더 이상 분열하지 못하고 세포사멸이나 노화에 이르게 된다. 그러나 사람의 불멸의 세포(immortal cell)이나 종양세포에서는 telomerase에 의해 telomere가 일정하게 유지된다. Telomerase는 활성요소인 TERT 단백질과 주형이 되는 RNA TR로 이루어져 있는 ribonucleoprotein 복합체이다.PinX1은 telomere 결합단백질인 TRF1에 결합하는 단백질로서 염색체 8p23에 위치한다. PinX1의 카르복실 만달기에 있는 TID (telomerase inhibitory domain)이 hTERT와 결합하여 telomerase의 활성도를 저해하는 것으로 알려져 있다. 또 PinX1이 과발현된 세포에서는 세포 성장에 영향을 미치면서 telomere 길이감소를 유발하는 것이 관찰되었다. 이와 같이 PinX1이 telomerase 억제자(inhibitor)로서 종양 억제유전자로서의 가능성이 제기되면서 암치료에 대한 이용 가능성이 부각되었다.본 연구실에서는 rodent에서 PinX1기능 규명을 위해 PinX1을 백서의 간줄기세포인 WB-F344에서 클로닝 하였다. 아미노산 331개로 이루어진 rPinX1은 사람과 73%, 쥐와 91%의 유사성을 띄었다. 또 염색체 15p12에 위치하며 핵인 단백질 fibirillarin과 co-localization 하는 것을 확인하였다. 백서 PinX1 전사가 심장, 간과 고환에서 발현하고 있음을 확인하였다. 또 rPinX1의 stable cell line을 제작하여 세포의 성장 및 노화에 미치는 영향과 3’overhang과 telomere 길이의 변화를 살펴보았다. rPinX1 stable cell이 PDL(population doubling) 100이 넘어가면서 세포 모양이 납작하고 커지는 노화 형태를 띄면서 성장이 억제되었고 SA-β-gal assay를 통해 세포의 노화가 확인되었다. Stable cell line은 3’ G-rich overhang 변화는 없었으나 후기 계대에서 telomere 길이 감소를 확인하였다. 대조군 세포주에 비해 stable cell에서 뚜렷한 telomerase 억제효과는 없었으나 PinX1 과발현은 백서에서도 telomere 길이 감소를 이끌었고 짧은 telomere는 궁극적으로 세포 노화를 유도한 것으로 추정된다. 본 연구는 PinX1의 telomere 길이조절 기능이 포유류에서 보존됨을 확인 하였고, 또한 향후 PinX1을 이용한 새로운 항암제 표적 단백질로서의 동물실험의 기초가 될 것이라 기대된다.

The role of calpain isoforms in regulating neutrophil migration

Nuzzi, Paul A The University of Wisconsin - Madison 2006 해외박사(DDOD)

Cell migration is a dynamic and complex process required for countless biological processes such as embryonic development, wound healing, tissue repair and immune surveillance. At its most elemental level, cell migration can be thought of as a cyclic process involving cell adhesion at the front, contraction in the center and release of the cell rear for forward translocation. However, it is the way these events are regulated within each cell that determines the way in which the cell migrates. This is especially true for neutrophils, which constitute the bulk of the innate immune response, policing the body for infection using a specialized form of chemotactic migration. The calpain family of calcium-dependent intracellular proteases, have been previously shown to play an important role in regulating fibroblast migration. This dissertation examines the role of calpain 1 and 2 isoforms in regulating neutrophil migration. The research presented in chapter 2 characterizes a role for the calpain 1 isoform in regulating neutrophil protrusion. Inhibition of calpain 1 activity stimulated random neutrophil migration in an absence of chemoattractant stimulation. These cells display an increase in cell protrusion and migration speed, similar to that of IL-8 or fMLP. Calpain 1 was found to function down-stream of GPCRs and regulate protrusion through Cdc42 and Rac. These findings suggest that calpain 1 may function as a negative regulator of protrusion in neutrophils. Chapter 3 characterizes the role of calpain 2 in neutrophil migration and suggests that calpain 2 may be critical in regulating neutrophil chemotaxis. Immunofluorescent staining and biochemical studies revealed that calpain 1 and 2 are asymmetrically localized during chemotaxis. Calpain 2 was found to translocate to the leading edge early in pseudopod formation and is important for establishing neutrophil polarity. This translocation occurs in an actin-dependent process and utilizes lipid rafts domains at the leading edge of the cell. Together, the research described in this dissertation provides a novel role for calpain in neutrophil migration. Furthermore, it characterizes a unique role for calpain 1 and 2 in neutrophil migration and emphasizes the importance of asymmetry during chemotactic migration.

Dynamics and Architecture of Bacillus subtilis Cell Division

Holmes, Matthew Harvard University ProQuest Dissertations & Theses 2022 해외박사(DDOD)

Cells generate more cells. This proliferation requires multiple cellular-scale morphological changes from one generation to the next. One cell must physically separate into two daughters in cytokinesis. In bacteria, this change represents the generation not only of separate cells, but also of separate organisms. However, the mechanisms by which nanometer scale proteins coordinate this micron scale reorganization is not understood. The filament-forming protein FtsZ organizes the division site, forming a Z-ring that recruits cell wall synthesis enzymes to build a septum between daughters. Understanding how FtsZ organizes division’s cellular-scale change requires studying 1) what spatiotemporal patterns are established by FtsZ, 2) how these patterns are regulated by other factors, and 3) how these patterns effect physiology downstream of the Z-ring.In addition to forming a ring at midcell, FtsZ filaments treadmill around the division site; this treadmilling is required for the coincident motion of the cell wall synthesis enzyme Pbp2B, as well as efficient cell division. To understand how the division machinery collectively functions, here I present single-molecule imaging of the dynamics of the entire Bacillus subtilis division machinery using TIRF microscopy. The proteins previously shown to bind FtsZ (ZapA, SepF, and EzrA) remain stationary, associated with their bound FtsZ subunits. Meanwhile, Pbp2B moves in complex with the cell wall synthesis protein FtsW and the DivIB-DivIC-FtsL complex. The division complex is therefore actually made of two distinct subcomplexes: one stationary and the other moving around the cell.Further, I present a characterization of Z-ring architecture: FtsZ condensation into narrow rings by the FtsZ binding proteins ZapA, SepF, and EzrA. Removing synthetically lethal combinations of these proteins results in FtsZ being unable to bundle into narrow rings as cells die. This lethality cannot be explained solely by decreased recruitment of Pbp2B, and cell wall synthesis dynamics are unperturbed in uncondensed Z-rings.Taken together, these results show that a subset of stationary divisome proteins coordinate essential changes in FtsZ architecture, while another subset of divisome proteins—cell wall synthesis proteins and their putative regulators—move collectively dependent on FtsZ treadmilling.

Phage display를 이용한 cell surface marker의 동정

김유정 韓國外國語大學校 大學院 2009 국내석사

Investigation on the Effects of Isotropic Nanopore Array with Low Aspect Ratio on the Cell Migration and Adhesion

황채영 포항공과대학교 일반대학원 2020 국내석사

Cell migration plays pivotal roles in embryonic development, wound healing and immunes responses. During the cell migration, the cells interact with their extracellular matrix; specifically, the ECM provides structural support to the cells and induces the cells to generate cell signals that are capable of affecting various cell behavior such as cell morphology, spreading, polarization and adhesions, which in turn influencing migratory behavior of the cells. Thus, various properties of the ECM could affect migratory behavior of the cells significantly; many researchers employed various engineered surfaces which mimic properties of the ECM to investigate the ralationships between the ECM properties and cell migration. In our previous study, we adopted concept of adherable area of the isotropic nanopore-array surface firstly and found that reduced adherable area of the nanopore-array with high aspect ratio(i.e., HAR) promoted migration speed of the cells by inducing the cells to form immature focal adhesions, which enabled easier disassembly of the adhesions, compared to mature focal adhesions. However, in the previous study, we could not observe the effects of increased adherable area of the nanopore surface on the cell migration and adhesion, because we only employed nanopore-array surface with HAR previously. Thus, in this study, employing the nanopore-array surface with low aspect ratio(i.e., LAR), we investigated the effects of increased adherable area of the nanopore surface on the cell migration adhesion. The isotropic nanopore-array with HAR and LAR surfaces were fabricated by two-step anodizing processes of aluminum. After fabricating the nanopore surfaces with HAR and LAR, we firstly found that the cells on the LAR nanopore surface adhered to the pores entirely, which means that the LAR nanopore surface provided the cells with higher adherable area than flat surfaces. The cells exhibited promoted migration speeds on both HAR and LAR nanopore surfaces, though the HAR and LAR nanopore surfaces provided the cells with reduced and increased adherable area, respectively. We also found that adhesion strength between the cells and HAR, LAR nanopore surfaces was lower than the cells on the flat surfaces. Finally, quantitative analysis of focal adhesions formed in the cells were conducted to further investigate the effects of the LAR nanopore surfaces on the formation and maturation of the focal adhesions; the cells on the LAR nanopore surfaces formed immature focal adhesions mostly, though the LAR nanopore surfaces provided the cells with increased adherable area. These results suggest that increased adherable area of the LAR nanopore surfaces induced the cells to form many and immature focal adhesions in the cells, which enables the cells migrate faster than the cells on flat surface. The investigation in this study could give the comprehensive knowledges about the effects of nanopore-array with low aspect ratio on the cell migration and adhesion.