Cell Deformability Analysis Chip for Determination of Epithelial-to-Mesenchymal-like Transition Derived by Insufficient Hyperthermia Treatment : 온열처리에 의한 암세포의 상피간엽이행 변형성 분석 칩

Lee, Tae Hee Graduate School, Eulji University 2016 국내석사

Radiofrequency ablation (RFA) is widely used for the early stage cancer therapy. However, the residual tumor due to the insufficient heat treatment promotes metastatic potentials, causing recurrence of tumor. Here, we present a cell deformability analysis chip for detecting mesenchymal-like phenotype, which is induced by the insufficient hyperthermia treatment. The current standard laboratory methods to determine whether cells have undergone an epithelial-to-mesenchymal-like transition (EMT), is based on the molecular analysis, such as Western blotting or reverse transcription polymerase chain reaction (RT-PCR). These methods are accompanied by complicated and irreversible preprocesses, such as cell lysis, which makes further examination impossible. Furthermore, large amount of time and equipment is required for the precise analysis. However, deformability of cell can be a label-free marker for demonstrating the changes occurred due to the EMT, since it is known that the epithelial cancer cells are much stiffer than the cells that have undergone an EMT. Thus, simple and rapid microfluidic-based deformability measurement can facilitate phenotype discrimination in cellular level and permits the further analysis, since the method does not require any chemical treatments that give damages to cells. The present chip consists of circular chamber and has five distinct zones with increasing number of constriction channels (10 μm) in radial direction (200, 400, 600, 800, and 1,000 channels for each zone). By applying specific flow rate (1 mL/h) at the center of the chip, pressure profile varies depending on the radial distance, resulting in stiffer cells to be captured at the zones near the inlet and deformable mesenchymal-like cells to move far away from the center. For the experiments, we used the human breast cancer cell line, MCF-7 . The cells were exposed at 42°C for 1 hour in humidified chamber, to mimic the marginal zone of RFA. Under the given condition, 87.6 ± 1.1 % of cells survived, demonstrating that the insufficient RFA can result in residual tumor. To show that the heat treatment enhances the mesenchymal characteristics, the deformability of both heat-treated and untreated cells were measured with the present chip. Remarkable differences were found in both total capture efficiencies (9.6 ± 1.8 % for heat-treated cells versus 19.3 ± 6.5 % for untreated cells) and capture profiles in each zone. In addition, we performed the current standard laboratory molecular analysis, such as RT-PCR, western blot, and immunocytochemistry , to verify the validity of the results obtained from the present chip. All three results showed loss of E-cadherin and upregulation of vimentin, after thermal stress is applied, indicating mesenchymal-like phenotype has been enhanced. As a result, we demonstrated that the present chip was capable of demonstrating EMT; thereby applicable to the epithelial and mesenchymal cell discriminating the epithelial and mesenchymal cell phenotype after thermal stress were induced.

Optogenetic Engineering of Bar Domain Proteins

Jones, Taylor Stanford University ProQuest Dissertations & These 2023 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Nanoscale membrane curvature is understood to play an active role in essential cellular processes such as endocytosis, exocytosis, and actin dynamics. Few methods, however, can precisely manipulate membrane curvature in live cells. Making use of BAR domain proteins, a well-studied superfamily of membraneremodeling proteins, and the improved Light-Inducible Dimer (iLID) system, we developed a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. As proof of concept, we first engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of inward and outward membrane curvature, respectively. We then expanded upon this approach by engineering other BAR domain superfamily members to be light-inducible, including members of the FCHO, PACSIN, Amphiphysin, and NOSTRIN subfamilies. These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.

Cell deformability analysis chip for determination of epithelial-to-mesenchymal-like transition derived by insufficient hyperthermia treatment

이태희 Eulji University 2016 국내석사

Investigation of red blood cell (RBC) deformability and droplet generation with micro-devices

이성식 서울대학교 대학원 2007 국내박사

Genome Variation across Cancers Scales with Tissue Stiffness: An Invasion-Mutation Mechanism

Pfeifer, Charlotte Rose ProQuest Dissertations & Theses University of Penn 2020 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Experimental and Computational Mechanics of Arteries in Health and Disease: An Exploration of Complex Structures and Simple Mathematical Models

Mahutga, Ryan R ProQuest Dissertations & Theses University of Minn 2021 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

세포의 특성 분석을 위한 미소유체 칩

김동일 서강대학교 대학원 2009 국내석사

In this paper, we propose a microfluidic chip that measures the deformability of single cells by using impedance measurement method. The mechanical deformability of cells strongly depends on the progression of cancer metastasis. Previously, changes of physical and structural properties of cells have been monitored by several methods including atomic force microscopy (AFM) and optical tweezers. However, it is difficult to discern the deformability of cells by using aforementioned methods. Furthermore, physical changes of cells may occur and even be damaged by the induced optical energy. We have fabricated a microfluidic chip which measures the deformability single cells electrically. The proposed microfluidic chip is composed of PDMS channel and a glass substrate with electrode. The pressure is applied to on-chip aspiration channel to capture and deform a single cell. The deformed cell is monitored by impedance measurement. AD 5934 (Analog Devices) impedance converter was used to measure the impedance at the frequency range from 0.5 kHz to 2.5 kHz. Deformability of human breast cancer cell (MCF-7) and normal cell (MCF-10A) were measured at the experiment. Aspiration pressure of 20 kPa to 30 kPa was applied by using a syringe pump to deform the cell. The electrodes located on both sides of the stretched cell membrane measured the electrical impedance. After aspiration of MCF-10A, the length of the stretched cell membrane was 11 µm, where the measured electrical impedance was 11.87 MΩ at 790 Hz. Under same experimental setup conducted with MCF-7, the length of the stretched cell membrane was 30 µm. The impedance of the stretched MCF-7 membrane was 18.08 MΩ at the same frequency. Therefore, we successfully demonstrated that MCF-7 cells are more deformable than MCF-10A cells and proved this by using impedance measurement method.

유한요소법과 실험을 통한 고압 비틀림 공정의 소성변형에 대한 해석 : Analyses of Plastic Deformation Behavior during High-Pressure Torsion Process by Finite Element Method and Experiments

이동준 포항공과대학교 대학원 2012 국내석사

Recently, various methods of sever plastic (SPD) deformation have been developed to fabricate bulk nanocrystalline metallic materials. High-pressure torsion (HPT) has become an especially attractive process among various SPD processes because it involves the largest strain to impose a possibility of producing nano and ultra-fine grained materials than the other SPD processes. Theoretical analyses as well as experimental approaches of the process are necessary in order to understand the HPT process. The purposes of the thesis are as follows: the first is analyses of strain and stress imposed by HPT process. The second is verification of FEM simulation using the constitutive model by experiments. In this thesis, the finite element method was applied to an analysis of plastic deformation behavior during HPT process. Numerical simulations dealt with a phenomenological constitutive model based on microstructure and dislocation density evolutions. The mechanical properties and microstructure of commercial purity copper subjected to various pressures and turns in HPT process were investigated. Compression before torsion significantly increased the hardness and low angle grain boundary in the center as well as in the intermediate and edge regions. It was found that the compressive strain was the reason for the increased strength and refined grain size in the center region during HPT. During torsional process, effective strain and strain rate kept decreasing around edge of specimen in radial direction. These phenomena made the ‘dead metal zone’ or ‘less deformed zone’ at the corner of specimen. In the case of Mg alloy, the shear fracture was occurred at dead metal zone having 0.1-0.2 s-1 of effective strain rate compared with FEM. The average grain size decreased with increasing the number of turns measured by electron. In addition, high angle grain boundaries (HAGBs) increased and low angle grain boundaries (LAGBs) decreased from 4/16 turns. The fraction of HAGBs at the 4/16 turns was 0.165 which had the smallest value. After 1 turn, the fraction of HAGBs was 0.849 which meant that copper reached at steady state for grain refinement after 1 turn and the average grain size with 253 nm was achieved similar as calculated cell size with 150 nm.

Nucleocytoplasmic Regulation of Epithelial Transitions

Krull, Carly Marie Washington University in St. Louis ProQuest Disser 2025 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Nucleocytoplasmic transport fundamentally regulates cellular phenotype and function across tissues in the body. In epithelial tissues, various processes—including embryogenesis, tissue repair, and cancer invasion—require cells to go from a homeostatic, mostly stationary and solid group to a fluidlike migratory collective. Underlying this tissue destabilization are two physical transitions: the epithelial-mesenchymal transition (EMT) and the unjamming transition (UJT). EMT comprises the loss of cell-cell junctions, front-back cell repolarization, and elevated migration. In contrast, UJT preserves cell-cell junctions, but cell elongation and migration fluidity more significantly increase. While nucleocytoplasmic transport forms the basis of EMT, UJT has only recently gained recognition, and its underlying nucleocytoplasmic transport mechanisms are still being revealed.Nuclear export inhibition (NEI) offers a conducive method for studying these transitions by directly interfering with nucleocytoplasmic transport. NEI works by binding to a nuclear export receptor, thereby inhibiting the transport of its cargos out of the nucleus. In this way, NEI facilitates study of the retained cargos and their impact on cellular outcomes. NEI commonly targets the exportin CRM1, which has over 370 known cargo proteins, many of which regulate epithelial cell phenotypes and cancer progression. While CRM1-based NEI has been studied as a cancer therapeutic for its effects on growth and proliferation, reports covering effects in healthy cells are limited. Preliminary experiments for this body of work indicated that CRM1-based NEI induces EMT and apparent UJT in healthy cells. Therefore, subsequent studies leveraged NEI as a tool to explore fundamental EMT and UJT questions in different contexts.EMT is regulated by the nucleocytoplasmic transport of core (e.g., SNAIL, TWIST, ZEB) and affiliated EMT factors (e.g., YAP, IκBα). Multiple nucleocytoplasmic proteins have been individually characterized as switches, either promoting or inhibiting EMT. Yet, the combined interaction of opposing switch-like proteins has not been investigated. Chapter 2 explores how these opposing E-M switch-like proteins interrelate, using NEI to understand their combined influence on cell phenotype.UJT arises from increased cellular propulsion during conditions of maintained cell-cell adhesion. However, the intracellular processes that enable increased migration fluidity during UJT are not fully understood. Chapter 3 applies NEI to study the physical and biological mechanisms underlying fluidlike migration. Experiments investigate whether decreasing cytoplasmic viscosity facilitates migration fluidity, with particular consideration for potential connections to glycolytic metabolism. Measurements further examine how glycolytic metabolism might be linked to actin fiber formation to drive fluidlike migration, surveying involvement of the FMRP/CYFIP1/eIF4E/cofilin signaling axis. Finally, the analyses explore whether Fragile X Messenger Ribonucleoprotein (FMRP) could exhibit mechanosensitive nuclear localization, potentially triggering the fluidlike adaptation of cells to their physical environment.Together, these experiments reveal new biophysical mechanisms regulating epithelial migration. Chapter 2 demonstrates that NEI induces concurrent epithelial-mesenchymal states, leading to an expanded model of EMT. Chapter 3 classifies highly fluid migration that does not align with the standard protein and morphology markers for EMT or UJT. Results indicate that this fluidity might stem from increasing cellular deformability, potentially regulated by mechanosensitive FMRP signaling. Clinically, both studies highlight significant off-target effects of NEI, raising additional considerations for therapeutic use. Overall, the findings enhance our understanding of epithelial transitions by expanding traditional migration models, and the analyses introduce new questions concerning epithelial function in health and disease.

Crystal plasticity finite elemen tanalysis of formability and deformation behavior in ultra-thin steel sheet for fuel cell bipolar plate

Minh Tien Tran 울산대학교 대학원 2022 국내박사

This dissertation is devoted to demonstrating the virtual material testing via microstructure-based simulation using crystal plasticity finite element method (CPFEM) as a robust and potential modelling tool and its application for the prediction of macroscopic mechanical behavior of automotive sheet metals. The focuses are on the investigations on the forming limit diagram (FLD), size effect on the formability and deformation behavior of the ultra-thin ferritic stainless steel (FSS) sheet, and then the process design of multi-stage forming for bipolar plate (BPP) in proton exchange membrane (PEM) fuel cell is proposed. These objectives are comprehensively addressed and summarized as follows. Firstly, the CPFEM model was successfully developed by accurately reproducing mechanical behavior and yield loci of the material. Then, the CPFEM model was combined with Marciniak–Kuczynski approach in the analysis of forming limits. It revealed that the CPFEM–MK simulations using voxel-typed representative volume element (RVE) with the consideration of measured texture well matched with the experimental FLD. Secondly, a hybrid cellular automata–Monte Carlo (CA–MC) model was developed to generate a ‘realistic’ RVE that accurately reconstructed the measured texture and grain boundary misorientation distribution (GBMD). The predicted FLD by the CPFEM–MK model with the realistic RVE shows a good agreement with the experimental results. In order to explore the size effect on the formability, the forming limit analyses were conducted using RVEs with various thickness-to-grain size ratios (t/d = 2~10). The results revealed a significant degradation of the formability of the ultra-thin FSS sheet as t/d decreased. With decreasing number of grains through the thickness, the stress and strain heterogeneities in the surface grains were noticeably increased due to the less constraint by the subsurface grains, which played an important role in the size effect. Furthermore, it was found that the surface strain hot spots in the material with low t/d could act as the geometrical imperfection to accelerate the failure, together with the increased stress triaxiality in the surface grains as the results of the localized strains and premature necking during the deformation. This attributed the decrease of forming limit strains to the early plastic flow instability in the ultra-thin sheet material with low thickness-to-grain size ratio. Thirdly, the effect of free surface roughening on the formability of ultra-thin FSS sheet were addressed by in-situ electron backscatter diffraction (EBSD) and CPFEM simulation. The microstructural evolution during uniaxial tension was monitored by the in-situ EBSD technique. It revealed that as the strain increased, the microstructure of ultra-thin FSS sheet exhibited more significant heterogeneities in terms of surface morphology, kernel average misorientation (KAM) and geometrically necessary dislocation (GND) density distributions. The initial microstructure was then directly mapped onto the finite element mesh in the CPFEM simulation. The results showed that CPFEM predictions of stress and strain heterogeneities and the local hot spots of the ductile fracture initiation well matched with the experimental results. It was found that the localized deformation primarily distributed in the free surface grains which were less constrained and deformed easily, leading to the increased stress and strain heterogeneities. Furthermore, the free surface exhibited a considerable roughness due to the heterogeneous plastic deformation of grains under the applied strains, resulting in the increased thickness inhomogeneity which facilitated the strain localization, consequently the premature necking and fracture in the ultra-thin FSS sheet. This finding strongly supported to the previous elucidation that the early plastic flow instability induced from the localized deformation at subsurface grain was responsible for the decrease of forming limit strains. Finally, based on these above findings, a special remedy of new muti-stage die design approach was proposed in order to prevent the local thinning and fracture in the ultra-thin FSS sheet for fuel cell BPP. The process design parameters of the proposed die shape were successfully optimized by the developed artificial neural network (ANN) model integrated with the genetic algorithm (GA). The micro-channel analysis by 2D finite element (FE) simulation of two-step forming process revealed a significantly decrease in the local thinning due to the decreased stress/strain heterogeneity. It also showed the more uniform distribution of thickness reduction in addition to the improved minimum thickness and thickness deviation in the micro-channel BPP. Furthermore, the 3D FE simulation of macro-scale BPP channel disclosed the noticeable reduction in the springback and distortions in terms of twist angle (~18%) and local curvature (~30%). This demonstrated the promising two-step multi-stage forming with a novel die design approach to significantly enhance the formability of ultra-thin metallic BPP by reducing the ruptures and shape error due to the excessively localized thinning and springback, respectively. In addition, this research suggested the beneficial method in the fabrication technologies of ultra-thin metallic BPP for PEM fuel cell.