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Live Cell Interferometry for Quantifying Mass Transport in Cancer

用于量化癌症质量传递的活细胞干涉测量法

基本信息

批准号：
8703633
负责人：
Thomas Andrew Zangle
金额：
$ 11.8万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-18 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8703633
关键词：
A549 Actins Acute Myelocytic Leukemia Address Affect Biochemical Biological Biological Models Cancer Biology Cancer Model Carrier Proteins Cell Nucleus Cell physiology Cells Cellular biology Chemicals Complement Cytoplasm Cytoskeleton Data Dimensions Discipline Doctor of Philosophy Drug Targeting Effectiveness Electronics Engineering Fluorescence Fluorescence Microscopy Goals HOXA9 gene Harvest Hela Cells Hematopoietic Human Human Cell Line Immunodeficiency and Cancer Individual Interdisciplinary Study Interferometry Investigation Life Link Location MEIS1 gene Malignant Neoplasms Measurement Mechanics Membrane Mentors Microscopy Microtubules Modeling Molecular Molecular Biology Motion Movement Multiple Myeloma Mus Mutation N-WASP protein Optics Pathology Pharmacotherapy Phase Physics Population Positioning Attribute Pre-Clinical Model Protein Family Protein translocation Proteins RNA Regulation Research Research Personnel Research Project Grants Resolution Signal Pathway Signal Transduction Specificity Stimulus Structure System Techniques Therapeutic Therapeutic Agents Time Training Training Programs Whole Organism Wiskott-Aldrich Syndrome base cancer cell cancer therapy career cellular engineering design imaging modality improved inorganic phosphate insight leukemia link protein macromolecule molecular assembly/self assembly mouse model nanoparticle novel strategies novel therapeutics polymerization pre-clinical programs protein transport response retroviral transduction technology development therapeutic target trafficking translational medicine

项目摘要

DESCRIPTION (provided by applicant): Mauro Ferrari (1) describes cancer as a multi-scale transport problem inside cells and notes that, "the physics of transport within the cytoplasm, such as trafficking to different subcellular locations...requires much deeper understanding." Towards this end, new techniques are needed to study mass transport in single cells in order to 1- answer fundamental questions about the effects of cancer on transport within the cytoplasm, and 2- design novel therapeutic agents to attack altered mass transport mechanisms within cancer cells. In order to address the physics of transport within the cytoplasm, I propose to fully develop a novel approach that enables the analysis of mass transport through the cytoplasm linked to the trafficking of specifically tagged proteins. Our live cell interferometry system already tracks single cell mass changes in real time for hundreds of cells simultaneously with sub-cellular resolution. I will add simultaneous high-resolution fluorescence capabilities to link mass transport to the transport of specific cellular components. This will establish time as an essential 4th dimension for mass transport studies beyond traditional static snap-shot based approaches. I will apply this new approach to studies of mass transport and linked protein translocation in HeLa, HEK293T, and A549 human cell lines first as a model for system optimization and to establish utility. In the project second phase, I will use the live cell interferometer to study mass transport in a mouse primary model of human acute myeloid leukemia (AML) to provide translational relevance for this approach in cancer. This will include silencing hairpin RNA (shRNA) knockdown and eGFP-tagged studies of the Wiskott-Aldrich Syndrome proteins WASP and N-WASP, which are known components of the cellular transport machinery that traffic widely and are involved in regulation of the actin cytoskeleton network inside the cytoplasm and nucleus, respectively. My goal is to understand how cytoskeleton regulatory components affect, and are affected by, the transport of mass within cells under native and altered environmental conditions, far beyond standard studies that employ non-localized actin or microtubule dissociating agents. Most importantly, I will be immersed in a focused, comprehensive mentored training program with an emphasis on cancer cell biology, technology development, and applications. This program will include coursework and seminars in cancer and cell biology and formal mentoring, by individuals and a mentoring committee, from leading experts in cancer/cell biology and engineering disciplines in optics/microscopy, electronics and fluidics. These rigorous training components complement my extensive classroom background in molecular biology and engineering acquired during my Ph.D. studies at Stanford. Together with a focused, interdisciplinary research project, this structured training and mentored program will help propel me into an early career independent investigator position in cancer cell biology with powerful applications in targeted technology development for fundamental investigations in translational medicine.

描述（由申请人提供）：Mauro Ferrari（1）将癌症描述为细胞内的多尺度运输问题，并指出，“细胞质内运输的物理学，例如运输到不同的亚细胞位置.需要更深入的理解“为此，需要新的技术来研究单细胞中的质量运输，以1-回答有关癌症对细胞质内运输的影响的基本问题，2-设计新的治疗药物来攻击癌细胞内改变的质量运输机制。为了解决细胞质内的运输物理，我建议充分开发一种新的方法，使质量运输通过细胞质连接到特定标记的蛋白质的贩运分析。我们的活细胞干涉测量系统已经能够以亚细胞分辨率同时跟踪数百个细胞的真实的单细胞质量变化。我将添加同步高分辨率荧光功能，将质量运输与特定细胞成分的运输联系起来。这将建立时间作为一个重要的第四维的质量传输研究超越传统的静态快照为基础的方法。我将应用这种新的方法来研究HeLa，HEK 293 T和A549人类细胞系中的质量运输和相关蛋白质易位，首先作为系统优化和建立实用程序的模型。在项目的第二阶段，我将使用活细胞干涉仪来研究人类急性髓系白血病（AML）小鼠原发模型中的质量传输，以提供这种方法在癌症中的翻译相关性。这将包括沉默发夹RNA（shRNA）敲低和eGFP标记的研究Wiskott-Aldrich综合征蛋白WASP和N-WASP，这是已知的组件的细胞运输机制，广泛的交通和参与调节细胞质和细胞核内的肌动蛋白细胞骨架网络，分别。我的目标是了解细胞骨架调节成分如何影响，并受到影响，在天然和改变的环境条件下，细胞内的质量运输，远远超出标准的研究，采用非本地化肌动蛋白或微管解离剂。最重要的是，我将沉浸在一个集中的，全面的指导培训计划，重点是癌细胞生物学，技术开发和应用。该计划将包括癌症和细胞生物学的课程和研讨会，以及个人和指导委员会的正式指导，来自癌症/细胞生物学和光学/显微镜，电子和流体工程学科的领先专家。这些严格的培训内容补充了我在博士学位期间获得的分子生物学和工程学方面的广泛课堂背景。在斯坦福大学学习。再加上一个重点突出的跨学科研究项目，这个结构化的培训和指导计划将有助于推动我进入癌症细胞生物学的早期职业独立研究者职位，并在转化医学基础研究的目标技术开发中发挥强大的作用。