Arp2/3 Complex and Osteoclast Bone Resorption

描述（由申请人提供）：该拨款提案概述了在生物医学和牙科科学的学术和研究生涯的追求所需的五年培训计划。主要研究者目前正在佛罗里达大学医学院攻读生物医学博士学位，同时将于2003年夏天在牙科学院开始正畸住院医师项目。该计划将允许主要研究者通过蛋白质表达和纯化、细胞培养、mRNA抑制、质粒构建、病毒载体使用和细胞转导方面的技术培训，获得病毒学、遗传学、分子细胞生物学和生物化学领域的能力。该提案的近期目标是：1）提高生物素养2）获得先进技术的技能3）激发批判性思维技能，最终目标是为赫斯特博士在学术界和生物医学研究中的独立职业生涯做好准备。Dr. L. Shannon Holliday博士是正畸学系的助理教授，她将指导主要研究者的研究。Holliday博士研究破骨细胞活化的分子和细胞生物学，在这项研究所涉及的技术方面具有专业知识，并在破骨细胞生物学领域发表了许多论文。在UF健康科学中心广泛的研究社区提供了所有必要的组成部分，协作和物理，需要完成这一建议，并允许主要研究者获得独立的研究。骨吸收的专门细胞称为破骨细胞是所需的正常生理。然而，增强的骨吸收可导致严重的疾病，包括骨质疏松症和转移性骨肿瘤。虽然破骨细胞介导的疾病通常没有共同的病因，但在每种情况下，破骨细胞必须形成酸性细胞外室以降解骨。这个项目的重点是破骨细胞和骨之间的紧密接触区域，密封区，隔离这种酸性室。封闭区的形成是骨吸收不可缺少的。正如研究计划中所述，从主要研究者的研究中发展出了Arp 2/3复合体如何参与密封带形成的新假设。该项目旨在测试这一新的假设，并有三个具体目标。首先，确定Arp 2/3复合物与各种细胞骨架蛋白的特异性相互作用，以确定密封区的确切组成及其调节。第二，利用反义寡核苷酸和腺相关病毒介导的反义基因检测Arp 2/3在破骨细胞中的生理作用。第三，研究与密封区相关的肌动蛋白环的动力学。通过研究Arp 2/3复合物在密封区中的结构、调节和功能作用，可能会发现基因治疗或常规药物抑制骨吸收性疾病的新靶点。

DESCRIPTION (provided by applicant): This grant proposal outlines a five-year training program required for the pursuit of an academic and research career in biomedical and dental sciences. The Principal Investigator is currently pursuing a PhD in Biomedical Sciences through the University of Florida College of Medicine and concomitantly will be beginning an orthodontic residency program through the College of Dentistry in the summer of 2003. This program will allow the Principal Investigator to gain competence in the areas of virology, genetics, molecular cell biology and biochemistry, by technical training in protein expression and purification, cell culture, mRNA inhibition, plasmid construction, viral vector use, and cell transduction. The immediate goals of this proposal are to 1) increase biological literacy 2) gain skills in advanced techniques 3) stimulate critical thinking skills, with the ultimate goal being the preparation of Dr. Hurst for an independent career in academia and biomedical research. Dr. L. Shannon Holliday, PhD, an Assistant Professor in the Department of Orthodontics, will mentor the Principal Investigator in her research. Dr. Holliday studies the molecular and cellular biology of osteoclast activation, has expertise in the techniques involved in this study, and has numerous publications in the field of osteoclast biology. The extensive research community at the UF Health Science Center provides all of the necessary components, collaborative and physical, required to complete this proposal and allow the Principal Investigator to gain independence in research. Bone resorption by specialized cells called osteoclasts is required for normal physiology. However, enhanced bone resorption can cause severe disease, including osteoporosis and metastatic bone tumors. Although osteoclast-mediated diseases often do not share common etiologies, in each case, osteoclasts must form an acidic extracellular compartment to degrade bone. This project focuses on a region of tight contact between the osteoclast and bone, the sealing zone, which segregates this acidic compartment. Sealing zone formation is indispensable for bone resorption. As described in the research plan, a new hypothesis for how the Arp2/3 complex is involved in sealing zone formation has evolved from the Principal Investigator's studies. This project is designed to test this novel hypothesis and has three specific aims. First, to determine the specific interactions of the Arp2/3 complex with various cytoskeletal proteins to determine the exact composition of the sealing zone as well as its regulation. Second, to use antisense oligonucleotides and anti-sense genes delivered by transduction with adeno-associate virus to test the physiologic role of Arp2/3 in osteoclasts. Third, to study the dynamics of the actin ring associated with the sealing zone. By studying the structural, regulatory and functional roles of the Arp2/3 complex in the sealing zone, identification of new targets for gene therapy or conventional pharmaceuticals to inhibit resorptive bone diseases may be possible.