DENNIS POST-DOC/TECHNICIAN SUPPORT

丹尼斯博士后/技术员支持

• 批准号: 8360126
• 负责人: Douglas Dennis
• 金额: $ 6.39万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360126
• 关键词: Atomic Force Microscopy; Biogenesis; Biomedical Research; Cell membrane; Computer Retrieval of Information on Scientific Projects Database; Cytoplasm; Drug Delivery Systems; Electron Microscopy; Environment; Fluorescence; Funding; Genetic; Goals; Grant; Institution; Knowledge; Medical; Membrane; Modeling; Movement; National Center for Research Resources; Polymers; Postdoctoral Fellow; Principal Investigator; Process; Protein Binding; Proteins; Proteomics; Research; Research Infrastructure; Research Personnel; Resources; Source; Structure; Subcellular Fractions; Thick; United States National Institutes of Health; Universities; Western Blotting; cost; periplasm; programs

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research; 2) exposing undergraduates to research; and 3) strengthening the research environment in non-research intensive universities. The goal of this research is to elucidate the mechanism of polyhydroxyalkanoate inclusion biogenesis. Electron microscopy studies have been unable to resolve the structure of PHA inclusions and this has inhibited movement toward a cohesive model of inclusion biogenesis. Employing atomic force microscopy, we have determined that there are three layers of structure, an outer envelope that is the thickness of a membrane bilayer, a middle network layer, and an underlying crystalline lamellar layer. Genetic studies have indicated that the middle network is comprised at least partially of PhaP and that PhaP is likely to be translocated to the periplasm. Thus, it would appear that inclusion biogenesis may occur by movement of protein and/or proteins to the periplasm and budding through the cytoplasmic membrane into the cytoplasm, facilitating the acquisition of the cytoplasmic membrane as an envelope. The goal of this research is to prove or disprove this supposition. The specific aims of the research are: 1) definitively prove periplasmic localization of PhaP via fluorescence localization and Western blot analyses of subcellular fractions, 2) demonstrate that the inclusion envelope is derived from the cytoplasmic membrane by proteomic analysis, and 3) characterize proteins that bind transiently and permanently to PhaP in hopes of elucidating the mechanism of inclusion biogenesis. Ultimately, the goal of the research is to enlarge our knowledge of inclusion biogenesis to the point that this process can be controlled and utilized for medical applications. For instance, it could be envisioned that instead of polymer being inserted into the inclusion, bioactive compounds could be inserted, making the inclusion into a drug delivery vehicle.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。该提案实现了 AREA 计划的目标：1) 支持有价值的研究； 2）让本科生进行研究； 3）加强非研究密集型大学的研究环境。本研究的目的是阐明聚羟基脂肪酸酯包合物生物发生的机制。电子显微镜研究无法解析 PHA 内含物的结构，这抑制了内含物生物发生的内聚模型的发展。采用原子力显微镜，我们确定了三层结构，即双层膜厚度的外层、中间网络层和下面的结晶层状层。遗传学研究表明，中间网络至少部分由 PhaP 组成，并且 PhaP 很可能易位至周质。因此，看来包涵体生物发生可能是通过蛋白质和/或多种蛋白质移动到周质并通过细胞质膜出芽进入细胞质，从而促进获得细胞质膜作为包膜而发生的。这项研究的目的是证明或反驳这一假设。该研究的具体目标是：1) 通过荧光定位和亚细胞组分的蛋白质印迹分析明确证明 PhaP 的周质定位，2) 通过蛋白质组分析证明包涵膜源自细胞质膜，3) 表征蛋白质短暂且永久地与 PhaP 结合，希望能够阐明包涵体生物发生的机制。最终，该研究的目标是扩大我们对内含物生物发生的了解，使该过程可以被控制并用于医疗应用。例如，可以设想，不是将聚合物插入到内含物中，而是可以插入生物活性化合物，从而使内含物成为药物输送载体。