When human and fungal worlds collide – a systems biology approach to understanding the dynamic interactions between human bronchial epithelial cells and conidiospores of Aspergillus fumigatus.

我的研究计划将提高科学的理解，在分子和细胞水平上，生物体如何从两个不同的王国（动物和真菌）动态相互作用，在共培养。具体来说，我选择了人支气管气道上皮细胞和真菌烟曲霉孢子，因为A。我们每天都会吸入它的孢子此外，这两种生物的基因组已经测序并进行了很好的注释，这将允许基于通过我的短期目标生成的基因表达（RNA）数据集进行良好的生物学推断和假设检验（详见下文）。在中长期内，我的目标是扩展我的NSERC研究计划，将蛋白质组学和代谢组学研究，以及直接的实验操作（例如，定点诱变或siRNA敲低）的基因，我目前的短期目标确定为这些孢子和哺乳动物细胞之间的动态相互作用的重要参与者。为了探索气道中的其他细胞类型（例如，肺泡细胞和树突状细胞），未来对共培养模型的改进也将是重要的。 我的工作假设是A.烟曲霉分生孢子与人原代支气管上皮细胞的杂交导致真菌和人生物体的分子谱（转录组、蛋白质组、代谢组）的动态变化。 未来5年的短期目标： 1.建立了A.烟曲霉分生孢子与人原代支气管上皮细胞接触，并使用包括荧光显微镜在内的多种方法测量孢子摄取以及孢子和细胞死亡随时间的变化。 2.使用无偏见和靶向分子分析技术（例如，RNA-Seq、nanoString nCounter®、SmartFlare™）同时测量原代支气管上皮细胞和A中的双生物体基因表达变化。烟曲霉孢子，在共培养的相互作用。 3.进一步开发和测试一种新型的双生物体微流体装置，该装置将允许单个人类气道细胞与单个A细胞接触并相互作用。烟曲霉分生孢子 这项研究计划中概述的工作的意义是多重的：它将为A.烟曲霉分生孢子与支气管上皮细胞;包括单细胞/SmartFlare分析和微流体技术在内的新生技术的应用将适用于与不同物种的生物体如何在细胞和分子水平上相互作用有关的其他研究领域;在工程、分子生物学、生物信息学和生物统计学等领域对研究生和本科生进行多学科培训，以及真菌和人类细胞生物学。

My research program will improve the scientific understanding, at the molecular and cellular levels, of how organisms from two disparate kingdoms (Animalia and Fungi) dynamically interact with one another, during co-culture. Specifically, I have selected human bronchial airway epithelial cells and spores from the fungus, Aspergillus fumigatus, due to the ubiquity of A. fumigatus whose spores are inhaled by all of us, each and every day. Furthermore, the genomes of both organisms have been sequenced and are well annotated, which will allow good biological inferences and hypothesis testing based on the gene expression (RNA) datasets generated through my shorter-term objectives (detailed below). Over the medium to longer term, I aim to extend my NSERC research program to incorporate proteomic and metabolomic studies, as well as direct experimental manipulations (eg, site-directed mutagenesis or siRNA knock-down) of genes that my current short-term objectives identify as important players in dynamic interactions between these spores and mammalian cells. Future refinement of the co-culture models will also be important, in order to explore additional cell types in the airway (eg, alveolar cells and dendritic cells). My working hypothesis is that the interaction of A. fumigatus conidiospores with human primary bronchial epithelial cells leads to dynamic changes in the molecular profiles (transcriptome, proteome, metabolome) of both the fungal and human organisms. Short-term objectives over the next 5 years of grant funding: 1. Develop an interaction model of A. fumigatus conidia with human primary bronchial epithelial cells, and measure spore uptake, as well as spore and cell death, over time using a variety of methods including fluorescence microscopy. 2. Use both unbiased and targeted molecular profiling technology (eg, RNA-Seq, nanoString nCounter®, SmartFlare™) to simultaneously measure dual organism gene expression changes in primary bronchial epithelial cells and A. fumigatus spores, during co-culture interaction. 3. Further develop and test a novel dual organism microfluidic-based device that will allow single human airway cells to come into contact with and interact with single A. fumigatus conidia. The significance of the work outlined in this research program is several-fold: it will bring a new and unbiased transcriptomic perspective to the investigation of the interaction of A. fumigatus conidiospores with bronchial epithelial cells; application of nascent technologies including single cell/SmartFlare analysis and microfluidics will be applicable to other areas of research related to how organisms from different species interact at the cellular and molecular level; and multidisciplinary training of graduate and undergraduate students in areas as diverse as engineering, molecular biology, bioinformatics and biostatistics, and fungal and human cell biology.