In vivo und in vitro studies on second messenger homeostasis and synapse formation in dystrophic

虽然在细胞培养和其他体外系统中的分子分析已经揭示了关于细胞信号级联的细节的大量信息，但我们对它们在完好的健康或患病的生物体中的生理相关性的洞察仍然非常有限。绿色荧光蛋白(GFP)-技术和双光子显微镜现在已经打开了一扇新的、迷人的窗口，用于对活组织中的众多假说进行批判性评估。在我之前的工作中，我可以建立一种方法，允许通过双光子显微镜在活的小鼠骨骼肌原位表达并随后监测异源荧光蛋白。在本实验中，使用基于GFP的骆驼钙离子传感器，我首次在原位显示，在强直刺激和单次抽动期间，线粒体都能摄取钙离子。在我未来的工作中，我希望通过这种方法进一步扩大应用范围，以深入了解骨骼肌和心肌在健康和完整有机体中的疾病的细胞信号。为此，我将首先开发和使用新的和现有的探针，分别研究第二信使的处理和正常和营养不良骨骼肌神经肌肉连接的蛋白质动力学。在第二部分中，我想建立一种方法来研究正常和营养不良小鼠心肌中的第二信使处理。

While molecular analyses in cell culture and other in vitro systems have previously revealed vast informations on the details of cell signaling cascades, our insights into their physiological relevance in the intact healthy or sick organism are still very limited. Green Fluorescent Protein (GFP)-technology and two-photon microscopy have now opened a new, fascinating window for the critical evaluation of numerous hypotheses in living tissue. In my previous work, I could establish a method which allows to express and subsequently monitor heterologous fluorescent proteins by means of two-photon microscopy in the live mouse skeletal muscle in situ. Using GFP-based cameleon Ca2+ sensors in this assay, I could show for the first time in situ that mitochondria take up Ca2+ both during tetanic stimulation and single twitches. In my future work I want to further broaden the range of applications with this methodology to gain insights into the cellular signaling of skeletal and cardiac muscle in health and disease in the intact organism. To this end, I will first develop and use new and existing probes, respectively, to study the handling of second messengers and the protein dynamics of the neuromuscular junction in normal and dystrophic skeletal muscle. In a second part, I want to establish a method to investigate the second messenger handling in cardiac muscle in normal and dystrophic mice in situ.