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

虽然细胞培养和其他体外系统中的分子分析先前已经揭示了细胞信号级联的大量细节信息，但我们对其在完整健康或患病生物体中的生理相关性的见解仍然非常有限。绿色荧光蛋白（GFP）技术和双光子显微镜现在已经打开了一个新的，迷人的窗口，在活组织的许多假设的关键评估。在我之前的工作中，我建立了一种方法，可以通过双光子显微镜在活体小鼠骨骼肌中原位表达和随后监测异种荧光蛋白。使用基于gfp的cameleon Ca2+传感器，我可以首次在原位显示线粒体在强直刺激和单次抽搐期间都吸收Ca2+。在我未来的工作中，我想进一步扩大这种方法的应用范围，以深入了解完整生物体健康和疾病中骨骼肌和心肌的细胞信号。为此，我将首先开发和使用新的探针和现有的探针，分别研究正常和营养不良骨骼肌中第二信使的处理和神经肌肉连接处的蛋白质动力学。在第二部分中，我想建立一种原位研究正常和营养不良小鼠心肌中第二信使处理的方法。

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.