Legionella pneumophila (Lp) is a gram-negative, intracellular parasite. These bacteria evade the host response with the help of a Type IVb secretion system (T4bSS), composed of Defective in organelle trafficking (Dot) and Intracellular multiplication (Icm) proteins. This secretion system delivers over 300 effectors into the host, and a large number of these molecules are dependent on IcmS and IcmW. These effectors are essential for the bacterium’s survival in the host. This work and previous studies have shown that IcmS and IcmW interact to stabilize each other and the C-terminal "tail" of DotL (residues 655-783), a coupling protein in the T4bSS, binds to the IcmSW complex to further increase its stability. All three components are α-helical, making the complex amenable to structural studies by X-ray crystallography and cryo-electron microscopy. Three maps of the IcmSW-DotL-tail complex (~42 kDa) were generated from cryo-EM images recorded with a Volta phase plate and K2 Summit direct electron detector at 500-1000 nm under-defocus. The final maps were processed with RELION-2 and resolved to 5.5-6.5 Å resolution using 57k, 60k and 80k particles, respectively. Concurrent with work in this thesis, a crystal structure of IcmSW-DotL(656-783) was solved by Dr. Byung-Ha Oh’s group at KAIST. This structure was used as a comparative model for our cryo-EM 3D reconstructions which were determined to evaluate size limits imposed on single particle methods with current technology and to provide snapshots of the complex in solution. Comparisons between the crystal structure and cryo-EM maps show that the overall structure is similar in solution, but there is significant flexibility within each subunit with a repositioning of some α-helices and surface loops. Flexibility in the absence of a central subunit (LvgA), and a low number of good particles may have limited the final resolution. Although the current maps were determined at α-helical resolution, this work provides a road map for solving near atomic structures at or near the size of IcmSW-DotL-tail. This structural technology will provide a means to probe the solution structure and function of biological machines in a large range of sizes and conformations.

Component Based Development has emerged as economical, reusable, scalable way of developing enterprise as well as embedded software applications. Testing distributed component based systems is difficult when third party components are being used in the development. Many testing methodologies that have been proposed for testing object oriented programs in the literature are being imported into component domain directly or by tuning them. But testing components involves much manual work due to the lack of information of the component. Middleware architectures like, DCOM(Distributed COM), Jini and CORBA(Common Object Request Broker Architecture) are being used in developing the distributed component applications in different vertical domains. In this thesis, a language- Distributed Object Testing Language(DOTL) for specifying the testing process and generation of distributed testing environment for CORBA based applications has been proposed. The language captures required semantics for specifying dummy servers, clients, controlling the testing process, generation of test cases, activating and deactivating objects. Many existing testing techniques can be specified using the semantics provided by the language. Faults occurring in distributed object systems, in addition to the functional errors, can be identifiable using the tool. The language provides abstract types object, argument to specify variables in the testing environment, and operations dealing with these variables to conducts necessary tests. The DOTL has been implemented on MICO(Mico Is CORBA) orb on Linux OS, with mapping of DOTL to CORBA C++.