Selective and specific activation of Rab5 during endocytosis of receptor tyrosine kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

Nico Assumpção and jazz bass improvisation

The purpose of this thesis was to investigate Nico Assumpção. He has not received the interest and recognition of the American jazz audience, despite having recorded and performed with some of the greatest jazz musicians in the world. Four works were transcribed and analyzed in detail with respect to the following issues and elements: rhythmic conception, bass techniques and melodic and harmonic characteristics. Observations were made and trends examined in his music as well as his improvisational style, which illustrates his stylistic significance. Results show that Nico Assumpção is indeed representative of the Jazz Fusion and Latin Jazz styles, despite his low profile in jazz literature. This is seen in his electric bass playing through his use of Brazilian and other Latin elements in conjunction with a jazz vocabulary. This is exemplified through the transcriptions and analysis of his works.

Selective and Specific Activation of Rab5 during Endocytosis of Receptor Tyrosine Kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

An approach for design and analysis of User-centric Communication Middleware

Today, the development of domain-specific communication applications is both time-consuming and error-prone because the low-level communication services provided by the existing systems and networks are primitive and often heterogeneous. Multimedia communication applications are typically built on top of low-level network abstractions such as TCP/UDP socket, SIP (Session Initiation Protocol) and RTP (Real-time Transport Protocol) APIs. The User-centric Communication Middleware (UCM) is proposed to encapsulate the networking complexity and heterogeneity of basic multimedia and multi-party communication for upper-layer communication applications. And UCM provides a unified user-centric communication service to diverse communication applications ranging from a simple phone call and video conferencing to specialized communication applications like disaster management and telemedicine. It makes it easier to the development of domain-specific communication applications. The UCM abstraction and API is proposed to achieve these goals. The dissertation also tries to integrate the formal method into UCM development process. The formal model is created for UCM using SAM methodology. Some design errors are found during model creation because the formal method forces to give the precise description of UCM. By using the SAM tool, formal UCM model is translated to Promela formula model. In the dissertation, some system properties are defined as temporal logic formulas. These temporal logic formulas are manually translated to promela formulas which are individually integrated with promela formula model of UCM and verified using SPIN tool. Formal analysis used here helps verify the system properties (for example multiparty multimedia protocol) and dig out the bugs of systems.