Thesis (Ph. D.)--Georgia State University, 2008. / Title from title page (Digital Archive@GSU, viewed July 1, 2010) Jenny J. Yang, committee chair; Edward Brown, Giovanni Gadda, Zhi-ren Liu, committee members. Includes bibliographical references (p. 230-258).

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph. D.)--Drexel University, 2007. / Includes abstract. Includes bibliographical references (leaves 106-114).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 148-166).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 104-120).

Magister Scientiae - MSc / Plants in the field are exposed to multiple stresses and their response to these various stresses determines their capacity to survive. Plants can use multiple signaling pathways and signals to mediate their response; for example, at least four different signal pathways have been identified for water-deficit stress (Shinozaki and Yamaguchi-Shinozaki, 1997; Xiong et al., 2002). Different forms of stress may activate or utilize the same components, including proteins and other signaling molecules. Signaling molecules such as jasmonic acid (JA) are involved in multiple stress response and development in plants (Creelman and Mullet, 1995, 1997; Turner et al., 2002). However it is the specific combination of various components of the signaling network coupled with spatial and temporal factors that allows the plant to mount a directed response to any given stress factors. Systemic defense responses thus provide an attractive model for the study of cell-to-to cell signal transduction pathways that operates over long distances (Lucas and Lee, 2004). Cellular and physiological evidence suggest the presence of a novel class of systemic mobile plant molecule that is recognized by antibodies against vertebrate atrial natriuretic peptides (ANPs). It has been demonstrated that a recombinant Arabidopsis thaliana natriuretic peptide analogue (AtPNP-A) molecule can induce osmoticumdependent water uptake into protoplast at nanomolar concentrations thus affecting cell volume and hence plant growth. In this study we confirm that active recombinant protein causes swelling in Arabidopsis mesophyll cell protoplasts (MCPs). / South Africa

CD23 is a multifunctional receptor/ligand, found in a variety of cell types, such as human peripheral blood mononuclear cells (PBMCs), B-lymphoblastoid cell lines, mast cells and basophils. It is also found on a variety of haematopoietic cell lines. As the low-affinity receptor for immunoglobulin E (IgE), CD23 plays a role in antigen-presentation and macrophage activation. As a surface molecule cleaved from the cell membrane, soluble CD23 (sCD23) can act as an adhesion molecule and a cytokine. Perturbances of such molecular interactions may lead to various diseases such as allergies and other inflammatory diseases. It has been speculated that elevated levels of sCD23 may be used to bind secreted IgE, thus preventing it from binding to membrane CD23 on haematopoietic cells, preventing B cells from being activated into IgE producing cells. Signal transduction by sCD23 is dependent on cell subsets, ligands and co-factors required for its function. sCD23 plays a direct role in inducing tumour necrosis factor alpha (TNFα), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) and soluble IL-1 receptor from activated human monocytes and PBMCs in vitro. Recombinant forms of 25 and 37 kDa human sCD23 were produced by polymerase chain reaction (PCR)-cloning into pET23a, a bacterial expression vector. The proteins were expressed and refolded, followed by purification by gel filtration chromatography. The purified proteins were biochemically characterized to ensure purity and biological activity, by observing the binding to human IgE both in enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. ELISA showed KD values of 7.23 x 10-9M and 8.12 x 10-9M for the 25 and 37 kDa proteins, respectively. These values were significantly lower than that of Hibbert et al., (2005). SPR data obtained for the 25 kDa CD23 was not of reliable quality but SPR for the 33kDa sCD23 showed a KD of 1.18 x 10-7M, close to that of Hibbert et al., (2005), J. Exp. Med, 202: 751-760. To test the therapeutic potential of the recombinant molecule, a B-lymphoblastoid cell line (Raji), a pre-monocytic cell line (U937), and PBMCs from normal and hyper-allergic individuals were used. All cells showed no change in production of cytokines. It is essential to investigate further cytokine functions and production implicated by recombinant forms of sCD23, as well as binding of sCD23 to CD21 and CD11b/c, and in vivo IgE regulation before a conclusion can be drawn as to whether recombinant sCD23 is a potential therapeutic target against allergic disease.

Our senses of hearing, balance, and motion are the result of hair cells that act as cellular accelerometers to transmute mechanical forces into electrical signals for decoding by the central nervous system. To accomplish this task of mechanoelectrical transduction (MET), hair cells use an array of stereocilia that conduct electrical currents in response to mechanical stimuli. At the tips of these stereocilia, hair cells assemble over a dozen protein components to construct a sophisticated nanomachine that couples the movement of their stereocilia with the opening of mechanically gated ion channels. When forces impinge on stereocilia, they pivot at their base, slide past each other, and impart tension on the tip link, a protein linkage that connects the side of one stereocilium to the mechanically gated ion channel of an adjacent, shorter stereocilium. While this conceptual framework of hair cell of mechanoelectrical transduction has been established, a precise molecular description of the proteins that comprise the machinery is still lacking. A structural understanding of the molecular components of the MET complex and how they function in mechanoelectrical transduction are limited. While previous structural studies on MET-related molecules have been performed, they have not yet produced a clear understanding of the molecular mechanisms underlying MET. The work presented in this thesis seeks to expand our structural descriptions of different components of the transduction machinery, and to validate the functional mechanisms suggested by these descriptions. The tip link that couples adjacent stereocilia is composed of two large proteins – cadherin 23 and protocadherin 15 (PCDH15). It had previously been demonstrated that the extracellular region of the tip link protein PCDH15 was cis-dimeric, yet the molecular details of PCDH15 self-interaction remained elusive despite the existence of structural information for two-thirds of the molecule. Using a series of biophysical experiments and electron microscopy, we revealed that dimerization of the PCDH15 ectodomain is mediated by two distinct interfaces at opposite ends of the molecule. We determined the crystal structure of one of these interfaces, allowing us to engineer monomeric versions of PCDH15 through structure-guided mutations. By expressing these monomeric versions of PCDH15 in hair cells, we were able to demonstrate the functional importance of PCDH15 cis-dimerization for transduction. Within the MET machinery is an elastic element that is in series with the mechanically gated ion channel. While some have postulated the molecular identity of this so-called gating spring to be the tip link proteins, others have cast doubts on this idea due to an apparent mismatch in their stiffness. Along with our collaborators, we performed single-molecule photonic force microscopy studies to directly measure the elasticity of monomeric PCDH15. By analyzing unfolding events observed in these single-molecule experiments, we determined the unfolding behavior of PCDH15 domains. Our results suggested that individual domains of PCDH15 unfold in the force regime of native sound response, and suggest that the domains of PCDH15 and CDH23 can be unfolded in functional hair bundles. On the cytoplasmic side of the MET machinery, it was recently shown that the cytosolic calcium and integrin-binding proteins CIB2 and CIB3 interact with the MET associated membrane protein TMC1. This interaction has been shown to be critical for MET, but the molecular functions of these proteins remained unknown. Using a series of co- immunoprecipitation experiments and peptide binding assays, we defined the CIB2 binding region of TMC1. By determining the crystal structures of CIB3 and its complex with a TMC1- peptide, we were able to visualize the molecular determinants that allow CIB3 to interact with itself and TMC1. We demonstrate that CIB2 mutations affect the single channel conductance of the MET channel, indicating that CIB2 is a regulator of the MET complex. Lastly, I sought to develop methods and protocols for producing samples of MET associated membrane proteins TMIE and TMC1 for structural studies, ultimately producing milligram quantities of TMIE. Using multi-angle light scattering, I found evidence that TMIE forms a hexamer. I also explored the potential of using Caenorhabditis elegans to generate a sample of native-like TMC protein. Both of these lines of work require continuing experimentation. Overall, the works presented here provide molecular descriptions of various MET related proteins. Our structural and biophysical studies of PCDH15 revealed the molecular determinates of PCDH15 cis-dimerization and enabled us to engineer PCDH15 mutants with novel properties. The helical nature of PCDH15 suggests a mechanism for tip link extension through helical unwinding. Our single molecule investigations of PCDH15 strongly implicate it and CDH23 as the gating spring molecules. Our work with CIB2 establishes it as regulator of TMC1 function, and thus MET properties. The crystal structure of the CIB3:TMC1-peptide complex suggests potential mechanisms for this regulation that need to be investigated further.

No description available.