No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

by Anskar Yu-hung Leung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 147-163). / Chapter Section I --- Literature review / Chapter Chapter I.1. --- The epididymis - its structures and functions --- p.1 / Chapter Chapter I.2. --- Cellular mechanisms of transepithelial electrolyte transport in epididymis and other exocrine tissues --- p.5 / Chapter Chapter I.3 --- Signal transduction mechanism of chloride secretion in epididymis and other exocrine tissue --- p.11 / Chapter Chapter I.4 --- Significance of chloride secretion by the epididymal epithelium and the objectives of the study --- p.17 / Chapter Section II --- General methods / Chapter Chapter II.1. --- Tissue culture from the rat cauda epididymis --- p.20 / Chapter Chapter II.2. --- The short-circuit current technique --- p.30 / Chapter Chapter II.3. --- The immunofluorescence technique --- p.39 / Chapter Chapter II.4. --- Intracellular adenosine 3':5' cyclic monophosphate (cAMP) measurement --- p.43 / Chapter Chapter II.5 --- Intracellular Ca2+ measurement using the microfluorimetric technique --- p.49 / Chapter Section III --- Results / Chapter Chapter III.1. --- Studies on the neural and humoral controls of eletrogenic chloride secretion in rat epididymis --- p.55 / Chapter Chapter III.2. --- Local control of electrogenic chloride secretion in rat epididymis 226}0ؤThe role of the calcitonin gene-related peptide --- p.80 / Chapter Chapter III.3. --- Characterization of intracellular Ca2+ store using ATP as the calcium mobilizing agonist --- p.94 / Chapter Chapter III.4. --- Ca2+ handling mechanisms in single cultured rat epididymal cells --- p.106 / Chapter Chapter III.5. --- Studies on the effector process in the stimulus-secretion coupling- characterization of apical Cl- conductance in cultured rat cauda epididmal cells --- p.114 / Chapter Chapter III.6. --- Effects of secretory agonists on transepithelial Cl- transport and intracellular Ca2+ concentration in cultured human epididymal epithelium --- p.132 / Chapter Section IV --- General discussion --- p.142 / Chapter Section V --- References --- p.147 / Appendix --- p.164

Intracellular signaling cascades can no longer be viewed as linear pathways that relay and amplify information. Often, components of different pathways interact, resulting in signaling networks. The interactions of different pathways and the dynamic modulation of the activities of the components within signaling pathways can create a multitude of biological outputs. The cell appears to use these pathways as a way of integrating multiple inputs to shape a uniquely defined output. These outputs allow the cell to respond to and adapt to an ever-changing environment. Understanding how biological systems receive, process and respond to complex data inputs has important implications for the design and utilization of sensors for a variety of applications, including toxicology, pharmacology, medical diagnostics, and environmental monitoring. This study uses the elicitor sets method, which is an experimental framework designed to monitor information flows through signal transduction pathways. The elicitor set approach has been used to derive mechanistic interpretations from the action of Phenylmethylsulfonyl Fluoride (PMSF), a serine protease inhibitor and nerve agent analog. The elicitor panel comprises of signal transduction network effectors namely forskolin, clonidine, cirazoline and H89, each of which targets the signaling pathway at known specific points. The elicitor set experiments enable compartmentalization of the cAMP signaling pathway, examining the role played by each segment and identifying possible cross-talk mechanisms. Our experiments substantiate that selection of adenyl cyclase as the reference node and 10 [mu]M forskolin as the primary elicitor, segments the upper portion of the G-Protein Coupled Receptor (GPCR) pathway associated with the G[sub q] and G[sub i] proteins. Application of the secondary elicitors, 100 nM clonidine (a2-adrenergic receptor agonist), 1 pM and 100 pM cirazoline (al-adrenergic receptor agonists), and 1 [mu]M and 100 [mu]M H-89 (PKA inhibitor) fortifies the decoupling, as the system is unresponsive to clonidine and cirazoline in the presence of forskolin, while continuing to respond to H-89. Exposure of the cells to 1 mM PMSF subsequent to forskolin addition restricted the quantifiable impact of PMSF to regions of the signaling pathways below adenyl cyclase. Triggering the system by use of secondary elicitors augmented the information resolution which is reinforced by the increased sensitivity of cells to 100 [mu]M H-89 that acts at an important checkpoint below adenyl cyclase. / Graduation date: 2004

This study focuses on the mechanism of transmembrane signaling by Tar, the aspartate chemoreceptor of Escherichia coli. Like other bacterial chemoreceptors, Tar localizes to the cell membrane and relays information about the external chemical environment through the membrane to a cytoplasmic signaling domain. The output of the signaling domain controls the directional bias of the rotary flagellar motors of the cell. Net movement of a cell in a chemical gradient involves temporal comparison of the current concentration with the concentration in the recent (a few seconds) past. The current concentration is measured as the percent occupancy of the extracellular ligand-binding domain of the receptor, and the past is represented by the extent of covalent methylation of four conserved glutamyl residues in the cytoplasmic domain. Under steady-state conditions, the methylation level corresponds to ligand occupancy. Tar is a dimer, and much evidence suggests that dimers associate into trimers of dimers. Higher-order arrays of receptors form in the presence of the cytoplasmic proteins CheA and CheW. The conformational change generated by ligand binding is transmitted through the membrane by one subunit of a dimer. To examine whether this initially asymmetric signal becomes symmetric within the cytoplasmic domain, I examined aspartate-induced adaptive methylation of the two subunits of mutant Tar receptor heterodimers. In the presence of CheA and CheW, adaptive methylation after addition of aspartate was symmetric, but in their absence, although the level of methylation increased, the rates were different for the two subunits. I also found that cross-talk, at the level of adaptive methylation, occurs between different receptor types even in the absence of CheA and CheW. These results provide support for the idea that a tight association of receptor dimers within trimers of dimers allows for an actively signaling receptor to affect the methylation state, and thus presumably the signaling state, of receptors within a trimer that are not bound to an attractant ligand.