We have developed a novel framework that can be applied for the analysis of signal transduction networks, both to facilitate reconstruction of the network structure and quantitatively characterize the interaction between network components. This approach, termed activation ratio analysis, involves the ratio between active and inactive forms of signaling intermediates at steady state. The activation ratio of an intermediate is shown to depend linearly upon the concentration of the activating enzyme. The slope of the line is defined as the activation factor, and is determined by the kinetic parameters of activation and inactivation. When activation ratios for simple signaling systems are considered, a set of rules develop that can be used to transform a set of experimental data to a proposed model network structure, with activation factors yielding a measure of activation potential between intermediates. / Singapore-MIT Alliance (SMA)

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 161-195). Also available in electronic version.

Phototransduction is a well characterized system for study of G protein coupled receptor (GPCR) signaling. The GPCR rhodopsin couples to the heterotrimeric G protein transducin. Light-stimulated activation of transducin in turn activates phosphodiesterase (PDE), leading to closure to cGMP-gated channels and inhibition of glutamate release. Rod and cone photoreceptors are highly polarized neurons consisting of the outer segment (OS) where phototransduction biochemistry occurs, the inner segment containing mitochondria and other organelles, the nuclear layer, an axon, and a glutamatergic synapse. Upon illumination, activated G protein transducin redistributes from the rod OS (where it is localized in the dark) to the inner compartments of the cell. Interestingly, cone transducin does not translocate in light. Opposite to this, visual arrestin migrates from the inner compartments to the OS, where it binds to rhodopsin. Previous reports from other groups and our lab argue for either an active or passive mechanism for transducin and arrestin redistribution. Our lab has shown that arrestin migration occurs by diffusion which is restricted by molecular sinks (Nair et al, 2005b). The focus of my dissertation was to unravel the molecular mechanism of rod transducin translocation. Specifically, I found energy (ATP) was not required for transducin movement within photoreceptors. Also, I found that the disc membranes of the rod outer segments as well as protein-protein interactions with retinal guanylate cyclase serve to restrict transducin diffusion through the cell. In addition, I used the insights gained from these studies of transducin to re-examine the relationship of other G proteins' subcellular localization and signal transduction. Ultimately, I found that most G proteins do not undergo subunit dissociation under physiological activating conditions.

Regulators of G protein signaling (RGS) are a diverse group of proteins, which play a fundamental role in modulation of G protein coupled receptor signal transduction. RGS proteins are primarily known as GTPase activating proteins (GAPs) for Gá subunits. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins also contain other structural motifs. The R7 family of RGS proteins for example, which consists of RGS-6, 7, 9 and 11 gene products, also contains DEP, DHEX and GGL domains. All R7 RGS proteins are obligatory binding partners with G protein beta subunit, G beta sub five, which binds to the GGL domain. In my dissertation work, I provide insights into significance of the multi-domain architecture of G beta sub five-RGS7. I have identified a novel intramolecular interaction within the G beta sub five-RGS7 complex; between the DEP domain of RGS7 and G beta sub five subunit. My experimental evidence supports the idea that G beta sub five-RGS7 can exist in at least two hypothetical conformations: "closed" where the DEP domain and G beta sub five subunit are bound to each other, and "open" where DEP and G beta sub five are not interacting, and as a result both these proteins can associate with other binding partners. My results indicate that in its "open" conformation, G beta sub five-RGS7 can selectively inhibit calcium mobilization elicited by stimulated muscarinic acetylcholine receptor type 3 (M3R). This inhibition is mediated by direct interaction between the third intracellular loop of M3R and the DEP domain of RGS7. In addition to the effect on M3R signaling, I observed that the G beta sub five-RGS7 complex redistributes from the cytosol to endocytic vesicles in an M3R-specific manner. These results identify a novel molecular mechanism that can impart receptor-subtype selectivity on signal transduction via G protein-coupled receptors. Lastly, I have identified a small group of compounds that inhibits the DEP-G beta sub five interaction. These compounds may serve as starting points for design of G beta sub five-RGS7 modulators in the future.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 100-124).

Thesis (M.Sc.)--York University, 20uu. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 105-113). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL:http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99332

Thesis (M.S.)--University of Montana, 2006. / Title from title screen. Description based on contents viewed Mar. 9, 2007. Includes bibliographical references (p. 21-22).

The ability to swim and navigate the surrounding environment confers an advantage to motile bacteria, allowing the occupation of niches that are optimum for survival and growth. Bacteria are too small to sense their environment spatially, so they must sense the environment temporally by comparing the past and present environments and altering their motility accordingly. Chemotaxis systems coordinate flagellar motility responses with temporal sensing of the environment. Chemotaxis is proposed to be involved in plant root colonization by directing soil bacteria toward root exudates of various cereals, promoting growth. The nitrogen-fixing alpha-proteobacterium Azospirillum brasilense utilizes chemotaxis to navigate its environment by integrating various environmental signals into a chemotaxis signal transduction pathway. In chemotaxis, transducers receive environmental sensory information and transmit the signal to the histidine kinase CheA, which relays the signal to the response regulator CheY. A novel chemotaxis transducer, Tlp1, has been identified and characterized as an energy sensor by constructing a tlp1 mutant and performing behavioral and root colonization assays. In order to adapt to changing environmental conditions, chemotactic microorganisms must employ a molecular “memory” comparing present environmental conditions to ones previously experienced and resetting the chemotaxis transducer to a prestimulatory status. A recently identified chemotaxis operon revealed a methyltransferase CheR and methylesterase CheB, comprising an adaptation system, suggesting that A. brasilense undergoes methylation-dependent taxis responses, contrary to previous reports. Chemotaxis and methanol release assays suggest that adaptation by methylation in locomotor behavior involves the presence of other unknown methylation systems, and the contribution of CheR and CheB to chemotactic and aerotactic responses is complex. There is growing evidence that chemotaxis-like signal transduction pathways control a myriad of other cellular processes regulated in a temporal fashion. This would convey an advantage to cells by allowing modulation of cellular processes based on slight changes in environmental conditions and provide checkpoints for energetically consuming processes. Mutations in components of the chemotaxis-like signal transduction system revealed differences in cell size and exopolysaccharide production. This work shows that the signal transduction pathway of A. brasilense modulates cell length in response to changes in nutrient conditions, independently of growth rate.

Many problems in bioinformatics are inference problems, that is, the problem objective is to infer something based upon a limited amount of information. In this work we explore two different inference problems in bioinformatics. The first problem is inferring the structure of signal transduction networks from interactions between pairs of cellular components. We present two contributions towards the solution to this problem: an mixed integer program that produces and exact solution, and an implementation of an approximation algorithm in Java that was originally described by DasGupta et al. An exact solution is obtained for a problem instance consisting of real data. The second problem this thesis examines is the problem of inferring complete haplotypes from informative SNPs. In this work we describe two variations of the linear algebraic method for haplotype prediction and tag SNP selection: Two different variants of the algorithm are described and implemented, and the results summarized.

Plants activate an array of defense mechanisms upon pathogen attack. Systemic acquired resistance (SAR) is an induced disease resistance phenomenon deployed after infection by a necrogenic pathogen and is dependent on endogenous accumulation of salicylic acid. The objectives of my research were to characterize SAR in the crop plant, <i>Brassica napus</i> (canola), and study the effects of overexpressing genes involved in SAR on disease resistance. Biological induction of SAR using necrogenic Pseudomonas syringae and chemical induction using benzo (1,2,3) thiadiazole-7-carbothionic acid reduced growth of the bacterial pathogen P. syringae and the fungal pathogen Leptosphaeria maculans. This growth reduction was associated with an increase in transcript levels of pathogenesis-related (PR) genes, one of the characteristic features of SAR. Transgenic plants expressing a bacterial salicylate hydroxylase gene (NahG), were more susceptible to the above pathogens and were delayed in accumulating PR gene transcripts, indicating a need for SA accumulation for SAR in B. napus. Expression of two SAR genes from Arabidopsis, DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1) and NON EXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1), in <i>B. napus</i> enhanced resistance against virulent P. syringae without SAR pre-treatments. Putative orthologs of DIR1 and NPR1 (BnDIR1 and BnNPR1) were isolated from B. napus based on EST sequences. BnDIR1 and BnNPR1 display 71% and 66% amino acid sequence similarities, respectively, to the corresponding Arabidopsis proteins. Expression of BnNPR1 in Arabidopsis npr1 mutant backgrounds indicated that it was able to functionally complement these mutations. Expression of BnDIR1 enhanced disease resistance in both Arabidopsis wild-type and dir1-1 mutant backgrounds. Expression of DIR1, NPR1, BnDIR1 and BnNPR1, separately, in <i>B. napus</i> plants enhanced resistance against P. syringae. SAR pre-treatments further enhanced resistance of transgenic <i>B. napus</i> plants expressing DIR1 and BnDIR1 to <i>P. syringae</i>, indicating an additive effect. Expression of DIR1 in B. napus did not provide resistance against <i>L. maculans</i>. These results provide the first in-depth molecular characterization of SAR in B. napus, and in particular, provide new insight into DIR1 function not previously reported in Arabidopsis.