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.

Abstract Neutrophils are the major cellular component of acute inflammatory response. The mechanism by which fMLP or PAF activates neutrophils is not fully elucidated. Stimulation of MAPKs and activation of NF-kappa B in neutrophils regulate various cell functions, including superoxide production. Neutrophils isolated from blood taken from healthy donors, were incubated with specific inhibitors, GF109203X (PKC inhibitor), calphostin C (PKC-gamma isoform inhibitor), wortmannin (PI3K inhibitor), U73122 (PLC inhibitor), aristolochic acid (PLA2 inhibitor), SKF96365 (SOC channel inhibitor), EGTA (extracellular calcium chelator), SB203580 (p38 MAPK inhibitor), and PD98059 (MEK inhibitor), followed by fMLP or PAF treatment. MAPK activation by fMLP or PAF is based on immunoblot analysis. NF-kappa B activation is detected by EMSA, and superoxide production is measured by flow cytometry. The data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK MAPK phosphorylation was involved PI3K, PKC, PLA2, PLC, and extracellular calcium, wheres fMLP-induced phosphorylation doesn¡¦t involve PKC

The plant hormone ethylene plays an important role in plant growth, development, and physiology. One of the critical components of the ethylene signal transduction pathway, ctr1 (constitutive triple response), was identified using a particularly useful seedling screen that takes advantage of the profound effects ethylene has on etiolated seedlings, known as triple response. CTR1 is one of six Arabidopsis MAPKKKs that are related to the Raf kinases, and acts as a negative regulator of ethylene response. In this study, isolation and characterization of a family of CTR1-like genes in tomato is reported. Based on amino acid alignments and phylogenetic analysis, the tomato CTR1-like (LeCTR) genes are more similar to Arabidopsis CTR1 (AtCTR1) than any other MAPKKK sequences in the Arabidopsis genome. The capacity of the LeCTR genes to function as negative regulators in ethylene signal transduction was tested through complementation of the Arabidopsis ctr1-8 mutant. Quantitative real-time PCR was carried out to generate an expression profile for the CTR1-like gene family during different stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1-like genes in other species besides tomato was examined through mining of EST and genomic sequence databases. Based on nucleotide and amino acid identity, At4g24480 is most similar to AtCTR1 and could potentially represent a CTR1-like gene in Arabidopsis. Arabidopsis plants carrying a T-DNA insert in the At4g24480 locus were examined for abnormal ethylene response phenotypes including sensitivity to other hormones, signal molecules and abiotic stresses. Two mutant alleles, ctr1-1 and ctr1-8, containing mutations that disrupt kinase activity and receptor association, respectively, were examined for sensitivity to these same treatments in an effort to better characterize ethylene hormone and non-hormone interactions. They also served as controls to determine if At4g24480 indeed possessed CTR1-like function. Arabidopsis and tomato represent species with very distinct fruit ripening/maturation programs. The critical dependence on ethylene for fruit ripening in tomato might have resulted in alteration or modification of the ethylene signal transduction pathway relative to Arabidopsis. Plans to characterize individual functions of the LeCTR genes through over-expression and reduced expression in tomato are outlined.

This work develops microfluidic technologies to advance the state-of-the-art in living cell-based assays. Current cell-based assay platforms are limited in their capabilities, particularly with respect to spatial and temporal control of external signaling factors, sample usage, and throughput. The emergence of highly quantitative, data-driven systems approaches to studying biology have added further challenges to develop assay technologies with greater throughput, content, and physiological relevance. The primary objectives of this research are to (i) develop a method to reliably fabricate 3-D flow networks and (ii) apply 3-D flow networks to the development and testing of microfluidic chamber arrays to query cellular response to soluble-matrix signal combinations and gradient signaling fields. An equally important objective is for the chamber arrays to be scaled efficiently for higher-throughput applications, which is another reason for 3-D flow networks. Two prototype chamber arrays are designed, modeled, fabricated, and characterized. Furthermore, tests are performed wherein cells are introduced into the chambers and microenvironments are presented to elicit complex responses. Specifically, soluble-matrix signaling combinations and soluble signal gradients are presented. The study of complex biological processes necessitates improved assay techniques to control the microenvironment and increase throughput. Quantitative morphological, migrational, and fluorescence readouts, along with qualitative observations, suggest that the chamber arrays elicit responses; however further experiments are required to confirm specific phenotypes. The experiments provide initial proof-of-concept that the developed arrays can one day serve as effective and versatile screening platforms. Understanding the integration of extracellular signals on complex cellular behaviors has significance in the study of embryonic development, tissue repair and regeneration, and pathological conditions such as cancer. The microfluidic chamber arrays developed in this work could form the basis for enhanced assay platforms to perform massively parallel interrogation of complex signaling events upon living cells. This could lead to the rapid identification of synergistic and antagonistic signaling mechanisms that regulate complex behaviors. In addition, the same technology could be used to rapidly screen potential therapeutic compounds and identify suitable candidates to regulate pathological processes, such as cancer and fibrosis.

Dissertation (Ph.D.)--University of Toledo, 2009. / "Signaling" misspelled as "singaling" on title page of document. "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Non-Latin script record Bibliography: p. 94-108.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves [202]-230). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 166-185). Also available in print.

Thesis (Ph. D.)--University of Chicago, Committee on Human Nutrition and Nutritional Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 163-175). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves [202]-230). Also available online.