Systemic acquired resistance (SAR) is a defense response induced by an initial localized infection that leads to the generation of long-distance immune signals that travel to distant leaves to provide enhanced resistance to subsequent infections. The lipid transfer protein (LTP) DEFECTIVE IN INDUCED RESISTANCE1 (DIR1) travels via the phloem from induced to distant leaves during SAR and may chaperone several long-distance signal candidates. In this thesis, the role of DIR1 during SAR is explored by examining the route of DIR1 movement, investigating the conservation of DIR1 structure and function, and by identifying DIR1-interacting proteins. I demonstrate that Arabidopsis plant lines with restricted cell-to-cell movement through plasmodesmata are negatively impacted in long-distance DIR1 movement, suggesting that cell-to-cell movement is important for DIR1 to access distant leaves. To elucidate the molecular function of DIR1, orthology analysis was performed with putative DIR1 orthologs. Structurally important amino acid residues that contribute to the hydrophobicity of the LTP cavity were identified, supporting the idea that DIR1 binds a hydrophobic ligand during SAR. RNAi-mediated knockdown of the DIR1 paralog DIR1-like did not impact the SAR response, supporting the idea that DIR1- like plays a lesser role in SAR. In addition, targeted protein-protein interaction assays determined that LTP1 and LTP2 interact with DIR1, and SAR phenotypic analysis of an ltp2-1 mutant supported a role for LTP2 in SAR. Lastly, a comparative proteomics approach identified several proteins with differential abundance in phloem exudates collected during the induction of SAR. Of these proteins, m-type thioredoxins, a major latex protein-like protein, and the UV-B photoreceptor UVR8 were essential for the manifestation of SAR. Together, these data provide insight into DIR1 function by identifying the importance of cell-to-cell movement through plasmodesmata, the DIR1 hydrophobic cavity, and DIR1-interacting proteins for DIR1-mediated SAR. In addition, this work identifies new phloem-localized proteins that contribute to the SAR response, providing fundamental knowledge on protein composition within the phloem during biotic stress. / Dissertation / Doctor of Philosophy (PhD)

We develop a coarse-grained model of lipids and proteins in which the lipids are modelled as diblock copolymers and the proteins as rigid cylinders. The generic protein model allows the possibility of amphipathic proteins with intrinsic curvature. Self-consistent field theory (SCFT) is used to determine the morphology of the lipid bilayer in the vicinity of the proteins. In particular, we focus on the case of a long transmembrane protein inserted perpendicular to the bilayer. For this system we use SCFT to determine the mechanical properties of the membrane and the thickness profile as a function of distance from the protein inclusion. The mechanical constants are also used in an elastic theory to predict the thickness profile. Good agreement between the full SCFT and elastic theory is obtained. We also use SCFT to determine systematic trends of the boundary conditions for the thickness profile at the protein interface. Such results could be used as boundary conditions for the description of bilayers using elastic theory. We show that this system undergoes a second order wetting transition as the interaction strength between the protein and membrane is varied. / Thesis / Master of Science (MSc)

Improvements in mass spectrometry (MS)-based strategies for characterizing the plant lipidome through quantitative and qualitative approaches such as shotgun lipidomics have substantially enhanced our understanding of the structural diversity and functional complexity of plant lipids. However, most of these approaches require chemical extractions that result in the loss of the original spatial context and cellular compartmentation for these compounds. To address this current limitation, several technologies were developed to visualize lipids in situ with detailed chemical information. A subcellular visualization approach, direct organelle MS, was developed for directly sampling and analyzing the triacylglycerol contents within purified lipid droplets (LDs) at the level of a single LD. Sampling of single LDs demonstrated seed lipid droplet-to-droplet variability in triacylglycerol (TAG) composition suggesting that there may be substantial variation in the intracellular packaging process for neutral lipids in plant tissues. A cellular and tissue visualization approach, MS imaging, was implemented and enhanced for visualizing the lipid distributions in oilseeds. In mature cotton seed embryos distributions of storage lipids (TAGs) and their phosphatidylcholine (PCs) precursors were distribution heterogeneous between the cotyledons and embryonic axis raising new questions about extent and regulation of oilseed heterogeneity. Extension of this methodology provides an avenue for understanding metabolism in cellular (perhaps even subcellular) context with substantial metabolic engineering implications. To visualize metabolite distributions, a free and customizable application, Metabolite Imager, was developed providing several tools for spatially-based chemical data analysis. These tools collectively enable new forms of visualizing the plant lipidome and should prove valuable toward addressing additional unanswered biological questions.

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My Ph.D. research project in the Bhagwati Gupta lab focuses on understanding the mechanism by which the Axin family of scaffolding proteins functions to regulate biological processes in multicellular eukaryotes. Towards this, I am using the nematode (worm) Caenorhabditis elegans as an animal model to investigate the role of one of the Axin homologs, PRY-1. Studies in various model systems and humans have shown that the Axin family of proteins plays crucial roles during cell proliferation, cell differentiation, and organ formation. Such a role of Axin depends on the negative regulation of the WNT signaling cascade. Consistent with these, alterations in Axin function are associated with developmental abnormalities and age-associated diseases such as axis duplication, neuroectodermal defect, and muscle degeneration. As a scaffolding protein, Axin family members bind to and recruit multiple protein partners that are both WNT dependent and independent. However, how Axin interacts with these factors to regulate molecular events is not well understood. While some Axin-interacting factors have been identified, many more remain to be discovered. My project deals with the identification and functional characterization of pry-1/Axin interactors in C. elegans. The key findings of my Ph.D. research are published in five peer-reviewed papers. Collectively, the results demonstrate that PRY-1 is necessary to regulate lipid metabolism, stress response, muscle health, and aging. I have shown that PRY-1 utilizes multiple pathways to control these diverse processes. Specifically, PRY-1 functions via the SREBP transcription factor homolog SBP-1 to regulate yolk lipoprotein expression to promote lipid synthesis. The analysis of pry-1’s role in aging and muscle health has revealed its interactions with the energy sensor AMPK homolog AAK-2, thereby affecting the function of the Insulin/IGF1 signaling (IIS) transcriptional regulator DAF-16/FOXO. Moreover, I have identified several mRNA genes and microRNAs that function downstream of PRY-1/Axin signaling to either suppress or enhance pry-1 mutant defects. All these novel interactors have mammalian homologs. Altogether, these findings form the basis to pursue future work to investigate the conserved mechanism of Axin signaling and hold the potential for effective intervention to delay aging and age-associated muscle deterioration. / Thesis / Doctor of Philosophy (PhD)

Carboxylesterases (CEs) are nonspecific hydrolytic enzymes and responsible for the metabolism of xenobiotics and endobiotics that contain ester bonds. There are two human CE isoforms found in liver, CES1 and CES2. In this study it is shown that the mere abundance of CES1 protein expression in human liver does not predict its biochemical activity. The human interindividual variation in CES1 activities may attribute to several mechanisms. One possibility is the presence of endogenous inhibitors in liver, arachidonic acid (AA) and 27-hydroxycholesterol (27-HC). CES1 is also expressed in human monocytes/macrophages and is proposed to catalyze the rate-limiting step of cholesterol ester mobilization in macrophages. It is of interest to determine whether CES1 can degrade the lipid mediators, 2-arachidonoylglycerol (2-AG), prostaglandin E2-1-glyceryl ester (PGE2-G), and prostaglandin F2α-1-glyceryl ester (PGF2α-G), in monocytes/macrophages and to determine if this metabolism is inhibited by organophosphate pesticide exposure.

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