SLAP Is a Negative Regulator of FcεRI Receptor-Mediated Signaling and Allergic Response

Binding of antigen to IgE-high affinity FcεRI complexes on mast cells and basophils results in the release of preformed mediators such as histamine and de novo synthesis of cytokines causing allergic reactions. Src-like adapter protein (SLAP) functions co-operatively with c-Cbl to negatively regulate signaling downstream of the T cell receptor, B cell receptor, and receptor tyrosine kinases (RTK). Here, we investigated the role of SLAP in FcεRI-mediated mast cell signaling, using bone marrow derived mast cells (BMMCs) from SLAP knock out (SLAP KO) mice. Mature SLAP-KO BMMCs displayed significantly enhanced antigen induced degranulation and synthesis of IL-6, TNFα, and MCP-1 compared to wild type (WT) BMMCs. In addition, SLAP KO mice displayed an enhanced passive cutaneous anaphylaxis response. In agreement with a negative regulatory role, SLAP KO BMMCs showed enhanced FcεRI-mediated signaling to downstream effector kinases, Syk, Erk, and Akt. Recombinant GST-SLAP protein binds to the FcεRIβ chain and to the Cbl-b in mast cell lysates, suggesting a role in FcεRI down regulation. In addition, the ubiquitination of FcεRIγ chain and antigen mediated down regulation of FcεRI is impaired in SLAP KO BMMCs compared to the wild type. In line with these findings, stimulation of peripheral blood human basophils with FcεRIα antibody, or a clinically relevant allergen, resulted in increased SLAP expression. Together, these results indicate that SLAP is a dynamic regulator of IgE-FcεRI signaling, limiting allergic responses

Identification of Novel Substrates of the Ubiquitin E3 Ligase RNF126 and Characterization of its Role in Lipid Droplet Homeostasis

Ubiquitin E3 ligases confer specificity by recognizing target substrates and mediating the final step of the ubiquitination process. Despite this critical role, our understanding of their physiological partners and biological functions remains limited. Here we use a proximity-based protein screen called BioID to explore novel interacting proteins and substrates of the ubiquitin E3 ligase RNF126. We demonstrate that RNF126 can associate with p97 as well as its co-factors UBXD1 and UBXD8 and show that UBXD1 and UBXD8 are bonafide substrates of RNF126. To determine a functional role, we explored the role of RNF126 in UBXD8 mediated functions. We found that stable knockdown of RNF126 in HeLa cells results in smaller LDs following oleate stimulation and that this is due to defective expansion. Together, this work demonstrates the successful use of BioID to identify substrates of ubiquitin E3 ligases and furthers our understanding of the role of ubiquitin in LD biology.M.Sc

Development of Catalytic C–CN, C–O, and N–CN Sigma-Bonds Activation and Alkene Addition Reactions

University of Minnesota Ph.D. dissertation. November 2016. Major: Chemistry. Advisor: Christopher Douglas. 1 computer file (PDF); xiii, 284 pages.This Thesis describes the development of transition-metal-catalyzed alkene additions reactions through the activation of C–C and C–heteroatom σ-bonds. In these transformations, a C–C or C–heteroatom σ-bond is activated, cleaved, and added across an alkene double bond without inducing fragmentation, allowing addition of an alkene with vicinal functional groups and rapid construction of carbo- and heterocycles in an atom-economical manner. Chapter 1 provides an overview of catalytic alkene addition reactions and C–C σ-bond activation reactions. Chapters 2, 3, and 5 describe alkene addition reactions by catalytic activation of C–CN, C–O, and N–CN σ-bonds, respectively. An unprecedented metal-free N–CN bond cleavage process is discussed in Chapter 4.Pan, Zhongda. (2016). Development of Catalytic C–CN, C–O, and N–CN Sigma-Bonds Activation and Alkene Addition Reactions. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/185147

Synthesis of ZnO Nanoparticles with Controlled Shapes, Sizes, Aggregations, and Surface Complex Compounds for Tuning or Switching the Photoluminescence

The electronic energy transfer (EET) usually induces the fluorescence self-quenching, but it has been positively used here to tune and/or switch the photoluminescence (PL) of ZnO nanoparticles (NPs). Monodisperse ZnO nanospheres, rods, tripods, and clusters with tunable sizes have been synthesized to reproducibly and finely control the NP aggregation because EET is sensitive to the interparticle separation. The complex reactions between these NPs and their dispersion media have been used to further control the EET for tuning the ZnO PL. By changing the NP concentrations, shapes, and/or the cluster sizes, the band-edge UV PL of the ZnO NPs dispersed in alcohol or water is modified in both intensity and peak position, and new blue emissions with tunable intensity around 418, 435, and 468 nm are induced. As confirmed by the X-ray diffraction patterns and the infrared, PL, absorption, and Raman spectra, the ZnO NPs made here can slowly react with ethanol to form a new composite ZnO–(C₂H₅OH)_<i>n</i>, which changes the EET between NPs and leads to strong blue PL around 435 nm. By simply using different dispersion media (such as ethanol or water) to modify the surface complex compounds of ZnO NPs, the 435 nm blue PL can be turned on or off

Palladium and Lewis-Acid-Catalyzed Intramolecular Aminocyanation of Alkenes: Scope, Mechanism, and Stereoselective Alkene Difunctionalizations

An expansion of methodologies aimed at the formation of versatile organonitriles, via the intramolecular aminocyanation of unactivated alkenes, is herein reported. Importantly, the need for a rigid tether in these reactions has been obviated. The ease-of-synthesis and viability of substrates bearing flexible backbones has permitted for diastereoselective variants as well. We demonstrated the utility of this methodology with the formation of pyrrolidones, piperidinones, isoindolinones, and sultams. Furthermore, subsequent transformation of these motifs into medicinally relevant molecules is also demonstrated. A double crossover ¹³C-labeling experiment is consistent with a fully intramolecular cyclization mechanism. Deuterium labeling experiments support a mechanism involving <i>syn</i>-addition across the alkene

Intramolecular Oxyacylation of Alkenes Using a Hydroxyl Directing Group

Alkene oxyacylation is a new strategy for the preparation of β-oxygenated ketones. Now, with Ir catalysis and low-cost salicylate esters, alkene oxyacylation can be promoted by simple and versatile hydroxyl directing groups. This paper discusses catalyst optimization, substituent effects, mechanistic experiments, and the challenges associated with asymmetric catalysis. Crossover experiments point to several key steps of the mechanism being reversible, including the most likely enantiodetermining steps. The oxyacylation products are also prone to racemization without catalyst when heated alone; however, crossover is not observed without catalyst. These observations account for the low levels of enantioinduction in alkene oxyacylation. The versatility of the hydroxyl directing group is highlighted by demonstrating further transformations of the products

Synthesis of ZnO Nanoparticles with Controlled Shapes, Sizes, Aggregations, and Surface Complex Compounds for Tuning or Switching the Photoluminescence

The electronic energy transfer (EET) usually induces the fluorescence self-quenching, but it has been positively used here to tune and/or switch the photoluminescence (PL) of ZnO nanoparticles (NPs). Monodisperse ZnO nanospheres, rods, tripods, and clusters with tunable sizes have been synthesized to reproducibly and finely control the NP aggregation because EET is sensitive to the interparticle separation. The complex reactions between these NPs and their dispersion media have been used to further control the EET for tuning the ZnO PL. By changing the NP concentrations, shapes, and/or the cluster sizes, the band-edge UV PL of the ZnO NPs dispersed in alcohol or water is modified in both intensity and peak position, and new blue emissions with tunable intensity around 418, 435, and 468 nm are induced. As confirmed by the X-ray diffraction patterns and the infrared, PL, absorption, and Raman spectra, the ZnO NPs made here can slowly react with ethanol to form a new composite ZnO–(C₂H₅OH)_<i>n</i>, which changes the EET between NPs and leads to strong blue PL around 435 nm. By simply using different dispersion media (such as ethanol or water) to modify the surface complex compounds of ZnO NPs, the 435 nm blue PL can be turned on or off

Intramolecular Oxyacylation of Alkenes Using a Hydroxyl Directing Group

Alkene oxyacylation is a new strategy for the preparation of β-oxygenated ketones. Now, with Ir catalysis and low-cost salicylate esters, alkene oxyacylation can be promoted by simple and versatile hydroxyl directing groups. This paper discusses catalyst optimization, substituent effects, mechanistic experiments, and the challenges associated with asymmetric catalysis. Crossover experiments point to several key steps of the mechanism being reversible, including the most likely enantiodetermining steps. The oxyacylation products are also prone to racemization without catalyst when heated alone; however, crossover is not observed without catalyst. These observations account for the low levels of enantioinduction in alkene oxyacylation. The versatility of the hydroxyl directing group is highlighted by demonstrating further transformations of the products