Chitin Microparticles (CMPs, 1-10um), a special form of the ubiquitous and nontoxic polysaccharide Chitin (GlcNAc), is capable of inducing a switch in macrophages from the wound-healing M2 phenotype to the classically activated pro-inflammatory M1 phenotype; which has therapeutic implications in allergy and cancer. We hypothesized that TLR2 forms a complex with CMPs and Chitin-Binding Proteins (CBPs) at the surface of peritoneal macrophages and remains with that complex after internalization to initiate downstream signaling events, leading to the production of the M1 cytokine, TNFalpha. Our results from experiments performed in RAW 264.7 cells show that TLR2 and TLR1, but not TLR6, are associated with the CMP binding fraction, and that both TLR1 and TLR2 might be important for M1 activation as a result of CMP phagocytosis. This project sheds light on CMP as a potential therapeutic agent and provides more evidence for a phagocytosis-dependent TLR2 signaling pathway. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

In a phage binding assay, phage clone (3p5A190) expressing a surrogate mas ligand displayed punctate binding and were internalized in cell expressing native mas and GFP-tagged variants. However, the number of bound and internalized phages in cells expressing mas-GFP was substantially less than the cells expressing mas-(Gly10Ser5)GFP and native mas. In parallel, biotinylation experiment quantitatively showed that the extent of mas-(Gly10Ser 5)-GFP translocation was higher than that of mas-GFP. Consistently, cells expressing mas-(Gly10Ser5)-GFP and native mas showed a rapid and sustained increase of intracellular calcium levels upon MBP7 stimulation. By contrast, cells expressing mas-GFP only response to higher concentration of MBP7 challenge and showed a delayed increase of intracellular calcium level. Moreover, cells expressing native mas had a higher proportion (80%) of cells responsive to MBP7 stimulation; in contrast to only 10∼20% of cells expressing mas fusion proteins. / MBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1. / Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas. / To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized. / Sun, Jingxin. / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-170). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

The interaction between molecules is essential in a wide range of biological processes. A detailed knowledge of these interactions is necessary for understanding these processes. Among the systems that involve important interactions is the immune system. NMR spectroscopy has a large number of spectral parameters that were used in this work to study antibody-antigen interactions. These same parameters were also used to begin a structural analysis of a medium-sized protein, neurophysin, that has important interactions with neurohormones, and served here as a model antigen. A set of ligands differing in size and charge was designed and used to probe the binding site of anti-phosphocholine antibodies. These ligands ranged from small organic species to medium sized proteins. Amino acids, peptides and proteins were homogeneously linked to phenyl phosphocholine and analyzed by NMR techniques. Transferred nuclear Overhauser effect measurements were used to determine the conformation of bound ligands. The conformational change observed in some ligands was explained as either due to the antibody selecting one conformation that already exists, or the antibody binding inducing a conformational change. Titration data and detailed NMR analysis showed a more rigid M3C65 antibody fragment upon binding. In summary, with eight examples of ligands and four examples of antibodies studied by NMR, a spectrum of effects was seen, including a lock-and-key model and limited local induced fit. The contribution of the carrier molecule to antibody binding was in restricting the conformation of the ligand. Bigger ligands that are expected to be more immunogenic, showed less binding avidity as determined by immunological assays. Fluorinated ligands were synthesized to determine the kinetics of binding using 19F NMR spectra. Higher concentration of a fragment of the antibody M3C65 was analyzed to determine assignments of some residues in the combining site of the antibody. High resolution NMR techniques were used to assign resonances in neurophysin. The physiological role of neurophysin includes hormone storage and stabilization of oxytocin and vasopressin against proteolytic degradation within the posterior pituitary. Neurophysin is a 10 KD protein that dimerizes at high concentrations needed for NMR studies. An organic cosolvent was used to lower the dimerization constant, and hence inrease the spectral resolution. This permitted sequence-specific assignments that were then used to identify residues in the neurophysin-hormone binding site. Chemical shift differences and conformational changes were observed for the residues glutamate 47 and leucine 50. The 3₁₀ helix was further stabilized towards a more ideal helix upon hormone-analog peptide binding. Some of the residues contributing to the monomer-monomer interface were also assigned. Dimerization ill1duced chemical shift differences and conformational changes were observed for phenylalanine 35, threonine 38, and alanine 69. Tyrosine: 49 and phenylalanine 22 were affected but to a lesser extent. One characteristic of neurophysin in all studied cases was dynamic equilibrium between different folding states.

Src Homology 3 (SH3) domains are small protein- protein interaction domains that bind to proline-rich peptides, mediating a range of important biological processes. Because the deregulation of events involving SH3 domains forms the basis of many human diseases, the SH3 domains are appealing targets for the development of potential therapeutics. Previously in the field, no examples of entirely small-molecule ligands for the SH3 domains have been identified. However, in our research group, we have discovered a class of heterocyclic compounds that bind to the Tec SH3 domain at conserved residues in the proline-rich peptide binding site, with weak to moderate affinity. The highest affinity of these was 2- aminoquinoline (Kd = 125 mM). In this thesis, a range of approaches are described, that were intended to contribute towards development of higher affinity small-molecule ligands for the Tec SH3 domain. Preliminary experiments, involving testing a variety of compounds structurally related to 2- aminoquinoline, provided new structure activity information, and led to a better understanding of the 2-aminoquinoline/SH3 domain binding event. The major component of this thesis is a thorough investigation into the synthesis of a range of 2- aminoquinoline derivatives. N-Substituted- 2-aminoquinolines were synthesised, however these compounds bound the SH3 domain with slightly lower affinity than 2-aminoquinoline. 6- Substituted-2-aminoquinolines were subsequently prepared, and ligands were identified with up to six-fold improved affinity relative to 2-aminoquinoline, and enhanced selectivity for the Tec SH3 domain. The techniques used for the ligand binding studies were Nuclear Magnetic Resonance (NMR) chemical shift perturbation and Fluorescence Polarisation (FP) peptide displacement assays. As part of the ligand binding studies, it was intended that the 3D tructure of a 2- aminoquinoline ligand/SH3 complex would be obtained using NMR methods, provided that a ligand was identified that bound the SH3 domain in slow exchange on the NMR timescale. However, this goal was not fulfilled. Despite this, the work presented in this thesis provides a solid foundation for the development of potent 2-aminoquinoline ligands for SH3 domains, with engineered specificity. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

This dissertation investigates the photophysics of bis(diarylamino)biphenyl (TPD) and silver nanoparticles (AgNP). A main goal of this work was to develop an understanding of the relaxation pathways involved in the deactivation of photoexcited TPD chromophores in close proximity to silver nanoparticles. The TPD chromophores were attached to the silver nanoparticle core via an alkylthiol group. The TPD-AgNP systems were synthesized and characterized using Transmission Electron Microscopy (TEM), UV-visible absorption, infrared spectroscopy, and Nuclear Magnetic Resonance (NMR) spectroscopy, Inductively Coupled Plasma - Emission Spectroscopy (ICP-ES) and Thermogravimetric Analysis (TGA). Time-resolved photophysical processes in these systems were studied using femtosecond transient absorption spectroscopy. Initial studies of the interaction of the TPD and AgNP addressed the linker length dependence of the dye excited state decay kinetics, wherein alkyl linker chains of 3, 4, 8 and 12 carbon atoms were used. These results showed that an ultrafast deactivation of the excited state of the TPD chromophore, which is three orders of magnitude faster than that of the free chromophore in solution, occurred in all of the systems. However, an unexpected new transient species was observed for the systems with three and four carbon linker chains. Further studies showed this species to be spectroscopically very similar to the TPD radical cation, suggesting a charge separation pathway in the excited state relaxation. Possible pathways for formation of the cation-like state were examined through comparisons to the photophysics of alkyl substituted TPD in solution and in solid films, investigation of the pulse energy and TPD surface coverage dependence of the yield of the cation-like TPD species, transient absorption anisotropy decay dynamics, and kinetic modeling studies. Taken together, these investigations provide support for exciton-exciton annihilation being responsible for the formation of cation-like species. The packing of the TPD chromophores is concluded to be of critical importance in the generation of the cation like species but it is also possible that proximity to the silver nanoparticle plays a role in facilitating charge separation as well.

Thesis (M. Sc.)--University of Alberta, 2010. / Title from pdf file main screen (viewed on Jan. 27, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Physics, University of Alberta. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 150-159). Also available on the Internet.

Thesis (Ph. D.)--Uppsala universitet, 2004. / Available also in print. Description based on contents viewed Aug. 12, 2004; title from title screen. Includes bibliographical references (p. 69-77).

Includes bibliographical references.

Thesis (M.S.)--University of North Carolina at Wilmington, 2003. / Vita. Includes bibliographical references (leaves : [37]-40).