Purge

This thesis statement describes the eight hanging veil-like structures and sculptures that constitute my thesis exhibition work; it further comments on the movements, philosophy, and personal sensibility that most influenced the art making: Process Art, Taoism, and my own empathetic experiences. The movement of Process Art is discussed in reference to materiality and the physicality that goes into making these pieces. The influence of the Taoist philosophy is discussed in light of the unity and balance found in the combination of dualistic materials, and their relationship to one another, in these sculptural forms. Lastly, this statement expresses how the emotional states brought about from my own empathetic experiences and interpersonal relationships have had an effect on the way I manipulate the materials and on my construction process. My intention is to invite the viewer to follow a journey through the process of making these objects. This is meant to engage the viewer to investigate the multiple fragmented constructions that are unified into one form. As this exploration occurs, tensions created from the dualistic nature of the materials used and the emotional tension that is “recorded” from the action upon those materials elicits in the viewer their own emotional response. It is through intrigue that I stimulate the viewers\u27 contemplation of their own emotional states

Structural and Functional Studies on IroB: A Pathogen-Associated C-glycosyltransferase

Bacterial iron acquisition by the means of enterobactin (ENT) is constrained in mammalian hosts due to ENT-binding proteins such as siderocalin and serum albumin. To evade sequestration by these proteins, ENT can be modified by the C glycosyltransferase IroB, which is located in the iroA locus of Salmonella and certain extraintestinal E. coli strains such as uropathogenic E. coli CFT073. The glycosylation of ENT has been reported to be a bacterial evasion mechanism to restore the iron scavenging ability of ENT in the presence of mammalian ENT-binding proteins by the installation of a steric impediment. The C glycosyltransferase IroB catalyses the transfer of a glucose moiety to the DHB subunit of ENT under formation of a C-C bond between the anomeric C1 of the glucose moiety and the C5 of the 2,3-DHB subunit of ENT. The formation of mono-, di- and triglycosylated Ent (MGE/DGE/TGE) products where observed in vitro. The formation of a C-C bond is remarkable because of its chemical stability and resilience against enzymatic degradation. In this M.Sc. thesis, we initially identified the iroB gene product in the iroA harbouring E. coli strain Nissle 1917 on transcriptional and translational level and expressed and purified IroB recombinant. Then, we investigated the mechanism of the C-C bond formation catalysed by IroB in vitro. Based on the hypothesis that deprotonation of the catechol 2 hydroxyl renders the catechol C5 para to the 2-hydroxyl nucleophilic, the C-C bond would then be formed in a general SN2 reaction between the attacking nucleophile and the anomeric carbon of glucose, which is further facilitated by the excellent phosphate leaving group of the UDP-glucose donor. By the means of homology modelling and superposition strategies, we were able to identify the binding sites of the glycosyl donor UDP-glucose and the glycosyl acceptor ENT and to locate residues that could potentially act as base catalysts to increase the phenolate anionic character of the 2,3-DHB subunit during catalysis. We established an activity assay for IroB, separated products arising from IroB activity by reversed phase chromatography and compared so the activity of wild-type IroB and several variants. Additionally, all variants were characterized biophysically, mainly to confirm that the structural integrity was not impaired by mutations. Ultimately, our results enable us to propose a mechanism for C-glycosylation of IroB that is consistent with other glycosyltransferases found in nature

The C-glycosyltransferase IroB from Pathogenic Escherichia coli: Identification of Residues Required for Efficient Catalysis

The E. coli C-glycosyltransferase IroB catalyzes formation of a C-C bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65-67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation

Genetic Variants in Immune Related Genes as Predictors of Responsiveness to BCG Immunotherapy in Metastatic Melanoma Patients.

Adjuvant immunotherapy in melanoma patients improves clinical outcomes. However, success is unpredictable due to inherited heterogeneity of immune responses. Inherent immune genes associated with single nucleotide polymorphisms (SNPs) may influence anti-tumor immune responses. We assessed the predictive ability of 26 immune-gene SNPs genomic panels for a clinical response to adjuvant BCG (Bacillus Calmette-Guérin) immunotherapy, using melanoma patient cohorts derived from three phase III multicenter clinical trials: AJCC (American Joint Committee on Cancer) stage IV patients given adjuvant BCG (pilot cohort; n = 92), AJCC stage III patients given adjuvant BCG (verification cohort; n = 269), and AJCC stage III patients that are sentinel lymph node (SLN) positive receiving no immunotherapy (control cohort; n = 80). The SNP panel analysis demonstrated that the responder patient group had an improved disease-free survival (DFS) (hazard ratio [HR] 1.84, 95% CI 1.09-3.13, p = 0.021) in the pilot cohort. In the verification cohort, an improved overall survival (OS) (HR 1.67, 95% CI 1.07-2.67, p = 0.025) was observed. No significant differences of SNPs were observed in DFS or OS in the control patient cohort. This study demonstrates that SNP immune genes can be utilized as a predictive tool for identifying melanoma patients that are inherently responsive to BCG and potentially other immunotherapies in the future

Raman Spectroscopy of the Sampleite Group of Minerals

Raman and infrared spectroscopy has enabled insights into the molecular structure of the sampleite group of minerals. These minerals are based upon the incorporation of either phosphate or arsenate with chloride anion into the structure and as a consequence the spectra refect the bands attributable to these anions, namely phosphate or arsenate with chloride. The sampleite vibrational spectrum reflects the spectrum of the phosphate anion and consists of ν1 at 964, ν2 at 451 cm-1, ν3 at 1016 and 1088 and ν4 at 643, 604, 591 and 557 cm-1. The lavendulan spectrum consists of ν1 at 854, ν2 at 345 cm-1, ν3 at 878 cm-1 and ν4 at 545 cm-1. The Raman spectrum of lemanskiite is different from that of lavendulan consistent with a different structure. Low wavenumber bands at 227 and 210 cm-1 may be assigned to CuCl TO/LO optic vibrations. Raman spectroscopy identified the substitution of arsenate by phosphate in zdenekite and lavendulan