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Anterior gradient-2 (AGR2) is an endoplasmic reticulum (ER)-resident protein that belongs to a member of protein disulphide isomerase (PDI) superfamily. AGR2 initially emerged as a dominant effector of basic biological properties in vertebrates such as specifying forebrain integrity and limb generation. Subsequent studies in mammals implicated the role of AGR2 as a pro-metastatic protein essential to cancer progression and drug resistance, asthma and inflammatory bowel disease. AGR2 protein is mainly overexpressed in a number of human cancers and involved in pathways for ER stress, protein folding, transcription regulation, and exosome formation. Hence, AGR2 protein represents a clinically-relevant oncoprotein in tumour emergence and survival. The aim of this thesis is to shed more light on the role of AGR2 in cancer development. AGR2 was previously shown that it could bind sequence-specifically to a linear peptide motif. In this study, hydrogen/deuterium exchange mass spectrometry was used to identify the dominant peptide-binding site on AGR2 by comparing the deuterium uptake between AGR2 and AGR2 with its ligand (linear peptide motif). The binding of the peptide was probed by making mutant series in the identified peptide ‘docking site’ region on AGR2. A consensus peptide-binding motif was then developed to identify potential cellular proteins that harbour this motif as potential AGR2 client proteins. Database mining using this consensus binding peptide demonstrated that transmembrane proteins were dominant class of proteins. Epithelial cell adhesion molecule (EpCAM), an oncogenic transmembrane protein, was chosen as putative AGR2 client proteins and their interaction was verified using both cell-free and cell-based assays. The AGR2 and EpCAM pathway dynamics were reconstituted and investigated in cells that do not endogenously express both proteins. Further, the expression of AGR2 and EpCAM were assessed in clinical tumour samples using immunohistochemistry. Proteomics screen using quantitative tandem mass tag (TMT) mass spectrometry on cells transiently overexpressing AGR2 were used to identify potential AGR2 client proteins and to find relevant dominant pathways affected by AGR2 signalling. Additionally, synthetic tools were devised to further dissect the function, regulation and ‘druggability’ of AGR2 protein. These tools include: i) isolation of high-affinity AGR2-binding synthetic antibodies from a phage-scFv (single-chain variable fragment) library; ii) engineering synthetic mini-protein (synPRO) containing copies of wild-type and mutated AGR2 linear peptide motif and; iii) engineering synthetic membrane protein model that bind to AGR2. In conclusion, the data presented hereby demonstrated a novel role of an ER-resident protein AGR2 which possess an intrinsic sequence-specific peptide binding for a subset of its client proteins and one function of this motif is to ensure proper maturation of client proteins to their final destination. Development of synthetic tools in this study can be further manipulated to disrupt AGR2 signalling and the fate of its binding proteins which in turn highlights a potentially ‘druggable’ stage in the oncogenic secretory pathway.

The design and synthesis of porous materials attracted great attention recently because of their potential use in many fields like clean energy and environmental protection. Herein, we introduced new synthetic approaches for the preparation for porous carbons and organic polymers for selective CO2 and iodine capture application. Regarding CO2 capture application, Two new series of porous carbons (PYDCs) and (TRI-Ps) were synthesized by thermally activating Pyrazole and Triazolo Pyridine monomers respectively using KOH as a catalyst, which leads to porous carbon. PYDCs exhibit high surface area according to Brunauer–Emmett–Tellertheory (SABET = 1266–2013 m2 g−1), high CO2 Isosetric heat Qst(33.2-37.1 kJ/mol) and significantly high CO2 uptakes 8.59mmol g−1 (1bar) at 273k. The reported porous carbons also show significantly high adsorption selectivity for CO2/N2 (128) and CO2/CH4 (13.4) according to Ideal adsorbed solution theory (IAST) calculation using pure gas isotherms at 298 K. TRI-Ps exhibit high surface area (SABET=1852- 2917 m2 g−1) with a combination of mesoporous and microporous pores. TRI-Ps exhibit CO2 capture capacity of 6.98mmol g−1 at 1 bar and 273K. The development of majority of microporous offered a high CO2 storage capacity to TRI-Ps. High CO2 uptake achieved as a result of preferable pore size, surface area, and high oxygen content and high Qst values. Based on IAST calculation, high CO2/N2 selectivity (113.9) at 298K was achieved. Regarding iodine capture application, two new series of porous organic polymers Benzamidizole Linked Polymers (BILPs) and nitrogen rich porous Polymers (NRPPs) were synthesized and tested. Iodine capture, sorption isotherm and kinetics of adsorption were studied. BILPs exhibit iodine uptake capacity of 227.8 wt.% and 202.8 wt. % for BILP-A and BILP-B respectively. BILP-A shows ability of releasing 95.8% of captured iodine, while, BILP-B released 82.22%. Sorption curves were fitted by Freundlich equation indicating a heterogeneity of adsorption process on the surface. NRPPs exhibit iodine capacity of 192.35 wt.% and 222.35 wt. % for NRPP-1 and NRPP-2 respectively. NRPPs shows ability of releasing the majority of adsorbed Iodine. Sorption curves were fitted by Langmuir equation indicating a heterogeneity of adsorption process on the surface.

Recent progress in drug discovery has enabled us to target specific intracellular molecules to achieve therapeutic effects. These next generation therapeutics are often biologics which cannot enter cells by mere diffusion. Therefore it is imperative that drug carriers are efficiently internalized by cells before releasing their cargo. Nanoscale polymeric carriers are particularly suitable for such intra-cellular delivery. Although size and surface-charge has been the most studied parameters for nanocarriers, it is now well appreciated that particle shape also plays a critical role in their transport across physiological barriers. Hence there is increasing interest in fabricating shape-specific polymeric nano and microparticles for efficient delivery of drugs and imaging agents. Nanoimprint lithography methods, such as Jet-and-flash imprint lithography (J-FIL), provide versatile top-down processes to fabricate shape-specific, biocompatible nanoscale hydrogels that can deliver therapeutic and diagnostic molecules in response to disease-specific cues. However, the key challenges in top-down fabrication of such nanocarriers are scalable imprinting with biological and biocompatible materials, ease of particle-surface modification using both aqueous and organic chemistry as well as simple yet biocompatible harvesting. Here we report that a biopolymer-based sacrificial release layer in combination with improved nanocarrier-material formulation can address these challenges. The sacrificial layer improves scalability and ease of imprint-surface modification due to its switchable solubility through simple ion exchange between monovalent and divalent cations. This process enables large-scale bio-nanoimprinting and efficient, one-step harvesting of hydrogel nanoparticles in both water- and organic-based imprint solutions. We also show that when shape is decoupled from volume, charge and composition, mammalian cells preferentially internalize disc-shaped nanohydrogels of higher aspect ratios over nanorods. Interestingly, unlike nanospheres, larger-sized hydrogel nanodiscs and nanorods are internalized more efficiently. Uptake kinetics, efficiency and internalization mechanisms are all shape-dependent and cell-type specific. Although macropinocytosis is used by all cells, epithelial cells uniquely internalize nanodiscs using caveolae pathway. On the other hand, endothelial cells use clathrin-mediated uptake along with macropinocytosis for all shapes and show significantly higher uptake efficiency compared to epithelial cells. We also study the effect of shape and surface properties for their tissue uptake and penetration using spheroids as a 3D tumor model and show that hydrophobic particles show no difference in penetration inside such models even after 125 fold reduction in volume. These results provide a fundamental understanding of how cell and tissue behavior is influenced by nanoscale shape and surface properties and are critical for designing improved nanocarriers and predicting nanomaterial toxicity. / text

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 98-107.

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