Hollow fiber membranes (HFMs) formed through phase inversion methods exhibit specific physicochemical characteristics and generally favorable surface and mechanical properties, supporting their use in diverse applications including ultrafiltration, dialysis, cell culture, bioreactors, and tissue engineering. Characterization of, and modifications to, such membranes are important steps in achieving desired characteristics for specific applications. HFMs subject to gas, irradiation, and chemical sterilization techniques were characterized based on several analytical techniques. It was revealed that these common sterilization techniques can cause inadvertent changes to HFM properties. While these changes may cause detrimental effects to HFMs used in filtration, the methods of sterilization are also presented as a facile means of tuning properties toward specific applications. Modifications to HFM surface chemistries were also sought as a method of adsorbing bacterial lipopolysaccharide (LPS) from solutions used in hemodialysis treatments and bioprocessing applications. It was found that additives such as polyvinylpyrrolidone (PVP), polyethyleneglycol (PEG), and poly-L-lysine (PLL) can facilitate adsorption capacities of HFMs toward LPS. Additionally, chemical changes are presented as a means of preferentially adsorbing LPS to specific locations on the HFM surface.

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Several Gram-negative bacteria modify their outer most surface structure, lipopolysaccharide (LPS), to evade immune surveillance and survive within the host. Many of these changes occur within the lipid A domain, a region that is recognized by the innate immune system via Toll-like receptor-4 (TLR4). One such pathogen, Porphyromonas gingivalis, orchestrates chronic inflammatory disease by disrupting immune homeostasis. P. gingivalis initially synthesizes a penta-acylated lipid A that functions as a weak TLR4 agonist but displays tetra-acylated forms that are either immunologically silent or TLR4 antagonists. The impact of lipid A modifications on downstream signaling and antigen-specific immunity are unclear. TLR4 signals from the plasma membrane through a MyD88-dependent pathway and intracellularly through a TRIF-dependent pathway. Here we show that expression of immunological silent or antagonistic lipid A enables P. gingivalis to evade TRIF-dependent signaling in dendritic cells (DCs). Evasion of TRIF signaling accelerated antigen degradation and impaired priming of pathogen-specific T cells. In contrast, a P. gingivalis strain expressing agonist lipid A potently activated TRIF signaling and delayed antigen degradation, thereby preserving peptides for optimal T cell activation. We propose that lipid A modifications control the endocytic activity of DCs and the efficiency at which microbe-specific T cells are primed. We next investigated the impact of purified P. gingivalis LPS on innate signaling and antigen presentation. All P. gingivalis LPS species induced a program of DC maturation that allowed for constitutive antigen uptake and cross-presentation. This was independent of TLR4 agonist activity and required CD14, a protein that transports TLR4 to endosomes where TRIF signaling can occur. Agonist LPS induced signaling through both MyD88 and TRIF and elicited T cell priming. Antagonistic LPS potently accelerated CD14 endocytosis and induced TRIF-biased signaling leading to comparable degree of cross-priming. Immunologically silent LPS promoted CD14 endocytosis but failed to activate signaling and induced T cell tolerance. Collectively, our results demonstrate that modification of lipid A structure enables Gram-negative bacteria to direct the host immune system towards tolerance or immunity. We propose that these findings can be harnessed for therapeutic modulation of the immune system to treat a variety of immune-mediated diseases. / 2021-06-14T00:00:00Z

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<p>The current rise in multi-drug resistant Gram-negative bacterial infections is of particularconcern. Gram-negative pathogens are difficult to treat due to their intrinsic resistome.The outer membrane (OM) of Gram-negative bacteria serves as a permeability barrier tomany antibiotics, due in large part to the lipopolysaccharide (LPS) component that isunique to these organisms, and in addition to, the OM is lined with a number of multidrugresistant efflux pumps. As the clinical effectiveness of first line therapies declines inthe face of this resistance, novel strategies to discover new antibiotics are required. Theidentification of new antibiotic targets is one method currently being applied to meet thischallenge. This work examines the permeability barrier of Escherichia coli as a possibletarget for antibiotic adjuvants. A structure-function analysis of GmhA and GmhB, whichcatalyze the first and third conserved steps in LPS ADP-heptose biosynthesis, wasperformed. The active site residues of each of these enzymes were identified viacrystallographic, mutagenic, and kinetic analyses. Potential mechanisms have beenproposed, offering insight into the function of these potential adjuvant targets. In addition,a whole screen of E. coli was performed to identify compounds that potentiatenovobiocin, an antibiotic with limited activity against Gram-negative pathogens due toOM permeability. Four small molecules were found that were able to synergize withnovobiocin. One of these, A22, is known to alter bacterial cell shape, suggesting a newpathway for antibiotic adjuvants to combat Gram-negative infection. Together, thesestudies highlight the varied targets available for novel therapeutic strategies.</p> / Doctor of Philosophy (PhD)