acase@tulane.edu / This work embraces the fabrication of functional materials through the interactions at interfaces and covers two topics. First, we investigate the assembly of hydrophobically modified polypeptoids (HMPs) with lipid bilayers and the structural transition of lipid bilayers induced by HMPs. Polypeptoids are a class of pseudo-peptidic polymers. Properties such as biocompatibility, biodegradability, enzymatic resistance, and good processibility make polypeptoid a promising material for biotechnological applications. With decyl groups as hydrophobic side chains, HMPs interact with lipid bilayers by inserting the hydrophobes into the lipid bilayers through hydrophobic interaction. The hydrophobe insertion results in the disruption of lipid bilayers and the formation of lipid bilayer fragments containing HMPs and HMP-lipid complexes depending on HMP-to-lipid ratio. These polymer-lipid “rafts” can attach to the bare lipid bilayers forming close-spaced multilamellar structures, with HMPs connecting across the adjacent lipid bilayers. Also, HMP-lipid rafts can significantly increase the solubility of sorafenib in aqueous solution through hydrophobic interaction. The ability to assemble to lipid bilayers and to encapsulate hydrophobic drugs demonstrate the promise of HMP-lipid rafts in the application of drug delivery. The second topic is the rapid fabrication of hollow and yolk-shell functional materials through an aerosol assisted synthesis. Materials with hollow and yolk-shell structures have potentials in many applications, such as catalysis and energy storage. But the fabrication of such materials often suffers time-consuming and tedious procedures, which limits the practical application of these materials. We first demonstrated the rapid fabrication of hollow and yolk-shell Fe2O3 microspheres for enhanced photo-Fenton reactions. The placement of iron salts on the external surface of a carbonaceous microsphere generated from an aerosol droplet allows the formation of a Fe2O3 capsule by calcination, where colloid particles can be encapsulated. The Fe2O3 microspheres show significant enhancement in the photo-Fenton reaction. We then illustrated the fabrication of hollow and cage-like mesoporous Fe2O3/SiO2 microspheres. Through salt bridge effect, iron salts and cationic surfactants form colloidal aggregates which are locked within a rapidly formed SiO2 shell during the aerosol process. Sucrose as a pore generating agent leads to the formation of mesopores in the shell, which was proven favored features for CO2 capture. / 1 / Yueheng Zhang

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In Escherichia coli, the integral outer membrane (OM) enzyme PagP covalently modifies lipid A by incorporating a phospholipid-derived palmitate chain to fortify the OM permeability barrier. We perturbed the bacterial OM in order to activate PagP and examined if it exerts transcriptional regulation through either of its extracellular or periplasmic active sites. Data from RNA-seq revealed the differential expression of 50 genes upon comparing the E. coli imp4213 (lptD4213) strain NR760∆pagPλInChpagP (shortened to NR760λp in this work), in which PagP is constitutively activated, and the mutant NR760λpY87F carrying the periplasmic residue mutation Y87F. 40 genes were upregulated, and encoded proteins related to anaerobic processes, whereas 10 genes were downregulated, and encoded proteins related to aerobic processes. RNA-seq was followed by a study of differential gene expression using the NanoString nCounter system. Results confirmed a 2.7-fold upregulation of dmsA when we compared the strains NR760λp to NR760λpY87F. We also found a 2.5-fold repression of dmsA transcription when we compared the lptD+ parental strain NR754λp to NR754λpS77A carrying the mutation S77A in the extracellular active site of PagP. We then investigated dmsA transcription using a lacZ reporter gene in plasmid pRS551-lacZ. High basal β-galactosidase activity became attenuated in pagP null mutants. OM perturbation using pentamidine showed that dmsA transcription was repressed. Complementation of the chromosomal ∆pagP deletion with a single copy pBADGr plasmid, expressing PagP under the control of an arabinose-inducible promoter, restored dmsA β-galactosidase activity. We observed partial complementation with the downstream cspE gene, which identified a polar effect of the ∆pagP allele. Through deletion of rpoS, rcsB, cpxA, cpxR, pmrA, pmrB, and fadD in E. coli MC4100, we showed that β-galactosidase activity of pdmsA-lacZ was affected by all of these regulators. Our results indicate that these regulators are involved in PagP-mediated regulation of dmsA transcription under aerobic conditions. / Thesis / Doctor of Philosophy (PhD)

No / Dimethylsulfoxide (DMSO) is an aprotic solvent that has the ability to induce cell fusion and cell differentiation and enhance the permeability of lipid membranes. It is also an effective cryoprotectant. Insights into how this molecule modulates membrane structure and function would be invaluable toward regulating the above processes and for developing chemical means for enhancing or hindering the absorption of biologically active molecules, in particular into or via the skin. We show here by means of molecular simulations that DMSO can induce water pores in dipalmitoyl-phosphatidylcholine bilayers and propose this to be a possible pathway for the enhancement of penetration of actives through lipid membranes. DMSO also causes the membrane to become floppier, which would enhance permeability, facilitate membrane fusion, and enable the cell membrane to accommodate osmotic and mechanical stresses during cryopreservation.

Nanoscale drug delivery systems have a great impact in current medical field. These carriers have the potential to improve the efficacy and reduce the toxicity of various medicinal products. A broad variety of different lipid based carriers had been developed and used as delivery systems in the past decades. This dissertation focused on the development of solid lipid nanoparticles (SLN) as delivery systems for a chemotherapeutic agent, docetaxel, and the use of liposomes as a carrier for recombinant protein vaccines. Docetaxel is a potent anticancer drug. However, there continues to be a need for alternative docetaxel delivery systems to improve its efficacy. Docetaxel nanoparticles comprised of lecithin as the main component were engineered using two methods, the emulsion precursor method and the solvent emulsification/evaporation method. Docetaxel in nanoparticles were more effective in killing tumor cells in culture than docetaxel solution. The intravenously injected docetaxel-nanoparticles increased the accumulation of docetaxel in tumors in mice. When administered by intravenous injection or oral routes, docetaxel-nanoparticles showed antitumor activity in tumor-bearing mice. The lecithin-based nanoparticles have the potential to be a novel biocompatible and efficacious delivery system for docetaxel. Liposomes, a well-known lipid based carrier, have been investigated extensively as a vaccine delivery system. The adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when simply admixed with protein antigens. Immunization study in mice after subcutaneously injection of different net charged liposomes showed different antibody responses, depending on the protein antigens. Antigens (OVA, PA) admixed with the negatively charged liposomes prepared with phospholipid, DOPA, induced a strong and functional antibody response comparable to the positively charged liposomes prepared with DOTAP lipid. The negatively charged DOPA liposomes admixed with OVA also induced OVA-specific CD8��� cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA melanoma in a mouse model. The adjuvant activity of the negatively charged liposomes may be related to the liposome's ability (i) to upregulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Sept. 6, 2011 - Sept. 6, 2012

Pik1p, one of three phosphatidylinositol 4-kinases in the fission yeast, <i>Schizosaccharomyces pombe</i>, was found previously to interact with Cdc4p, a myosin essential light chain that is required for cytokinesis. The involvement of pik1 in cell cycle control was investigated. A fluorescently tagged Pik1p fusion protein was associated with Golgi throughout the cycle, and was found at the medial division plane of the cell during late cytokinesis. This latter distribution has not been reported previously. Gene deletion in diploid cells and tetrad analysis revealed that pik1 is essential for cell viability and is required for spore germination. The terminal phenotype of a temperature-sensitive, loss-of-function allele (pik1-td) indicated that pik1 is involved in cytokinesis: particularly for suppression of secondary septum material deposition, for suppression of initiation of supernumerary septa, and for cell separation. Contractile ring formation was normal in pik1-td cells at the restrictive temperature although the pattern of F-actin patches was disrupted. The F-actin patches were dispersed throughout the cytoplasm. Accumulation of extra inner membranous or vesicle-like structures was observed in these cells. The <i>S. pombe</i> nmt1 promoter and attenuated versions of it were found to be useful for complementation studies in <i>S. cerevisiae</i>. Heterologous expression of <i>S. pombe</i> pik1 complemented the essential functions of a temperature-sensitive allele (pik1﷓101) of its orthologue in <i>Saccharomyces cerevisiae</i> that were lost at the restrictive temperature. A residue required for <i>S. pombe</i> Pik1p lipid kinase activity, D709, was also required for this complementation. A residue, R838, which is required for interactions between Pik1p and Cdc4p was not required for this complementation. The timing and localization of Pik1p to the division plane of the cell late in cytokinesis combined with analysis of the terminal phenotype of a loss-of-function allele, indicate that Pik1p and/or its derived phosphoinositides are required for regulation of septation and cell separation. Pik1p may be involved in the transport, possibly via vesicular transport, of enzymes required for hydrolysis of the primary septum. It may be involved in signaling pathways that lead to the initiation of septation and to the cessation of the deposition of secondary septum material.

Lipid rafts are nanoscale entities in the membranes of eukaryotic cells which provide a mechanism for the functional membrane segregation vital for several cellular processes. This lateral segregation of specific lipid and protein components provides the facilitative platforms for a variety of signaling and trafficking events at the plasma membrane and in the Golgi. Rafts are distinguished from the surrounding membranes by their physical properties and composition - they are relatively tightly packed and enriched in saturated lipids, sterols, and lipid-anchored proteins. Although the existence of rafts has been conclusively confirmed by several independent techniques, questions concerning various aspects of membrane heterogeneity are still to be addressed. Typical experiments investigating raft composition have been designed to evaluate the affinity of a given component for raft domains. In such experiments, the results are usually interpreted in a Boolean fashion, i.e., the component is either a raft molecule, or not. However, this binary point of view overlooks potential complexity that may underlie the nature of membrane heterogeneity. In this work, we systematically investigated the nature of functional cellular membrane heterogeneity. We started by characterizing the model membranes and fluorescent lipid analogs widely used in research into membrane domains. After extensively evaluating the potentials/limits of these approaches and the artifacts that must be avoided or alternatively could be exploited, we applied these tools to understand whether the cell membrane has multiple kinds of raft domains with distinct compositions and physical properties, rather than only one. We found that cell membranes have the potential to form various kinds of functional domains having different physicochemical properties, compositions, and functional outputs. Therefore, we propose continuously variable lipid packing as the principle of the functional membrane lateral heterogeneity. According to this principle, the membrane is not composed of a single variety of raft domain with strictly defined properties coexisting alongside a specific and uniform non-raft environment; rather it is composed of entities having continuously variable lipid packing. Finally, we show that this spectrum of membrane packing modulates the orientation of membrane lipid receptors, which ultimately influences their specific bioactivity. Our results showing continuously variable lipid packing and its ability to fine-tune the activity of membrane molecules comprise a novel model for the structure and function of eukaryotic membranes.

Antimicrobial peptides (AMPs) are not only fast microbe-killing molecules deployed in the host defense of living organisms but also offer valuable lessons for developing new therapeutic agents. While the mode of action of AMPs is not clearly understood yet, membrane perturbation has been recognized as a crucial step in the microbial killing mechanism of many AMPs. In this thesis, we first present a physical basis for the selective membrane-disrupting activity of cationic AMPs. To this end, we present a coarse-grained physical model that approximately captures essential molecular details such as peptide amphiphilicity and lipid composition (e.g., anionic lipids). In particular, we calculate the surface coverage of peptides embedded in the lipid headgroup-tail interface and the resulting membrane-area change, in terms of peptide and membrane parameters for varying salt concentrations. We show that the threshold peptide coverage on the membrane surface required for disruption can easily be reached for microbes, but not for the host cell -- large peptide charge (≳4) is shown to be the key ingredient for the optimal activity-selectivity of AMPs (in an ambient-salt dependent way). Intriguingly, we find that in a higher-salt environment, larger charge is required for optimal activity. Inspired by membrane softening by AMPs, we also study electrostatic modification of lipid headgroups and its effects on membrane curvature. Despite its relevance, a full theoretical description of membrane electrostatics is still lacking -- in the past, membrane bending has often been considered under a few assumptions about how bending modifies lipid arrangements and surface charges. Here, we present a unified theoretical approach to spontaneous membrane curvature, C₀, in which lipid properties (e.g., packing shape) and electrostatic effects are self-consistently integrated. Our results show that C₀ is sensitive to the way lipid rearrangements and divalent counterions are modeled. Interestingly, it can change its sign in the presence of divalent counterions, thus stabilizing reverse hexagonal (H_II) phases.