The modeling of transport behavior of charged particles carried out in our laboratory is based on classical continuum electro kinetic theory. It is applied to a variety of systems from small electrolyte ions to macromolecules including peptides, DNA and nanoparticles. Systems range from weakly charged particles to highly charged ones. Transport properties studied include conductance, electrophoresis, and diffusion. In this dissertation, the conductance of polyvalent electrolytes ions is studied both by a “small ion” model [R.M. Fuoss, L. Onsager, J. Phys. Chem. 61 (1957) 668] and “large ion” model [R.W. O’Brien, L.R. White, J. Chem. Soc. Faraday Trans. 2 (74) (1978) 1607)]. Also, the coarse-grained continuum primitive model is developed and used to characterize the titration and electrical conductance behavior of aqueous solutions of fullerene hexa-malonic acid, which is a highly charged electrolyte with an absolute valence charge as high as 12. Free solution electrophoresis is closely related to conductance and a coarse-grained bead modeling methodology, BMM, developed in the Allison’s laboratory starting in 2006, is generalized to characterize peptide systems with respect to the charge, conformation, and possibly specific interactions with components of the BGE. For weakly charged peptides, the electrostatic potential is treated at the level of linear Poisson-Boltzmann equation, which predicts the electrophoretic mobility with considerable accuracy [S. Allison, H. Pei, U. Twahir, H. Wu, J. Sep. Sci., 2010, 33(16):2430-2438], but fails for highly charged systems. A new nonlinear Poisson-Boltzmann, NLPB-BM procedure is developed and applied to the free solution electrophoretic mobility of low molecular mass oligolysines. The difficulty of highly charged systems is twofold: more complex handeling of electrostatics and accounting for the relaxation effect. Both issues are addressed in this dissertation. A related problem we investigated deals with the retarding influence of a gel on the rotational motion of a macromolecule. This is investigated within the framework of the Effective Medium (EM) model and is applied to examine the electric birefringence decay of a 622 base pair DNA fragment in an agarose gel. Modeling is also compared with experiment.

Influenza infection remains a constant threat to human health and results in huge financial loss annually. Rapid and accurate detection of influenza can aid health officials to monitor influenza activity and take measurements when necessary. In addition, influenza detection in a timely manner can help doctors make diagnosis and provide effective treatment. Additionally, novel inhibitors of influenza virus are in high demand because circulating strains have started to develop resistance to currently available anti-viral drugs. Influenza virus has two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA), which play important roles in the influenza infection. The binding of HA to sialic acid-containing carbohydrates on cell surface initiates virus internalization, while cleavage of terminal sialic acid by NA facilitates viral particle release. In this dissertation, we focus on the development of a glycan microarray that is comprised of a panel of NA resistant sialosides, and demonstrate the application of the microarray to capture influenza virus at ambient temperature without the addition of NA inhibitors. We also describe a novel electrochemical assay for the detection of influenza virus. In addition, we have developed a new class of bivalent NA inhibitors that show promising inhibitory activities against influenza viruses.

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

Recently, there has been increasing focus toward the development of small molecules designed to target a specific sequences of double stranded DNA for therapeutic purposes1. Minor groove binding compounds have been shown to be capable of selectivity target GC sites in AT tract DNA2. In this research, binding selectivity was investigated using absorption, fluorescence and circular dichroic properties of selected DB minor groove binders in the presence of two unique DNA sequences. Further insight was gained by comparing the electrostatic potential maps and optimized structures of the compounds of interest. Using the results presented, potential selective minor groove binders can be selected for further investigation and kinetic studies.

Protein based contrast agents (ProCAs) developed by the Yang lab exhibit unique capabilities in enabling magnetic resonance imaging (MRI) with significantly improved sensitivity and targeting capabilities by utilizing biomarkers which can target various carcinomas in animals. Further clinical in vivo human applications require modifications of these designed contrast agents to further improve organ and tissue biodistribution, biomarker and cell targeting capabilities, and reduction of immunogenicity. The aim of this thesis is to develop a novel protein modification on ProCA by glycosylation to improve liver distribution by targeting liver receptor, asialoglycoprotein receptor (ASGPR). Rat and humanized first generation and humanized third generation ProCA were expressed and purified using either glutathione s-transferase (GST) tagged or taggless methods. Rat ProCA1, rProCA1, was then used to optimize glycan modification with glycosylation achieved at the highest level using a 100:1 molar ratio and three lysine residues. Similar to non-modified rProCA1 and PEGylated rProCA1, metal binding affinity of gadolinium for glycan modified rProCA1, Glyco-rProCA1, was found to be 9.49 x 10-12 M, and relaxivity was found to be greater than clinically available contrast agents with 34.08 and 42.67 mM-1s-1 for r1 and r2 respectively. Glycosylation of rProCA1 has significantly increased human serum stability, and we have achieved significant liver MRI enhancement via tail vein injection due to high ASGPR expression in the liver altering biodistribution of glycan modified ProCA, and we have also imaged uptake in the secretory glands. These biodistribution changes were noted by immunohistochemistry (IHC) staining which was found to stain liver sinusoid with spaces in between. The distribution to the liver was further confirmed via inductively coupled plasma optical emission spectrometry (ICP-OES) which shows Glyco-rProCA1 has significant uptake of gadolinium in the liver tissue. This study represents the first achievement of in vivo liver imaging by glycosylation using a lactose targeting moiety covalently bonded to protein contrast agents for MRI showing promise for future more specific targeting or whole body imaging capabilities.

Cyanine dyes are attractive photosensitizers for photodynamic therapy due to their ease of structure modification and intense absorption in the near-infrared range. Photosensitizers that can bind to DNA and absorb at long enough wavelengths of light to deeply penetrate biological tissue are in high demand for treatment of cancer and other diseases. The following study analyzes the DNA interactions of three pentamethine cyanine dyes with very similar structures, all of which absorb light at wavelengths longer than 800 nm. The work described involves an extensive study of the photocleavage abilities and DNA binding characteristics of these dyes. Our lead compound was a bromophenyl meso substituted symmetrical quinoline cyanine dye. Spectroscopic data, gel electrophoresis experiments and other studies were used to provide evidence of DNA binding mode, ROS production, and of dye-sensitized DNA photocleavage at the unprecedented wavelength of 850 nm.

The NF-κB family of transcription factors controls a number of essential cellular functions. Pirin is a non-heme iron (Fe) redox specific co-regulator of NF-κB (p65) and has been shown to modulate the affinity between the homodimeric p65 and the DNA. The allosteric effect of the active Fe(III) form of Pirin on the DNA is not known and has not been investigated. We carry out multiple microsecond-long molecular dynamics simulations of the free DNA, p65-DNA complex, and the Pirin-p65-DNA supramolecular complexes in explicit water. We show that, unlike the Fe(II) form of Pirin, the Fe(III) form in the Pirin-p65-DNA supramolecular complex enhances the interactions and affinity between p65 and the DNA, in agreement with experiments. The results further provide atomistic details of the effect of the Fe(III) form of Pirin on the DNA upon binding to p65 to form the supramolecular complex.

Among the peptidyl prolyl isomerases, the Cyclophilin family of proteins has been linked to various cellular activities such as regulation of homeostasis, mitochondrial permeability, and cell death. Their functionality spans throughout the cell and throughout all cell types as different isoforms. Previous studies done on Cyclophilin A revealed an interesting contact ensemble when bound to a substrate. Because of the similarity of CypA to its homologues, it is believed that they too will exhibit the same contact dynamics. We have defined the dynamics of cyclophilin isoforms through Molecular Dynamics simulations and determined their contact dynamics, characterizing their contact ensembles, and their relative dynamical conservation to each other.

Gram-positive pathogenic bacteria utilize cell-surface anchored proteins to bind and transport heme into the cell. These bacteria acquire iron from host proteins containing heme e.g., hemoglobin. Proteins like HmuT from Corynebacterium diphtheriae bind and help transport heme into the cell. Residues His136 and Tyr235 are utilized as the axial ligands, with a conserved Arg237 residue acting as the hydrogen bonding partner to the axial Tyr235. Similarly, Streptococcus pyogenes utilizes the cell anchored protein Shr to transfer heme into the cell. Shr-NEAT2 is hexacoordinated by two axial methionines and is prone to autoreduction where lysines are the most likely source of electrons. Lastly, PefR of Group A Streptococcus is a DNA transcription factor which regulates protein expression. Preliminary studies indicate a cysteine may coordinate the heme. A combination of UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies shows these proteins play a crucial role heme transport and regulation.

CXCR4 is a chemokine receptor that has been linked to several disease related pathways including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer metastasis. The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C chemokine ligand 12 (CXCL12) plays a key role in triggering these disease related pathways. Various antagonists for these receptors have been synthesized and tested, but many are not useful clinically either because of toxicity or poor pharmacokinetics. Some of the most extensive CXCR4 antagonist libraries stem from a class of compounds, p-xylyl-enediamines, which all feature a benzene ring as the core of the compound. This work focuses on the design and synthesis of a new class of compounds that show potential as CXCR4 antagonists by using heterocyclic aromatic rings (2,6-pyridine, 2,5-furan, 2,5-pyrazine and 3,4-thiophene) as the core of the scaffold. After synthesis, these analogues were probed through a variety of assays and techniques by our collaborators in the Shim lab at Emory University including: preliminary binding assays, Matrigel invasion assays, carrageenan mouse paw edema tests, and in silico analysis. In silico analysis also probed 2,5-thiophene-based analogues previously synthesized. This work has produced the beginnings of a new library of CXCR4 antagonists and has identified fifteen hit compounds that are promising leads for further testing and modification.