Nitric oxide plays a major role in physiology and disease pathology. There are many available methods for the detection of NO; however, these techniques typically detect products of nitric oxide decomposition. Herein, I present a novel method of direct nitric oxide detection using a nitrophorin mutant to capture NO on an optical waveguide platform. Nitrophorins are unique among ferric proteins for their ability to bind NO strongly. The spectral shift of the Soret band of the Nitrophorin was used to monitor NO concentration. The limit of detection was found to be 18 nM, and a linear response to 225 nM. The sensor is highly specific, non-destructive and reusable. Detection of NO was demonstrated in a solution of BSA at serum level concentration, and cell culture solution containing 10% FBS. This method allows for direct NO with high specificity, low detection limit and good temporal resolution. Also described herein are the investigations of the structure of the NO receptor, soluble guanylate cyclase (sGC). sGC is a 150 kDa heterodimeric protein that catalyzes the production of cyclic guanosine monophosphate from guanosine triphosphate, which leads to many downstream affects such as vasodilation. The structural analysis was performed with transmission electron microscopy (TEM). Data presented indicate that the protein is too heterogeneous to be reconstructed with TEM. This is either the result of the sample preparations examined, the purity of the sample, the inherent flexibility or conformational heterogeneity of the protein after applying the sample to the TEM grid.The final project presented describes the use of silica colloidal crystals for the enhancement of the sensitivity of protein microarrays as a function of silica particle size. Protein microarrays are a tool used to discover biomarkers of diseases, and increasing the sensitivity lowers the limit of detection of the method. This is accomplished by an increase of the surface area available for proteins to bind, and by using silica so that the surface chemistry of the microarray is maintained. It was concluded that 510 nm colloids provide the greatest enhancement of the fluorescence of a BSA ant-BSA-FITC system, providing a 17-fold enhancement over the control.

The majority of the work in this dissertation describes the characterization of the nitrophorins (NPs) by NMR spectroscopy.NP7 was studied and compared to NP2 and relevant mutants.NP7 is shown to have three additional amino acids at the N-terminus, which contribute significantly to the protein fold stability and maintenance of NP7 function. The paramagnetic form of the protein has large 1H NMR chemical shifts and broad signals due to protein aggregation. The 1H NMR and CD spectroscopy both revealed that NP7 has mainly the A heme orientation, and the mutation of Glu27 to Val27 leads to an A:B ratio change, from mainly B to mainly or almost exclusively A.NMR investigation of the ferriheme resonances of the low-spin complexes of NP2 and mutant NP2(V24E) with imidazole (ImH), histamine (Hm) and cyanide (CN-) ligands as a function of pH has been completed. Strong chemical exchange cross peaks were observed in the NOESY/EXSY spectra at low pH (pH* 5.5 to 4.0) for the three wt NP2 complexes, which indicate an interchange between two ruffling distortions of the heme. A dramatic change of A:B ratios with pH was observed for the three NP2(V24E) ligand complexes. They both are believed to be a result of a change in protein structure near E53 when it is protonated at low pH.Homo-dimers of NP1 and NP4 were investigated. 1H{15N}-HSQC NMR experimental results revealed that holo-NP4 is a homo-dimer at low pH and a mixture of dimer and monomer at high pH. In contrast, holo-NP4-Hm is monomeric at high pH (6.5 and 7.5). A H-bond between Asp30 and the Hm ligand is responsible for destabilization of the homo-dimer. NP1 was also shown to be a mixture of dimer and monomer at high pH.Heme d1 model complexes were studied. The high-spin form and low-spin forms of the Fe(III) complexes with 2-methylimidazole or imidazole, for monooxo-octaethylchlorin, trans-dioxo-octaethylisobacteriochlorin and 2,7-dioxo-octaethyliso- bacteriochlorin, were characterized by NMR spectroscopy. Spin density maps that show the orbital used for spin delocalization have been generated from Hückel calculations.Preliminary results of additional studies of the nitrophorins and future directions of study are presented.

No description available.

Hematophagy, or blood-feeding behavior, has independently evolved multiple times in the class Insecta and results in the transmission of vector-borne parasites, bacteria and viruses causing 17% of all infectious diseases and over 700,000 human deaths annually. One of the major challenges in studying the discrete mechanisms involved in host-response to hematophagy or the host seeking behavior of hematophagous insects, is the vast amount of signaling processes and genes that are inextricably tied to these interactions. These experiments used the clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated (Cas) and dsRNA knock-down technologies to examine mechanisms of immunological reactivity and host-seeking behavior. Using Illumina next-generation sequencing, 19,269 unique genes were found to be expressed in the salivary glands of C. lectularius. Of those genes, a total of 10,587 were differentially expressed. There were 6 genes definitively characterized as nitrophorin or nitrophorin-like, which were targeted for RNAi experiments. Silencing two nitrophorin genes, LOC106662976 and LOC106662977, which were the most highly expressed, did not result in any significant changes in feeding behavior or dermal reaction in the host. While RNAi-based knock-down experiments can gauge a proteins function, CRISPR/Cas-based knock-out experiments more definitively assess functionality. The second part of the study is thus focused on performing the first homology directed repair knock-in in Ae. albopictus using a plasmid, Cas9 protein and sgRNA targeting the key olfactory receptor gene, AealbOR4. An injection plasmid was constructed targeting AealbOR4 using standard molecular methods. Preliminary injections of the plasmid yielded no viable embryos. To determine if it is the plasmid or injection components that are lethal to developing Ae. albopictus embryos, further studies are required. Despite this, the development of this technique is a first step to be able to effectively interrogate gene function of an olfactory receptor and its role in host-seeking behavior. Taken together, these studies indicate that both RNAi and CRISPR/Cas-based gene editing technologies can effectively be utilized to answer some of the intricate questions related to insect-host interactions in the future.