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RIG-I-like receptors (RLRs), RIG-I, MDA5, and LGP2, are a family of innate immune receptors that recognize viral RNA in the cytoplasm and initiate antiviral responses including the induction of type I interferons and other pro-inflammatory cytokines. All three proteins have both an RNA helicase domain with ATPase activity and a C-terminal domain (CTD) which is responsible for RNA binding. RIG-I and MDA5 also have two tandem caspase activation and recruitment domains (CARDs) at the N-terminus which are involved in downstream signaling. To understand the structural basis of viral RNA recognition by the RLRs, especially RIG-I, we have performed extensive biochemical studies to determine the binding properties of RIG-I with different forms of RNA, including dsRNA with and without 5'-triphosphate (5'-ppp) groups, and 5'-ppp ssRNA. RIG-I CTD binds to these forms of RNA, and exhibits the highest affinity for 5'-ppp dsRNA. We also determined the crystal structures of RIG-I CTD in complex with dsRNA with and without 5'-ppp by X-ray crystallography. The structures showed that RIG-I recognizes the termini of the dsRNA and interacts with the two types of RNA in different orientations. By comparing these complex structures together with mutagenesis studies, we conclude that RIG-I CTD is a versatile binding module capable of recognizing different RNA ligands. Similar but partially differing sets of residues are involved in the recognition of dsRNA with and without 5'-ppp. Mutations of key residues at the RNA binding surface also abolished RIG-I signaling in cells. In order to compare the RIG-I/RNA interactions with other RLRs, we also determined the dsRNA binding surface of MDA5 CTD by NMR titration studies. MDA5 CTD has a similar binding surface to that of RIG-I CTD, however with slightly different surface electrostatic potentials which indicate different interactions with RNA. This may explain how MDA5 senses differing types of viruses compared to RIG-I. The current RIG-I activation model suggests that after stimulation by RNA binding, RIG-I undergoes an ATP-dependent conformational change, exposing the CARDs for downstream signaling. To understand the critical role that the helicase domain plays in RIG-I activation by structural approach, we also attempted to crystallize the dsRNA-bound helicase domain together with CTD.

<p> An X-ray study of the various phases of anhydrous sodium pyrophosphate has been carried out. The system began with an ordered structure in orthorhombic space group P212121 and ended with a completely disordered structure in hexagonal space group P63/mmc, All the intermediate phases were partially disordered.</p> <p> The crystal structure of the first two phases of the system were determined while the rest were only partially solved due to the complexity of the disorder involved. A discussion on the symmetry aspects of the phase transformations was given and a model for the disorder of the hexagonal phase was proposed.</p> <p> As an integral part of the study the room-temperature phase of sodium diarsenate was also investigated.</p> / Thesis / Doctor of Philosophy (PhD)

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“Matrix effects” in Near Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy are the spectroscopic changes induced by intermolecular interactions, providing sensitivity to local structure and order in solids and liquids. This project aims to identify the effect of intermolecular interactions caused by different n-alkane solid state structures on their NEXAFS spectra. Changes to the carbon 1s NEXAFS spectra are studied as a function of their solid state structure and organization. Two experimental approaches were proposed in this project. In the first approach, different n-alkane crystal structures (orthorhombic, monoclinic, etc.) are examined which arise as a function of n-alkane chain length. In the second approach, changes observed through the pre-melting point order-disorder phase transition found in n-alkanes are examined. This work will explore the characteristic spectroscopic differences observed between n-alkanes in different crystalline forms as well as in the disordered phase below the melting points. In this project, well-ordered diamond shape n-alkane single crystals were obtained by solution casting, using experimental conditions optimized for each n-alkane. As circularly polarized radiation will average the effect of molecular orientation, circularly polarized radiation was used to obtain the NEXAFS spectra of n-alkanes. However, in the analysis of the NEXAFS spectra of n-alkanes recorded with the left circularly polarized X-rays, a significant linear polarization contamination was found. Therefore, linearly polarized X-rays were used to acquire angle dependent NEXAFS spectra, where the X-ray polarization was deliberately aligned along the principal axes (X, Y) of the n-alkane crystal. It was observed that the room temperature carbon 1s NEXAFS spectrum of n-octacosane (C28H58) was different from that of the other n-alkanes, n-tetracosane (C24H50), n-tricosane (C23H48) and n-tetracontane (C40H82). This difference can be attributed due to the different crystal packing of n-octacosane (C28H58 - monoclinic) relative to the other n-alkanes (triclinic and orthorhombic), suggesting different intermolecular interactions (matrix effects) in n-octacosane (C28H58). The analysis of the temperature dependent NEXAFS spectra of n-alkanes reveals that samples of the short chain n-alkane crystals, specially n-tricosane (C23H48), n-tetracosane (C24H50) and n-octacosane (C28H58), sublimed in the STXM microscope. Changes observed in the carbon 1s NEXAFS spectra of n-tetracontane (C40H82) with temperature were attributed to the order-disorder transition. This further illustrates the existence of matrix effects in the NEXAFS spectra of n-alkanes.