Brain tumors are one of the leading death causes worldwide. Magnetic resonance imaging is the most commonly applied imaging technique for brain tumor detection. However, magnetic resonance imaging loses its power when it comes to detect early stage tumor differs from normal tissue in terms of relaxation properties. To overcome this difficulty in early tumor detection, I investigated chaotic spin dynamics generated by radiation damping and utilized a novel MRI technique called the active-feedback to enhance the imaging contrast between normal tissue and early stage brain tumor. The theme of my Ph.D. thesis is summarized as following:

(1) Fixed Point Imaging with Continuous Wave in Presence of Active-feedback Field. Active-feedback field is a time-dependent magnetic field irradiation inspired by a natural radiation damping. It is shown that active-feedback field yields unique fixed points in spin evolution. Spins are excited to opposite directions by the field until they reach their corresponding fixed points.

(2) Contrast enhancement by selective self-excitation with active-feedback magnetic resonance imaging. It is shown by both theory and simulation that active-feedback magnetic resonance technique generates a selective self-excitation process that selectively excites spins and thus makes this technique very sensitive to local magnetic field inhomogeneity. Active-feedback shows potential in detecting tumors at an early stage.

(3) Local-magnetic-field-dependent active-feedback magnetic resonance imaging and its application in early glioblastoma detection. The active-feedback technique is further developed and an advanced technique called local-field-dependent active-feedback magnetic resonance imaging is introduced. This technique is tested on an early stage glioblastoma multiforme model to show its potential in detecting brain tumors at a very early stage.

A virus has one main goal: to replicate itself. To achieve this goal, many kinds of viruses must encode their own replication machinery to amplify their genomes in the host cell. This study focuses on a simple, positive-sense, single-stranded RNA virus, Nodamura, which has a genome consisting of two mRNA molecules. The translation product of RNA1 is an RNA-dependent RNA polymerase (RdRp), while its RNA2 codes for the capsid protein (CP); the RdRp binds to and replicates each of these molecules, and the CP packages the two of them into a single capsid. The focus of the current study is to assess the relative levels at which these two viral RNA molecules compete to be bound and replicated by the RdRp. Understanding replication phenomena such as this has important implications in numerous other many-molecule-mRNA-genome virus infection scenarios, in addition to potential translational medicine applications where it is useful to amplify therapeutic mRNAs using an RdRp. In vitro transcribed Nodamura RNA constructs were created in which a fluorescent reporter gene is added to RNA1, and the CP gene in RNA2 is replaced entirely by a different fluorescent reporter. These two constructs were then transfected into mammalian (BHK-21) cells. Fluorescence intensity assays and quantitative PCR experiments were performed at various time points post-transfection to study the replication competition between Nodamura’s RNA1 and RNA2 in the absence and presence of one another. These assays showed that Nodamura RNA1 replication decreases by as much as a factor of two when in the presence of RNA2, while RNA2 is not amplified at all in the presence of the RdRp encoded by RNA1. These results indicate how in cis and in trans replication dynamics determine the differential expression of the viral genomic RNAs.

Crystallization and phase retrieval are the two perennial problems that challenge any endeavor to retrieve structural information from a crystal. Micro-electron diffraction has emerged in recent years as a complementary method to X-ray diffraction by allowing the diffraction of crystals previously too small for routine X-ray techniques. While micro-electron diffraction has great potential to address barriers in crystallization, issues with phase retrieval precludes the technique’s broad utilization. As a nascent technique, many X-ray phasing methods have not been replicated in electron diffraction. In this work, we demonstrate the development and application of racemic crystallography and fragment-based phasing methods for electron diffraction.

We have genetically engineered the capsid proteins (CPs) of two RNA-packaging plant viruses, BMV and TYMV, to facilitate the presentation of arbitrary protein ligands on the exterior surface of the virus-like particles (VLPs) that they form. This is important for the targeting of in vitro self-assembled mRNA-containing VLPs for in vivo gene delivery purposes. In particular, this platform will provide an alternative to lipid nanoparticle systems, with the advantages: of ensuring definite stoichiometry – one mRNA molecule per 180-protein capsid (as opposed to an indeterminate number of mRNAs in polymorphic/mixed-component liposomes); and of precise control over the site of conjugation of protein ligands, introducing genetic fusions (as opposed to promiscuous N-Hydroxysuccinimide (NHS) esters or sulfhydryl-maleimide chemistry).More explicitly, we have developed two novel approaches to the functionalization of in vitro reconstituted VLPs with protein ligands. The first involves the genetic insertion of a poly-Glycine (polyG) moiety into an exterior loop of a CP, just downstream of a protease cut site. Cutting of the loop, followed by the addition of the Sortase A enzyme, will result in covalently linking – to the newly created polyG N-terminus – an arbitrary protein to whose C-terminus contains the Sortase A binding motif (LPXTG). The second strategy involves exploiting an analogy between the beta barrels of viral CPs and of green fluorescent protein (GFP) “split proteins.” Here the idea is to make the split portions of CPs, genetically fuse the N-terminus of the C-terminal part (i.e., the tail end of the CP) to a protein ligand of interest, and then mix the two CP portions together so that they associate into a nicked mutant CP that, in turn, self-assembles into VLPs with protein ligands already displayed on their exterior surfaces. These two strategies are demonstrated for BMV and TYMV, respectively, and offer “proof-of-principle” examples of a potentially powerful means for in vitro synthesis of targeted mRNA-containing particles for gene delivery.

Supercritical fluids in rock-H2O systems have been proposed to be important agents of mass transfer in high-pressure environments such as subduction zones. For the first study, experiments were conducted on the model system NaAlSi3O8 (Ab)-H2O to investigate phase relations at pressure (P) and temperature (T) in the vicinity of critical mixing between aqueous fluid and silicate melt. Isobaric equilibrium phase relations were determined at 1.0-1.7 GPa, 600-1060 °C, and H2O mole fractions (x_(H2O)) of 0.035-0.99. A subregular solution model was used to describe the solvus curves. P- and T-dependent Margules coefficients, W_Ab and W_(H2O), and activities of H2O and Ab were formulated using compositions at the solidus and critical point at each pressure as input. The results provide a comprehensive account of the solution properties of subcritical and supercritical fluids in the Ab-H2O system at temperatures and pressures corresponding to the deep-crust regions of granite magma generation. This work may have considerable bearing on element transport, extractability of partial melt, and ultimate level of melt emplacement in the middle and upper crust.

The second study is an experimental determination at deep-crustal conditions of liquidus-H2O contents of more complex granitic systems containing albite, potassium feldspar and quartz. The liquidus water content of a granitic melt at high-P and T is important because it constrains the volume of granite that could be produced by dehydration melting of the deep crust and it strongly influences physical properties that control the ability of granitic liquids to accumulate and ascend. The high liquidus-H2O contents determined in this study present a challenge for producing voluminous amounts of metaluminous granites from lower crustal biotite-amphibole gneisses by dehydration melting. Additionally, rapid undercooling of granitic melts may lead to granophyric textures described in volcanic and impactitie surge deposits and the formation of a solid solution of coesite and feldspar.

Viruses are self-replicating nucleoprotein complexes that have the ability to cause diseases and are composed of, at least, a single copy of genetic material (either RNA or DNA) and a virally encoded proteinaceous (capsid protein [CP]) protective shell. In single-stranded RNA (ss-RNA) viruses genome packaging is unequivocally coupled to capsid assembly. This spontaneous process is driven by CP-CP and CP-RNA interactions. This thesis is concerned with the in vitro self-assembly of a ss-RNA virus, cowpea chlorotic mottle virus (CCMV), and the effects of the length of the nucleic acid on capsid assembly. We show that the size of a capsid depends not only on the preferred curvature of the CP, but also on the length and number of packaged RNA molecules. By carrying out a series of titration curves of CP with RNAs of varying lengths we find that such high encapsidation yield is possible if and only if the assembly reaction is carried out under very specific experimental conditions, namely at a CP:RNA mass-ratio of 6:1 ("Magic ratio"). By doing head-to-head competition experiments, between a reference and a competitor RNA, under limiting assembly conditions we show that the relative packaging efficiency of ssRNA by CCMV CP is a non-monotonic function of RNA length. The relative packaging efficiency increases as the length of the competitor RNAs approaches that of the wild-type RNA (3.2 kilobases [kb]), while for equal-length RNAs it depends on the secondary and tertiary structure of the RNAs. We demonstrate that the length and number of packaged RNAs determines the assembly pathway; unlike the case of long RNAs (> 1.0 kb), when multiple short RNAs (0.5 kb) are packaged by CCMV CP, to virus assembly is a highly cooperative process. This is the first study in which it is shown that in contrast to the accepted dogma the degree of cooperativity of virus assembly depends not only on the relative strength of the CP-CP and CP-RNA complexes but also on the length and number of packaged RNA molecules.

We have developed coarse-grained models of RNA bound by capsid protein (CP) in response to recent in vitro studies on the self-assembly of viral RNA by capsid protein. Under typical in vitro self-assembly conditions and in particular for the case of many ssRNA viruses whose CP have cationic N-termini, the adsorption of CP onto the (anionic) RNA is non-specific because the CP concentration exceeds the Largest dissociation constant for CP-RNA binding.

Following an introductory chapter, which recounts the history and physics of the in vitro self-assembly experiments, Chapters 2-4 of this dissertation explore simple lattice models of single-stranded (ss) RNA in the presence of interacting bound particles. In Chapter 2 our investigation opens with a simple model to account for the measured yield of packaged RNA (nucleocapsids) by CP. We treat the RNA as a 1D lattice, whose sites represent CP binding sites, to calculate the yield of RNA packaged as a function of

the CP:RNA mol ratio. The measured yield of in vitro assembled nucleocapsids agrees exceptionally well with our simple 1D model.

In Chapters 3 and 4 we extend our 1D model to 2D to better account for the branched nature of RNA. Our theoretical work provides the statistical-thermodynamic grounding for understanding the in vitro “competition” experiments. In these experiments two RNAs compete for a limited amount of CP. The exchange of CP between the RNAs was found to be reversible at pH 7 and, separately, it was found that long RNAs were able to strip CPs from shorter ones. Our Monte Carlo simulations demonstrate that, for a given RNA mass, the sequence with the highest affinity for protein is the one with the most compact

secondary structure arising from self-complementarity; similarly, a long RNA steals protein from an equal mass of shorter ones because of the energetic preference of forming one large cluster of CPs over forming two smaller clusters.

Finally, in chapter 5 we turn our attention away from the self-assembly experiments to focus on branched polymers. We show there that the 3D size of a branched polymer with N monomers can be directly calculated from a sequence of N − 2 integers, known as the Pruefer sequence. The calculation was performed numerically and shown to be far more efficient (memory-wise) than typical calculations of the 3D size.

The evolved strategies of viruses are wondrous and should be investigated and understood better to exploit their gene delivery “tricks” for the development of effective therapeutics. Simple RNA viruses – those consisting of a positive-sense/ready-to-translate RNA genome inside an ordered capsid made up of multiple copies of a single protein – have been used as platforms for delivery of therapeutic cargo to cells. For example, early work by several groups established that spontaneous self-assembly of virus-like particles (VLPs) can be carried out in vitro from purified components of RNA and the capsid protein (CP) from the plant viruses tobacco mosaic virus (TMV), cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV). CCMV and BMV CP can encapsulate non-viral RNAs into monodisperse, RNase-resistant spherical capsids if the length of the RNA is between 2,500 and 4,500 nucleotides (nts); longer RNAs are packaged into “multiplets” – multiple 28-nm VLPs, where RNA is shared between two or more particles. Additionally, researchers have exploited the self-replicating nature of viruses, specifically nodamura virus (NOV), to amplify expression of genes of interest (GOIs). The GOI can vary widely from reporter genes to RNA encoding for cancer or viral antigens. This self-replicating RNA, or replicon, can be packaged into CCMV VLPs using an in vitro assembly method. In this study, I investigate viral strategies such as self-assembly and self-replication with the goal of delivering self-replicating functional RNAs encapsulated in spherical VLPs to mammalian cells as therapeutics. This thesis work reports on a systematic series of experiments elucidating the physical chemical properties of self-assembled VLPs as delivery platforms for mRNA replicon therapeutics. I am interested in understanding the physical chemical parameters governing successful self-assembly – solution ionic strength, pH, protein concentration, charge and size of cargo being packaged – to develop robust therapeutics. To probe the role of these in determining RNA-protein and protein-protein interactions and ensuing assembly of nucleocapsids, interferometric scattering (iSCAT) microscopy was used to follow the assembly kinetics of individual BMV VLPs around BMV RNA1 using a range of solution ionic strengths and protein concentrations. The iSCAT experiments revealed that nucleation times become longer and more broadly distributed when RNA-protein interactions are reduced (high ionic strength) and/or protein concentrations are decreased, corresponding to an increase in nucleation barriers. To further explore the role of RNA-protein and protein-protein interactions, assemblies were carried out in a variety of assembly buffers spanning ranges of pH from 5 to 7 and of ionic strengths from 0.1 to 0.5 M, and assembly products were imaged using negative-stain electron microscopy (EM). Additionally, the salt- and pH-dependence of protein-protein interactions were characterized by differential scanning fluorimetry. To quantify the dependence of GOI protein expression on the numbers of GOI replicons (derived from NOV RNA1) transfected into mammalian cells, cell lines were transfected with increasing masses of replicon RNA mixed with an inert carrier RNA, with the total mass of the transfected RNA held constant while increasing the fraction of replicon. Fluorescence microscopy, flow cytometry, and trypan-blue exclusion assays show a non-monotonic dose-dependence of protein expression – i.e., “less is better” – in cells transfected with replicon RNA. To package long replicon molecules – those with GOI > 1,000 nts – into RNase-resistant VLPs, the RNA needs to be compacted to fit into a BMV VLP. To probe the role of charge and size of cargo on self-assembly, long RNA molecules were pre-compacted with increasing concentrations of polyvalent cations and assembled into BMV VLPs. VLPs were analyzed by negative-stain EM and gel electrophoresis. Finally, the same replicon molecule was packaged into spherical and rod-like VLPs using the CP of CCMV and TMV, respectively, with the goal of comparing how the shape of these particles impacts their efficacy as a therapeutic delivery vehicle.

The delivery of RNA genes has great potential in a range of therapeutic applications. A couple of main limitations for RNA-based therapies include its vulnerability to ribonuclease (RNase) digestion, and its transient and low expression levels due to its lack of amplification. Accordingly, a gene delivery platform that includes self-amplifying mRNA inside a protective capsid allowing for cell targeting and uptake could represent a major step forward in mRNA-based gene therapy.

The self-amplifying nature of viral-encoded RNA-dependent RNA polymerases (RdRp) of Nodamura virus holds promise in the development of a platform for self-amplifying RNA gene delivery. To protect and assist in uptake of mRNA encoding an RdRp coupled to a gene of interest, plant virus-like-particles (VLPs) are a strong candidate. Cowpea chlorotic mottle virus (CCMV) capsid protein has been shown to be able to package in vitro heterologous mRNAs, and also to make the genes available to mammalian cells. To better characterize the uptake and amplification in mammalian cells of RdRp-associated genes (also called a replicon, because of its self-amplifying nature), both when naked or encapsidated, reporter genes such as enhanced yellow fluorescent protein (EYFP) or luciferase were used.

In collaboration with the German pharmaceutical company Boehringer Ingelheim (B-I), evaluation of possible efficacy of this platform for a cancer vaccine was explored by incorporation of a model antigen (Ovalbumin epitope, SIINFEKL) sequence into the RNA1 of Nodamura virus. From in vitro studies involving incubation of OVA-replicon-VLPs, we observed that CCMV VLPs can be taken up by dendritic cells, where replication then occurs to promote increased production of antigen. Flow cytometry analysis revealed increased expression of maturation markers, and assaying the dendritic cell content by qPCR showed a significant number of SIINFEKL epitope-containing RNA. After these OVA-replicon-VLPs are injected into mice, a population of SIINFEKL-specific CD8+ (cytotoxic) T cells results.

Many viruses use long (thousands of nucleotides) single-stranded (ss)RNAs as their genomic material. Such viruses can be as simple as a single RNA molecule encapsidated inside a shell (capsid) composed of many copies of a single capsid protein (CP). Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV), the two sibling viruses studied throughout this work, are model ssRNA viruses that are capable of spontaneous self-assembly \textit{in vitro}. This work aims to elucidate how RNA content affects both the structure of the particles formed and their physical properties. In particular, particles containing one or another of a variety of RNA molecules are compared using physical and biological tools. The results of this work further our understanding of the self-assembly of ssRNA viruses, and the physical forces that result in their structure and dynamical properties.

Chapter one is an introductory chapter, introducing the reader to viruses, ssRNAs and their secondary/tertiary structures, CCMV and BMV, and the \textit{in vitro} self-assembly of these viruses. Chapters two and three discuss the production of viral-length polyU, an RNA molecule that lacks secondary structure due to its inability to base-pair or base-stack, and the packaging of this unique, structureless RNA with CCMV CP. Chapter four discusses the cryo-electron microscopy (cryoEM) asymmetric reconstruction of BMV virions, which allows for unprecedented visualization of the RNA genome inside a multipartite virus--a virus with a genome comprised of more than one RNA packaged into several indistinguishable particles. Chapter five summarizes the results of this work, and provides some future experiments related to the work discussed herein.