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Chapter 1 presents the development of an efficient method for the expression and purification of aggregation-prone amyloid-β (Aβ) peptides, including Aβ(M1-42), 15N-labeled Aβ(M1-42), and Aβ(M1-42) familial mutants. Aβ peptides are central to the pathogenesis of Alzheimer’s disease. Aβ peptides are highly aggregation-prone, making them challenging to prepare and purify. Advances in amyloid research rely on improved access to Aβ peptides. Chemical synthesis of Aβ can lead to impurities, such as amino acid deletion products, that are difficult to eliminate during purification. Expression of Aβ(1-42) peptide requires the generation of fusion protein and cleavage by protease to remove the N-terminal methionine group that originates from the translational start codon, which can make the preparation of Aβ costly and time-intensive. In this chapter, I collaborated with fellow graduate student Stan Yoo to express Aβ(M1-42), a widely used form of Aβ with properties comparable to those of the native Aβ(1−42) peptide. Expression of Aβ(M1−42) is simple to execute and avoids the expensive and difficult enzymatic cleavage step associated with expression and isolation of Aβ(1−42). We then developed an efficient method to afford Aβ(M1-42) and 15N-labeled Aβ(M1-42) at around 19 mg per liter of bacterial culture with high purity using simple and inexpensive steps in three days. This method relies on the combination of protein biology tools such as bacterial expression and inclusion body solubilization, as well as peptide chemistry tools such as preparative HPLC purification of the solubilized inclusion bodies. This chapter also describes a simple method for the construction of recombinant plasmids and the preparation of Aβ peptides containing familial mutations. These methods may enable experiments that would otherwise be hindered by insufficient access to Aβ, such as NMR experiments with 15N-labeled Aβ. We anticipate that this method can be adjusted for the expression and purification of other amyloidogenic proteins.Chapter 2 presents the preparation of Aβ peptide with an N‑terminal cysteine [Aβ(C1–42)], the development of tailored chemical reaction conditions for the conjugation of aggregation-prone Aβ(C1–42) peptide with fluorophores or biotin, and the biophysical studies of labeled Aβ peptides.N-terminally functionalized Aβ peptides are important in amyloid and Alzheimer’s disease research. Site-specific labeling on the N-terminus of Aβ minimizes perturbation in the structure and function of the peptide, as the central and C-terminal regions of Aβ are more involved in fibril and oligomer formation. Although synthetic N-terminally functionalized Aβ peptides are commercially available, these peptides are expensive and limited to biotin and a few common fluorophores. Expressed Aβ peptides offer advantages over synthetic Aβ because they are free from amino acid deletions and epimeric contaminants, and they have been found to be more biological relevant because they aggregate more quickly and are more neurotoxic than synthetic Aβ. The method of preparing Aβ(C1–42) relies on the hitherto unrecognized observation that the expression of the Aβ(MC1-42) gene yields the Aβ(C1–42) peptide, because the N-terminal methionine is endogenously excised by E. coli. This observation is significant, because it allows the preparation of a useful Aβ(C1–42) peptide without the additional N-terminal methionine that originates from the translational start codon. The Aβ(C1–42) peptide represents a minimal modification of native Aβ(1–42), and the addition of a single cysteine residue at the N-terminus enables labeling of the expressed Aβ with complete site specificity using widely available maleimide-based reagents.The Aβ peptide is challenging to handle and label because of its strong propensity to aggregate. This chapter details the development of tailored chemical reaction conditions for handling and labeling of this aggregation-prone peptide. The labeling chemistry was optimized to be performed at pH 9 in HPLC fractions Aβ(C1–42), where Aβ remains mostly monomeric. Biophysical studies show that the labeled Aβ peptides behave like unlabeled Aβ and suggest that labeling of the N-terminus does not substantially alter the properties of the Aβ. This chapter also goes on to demonstrate the utility of the labeled Aβ peptides through fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. Ready access to labeled Aβ bearing fluorophores will advance amyloid and Alzheimer’s disease research by enabling experiments for investigating the pathogenic mechanism, transport, and clearance of Aβ, as well as for screening anti-Aβ antibodies.Chapter 3 presents the introduction of intramolecular disulfide bonds to Aβ peptides that stabilizes Aβ into oligomeric state and the discovery of a disulfide-stapled peptide, AβC18C33, that forms homogeneous dimers and does not fibrilize. Aβ aggregates rapidly and exists as three different forms in equilibria: monomers, oligomers, and fibrils. Although Aβ fibrils are the most commonly found species in the brain tissue of patients with Alzheimer’s disease, soluble Aβ oligomers are the toxic contributors to neurodegeneration. Aβ oligomers are heterogenous and metastable: they can rapidly aggregate into more thermodynamically stable fibrils, which makes it challenging to isolate them and study their structures and biological properties. No high-resolution structures of Aβ oligomers have been reported thus far. Filling this gap is necessary for an enhanced understanding of the molecular basis of Alzheimer’s disease.In this chapter, we aimed to generate stable, non-covalent Aβ oligomers by introducing intramolecular disulfide linkages to Aβ. The intramolecular linkages were designed to enforce a β-hairpin conformation, the key conformation that favors Aβ oligomer formation and disfavors fibrilization. We have designed, prepared, and studied a variety of mutant Aβ peptides containing intramolecular linkages. Among these mutant peptides, we discovered AβC18C33, an Aβ peptide containing a disulfide bond between positions 18 and 33, that forms stable, homogeneous dimers and does not fibrilize, as evidenced by the results of a series of biophysical studies. The Aβ dimer has been proposed to be the basic building block of many larger Aβ oligomers and is thought to be one of the most neurotoxic and pathologically relevant species in Alzheimer’s disease. We thereby anticipate that the AβC18C33 peptide to serve as a stable, non-fibrilizing, and noncovalent Aβ dimer model for the exploration of the structure and pathogenesis of Aβ dimers, the generation of Aβ-specific antibodies, and the screening of Aβ-targeted drugs. Our laboratory is exploring ways to obtain high-resolution structures of this dimeric Aβ model peptide, using techniques including cryo-EM, NMR, and X-ray crystallography. Chapter 4 presents the structure-based design of a cyclic peptide inhibitor towards SARS-CoV-2 using free molecular modeling and docking software and publicly available X-ray crystallographic structures. When the COVID-19 pandemic forced the temporary closure of our laboratory in March 2020, we began working on the structure-based drug design of inhibitors against the SARS-CoV-2 virus main protease. These efforts resulted in the design of a cyclic peptide inhibitor, UCI-1. Working with other group members, I published a tutorial paper based to describe the structure-based drug design process and teach others how to do it using the publicly available software UCSF Chimera and AutoDock Vina.SARS-CoV-2 is a highly infectious virus that causes COVID-19, a serious respiratory infection that has caused over 178 million infections and over 3.8 million deaths worldwide as of June 2021. Main protease is a crucial enzyme that SARS-CoV-2 utilizes for site-specifically cleaving the polyprotein that is translated from viral mRNA and generating mature proteins that are necessary for replication and infection. The essential role of main protease, as well as the success of HIV protease inhibitors in the treatment of AIDS, make main protease an attractive therapeutic target in the treatment of COVID-19.This chapter begins with the analysis of the X-ray crystallographic structure of the main protease of the SARS coronavirus (SARS-CoV) bound to a peptide substrate. This chapter then describes the structural modification of the peptide substrate using the UCSF Chimera molecular modeling software to create a cyclic peptide inhibitor. Finally, this chapter presents the use of molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV main protease and the highly homologous SARS-CoV-2 main protease.The supporting information section of this chapter provides an illustrated step-by-step protocol showing the inhibitor design process. These and other molecular modeling studies helped our laboratory decide to pursue the synthesis of the cyclic peptide and experimentally evaluate its promise as an inhibitor of SARS-CoV-2 main protease. The molecular modeling studies presented in this chapter may help students and scientists design their own drug candidates for COVID-19 and the coronaviruses that may cause future pandemics.

Molten Salt Reactors (MSRs) are a class of Generation IV nuclear reactors using molten salts as Heat Transfer Fluids (HTFs). MSRs have two major variants, namely, solid fuel reactor (salt-cooled reactor) and liquid fuel reactor (salt-fueled reactor). This study primarily focuses on the solid fuel MSRs, i.e., the Fluoride-salt-cooled High-temperature Reactors (FHRs). FHRs adopt TRistructural-ISOtropic (TRISO) fuel particles, low-pressure liquid fluoride salts, and passive decay heat removal systems, such as the Direct Reactor Auxiliary Cooling System (DRACS). Although FHRs bring these benefits, there are a number of key technology gaps/issues that need to be addressed for FHR development. In this study, two key technology gaps/issues have been addressed for FHRs: (1) molten salt natural circulation in a passive decay heat removal system and (2) management/control of tritium, whose production rate in FHRs is expected to be significantly higher than that in Light Water Reactors (LWRs). In this study, both the heat transfer and friction characteristics of molten salts in straight circular pipes were first numerically investigated by a Computational Fluid Dynamics (CFD) tool, STAR-CCM+. The CFD results were then validated by comparing with experimental data in the literature and correlation predictions. Our analysis showed that molten salts in the transitional and turbulent flow regimes acted as ordinary HTFs, for which conventional forced convective heat transfer correlations could be applied with ±20% uncertainties in general. As for molten salts in the laminar flow regime, however, both the correlation results and CFD predictions without considering the effects of buoyancy and radiative heat transfer in molten salts significantly underestimated the salt Nusselt number, which could be as large as 40% and 30% lower, respectively. These significant discrepancies were attributed to the fact that the buoyancy effect and radiative heat transfer effect were generally not negligible for laminar flows of molten salts, which was demonstrated by good agreement (within ±14% uncertainties) between experimental data and CFD predictions considering those two effects. The molten salt natural circulation in a single loop and multiple coupled loops, similar to the passive decay heat removal system DRACS, was then investigated by a one-dimensional (1D) NAtural Circulation COde, NACCO, developed in this research. The above finding, i.e., the non-negligible effects of buoyancy and radiative heat transfer in laminar flows of molten salts, was further confirmed by comparing the code results with two high-temperature natural circulation experiments using NaNO3-KNO3 and FLiBe as coolants, respectively. This validated 1D code NACCO was then applied to a FLUoride Salt Test FAcility, FLUSTFA, which was designed based on a scaling analysis and constructed in this research to experimentally investigate the DRACS performance under steady-state and various transient scenarios. In addition, another technology gap/issue, tritium management, was studied for FHRs. Two strategies were proposed for tritium mitigation in FHRs, namely, Double-Wall Fluted-Tube Heat eXchanger (DW-FTHX) with a tritium carrier and Single-Wall FTHX (SW-FTHX) with a tritium barrier options. These two options were then evaluated by a 1D coupled HEat and MAss Transfer code, HEMAT, developed and benchmarked in this study. It was found that both the optimum designs of the two options, DW-FTHX with a tritium carrier (helium) and SW-FTHX with a tritium barrier (silicon carbide), could effectively reduce the total tritium leakage rate to several curies per day, the same order of magnitude in a typical LWR.

Abstract: The growing consumption of fossil resources has rapidly depleted fuel availability and increased CO2 emission, which presents two of the most prominent crises in the modern era. CO2 reduction reaction (CO2RR) at ambient conditions using electrochemical (EC) and photoelectrochemical (PEC) methods are promising ways to reducing CO2 emission while producing storable fuels and chemicals that can be later consumed on demand. To date, highly selective electrochemical conversion of CO2 to CO, an essential precursor in Fischer-Tropsch processes, require heavily on the use of precious metals while operating at a significant high overpotential. Photocathode-driven PEC conversion of CO2 can achieve one step conversion of solar energy to chemical energy, but issues such as toxicity, poor selectivity and stability, large external bias potential remain unresolved. On the other hand, photoanodes are well studied in water splitting researches, which can be integrated with CO2RR electrocatalysts in a photoanode-driven CO2 reduction system. In this thesis, a novel, simple, and low-cost Cu2O-SnO2 core-shell nanocrystal electrocatalyst was first designed. The electrocatalyst achieved over 90% Faradaic efficiency for CO2-to-CO conversion at overpotentials of 890 mV and 390 mV in 0.1 M KHCO3 and 0.5 M KHCO3, respectively, which are comparable to those achieved on the precious metals. To further reduce the cell overpotential, a photoanode-driven PEC system is demonstrated through combining Cu2O-SnO2 electrocatalyst as a dark cathode and an n-Si/Ni photoanode, achieving a 400-mV reduction in overpotential at 5 mA cm-2 when compared to an electrochemical system. The designed PEC cell obtained a photo-assisted efficiency () of 3.5% while operated over 12 hours with minor degradation.

Abstract: Human migration is a global phenomenon driven by two factors: individual needs or desires, and changes in the economics and politics in the society from which the migration stems. Human migration usually involves people crossing international borders, but the impact is often felt locally, in the economy, and in social, political, and cultural spheres. During the 1980s and 90s, Hong Kong Chinese went through a mass migration wave to western countries. The migration was strongest during the period of the handover of Hong Kong, and a primary destination was Canada. The purpose of this research is to map a migration wave from Hong Kong to Canada and back to Hong Kong during two crucial periods: 1984, when the handover decision was made; and 1997, the lead-up to when Britain handed political control of Hong Kong to China. The other purpose of this research is to gain insight into the factors that reflect these migration trends. The project involves digital technology, specifically a quantum geographic information system (QGIS), to capture the nodal Hong Kong migrant patterns in Canada by destination, gender, age group, and immigration class. The thesis will address neo-institutionalism; social identity; transnationalism; sense of place; the strong relationship between transnational space, changing institutions, cultural identity values and conflicts; and the transition of the affective sense of place that impacts the motivations and practices of Hong Kong Chinese migrants. This study is part of a body of research on transnational migration that crosses national borders, and uses large-scale data to visualize the relationship networks between the place of origin (Hong Kong) and destination (Canada), and spatial distribution of immigration into Canada.

Intramolecular iron-mediated diene/olefin cyclocoupling reactions using vinyl ethers as the olefin partners have been investigated. Spirolactams with functionalized (alkoxymethyl) side chains can be formed under thermal conditions with high yields. Introduction of a methoxy group at the C3 position allowed the formation of one single diastereomer after the demetallation and hydrolysis of the cyclized products.An iron mediated tandem double [6+2] ene-type cyclization reaction has been expanded using a dienol ether as the pendant diene, resulting in a spirotricyclic molecule that has alkoxymethyl functionality at the cyclization terminus. Competing [4+4] cycloaddition that occurs during the thermally activated reaction can be ameliorated by using a photochemical procedure at moderately elevated temperature.Alkylation reaction of an ester containing iron tricarbonyl moiety has been found to be stereoselective during the approach towards synthesis of hispidospermidin’s spirocyclic core. The stereocontrol is resulting from the adjacent iron tricarbonyl group during the enolate alkylation process, which was shown by the crystal structure of the alkylation product, which was further converted to a substrate that can potentially undergo spirocyclization. Another all-carbon substrate was synthesized to test a one-pot all-carbon cyclization. Preliminary attempts cyclizing both substrates were not successful.

In order to address the challenges of capacitive sensing in harsh environment, self resonant passive wireless sensors are studied. The capacitive sensing elements based on interdigitated capacitor (IDC) sensor are used. A semi-empirical model providing accurate capacitance calculation for IDCs over a wide range of dimensions and dielectric constants is developed. An equivalent circuit model based on electric field distribution is proposed, leading to a closed form approximation for IDC capacitance calculation. The conductivity of the material under test is also considered and a model is proposed to calculate effective capacitance as a function of conductivity and measurement frequency. The model is used to study the design optimization of IDC sensor and suggested design procedure is proposed. To wirelessly interrogate the capacitive sensor, it is connected to an inductive element to form a resonant circuit, while the measurement is made at remote reader coil. Advantages and disadvantages of different type of resonant structure design are analyzed. In order to assist the design process, a SPICE circuit model is developed to estimate the resonant frequency of the self resonant sensor. Miniaturized sensors with different dimensions are designed, fabricated and tested. The sensor is integrated with silicon nanowire fabric coated with polymer. Measurements are made to illustrate the enhancement in sensing capability by integrating chemical selective material.

The objective of this thesis is to develop a complex dielectric properties sensor using interdigitated capacitor (IDC) structure. IDCs are easy to fabricate and because of its planar structure, it can be easily integrated with other sensing components and signal processing electronics. The design, fabrication, modeling, and testing of IDC sensors are presented in this thesis. Design parameters and their influence on sensor's output signals are discussed. Previous IDC models are reviewed and the limitations are studied. A new equivalent circuit model based on the fringing electric field distribution and a novel iterative data extraction algorithm combining Finite-Element Method (FEM) and the equivalent circuit model is studied. Results suggest that the algorithm can accurately extract relatively low dielectric constant and conductivity of material under test (MUT) from measured impedance data.

In response to a chemoattractant signal, amoeboid cells such as neutrophils, macrophages, and Dictyostelium cells are able to polarize and move towards the chemoattractant source. During Dictyostelium chemotaxis, it is known that cells amplify and differentially localize specific signaling responses at the future anterior and posterior of the cell leading to outwardly directed F-actin polymerization and myosin II-mediated contractility at the front and back, respectively. However, how cells initially detect and orient themselves in chemoattractant gradients remains largely unknown. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium and inhibition of Ras signaling results in directional migration defects. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2. To investigate the role of Ras in directional sensing, I studied the effect of its misregulation using cells with disrupted RasGAP activity. I identified an orthologue of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. nfaA- cells fail to spatially and temporally regulate Ras activity, which leads to misregulated downstream PI3K activity and F-actin polymerization. As a result, severe cytokinesis and chemotaxis defects in nfaA- cells are observed. Through both genetic and biochemical approaches, I identified RasG as the major target of DdNF1 GAP activity in chemotaxis. Using unpolarized, latrunculin-treated cells, I showed that tight regulation of Ras is required for directional sensing. It is speculated that the uniformly distributed DdNF1 functions as a global inactivator of Ras activity, coupled to putative local activators (e.g. RasGEFs), unidentified global inhibitors as well as positive feedback signaling, leads to persistence and amplification of the Ras signal at the front and promotes leading edge formation. Consequently, cells migrate up the gradients. Together, it is suggested that Ras is part of the cell's compass, and that the RasGAP-mediated regulation of Ras activity affects directional sensing. Further, growing nfaA- cells exhibit elevated Ras activity and display enhanced random cellular movement, consistent with the model that a G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility