The design of probes for the selective recognition of biopolymers (nucleic acids and proteins) is a fundamental task for studying, diagnosing, and treating diseases. Traditional methods utilize a single component (small molecule or oligonucleotide) that binds directly to the target biopolymer. However, many biopolymers are unable to be targeted with this approach. The overarching goal of this dissertation is to explore a new, binary approach for designing probes. The binary approach requires two components that cooperatively bind to the target, triggering a recognition event. The requisite binding of two-components allows the probes to have excellent selectivity and modularity. The binary approach was applied to design a new sensor, called operating cooperatively (OC) sensor, for recognition of nucleic acids, including selectively differentiating between single nucleotide polymorphisms (SNPs). An OC sensor contains two oligonucleotide probe strands, called O and C, each with two domains. The first domain contains a target recognition sequence, whereas the second domain is complementary to a molecular beacon (MB) probe. Binding of both probe strands to the fully matched analyte generates a full MB probe recognition site, allowing a MB to bind and report the presence of the target analyte. Importantly, we show that the OC sensor selectively discriminates between single nucleotide polymorphisms (SNPs) in DNA and RNA targets at room temperature, including those with stable secondary structures. Furthermore, the combinatorial use of OC sensors to create a DNA logic gate capable of analyzing DNA sequences of Mycobacterium tuberculosis is described. The binary approach was also applied to design covalent inhibitors for HIV-1 reverse transcriptase (RT). In this application, two separate pre-reactive groups were attached to a natural RT ligand, deoxythymidine triphosphate (dTTP). Upon binding of both dTTP analogs in the RT active site, the pre-reactive groups are brought into the proper proximity and react with each other forming an intermediate that subsequently reacts with an amino acid side chain from the RT. This leads to covalent modification of RT, and inhibition of its DNA polymerase activity. This concept was tested in vitro using dTTP analogs containing pre-reactive groups derived from ?-lactamase inhibitors clavulanic acid (CA) and sulbactam (SB). Importantly, our in vitro assays show that CA based inhibitors are more potent than zidovudine (AZT), a representative of the dominant class of RT inhibitors currently used in anti-HIV therapy. Furthermore, molecular dynamics simulations predict that complexes of RT with these analogs are stable, and point to possible reaction mechanisms. The inhibitors described in this work may serve as the basis for the development of the first covalent inhibitors for RT. Moreover, the pre-reactive groups used in this study can be used to design covalent inhibitors for other targets by attaching them to different ligands. Overall, the work presented herein establishes the binary approach as a straightforward way to develop new probes to selectively recognize nucleic acids and proteins.

Covalent organic frameworks (COFs) are a novel class of crystalline materials with regular porosity, high specific surface area, and various linkage chemistries. Conventional chemical syntheses of these materials lead to the formation of bulk powders characterized as polycrystalline aggregates. Synthesizing these materials as colloidal systems is an effective means to prevent aggregation and achieve larger single-crystalline domain sizes. In this thesis, I describe the effect of temperature and transimination catalyst strength on COF particle nucleation and particle growth. Morphology and crystallinity of the COF-300 particles were confirmed using X-ray Diffraction (XRD) and Scanning Electron Microscopy (SEM). The onset of colloidal light scattering, or the Tyndall Effect, was measured using Dynamic Light Scattering (DLS). Reaction temperature affects both the reaction rate and the solubility of the monomeric and oligomeric species. The higher solubility at higher temperatures is hypothesized to delay particle nucleation, or the onset of colloidal light scattering. DLS measurements confirmed these results. However, measurement of particle size using DLS and SEM showed little association between the particle size and reaction temperature. Stronger acids are similarly hypothesized to accelerate the chemical reaction, leading to a shorter induction delay and smaller particles. DLS measurements confirmed this hypothesis on the effect of acid catalyst on the induction delay; stronger acids led to a v shorter induction delay. However, preliminary SEM measurements suggest that stronger acid catalysts create larger COF-300 particles.

The quadrupolar interaction in sodium orthovanadate dodecahydrate, calcium orthovanadate, vanadinite, descloizite, zirconium divanadate, cadmium divanadate, potassium metavanadate and vanadium pentoxide, was studied by nuclear magnetic resonance. The v51 quadrupole coupling constants in these compounds show a strong correlation with the distortion of the tetrahedral symmetry around the vanadium ion. Vanadium pentoxide is an exception and shows a surprisingly small coupling constant. The point multipole model was found to be inadequate for the calculations of the electric field gradients in these compounds. A covalent model provides an explanation of the small magnitude of the coupling constant of vanadium pentoxide. However, because of the numbers of approximations in this model, only a qualitative correlation with the experimental results was achieved. In addition, the chemical shift tensor of the v51 resonance line in a single crystal of vanadium pentoxide was measured to be very large. This result was correlated with a large Van Vleck term in the magnetic susceptibility of vanadium pentoxide. / Thesis / Doctor of Philosophy (PhD)

Antibody-recruiting molecules (ARMs) are therapeutic tools that simultaneously bind a hapten-specific serum antibody and a cancer cell surface protein, resulting in the activation and recruitment of an immune cell to the cancer surface. However, ARM efficacy is limited by the ability of ARMs to form a quaternary complex with the immune cell receptor, antibody, and cancer cell surface. The Rullo lab has previously developed and characterized a covalent ARM (cARM) that irreversibly links the ARM to the antibody and simplifies the quaternary binding equilibria. cARMs have shown a marked increase in both target immune recognition and therapeutic efficacy. However, cARM efficacy is still limited by the affinity of the antibody for the immune receptor. We aim to investigate how direct covalent engagement of the immune receptor and elimination the antibody-immune receptor binding equilibria impacts immune activation and therapeutic efficacy. This thesis focuses on the chemical programming of macrophages through direct covalent immune receptor engagement. We have developed and characterized covalent immune programmers (CIPs), which are molecules that contain a macrophage targeting domain and a tumour targeting domain. The macrophage targeting domain binds the activating receptor CD64 on the macrophage surface and contains a chemical warhead that covalently labels the receptor once bound. The tumour targeting domain can promote macrophage tumour engagement resulting in tumoricidal function. Flow cytometry experiments have shown that CIPS are able to bind Fc receptors specifically and effectively on the surface of macrophages. Further, CIPs were able to induce macrophage activation and induce target specific phagocytosis. These experiments have also shown that direct engagement of the receptor by the CIP is more effective than antibody-mediated engagement, suggesting that overall immune complex stability affects immune cell activation. Taken together, these concepts can be used to guide future immunotherapeutic design. / Thesis / Master of Science (MSc)

The fluorination of porous materials often leads to the enhancement of properties such as stability, crystallinity and selective adsorption. Although there has been much interest in the fluorination of many types of porous materials, little research has been done on the fluorination of macrocycles, specifically trianglimine and leaning pillararene based materials. In this work, we introduce for the first time highly fluorinated trianglimine and leaning pillararene and show the enhancement effects brought about by the inclusion of fluorinated-phenyl moieties, such as increased stability, surface area, and tendency for self-assembly in our systems. We then show how our fluorinated macrocycles open the door for the formation of extended macrocycle-based polymetric materials simply and in high yields via nucleophilic aromatic substitution. We show for the first time the formation of a trianglimine-based cross-linked polymer and demonstrate its use for micropollutant and gas separation.

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<p> Collagen, due to its good biocompatibility and abundance in mammalian structures, has been widely applied in developing better biomaterials. There remains the need for yet more stable surfaces of biomaterials. One strategy to achieve this is improved binding to surfaces using covalent rather than physical linking. However, due to collagen's poor solubility in neutral or alkaline conditions, there are only a few papers describing covalently linked collagen so far, and they generally use acidic conditions to generate surfaces with only low collagen density. N-Hydroxysuccimide ester (NHS) chemistry has been widely used in covalently binding proteins, but the NHS activity and its preparation efficiency are plagued with undesired, premature hydrolysis. A two-step method was developed for making NHS functional surfaces with a non-fouling spacer, PEO. The process was more efficient and led to concentrated NHS surfaces. Collagen was successfully immobilized onto this NHS surface after optimizing the conditions for immobilization. The solubility problem was overcome by increasing the ionic strength of the solution. Abundant collagen molecules could then be immobilized on the silicone surface. ATR-FTIR was used as a diagnostic tool to prove the surface had been modified. The low water contact angle (40°) indicated the presence of collagen. XPS data showed a significant increase on the nitrogen content after tethering collagen molecules. Deep freezing ToF-SIMS displayed a decrease in the peak intensity for cationic fractions of collagen molecules when warming from -96 °C to room temperature, which suggested the surface rearrangement due to the hydrophilic character of collagen. Profilometer and tapping-mode AFM were used to investigate the surface morphology after modification. The latter showed a high density mesh work (immobilized collagen fibers) on the collagen-modified surface. Collagen stain with Sirius Red F3B allowed us to look into the tertiary structures of covalently tethered collagen on the surface. However, it was found that only some of them were still in their native form. Interestingly, a subsequent epithelial cell culture assay showed that the cells grew very well on this collagen rich silicone surface. This suggested collagen's tertiary structure may not be necessary to support cell growth on the silicone surface covalently modified with collagen through the PEO spacer. However, further biochemical experiments are required to establish the underlying source of this observation.</p> / Thesis / Master of Science (MSc)

Fouling properties of materials such as polyethylene glycol (PEG) have been extensively studied over the past decades. Traditionally, the factors believed to result in protein adsorption resistance have included i) steric exclusion arising from the compression of longer chains and ii) grafting density contribution which may provide shielding from the underlying material. Recent studies have suggested that PEG interaction with water may also play a role in its ability to resist protein adsorption suggesting that steric exclusion may not be the only mechanism occurring during PEG/protein interactions. Star-shaped PEG polymers have been utilized in protein adsorption studies due to their high PEG segment concentration, which allows to increase the PEG chain grafting density compared to that achieved with linear PEG chains. Most studies that have investigated the interactions of tethered linear and star-shaped PEG layers with proteins have considered linear PEG molecules with molecular weights several orders of magnitude smaller than those considered for star-shaped PEG molecules (i.e. 10 000 g/mol vs. 200 000 g/mol, respectively). Additionally, the star-shaped PEG molecules which have been considered in the literature had up to ~70 arms and were therefore modeled by hard-sphere like structures and low chain densities near the surface due to steric hindrance. This resulted in some difficulties to achieve grafted PEG chain overlap for star molecules. Here, triethoxysilane end-functionalized linear PEG molecules have been synthesized and utilized to form star-shaped PEG derivatives based on ethoxy hydrolysis and condensation reactions. This resulted in PEG stars with up to ~4 arms, which were found to result in grafted star-shaped PEG chains with significant chain overlap. Linear PEG derivatives were synthesized so that their molecular weight would match the overall molecular weight of the star-shaped PEG molecules. These 2 PEG molecular architectures were covalently self-assembled to hydroxylated silicon wafers and the thickness, grafting density, and conformation of these films were studied. The adsorption of human albumin (serum protein) on linear and star-shaped PEG films was compared to that obtained on control samples, i.e. uncoated silicon wafers. Both film architectures were found to significantly lower albumin adsorption. / Ph. D.

Cancer immunotherapy has long been proposed as a powerful approach to curing tumors, based on the natural function of the immune system in protecting its host with specificity, thus holding the potential for developing long-term memory that prevents tumor recurrence. However, the immunosuppressive feature of the tumor microenvironment prevents the patients' own immune system from functioning normally in the fight against cancer. As one of the most potent cancer immunotherapies, immunostimulatory cytokines have been shown to elicit anti-tumor immune responses in preclinical studies, but their clinical application is limited by severe immune-related adverse events upon systemic administration. None of the current delivery strategies can fully address issues of toxicities and sustainably supply cytokines over the course of a few days without compromising cytokines' structural integrity. Herein, we have developed a novel formulation to anchor potent cytokine molecules to the surface of large-sized particles (1 µm) for local cancer treatment. The cytokines are confined in tumors and have minimal systemic exposure over a few days following intratumoral injection, thereby eliciting anti-tumor immunity while avoiding the systemic toxicities caused by the circulating cytokines. Such particle-anchored cytokines can be synergistic with other immunotherapies, including immune checkpoint blockade antibodies and tumor antigens, to safely promote tumor regressions in various syngeneic tumor models and genetically engineered murine tumor models. / Doctor of Philosophy / Cancer immunotherapy is a promising method to treat cancer by harnessing the power of the body's immune system, which naturally fights off diseases and can remember and prevent diseases from returning. Unfortunately, cancers create a hostile environment that weakens the immune system's ability to combat the disease effectively. Among the treatments explored, immunostimulatory cytokines (unique proteins that boost the immune system) have shown great promise in laboratory studies for their ability to fight cancer. However, when these proteins are administered to patients, they can cause severe side effects due to their systemic dissemination throughout the body. Herein, by attaching the potent cytokines to large-sized particles (1 µm), and injecting them directly into the tumor, their cancer-fighting abilities are focused precisely where they are most needed. This targeted delivery minimizes the cytokines' presence in the rest of the body, dramatically reducing the risk of side effects associated with their systemic dissemination. This method not only shows promise on its own but also enhances the effectiveness of other cancer treatments. Our findings suggest a new, safer way to encourage the body's defense system to fight cancer more effectively.