Chemistry - Theses, Dissertations, and other Required Graduate Degree Essays

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Development of Novel Instrumentation and Methodologies for Particulate Bound Phthalate Measurements

Date created: 
2015-07-16
Abstract: 

Presented is the development of a method to generate reproducible gaseous phthalate standards, a setup for generating airborne particulate matter and a membrane introduction mass spectrometry system suitable for the online measurement of gaseous phthalates. Upon development of appropriate standards and instrumentation, particulate-phthalate adsorption/desorption experiments were performed using dimethyl phthalate and two particulate species. Initial adsorption/desorption experiments were conducted using C18 coated silica particles, followed by further experimentation with house dust particles. Results from the development stages of the gas phase standards, the membrane introduction mass spectrometry system and phthalate adsorption/desorption experiments are presented.

Document type: 
Thesis
File(s): 
Senior supervisor: 
George Agnes
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Development of Gamma-Ray Spectroscopy Techniques for Fundamental and Applied Research

Author: 
Date created: 
2015-09-23
Abstract: 

The subject of this thesis is the methodology used to characterize high-purity Germanium (HPGe) detectors used for gamma-ray spectroscopy. To this end results of the acceptance tests performed on Gamma-Ray Infrastructure For Fundamental Investigation of Nuclei (GRIFFIN) detectors completed at the SFU Nuclear Science Laboratory (SFU-NSL) are provided. GRIFFIN is a state-of-the-art HPGe gamma-ray array for decay spectroscopic studies at TRIUMF, Vancouver, BC. GRIFFIN's efficiency allows decay spectroscopy studies at TRIUMF to be extended deeper into regions far from stability.Individual GRIFFIN detectors were sent to SFU-NSL for testing purposes. To accommodate these detectors, an automatic cooling system and testing station with a digital and an analog data acquisition (DAQ) systems was set up. The digital DAQ was used for energy resolution and efficiency tests, the analog DAQ was used to examine the timing resolution of each crystal with respect to a BaF₂ scintillator. Based on the results discussed here, all 16 GRIFFIN detectors were accepted by the end of 2014. The GRIFFIN array is now complete and in operation.Several additional investigations of the GRIFFIN clover detector performance have been undertaken. Digital timing resolution with respect to a BaF₂ scintillator has been measured using 14-bit, 100 MHz digitizers. The improvement in photopeak efficiency via energy add-back of gamma rays which scatter between the four crystals of a clover has been measured with standard calibration sources. GRIFFIN detectors have also been used in conjunction with a wall of 24 CsI detectors to measure the angular correlation between the α particle and the gamma ray emitted in the decay of ²⁴¹Am.As a part of this thesis a novel method is proposed for establishing the absolute efficiency calibration of a HPGe detector including the confidence interval in the energy range of ~80-3450~keV. The calibrations were accomplished with the ¹³³Ba, ⁶⁰Co, ⁵⁶Co and ¹⁵²Eu point-like radioactive sources with only the ⁶⁰Co source being activity calibrated to an accuracy of 2% at the 90% confidence level. The proposed fit function accounts for scaling of the data taken with activity uncalibrated sources to the data taken with the high accuracy activity calibrated source.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Krzysztof Starosta
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) M.Sc.

Small molecule agents that target amyloid-β aggregation in Alzheimer's disease

Author: 
Date created: 
2015-07-23
Abstract: 

Alzheimer’s disease (AD) is the most common form of dementia and currently there is no cure. AD is characterized by the formation of two pathological hallmarks; aggregated forms of the amyloid-β (Aβ) peptide called Aβ plaques and hyperphosphorylated tau proteins, called neurofibrillary tangles (NFT). Aβ is enzymatically cleaved from the amyloid precursor protein (APP) to afford a 38-43 amino acid residue peptide with Aβ1-40 and Aβ1-42 being the most common. Plaque deposits have been shown to contain abnormally high concentrations of dysregulated metal ions, specifically Cu, Zn, and Fe. Metal-Aβ interactions have been shown to increase the rate of Aβ aggregation leading to increased neurotoxicity and oxidative stress. Specifically, Cu-Aβ species in stoichiometric amounts produce soluble, oligomeric species, which are hypothesized to play a role in AD. This thesis presents several strategies to influence metal-Aβ interactions in order to mitigate peptide aggregation and overall toxicity. Three triazole-based ligand scaffolds are presented that were designed to exhibit a range of properties including metal binding, peptide interactions, and antioxidant capabilities. Chapter 2 describes a series of pyridine-triazole ligands that have an affinity for the N-terminus region of the Aβ peptide where metal binding occurs. Chapter 3 builds from the previous chapter by extending the aromatic ring system to present a series of quinoline-triazole ligands. This framework demonstrated interactions with the peptide in the hydrophobic region (residues 17-21) of the peptide and was able to influence Cu-Aβ aggregation. Finally, chapter 4 describes a series of phenol-triazoles that compete with Aβ for binding of free Cu, interact in the hydrophobic region of the Aβ peptide, and exhibit antioxidant properties. Chapter 5 presents a new strategy to prevent Aβ aggregation through the use of KP1019, a Ru(III) anticancer agent. KP1019 readily binds to the Aβ peptide within 2 hours, modulating Aβ aggregation by producing non-toxic aggregates as demonstrated in a human neuroblastoma cell line (SH-SY5Y).

Document type: 
Thesis
File(s): 
Senior supervisor: 
Tim Storr
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Reversal of Multidrug Resistance Fluorescently Measured in Single Cancer Cells Captured in the Microfluidic Chip

Author: 
Date created: 
2015-08-12
Abstract: 

In chemotherapy, cancer cells may show resistance to a wide range of anticancer drugs, a phenomenon called multidrug resistance (MDR). A well-established cause of MDR in cancer cells involves transporter proteins, such as permeability-glycoprotein (P-gp), multidrug resistance associated protein-1 (MRP1) and breast cancer resistance protein (BCRP). These proteins usually cause efflux of anticancer drugs such as daunorubicin (DNR) and paclitaxel (PTX) from cells, leading to a reduction in cellular drug accumulation. Blocking the activity of transporter proteins using MDR inhibitors is an important approach to overcome drug efflux, a method called MDR reversal. The overall goal of this research was to develop microfluidic chips and methods to enhance intracellular drug accumulation in MDR cells or reverse MDR. To investigate MDR inhibition, a microfluidic method called same-single-cell analysis was used to trap and measure single cells. In this method, drug accumulation was first measured in the trapped cell treated with anticancer drug only (control experiment). Then, enhancement of drug accumulation was measured in the same single cell treated with the drug in the presence of a MDR inhibitor. Cancer cells are usually heterogeneous. This single-cell method allowed us to conduct same cell analysis that overcame the issue of cell heterogeneity. Our method also successfully demonstrated the enhancement of drug accumulation measured in the single cells from cryopreserved patient cells. In addition, the real-time measurement in single cells provided time-dependent kinetic information that correlated with the fold-increase of drug accumulation enhancement by MDR inhibitors. Most importantly, a new integrated microfluidic chip was developed to select and measure rare cancer cells among many blood cells as a model of circulating tumor cells (CTCs). Our label-free microchip was used to separate single prostate cancer cells and measure the drug accumulation enhancement by using MDR inhibitors. A method of capturing a prostate cancer cell among blood cells was developed with an ultimate goal to find the appropriate MDR inhibitors to enhance cellular accumulation of anticancer drugs, leading to more effective chemotherapy.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Paul C. H. Li
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) Ph.D.

Mechanism-based candidate inhibitors of galactopyranose mutase

Date created: 
2015-05-21
Abstract: 

The synthesis of candidate inhibitors of Mycobacterium tuberculosis cell-wall biosynthesis as potential therapeutic agents for the treatment of tuberculosis is presented. A critical component of the cell wall of M. tuberculosis is a D-galactan polymer, a polysaccharide chain consisting of D-galactofuranose (Galf) residues. Since Galf residues are not found in mammalian systems, inhibition of the biosynthesis of this polymer constitutes a very attractive and accessible target for new anti-TB drugs. A critical enzyme required for the biosynthesis of the galactan polymer is uridine diphosphate galactopyranose mutase (UGM), which catalyzes the interconversion of UDP-galactopyranose (Galp) and UDP-galactofuranose (Galf). The goal of the project was to synthesize compounds that inhibit growth of the cell wall by compromising the activity of the UGM enzyme. The compounds were intended to mimic not only the positive charge character of the galactopyranosyl cation (transition state in the UGM-catalyzed reaction) but also its shape (proposed 4H3 conformation).

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert N. Young
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Conformational dynamics of linked discotic mesogens

Date created: 
2015-08-17
Abstract: 

Disc-shaped molecules often assemble into columnar liquid crystal phases, which are a promising class of materials that can be utilized in applications such as organic light emitting devices (OLEDs) and photovoltaics. Discotic dimers, which consist of two tethered discotic mesogens, also form columnar phases, but only when the linking group exceeds a critical length. However, the effects of the linking group on the self-assembly and solution properties remain poorly understood. A strategy was developed in order to design discotic dimers that self-assemble into liquid crystals even when the linking group is short. These studies investigated the effects of varying this linking group and how it effects the observed solution and liquid crystal properties. Disc-shaped dimers were formed through functionalization of compounds prepared from the condensation of 2,3,6,7-tetrakis(alkoxy)phenanthrene-9,10-diones with 3,4-diaminobenzoic acid. The solution properties of these dimers were studied by a variety of NMR techniques, while the materials properties were characterized via polarized optical microscopy, differential scanning calorimetry, and variable temperature X-ray diffraction. It was confirmed that the flexibly-linked dimers adopted intramolecular folded conformations. This folded conformation facilitated the formation of a columnar hexagonal liquid crystal phase in all cases, despite the short linking group for some of the compounds studied. This folded geometry was found to be largely independent of concentration, temperature, and solvent, indicating that the folded geometry is robust. In order to determine if the flexibility of the linking group was essential to the folded geometry observed for the flexibly-linked dimers, a phenyl linker dimer was prepared in order to investigate the solution and phase behaviour of a conformationally-rigid dimer. This compound cannot adopt a folded conformation in solution or the liquid crystal phase, but nonetheless is able to form a columnar oblique liquid crystal phase. A dimer that had properties of both the rigid and flexibly linked dimers was synthesized by utilizing cyclohexane as a linking group in order to further investigate and quantify the folded conformation adopted by the flexibly-linked dimer. This compound was found to adopt predominately a diaxial conformation, despite the large substituents, which facilitated the formation of a columnar hexagonal liquid crystal phase. A flexibly-linked discotic trimer was prepared in order to investigate higher order oligomers and to investigate what effects the addition of a third disc would have to the solution and materials properties that were observed for the dimeric systems. This compound was found to adopt an intramolecular folded conformation in which only two discs are folded at any given time in solution. This dominant conformation resulted in the absence of liquid crystal phase formation.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Vance Williams
Department: 
Science: Department of Chemistry
Thesis type: 
(Thesis) Ph.D.

Gypsy Moth Pheromone Binding Proteins: Ligand Interactions and Antennal Mapping

Date created: 
2015-07-02
Abstract: 

Pheromone binding proteins (PBPs) are believed to be part of the transport of hydrophobic pheromone in the sensory hairs of insects. In this thesis, I have studied two gypsy moth (Lymantria dispar) PBPs for their interactions with the main sex attractant pheromone, (+)-disparlure, (7R,8S)-epoxy-2-methyloctadecane. I also studied interactions of the PBPs with the enantiomer of the pheromone, (-)-disparlure and both enantiomers of heteroatom substituted analogues. These studies were prompted by the fact that the interaction of these PBPs with these natural and artificial ligands is not completely understood. To address this problem, docking simulations of the protonated homology PBP models with the enantiomers of various ligands (disparlure, 5-oxadisparlure, 10-oxadisparlure, 5-thiadisparlure and 10-thiadisparlure) were performed together with a binding assay. A second question regarding the two PBPs of gypsy moth is how they are distributed on the moth’s antennae. Mapping the antennal PBP distribution on the moth was done through cutting the antennae into known sections and determination of PBPs by Western blotting. The result of molecular simulations revealed different amino acid residues in the binding sites of PBP, abrupt movement of specific amino acid residues at a certain pH and distinct amino acid-ligand interactions (sidechain donors/acceptors, H-arene bonding, backbone donors/ acceptors) of the best bound conformers of each protein-ligand complex. Overall, the results may correlate with the pKa values obtained from the binding assay, and explain the enantioselectivity of the gypsy moth PBPs. Mapping the antenna was accomplished and suggests that the two PBPs are present in similar general distributions. The symmetrical distribution of the PBPs is related to the number of sensilla found in the branches calculated by taking total and normalized PBP weights.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Erika Plettner
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Studies Toward the Total Synthesis of Biselide A

Author: 
Date created: 
2015-05-12
Abstract: 

The use of natural products for medicinal purposes is a tradition dating back to ancient times. To this day bioactive natural products continue to inspire a large proportion of the new pharmaceuticals that are developed each year. Many natural products serve as drug leads and inspire the synthesis and development of more potent analogues. The biselides and haterumalides are two members of a class of recently described bioactive polyketide natural products that have been found in Okinawan ascidians. Many of the biselides and haterumalides exhibit anticancer activity, yet very little material is available from the natural sources. Of particular interest is biselide A, which is the only member of this class to demonstrate selective killing of human cancer cells over healthy cells. It has been proposed that C20 oxygenation in biselide A confers this selectivity and thus, derivatives with C20 oxygenation are also of pharmaceutical interest. While multiple syntheses of haterumalides have been published, biselide A has not yet been synthesized. This thesis highlights recent efforts towards a scalable total synthesis of biselide A. Three different approaches have been explored: the first incorporates a Horner-Wadsworth-Emmons reaction as a key step, and the second and third use a cross metathesis key step. While the Horner-Wadsworth-Emmons approach was ultimately unsuccessful, our successes in the cross metathesis approach should now yield access to this potentially important natural product.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert Britton
Department: 
Science: Chemistry
Thesis type: 
(Thesis) M.Sc.

Development of Assays and Irreversible Inhibitors for ATG4B: A Key Cysteine Protease Implicated in the Process of Autophagy

Author: 
Date created: 
2015-04-21
Abstract: 

Autophagy is a biological process responsible for the sequestration and degradation of cellular components for the purposes of maintaining homeostasis. ATG4B is a cysteine protease that plays a key role in the initiation of autophagy by cleaving the protein proLC3 revealing a C-terminal glycine residue and to form LC3-I. This glycine then becomes lipidated with phosphatidylethanolamine (PE) to form LC3-II which is inserted into partial protomembrane fragments in the Golgi which stimulates formation of autophagosomes. ATG4B also cleaves PE from the membrane bound LC3-II to reform and recycle LC3-I and to facilitate fusion of the autophagosomes with liposomes where the contents are subsequently digested producing energy and amino acids for protein synthesis. As cancer cells frequently “hijack” autophagy as a survival and resistance mechanism against therapy, autophagy has recently emerged as a potential therapeutic target for treating cancer. The development of two different assay methods for measuring ATG4B activity are reported. The first, LC/MS-based, assay monitors the cleavage of proLC3 to LC3-I based on the ratio of peak heights detected. This method has the added utility of being able to detect enzyme integrity throughout the reaction. The second, FRET-based, assay involves a novel YFP-LC3B-EmGFP doubly fluorescent protein substrate developed for the purposes of large scale screening of compound libraries. The FRET-based assay was successfully used to screen 5000 compounds in the commercial LOPAC and KD2 compound libraries with an overall hit rate of 0.6% and 0.5% respectively. Irreversible inhibitors of ATG4B were designed and synthesized based on information from the screening results as well as previous work on putative peptide substrates conducted by Dr. Nag Kumar (Young Lab, SFU). Inhibitors of the halo-methyl ketone type were developed and tested for inhibitory activity against ATG4B. Fluorescent analogues of these irreversible inhibitors were further developed and optimized for in cellulo potency. Kinetic analysis and digestion studies revealed the inhibitors were indeed active-site directed ATG4B inhibitors.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert Young
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Gold Nanoparticle-Enhanced Detection of Single Nucleotide Polymorphisms in the NanoBioArray Chip

Author: 
Date created: 
2015-04-16
Abstract: 

In this thesis, we report the use of gold nanoparticles (AuNPs) to enhance the detection of single nucleotide polymorphism (SNPs) in the NanoBioArray (NBA) chip. A combination of gold nanoparticles (AuNPs) and nucleic acids has recently been used in many biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Our kinetic analysis shows that in the presence of AuNP-ssDNA interactions, mechanisms of DNA hybridization and dehybridization are altered. Our proposed mechanisms include a shift of the rate-limiting step of hybridization from mismatch-insensitive to the mismatch-sensitive zipping step. Furthermore, the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the mismatched (MM) duplex DNAs, destabilize the duplexes and accelerates the dehybridization process. We employ these alterations in mechanisms, both of which disfavor the formation of MM duplexes, to enhance the detection of SNPs in the NBA chip. In this technique, we load the target DNAs on the surface of AuNPs (i.e. AuNP targets) and then introduce them to the surface-immobilized probes for DNA hybridization. Our results show that AuNP targets, in contrast to the targets free in the solution (free targets), were able to discriminate between the perfectly matched (PM) probes and the mismatched (MM) ones. Using AuNP targets, we developed a room-temperature method for detection of SNPs in the KRAS gene codon 12 in the NBA chip. Then, a novel wash method based on AuNPs was developed to preserve the DNA hybridization signals in CD-NBA chip while discriminating MM duplexes from PM duplexes. In this method, AuNPs are suspended in the wash buffers to preferentially destabilize the MM duplexes, in presence of the PM duplexes. Enjoying this targeted mechanism, AuNP wash method enhances specificity without compromising signal intensity. This method is simple and compatible with multiplexed DNA hybridization settings. The findings in this thesis can be used to enhance the reliability of DNA biosensors (e.g. DNA microarrays) and might lead to new applications in DNA biosensing.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Paul C. H. Li
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.