Chemical Sciences

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Unveiling the metabolic network underlying mitochondrial and nuclear metabolism in a model differentiating stem cell
(Laurentian University of Sudbury, 2013-10-07) Han, Sungwon
Participation of metabolism in stem cell differentiation has been largely disregarded until recently. Here, functional proteomics and metabolomics were performed to unveil the mitochondrial and nuclear metabolism during dimethyl sulfoxide (DMSO)-induced differentiation of P19 cells. DMSO-treated cells were shown to exhibit increased glycolytic enzymes activities and fuel pyruvate into oxidative phosphorylation. Subsequently, enzymes of electron transport chain also had elevated activities upon differentiation. These changes in mitochondrial metabolism were concomitant with increased mitochondrial biogenesis as PGC-1α expression was higher in the differentiated cells. To study nuclear metabolism, particular focus was placed on delineating a potential role of nuclear lactate dehydrogenase (LDH). Nuclear LDH was found to exhibit higher expression in pluripotent cells. NAD+ generated from LDH reaction was discovered to promote histone deacetylation via sirtuin-1 (SIRT1). Drastic alterations in mitochondrial and nuclear metabolism during differentiation point to a pivotal role of metabolism in deciding the final destination of stem cells.
Evaluation of liposomal bismuth-ethanedithiol-tobramycin for treatment of cystic fibrosis pulmonary pseudomonas aeruginosa infection
(Laurentian University of Sudbury, 2013-10-08) Alhariri, Moayad Abdulaziz I.
The effectiveness of liposomes incorporating bismuth-ethanedithiol and loaded with tobramycin (LipoBiEDT-TOB) at sub-inhibitory concentrations to inhibit the production of quorum sensing signaling molecules and virulence factors induced by P. aeruginosa was evaluated in vitro. In addition, we evaluated the efficacy and safety of free and encapsulated tobramycin in liposomal formulations administered intratracheally to rats chronically infected with P. aeruginosa. LipoBiEDT-TOB significantly reduced the production of quorum sensing signaling molecules and virulence factor secretion compared to free tobramycin. The LipoBiEDT-TOB formulation significantly reduced the bacterial count in lungs, modulated the IL-8 level in blood and minimized the nephrotoxicity that is associated with aminoglycoside treatment. These results support the hypothesis that aerosolization of liposomal aminoglycosides may enhance the management of chronic lung infections caused by resistant P. aeruginosa in patients with cystic fibrosis.
Transvection is a plastic phenotype
(Laurentian University of Sudbury, 2013-10-30) Bing, Xinyang (David)
Transvection, a chromosome pairing-dependent form of trans-based gene regulation, is widespread in the Drosophila melanogaster genome. Recent studies demonstrate that transvection is sensitive to cell environment and type in D. melanogaster, implicating transvection as a complex trait. To test this possibility, we first established that trans-interactions previously documented at the Malic enzyme (Men) locus are transvection (i.e., pairing-dependent). We then characterized the sensitivity of transvection at the Men locus to changes in the environment (temperature) and genetic background (third chromosome). Transvection varied significantly across genetic backgrounds and was significantly reduced by changes in temperature, and the two factors interacted to further modify transvection, while cis-based gene regulation remained unchanged by temperature. To determine if differences in transvection observed across genetic background and temperature are related to their effects on transcription factor expression, and possibly the presence or absence of binding sites for these transcription factors within the Men locus, we tested the relationship between Men expression and five transcription factors with binding sites near the Men transcription start sit (TSS). We found correlations between the expression of at least one transcription factor, Abd-B, and the presence of binding sites for that factor, and Men expression across changes in the environment. We also determined that changes in Abd-B expression can directly affect Men expression in cis, suggesting that cis and trans-regulation can share regulatory components in at least some cases. Together, our findings stress the importance of studying genetic interactions from a dynamic perspective by incorporating both genetic and environmental variation.
Determination and speciation of tellurium in environmental samples using hydride generation atomic fluorescence spectroscopy (HG-AFS)
(Laurentian University of Sudbury, 2014-01-27) Alzahrani, Ali
This thesis focuses on developing a new method to measure trace tellurium (Te) in different environmental samples such as lake waters, mine tailings and sediments. The developed technique is based on Hydride Generation Atomic Fluorescence Spectroscopy (HG-AFS), a technique that can measure low concentration of Te and also allows for Te speciation at low cost and high efficiency in various environmental samples. To validate the method that could be used to determine Te speciation in various types of environmental samples, a series of tests has been designed for finding the best conditions to measure Te(IV) using HG-AFS and obtain accurate and reliable results. Those tests include the stability of the signal, the acidity of the solution, the volatility of Te after digestion of solids, the reduction from Te(VI) to Te(IV), the detection limit of the technique, and the validity of two digestion methods under the optimum (HG-AFS) instrumental settings. An interference study including the most common elements in the Earth’s crust such as (Ni, Fe, Pb, Cr, Cu, Co, Zn, Mn and Mo) was also performed. The results of this study showed that Cu(II) can severely interfere with Te quantification decreasing the Te signal to almost zero. Therefore, different masking agents such as 8-hydroxyquinoline, 1,10-phenanthroline, urea and thiourea were tested to reduce and eliminate this interference.
Deposition and characterization of mesoporous silica coatings on magnesium alloys
(Laurentian University of Sudbury, 2014-03-17) Al Hegy, Afrah
In recent years, magnesium and magnesium alloys have received much attention as a new biomaterial in orthopaedic applications due to their biodegradability, biocompatibility, and their mechanical properties that are similar to natural bone tissue. The most common problem associated with magnesium as a biomaterial is low corrosion resistance in physiological solutions. This decreases the mechanical integrity of the implants in the early stages of healing and has a negative impact on the overall biocompatibility. The main goal of this study was to create a multi-layered coating consisting of a silica sol-gel under-layer to protect the substrate from corrosion in body fluids and a mesoporous silica top-layer to enhance the bioactivity of the coated implant material. The results indicate that the deposited multi-layered coating enhances both the bioactivity and the corrosion resistance of the material.
Comparison of multiple drug and metabolite levels recovered from skeletonized remains following standard passive extraction, microwave-assisted extraction and ultrasonic solvent extraction and GC-MS or UPLC-DAD
(Laurentian University of Sudbury, 2014-03-17) Betit, Caroline
Fate of limestone dissolution products in acidic metalcontaminated soil mesocosms
(Laurentian University of Sudbury, 2014-03-17) Driscoll, Kendra
The impact of liming (10 t ha-1 of calcitic and dolomitic limestone, separately) on the soil solution and soil matrix was investigated in an acidic metal-contaminated soil from Sudbury, ON. A soil mesocosm experiment was performed; columns were leached with simulated rainwater and the soil solution collected at various locations throughout the soil column. The dissolution rate of calcitic limestone used for this experiment was found to be approximately double that of the dolomitic limestone investigated. Calcium and Mg released during limestone dissolution migrated through the soil profile to the Bf-BC interface. The addition of limestone increased the pH and decreases the bioavailability of Ni, Cu, Co, Cd, As, Ba, Mn, and Zn the LFH horizon. Amending acid, contaminated soils with calcitic or dolomitic limestone has profound effects on soil solution chemistry.
Determining the metabolic profiles in drosophila melanogaster: development and application of a novel ion-pairing liquid chromatography-mass spectrometry protocol
(Laurentian University of Sudbury, 2014-03-17) Knee, Jose
Genetic perturbations and foreign chemicals can result in a multitude of changes across a wide range of biochemical processes in a biological system. These perturbations may affect the metabolome, the small molecule metabolites in an organism. Recently, liquid-chromatography coupled to mass spectrometry (LC-MS) technology has been used to quantify large proportions of the metabolome, however standardized protocols are not yet available for use with Drosophila melanogaster. Here, I developed an ion-pairing LC-MS protocol for the metabolomic characterization of D. melanogaster and demonstrated its implementation in establishing the metabolomic profile of flies under oxidative stress and in the metabolic profiles of four different Drosophila species. I demonstrated that this new method allows for the detection of otherwise difficult metabolites and that it is repeatable and sensitive with acceptable levels of ionsuppression, matrix effects, limits of detection and quantification. I then used this method to determine and quantify the metabolomic fingerprints of loss of Superoxide dismutase activity and paraquat-induced stress. Comparing and contrasting the effects of these two sources of oxidative stress, I document both similarities and stressor-specific effects.
Re-examining temporal and seasonal microbial acid mine: drainage community variation
(Laurentian University of Sudbury, 2014-03-19) Auld, Ryan Richard
Acid Mine Drainage (AMD) is characterized by high metal concentrations and an extremely low pH, primarily generated by the microbial oxidation of iron sulfides from mine tailings. Research on the microbial AMD community has largely focused on Bacteria, while little information is known about the Archaeal and Eukaryote members or the seasonal patterns within the communities. Here I examined the Bacterial, Archaeal, and eukaryotic AMD seasonal microbial community, using direct sequencing techniques on AMD samples from the Copper Cliff Tailings AMD site in Sudbury, Ontario, Canada. I found large variation in the community profile and species composition between sampling times of both the Bacterial and Eukaryote communities, suggesting a dynamic community, both between and within seasons. Bacterial diversity was highest during the winter, with Acidithiobacillus dominating, while during the summer, Acidiphilium was the dominant genus. The winter Eukaryote community was dominated by classes of algae and fungi, while the majority of summer sequencing could not be classified to the class level. Few reads were obtained for the Archaeal domain, with low and similar biodiversity between seasons. Overall, the AMD community variation and abundance were found to largely correlate with drainage water and seasonal temperature.
Examining the distribution of ketamine and metabolites in decomposed skeletal tissues: development of a high throughput approach and application to an environmental and decomposition study
(Laurentian University of Sudbury, 2014-08-07) Cornthwaite, Heather
The use of skeletal tissues in forensic toxicology research has increased dramatically in the last 5 years. These studies have yielded valuable information pertinent to using skeletal tissues as a biological matrix in drug analysis. The majority of these studies have been completed on a small scale so a larger scale is needed for further research. Here, a high throughput microwave assisted extraction (MAE) and microplate solid phase extraction (MPSPE) protocol is developed and implemented to examine how body position and microclimate affect ketamine and metabolite distribution in decomposed skeletal tissue. Analytes were successful recovered in 30 min following MAE compared to 180 min for passive solvent extraction (PSE). Body proved to be significantly different in the observed drug levels at both microclimates. The sunlit microclimate had significantly higher drug levels, with less maggot activity, compared to the shaded microclimate.
Surface modification of magnesium alloy AZ31 for improved biocompatibility
(Laurentian University of Sudbury, 2014-08-21) Xiaoxi, Yang
Synthesis and characterization of oligo/polythiophenes bearing stable radicals
(Laurentian University of Sudbury, 2014-09-02) Almubayedh, Somaiah
Two categories of verdazyl radical functionalized oligothiophenes have been prepared: 1,5-diisopropyl-6-oxoverdazyl radical directly linked to terthiophene (2.6), and 1,5- diisopropyl-6-oxoverdazyl radical attached to terthiophene through a π system bridge, e.g., pyridine (2.11). Compound radical 2.6 was prepared by a two step process starting with the condensation of terthiophene carboxaldehyde and 2,4-diisopropylcarbonhydrazide bis-hydrochloride to give the tetrazane 2.5, which was subsequently oxidized chemically to give the corresponding verdazyl radical grafted at the 3’ position of the terthiophene moiety. The latter displays excellent stability toward organic solvents and moisture. The electropolymerization of radical 2.6 resulted in the formation of its polymer (poly(2.6)), which was characterized by cyclic voltammetry and infrared spectroscopy. The electrochemical oxidation of the tetrazane, 2.5, yielded surprisingly a poly(terthiophene) bearing verdazyl radical, with similar electrochemical and infrared properties of those found for the polymer produced in the electro-oxidation of the terthiophene bearing verdazyl radical (poly(2.6)). Moreover, the electrochemical oxidation of 2.5 (beyond its oxidation potential of the tetrazane motif) affords the radical 2.6. A similar strategy has been used for the synthesis and the characterization of tetrazane 2.10 and its corresponding verdazyl 2.11.
Self-organized inorganic-organic phyllosilicate hybrids: investigating bridging ligands and imprinting methods for heavy metal ion adsorption
(Laurentian University of Sudbury, 2014-10-17) Bizley, Thomas
The talc-like organoclay, MgSi-b-en, was synthesized using a bridged organosilane precursor containing the ethylenediamine moiety through the sol-gel process. Characterization of the material revealed disordered, interconnected phyllosilicate sheets, with a high surface area and a composition resembling natural talc. Ethylenediamine moieties were successfully incorporated into the structure of MgSi-b-en. The material performed well as an adsorbent of the heavy metal ions Cu(II), Ni(II), and Pb(II). The adsorption capacities of the adsorbent were very high. Multi-component adsorption tests revealed a specific adsorption preference of Pb > Cu > Ni. An ionimprinting technique was performed to alter the specificity of the adsorbent. The procedure produced organoclays with cavities matching the size of the metal ion used in the synthesis. In the presence of larger ions, the two imprinted adsorbents, MgSi-b-en(- Cu) and MgSi-b-en(-Ni), displayed higher adsorption capacities and selectivity coefficients towards Cu(II) and Ni(II) ions, respectively.
The inhibition of the VIM-2 and IMP-1 METALLO-β-LACTAMASES by cationic peptides
(Laurentian University of Sudbury, 2014-12-04) Rotondo, Caitlyn Michelle
Metallo-β-lactamases (MBLs) are major contributors to bacterial antibiotic resistance due to their ability to cleave most β-lactam antibiotics. At the present time there are no clinically approved inhibitors of MBLs. However, previous preliminary studies have demonstrated that cationic peptides have the ability to inhibit these enzymes. Herein, more thorough investigations into the structure-activity relationship between two MBLs, VIM-2 and IMP-1, and cationic peptides are described, demonstrating that the inhibitory potency of these peptides increased with the number of arginine residues (IC50 of 10 nM for the most potent VIM-2 inhibitor). The degree of inhibition also varied depending on the β-lactam substrate and the MBL employed. Furthermore, steady-state and stopped-flow spectrophotometric studies demonstrated that the inhibition mediated by these peptides followed a complex mode of inhibition. In addition, results from spectroscopic studies (including UV-Vis, tryptophan fluorescence and dynamic light scattering) showed that the inhibition of MBLs by cationic peptides results in partially reversible peptide-influenced MBL aggregation. The better understanding of the relationship between these peptides and MBLs may lead to the development of novel MBL inhibitors suitable for clinical use.
A generalized electronic diabatic model applied to two-state reactions.
(Laurentian University of Sudbury, 2015-01-27) Laverdure, Laura Bray
A generalized electronic diabatic model for chemical reactions includes a physical mechanism for the transition from a reactant-like to a product-like quantum state, namely, an external field. In our model, an external electric field couples states and modifies effective potential energy surfaces thereby allowing to treat a reaction as a fully quantum process. Through semi-classical models of two-state reactions, we show that we can control the identity of the most stable nuclear configuration by varying the form and intensity of the external field’s coupling potential. We group topologically equivalent potential energy curves in phase diagrams for a manifold of simple two-state models. We also illustrate the method’s implementation in a fully quantum-mechanical approach by considering two diabatic states in the radical HBN⇄BNH isomerization. To ensure diabaticity, these states are built on a grid of floating Gaussian orbitals and the potential energy curves are constructed by moving the nuclei.
A highway of pollution with no boundaries: a study using lead isotope ratios in wet deposition and lichen to trace atmospheric pollution
(Laurentian University of Sudbury, 2015-05-27) Akerman, Tamra
To fingerprint lead deposition over approximately a two year timeframe, possible variationsin the sources of lead pollution over Northern Ontario was investigated by collecting snowpack, rainwater and lichen. The collection of samples was along two main transects: A West East transect between Sault Ste. Marie and Sudbury; and a South North transect from Sudbury up through Timmins to Kapuskasing. Sample collection along these two transects was conducted during the summer of 2009 and during the Shutdown of Vale Inco’s Smelter in the winter month of January 2009. The objective was to determine if Pb anthropogenic pollution from major point sources in northern Ontario (Essar Steel Algoma, Xstrata Sudbury Smelter Operations, Xstrata Kidd Metallurgical Plant) could be traced using isotopic ratios. As a result the Pb isotope fingerprint in northern Ontario can be explained by a binary mixing of anthropogenic emissions from northern Ontario refining operations and coal fired power plant emissions that are transported with prevailing winds from the Ohio Valley. Major point sources showed distinct differences in signatures and samples that were not collected in proximity to a point source were diluted and greatly influenced by the U. S signature.
Induced pluripotent stem cells: where are we today?
(2015-10-02) Dénommé, Ginny Michelle
For many years, scientists have been trying to elucidate the molecular mechanisms that convey pluripotency and self-renewal to stem cells. With this valuable knowledge, they hoped to discover relevant information in the development, growth and regeneration of cells, tissues, and organisms that allow organisms to live as long as they do. These characteristics were once thought to be present only in embryonic stem cells (ESCs), and a few adult multipotent cells such as hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Recent studies have tapped into the potential of our once thought to be terminally differentiated adult cells to produce self-renewable, pluripotentiating cells, now termed induced pluripotent stem cells (iPSCs). This critical review will discuss the advantages and limitations of the methods developed to generate and characterize iPSCs. Advantages and challenges of the use of iPSCs in applications such as research and therapeutics will also be discussed.
Synthesis of peptide-derived tetraoxazole containing macrocycles
(2016-02-19) Al Radhwan, Azizah.
Natural macrocyclic peptides isolated from marine metabolites have shown interesting biological activities, including anticancer properties. In addition, they are highly pre-organized for metal inclusion due to their structural features, such as the presence of a cavity with perfectly located oxygen and nitrogen donors within the macrocyclic structure. This thesis pertains to the development of a synthetic approach towards the synthesis of natural macrocyclic structures. These macrocyclic peptides are made up of four oxazole rings connected by amide bonds with functionalized side chains. A synthetic strategy for the synthesis of natural peptide-derived macrocyclic analogues was developed using solution phase peptide synthesis. The synthesis of the tetraoxazole macrocycle is described in more detail where the synthesis started with the formation of a dipeptide by coupling two amino acids, followed by oxazole ring formation, and subsequent formation of the di-, tri-, and tetraoxazole peptide. All of the peptide bonds were obtained using O-(benzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate (HBTU) or 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) with hydroxybenzotriazole (HOBt) with the base N,N-diisopropylethylamine (DIEA). Finally, macrocyclization of the linear unprotected tetraoxazole gave the macrocyclic structure. The macrocyclization was achieved efficiently by using (ethyl cyano(hydroxyimino)acetato)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate (PyOxim) as a coupling reagent under very dilute conditions. All structures were analyzed by 1H and 13C NMR. With the structural similarities to the macrocyclic peptides isolated from marine sources, this tetraoxazole macrocyclic peptide might be used as a chelating agent.
An EPR and DFT investigation of the products formed in the reaction of group 11 metal atoms with three-membered heterocyclic compounds
(2016-03-29) Orsini, Chad
Transition metals have been shown to be efficient catalysts in a variety of organic reactions. In particular, Group 11 metals have emerged as essential components in the carboxylation or metal-catalyzed ring expansion of three-membered heterocyclic compounds. Previous reports have speculated the presence of short-lived metal-substrate complexes and metallacyclic intermediates along the reaction path, yet to date there is no direct evidence for their existence. Using cryogenic matrix isolation EPR spectroscopy, we were able to characterize several highly reactive intermediates formed in the reactions of Group 11 metal atoms, Cu, Ag, and Au, with small, strained, heterocyclic compounds, namely, oxiranes, thiiranes, and aziridines. Experimental data indicates that the major products formed are mononuclear metal complexes, M-XCH2CH2 (X = O, S or NH). In addition, reaction mixtures containing Cu atoms and oxiranes or thiirane yield novel metallacycles, i.e., cupraoxetane and cuprathietane, respectively. DFT calculations were also used to support the structural assignments of the M-XCH2CH2 complexes.
Formulation and efficacy of liposome-encapsulated azithromycin for pulmonary infection due to Pseudomonias Aeruginosa
(2016-05-02) Solleti, Venkata Saran Tejaswi
Cystic fibrosis is the most common life-threatening autosomal recessive disorder in Caucasians. Recurrent pulmonary infection and inflammation are the major risk factors associated with cystic fibrosis. Microbial infection with highly resistant pathogens such as Burkholderia cenocepacia and Pseudomonas aeruginosa, is principally associated with cystic fibrosis. The effective management of pulmonary infection in cystic fibrosis patients is not controllable due to the multidrug-resistant strains and potential side effects of antibiotics usage. Liposomal encapsulation of macrolide antibiotics such as azithromycin show increased drug concentrations at the site of infection, along with reduced toxic effects. In this thesis work, liposome-loaded azithromycin formulation was prepared by dehydration-rehydration vesicle method and related characterizations including cytotoxicity were identified. The effects of liposomal azithromycin on biofilm community, purified bacterial virulence factors were determined and motility studies were performed in clinical isolate of P. aeruginosa. We found that liposomal azithromycin reduced biofilm activity, virulence factors production and bacterial motility. The liposomal formulation confirmed interactions between liposomes and bacterial membranes besides insignificant hemolysis or A549 cell toxicity. The end results collectively indicate that liposomal drug delivery systems could be a promising model to enhance the efficacy of antibiotics against resistant bacterial strains in lung infections.

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