Doctoral Theses

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Ultraweak photon emission in cells: coupling to molecular pathways, applied magnetic fields, and potential non-locality
(Laurentian University of Sudbury, 2014-03-19) Dotta, Blake
The possibilities and implications of photons within the infrared, visible, and ultraviolet behaving as sources of intracellular and intercellular communication and information were investigated experimentally for melanoma cells during the 24 hrs following removal from incubation. Specific wavelengths during different intervals were associated with specific classes of biomolecules that were predicted based on the physical properties associated with their amino acid sequences. Application of a specific intensity and physiologically patterned magnetic field predicted from a model that applied the concept of magnetic moment to the whole cell resulted in photon emissions. They were detected at distances sufficient to allow intercellular communication. The occurrence of macroscopic entanglement or non-locality was shown between two loci of where simple chemically-based photons emissions were generated. Within all three experiments there was marked quantitative congruence between the energies associated with the power density of the photon emissions and the physicochemical variables involved with their reduction. These results indicate that photon emissions coupled with classic biomolecular pathways and processes may behave as intra- and inter-cellular sources of information that could control the complex dynamics of cells. The effect may not depend upon locality but exhibit non-local characteristics.
Biochemical adaptations in pseudomonas fluorescens exposed to nitric oxide, an endogenous antibacterial agent
(Laurentian University of Sudbury, 2014-05-21) Auger, Christopher
Nitric oxide (NO), a free radical released by macrophages (a subset of white blood cells) as a response to infection, is noxious to organisms due to its ability to disable crucial biomolecules such as lipids, proteins and DNA. Although normally effective at eradicating invading bacteria, several pathogens have developed mechanisms to detoxify NO and its toxic by-products, reactive nitrogen species (RNS). While some of these detoxification processes have been characterized, very little is known about the metabolic changes that enable microbes to survive this deleterious environment. Investigations into the effects of RNS on microbial physiology have shown that these harmful radicals inactivate the citric acid cycle and oxidative phosphorylation, the series of reactions responsible for making energy aerobically. The central aim of this thesis was to determine how the organism counteracts the detrimental effects of RNS, while bypassing the ineffective central metabolic pathways. The findings presented herein show that P. fluorescens engineers an elaborate metabolic network to generate ATP whilst withstanding the injurious effects of nitrosative stress. Crucial to this adaptation is the ability to produce energy via substrate level phosphorylation, a necessity that arises out of the cells’ inability to produce a substantial amount of ATP using the electron transport chain (ETC). The up-regulation of the enzymes citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate, phosphate dikinase (PPDK) helps the organism accomplish this feat. Blue native polyacrylamide gel electrophoresis (BN-PAGE), high performance liquid chromatography (HPLC) as well as co-immunoprecipitation (CO-IP) studies were applied to demonstrate that these proteins form a metabolon, a transient complex of enzymes that ensures citrate is converted into its desired end products, pyruvate and ATP. In order to gauge the individual contributions iv of phosphoenolpyruvate-dependent kinases, a novel in-gel activity assay was developed to probe these enzymes under disparate conditions. These results suggest that the organism switches from an ATP-dependent metabolism to one based on the utilization of pyrophosphate (PPi). The rationale for this appears to be energy efficiency, as pyrophosphate-dependent glycolysis can theoretically produce five ATP rather than the two yielded by Embden-Meyerhof glycolysis. Additionally, the up-regulation in activity of the enzymes adenylate kinase, nucleoside diphosphate kinase and acetate kinase seem to ensure that ATP generated by PPDK is properly shuttled and stored when aerobic metabolism is defective. The lower activity of inorganic pyrophosphatase likely ensures an adequate supply of pyrophosphate for the activity of PPDK. Taken together, this research reveals the critical role metabolism plays in the survival of microbes under the onslaught of NO and RNS. As several of these enzymes are absent in mammalian systems, they present themselves as novel targets for the development of new antibacterial agents. A comprehensive awareness of bacterial defense systems in response to NO may lay the groundwork to developing more effective treatments to impede microbial infections.
Flaxseed oil induces apoptosis in the aggressive murine melanoma cell line B16-BL6
(Laurentian University of Sudbury, 2014-11-10) Buckner, Alison
Flaxseed is classified as a functional food and is renowned for its exceptional nutrional value. These foods have the ability to either promote overall well-being and/or reduce the risk of certain diseases. Flaxseed, in the form of seed-derived oil,is the highest plant source of the omega-3 fatty acid, alpha linolenic acid. This nutraceutical has been credited with providing protective benefits against breast, colon and prostate cancers. Humans are unable to synthesize omega-3 fatty acids within the body and therefore must obtain sufficient amounts of this antioxidant through diet alone. Currently, the Western diet is grossly deficient in omega-3 fatty acids and this is in part due to the overwhelming presence of omega-6 fatty acids used in food processing and manufacturing techniques. As one of the highest sources of omega-3 fatty acids, flaxseed is often taken as a supplement to help balance the ratio of omega-3 to omega-6 fatty acids in Western cultures. Although flaxseed has been beneficial in alleviating certain symptoms in patients suffering from cardiovascular disease, diabetes, and cancer, the underlying mechanisms remain unclear. Previous animal studies have shown that a diet supplemented with 10% flaxseed, significantly decreased tumour growth in rodent cancer models. Our studies include the treatment of malignant cells with flaxseed oil in an in vitro model. We have shown that flaxseed oil has the ability to reduce cell growth in B16-BL6 cells, an aggressive murine melanoma. Furthermore, a total of seven different oils containing high concentrations of omega fatty acids, including flaxseed oil, olive, sunflower, canola, sesame, peanut and grapeseed, were characterized by HPLC and GC/MS analysis for fatty acid profiles, and these oils were also used to treat B16-BL6 cells. Although all seven oils contain various amounts of omega-3, -6 or -9 fatty acids, only treatment with flaxseed oil decreased the growth of the aggressive murine melanoma cell line B16-BL6. DNA laddering, acridine-orange staining, TUNEL staining, and FACS analysis using Annexin V and propidium iodide, showed that the flaxseed-treated cells were undergoing apoptosis, a type of cell suicide. Therefore, due to its ability to selectively inhibit malignant cell proliferation, flaxseed oil has significant potential as an anti-cancer therapeutic.
Effect of glutamine limitation on the behavior of Sp2/0-Ag14 mouse hybridoma cells.
(2014-12-16) Chénier, Andréane Simone.
Cancer cells often display a dependence toward the amino acid L-glutamine for their survival, a phenomenon termed glutamine addiction. The mouse hybridoma Sp2/0-Ag14 (Sp2/0) undergoes rapid apoptotic cell death upon glutamine deprivation, making it a useful model for uncovering the molecular and cellular processes through which glutamine controls cell survival. This work was aimed at gaining a better understanding of the molecular and cellular events triggered when Sp2/0 cells are exposed to limiting amounts of glutamine. First, the effect of glutamine limitation on Sp2/0 cell behavior was investigated. We found that a threshold concentration of 100 μM glutamine exists where Sp2/0 cell density does not increase, but cells remain viable. Under this threshold, Sp2/0 cells underwent apoptosis, but in a more protracted fashion than under conditions of acute glutamine deprivation. Unexpectedly, I found that exposure of Sp2/0 cells to 25μM glutamine triggered a biphasic activation of caspase-3. Interestingly, glutamine limitation, but not acute glutamine deprivation, was sufficient to maintain intact mitochondria for several hours and to trigger the expression of the stress-related transcription factor GADD-153. My results raise the possibility that glutamine limitation triggers a stress response which could enable Sp2/0 cells to adapt to its environment. Using microscopic and biochemical techniques, I also provided evidence for a reduction in autophagic processes in Sp2/0 cells exposed to glutamine-limiting conditions. Chemical inhibitors of autophagy caused Sp2/0 cell death even in the presence of adequate supply of glutamine. On the other hand, rapamycin, a known activator of autophagy, improved Sp2/0 viability under glutamine limitation conditions. Therefore, the loss of Sp2/0 cell viability when exposed to limiting amounts of glutamine could be the result, at least in part, of a reduction in the cell’s autophagic capabilities. Finally, I explored the effect of ammonium ions, a product of glutamine metabolism, on the behavior of Sp2/0 cells exposed to limiting amounts of glutamine. Ammonium ions treatment rescued Sp2/0 cell viability and proliferation in Sp2/0 cells cultured in glutamine-limiting conditions. Interestingly, ammonium acetate, but not ammonium chloride, caused a reduction in caspase-3 activity in Sp2/0 cells maintained under limiting glutamine conditions. Finally, my data suggest that ammonium salts led to a partial restoration of the autophagy process in Sp2/0 cells exposed to limiting amounts of glutamine, providing a potential explanation for the beneficial effect of ammonium ions on cell viability. All together, the results obtained in the course my studies argue in favor of a mechanistic link between autophagy and ammonium ions in the modulation of the viability of a Sp2/0 cells exposed to glutamine-limiting conditions.
Expression, subcellular localization and functional characterization of RBM5 and RMB10 during the differentiation of C2C12 skeletal myoblasts (skeletal myogenesis)
(2015-03-26) Masilamani, Twinkle Jasmine
RNA-binding proteins (RBPs) are a highly regulated, evolutionarily conserved and functionally distinct family of proteins involved in key RNA metabolic processes. The RNA-binding motif protein RBM5 is an anti-proliferative, pro-apoptotic, putative tumor suppressor. A paralogue of RBM5, RBM10, which shares 50% identity with RBM5, functions in development. RBM5 and RBM10 are spliceosomal components involved in alternative splicing. RBM5 and RBM10 are ubiquitously expressed with higher levels in muscle (heart and skeletal) and pancreas. Most of the studies on RBM5 and RBM10 have been focused on cancer cells. Several factors such as 1) abundance in muscle, 2) developmental and temporal regulation, 3) alternative splicing activity and 4) association with functional events related to muscle development led us to hypothesize that both RBM5 and RBM10 are involved in skeletal muscle differentiation. The mechanism of action through which these two RBPs effect differentiation is hypothesized to involve alternative splicing of muscle differentiation-specific mRNAs. RBM5 and RBM10 expression and intracellular distribution was analyzed during muscle differentiation in the C2C12 murine model using qPCR, end-point PCR, immunoblotting and confocal microscopy. Also, RBM5 and RBM10 levels were transiently down-regulated using siRNA either separately and/or together and the associated changes in cell phenotype, expression of myogenic proteins plus a few alternative splicing events were analyzed. We observed a decrease in RBM5 and RBM10 protein expression levels in the differentiated myotubes compared to the myoblasts and myocytes, which indicates a time-dependent potential regulatory role during differentiation. Further, changes in RBM5 and RBM10 protein expression without modulating the levels of mRNA variants suggests posttranscriptional and/or post-translational regulation. Stage-specific differential localization suggests multiple functions related to mRNA biogenesis. RBM5-depleted cells showed a reduction in the total cell number during differentiation, and exhibited a delay in differentiation, fusion and maturation with down-regulated expression of myogenin and myosin heavy chain (MyHC). This implies that RBM5 is necessary to maintain the cell population to execute the myogenic differentiation process in a timely manner. RBM10- depleted cells showed an increase in total cell number immediately after transfection, and exhibited a delay in differentiation with a decrease in inclusion of exon 11 in Dtna mRNA. This indicates that RBM10 is required to maintain the necessary cell population before induction and acts as a splicing regulator during differentiation. RBM5- and RBM10- depleted cells differentiated and matured slowly, and had an increase in Mef2c γ exon inclusion. Therefore, these two RBPs are associated with the alternative splicing of Mef2cγ during differentiation. This is the first study to analyze the expression and the function of these two RBPs in a murine skeletal muscle differentiation model, and has implicated them in myogenesis, paving a way for further characterization. Future studies can investigate the involvement of RBM5 and RBM10 in disease states such as muscular dystrophy and rhabdomyosarcomas, given the known functions of RBPs in tumorigenesis in other cell types.
Characterisation of CTR-17 and CTR-20, novel chalcone derivatives that inhibit tubulin polymerisation activity
(2015-12-17) Lindamulage, Indeewari Kalhari Silva
Agents targeting colchicine-binding sites are recognised as valuable lead compounds in the development of new anticancer drugs. Although colchicine can effectively inhibit cell proliferation, its use as an anticancer agent has not been approved by the FDA due to its inherent toxicity. To develop colchicine-binding site targeting agents with low or no toxicity, in collaboration with Rajiv Gandhi Technical University of India, several chalcone derivatives were created and examined. Preliminary studies at the Lee Lab identified CTR-17 and CTR-20 as promising leads. Their anti-proliferative activities using three human breast cancer cell lines (MDA-MB468, MDA-MB231 and MCF-7) and two matching noncancer breast cell lines (184B5 and MCF10A) were initially determined. Subsequently, nine other cancer cell lines were used to assess the broad spectrum anti-proliferative effects of the CTR compounds. Data from this study showed that CTR-17 and CTR-20 preferentially kill cancer cells 10-25 times over non-cancer cells. Data obtained from flow cytometry, confocal microscopy and Western blotting showed that CTR-17 induced a prolonged mitotic arrest, leading to cancer cell death probably via apoptosis. I also found that both CTR-17 and CTR-20 inhibited tubulin polymerisation and bound to purified tubulin fibers with a dissociation constant of 4.58±0.95 μM and 5.09±0.49 μM, respectively. CTR-17 and CTR-20 competitively inhibited the binding of colchicine to tubulin with an inhibitory concentration of 5.68±0.35 μM and 1.05±0.39 μM, respectively, suggesting that the CTR compounds bind to tubulin at a site partially overlapping the colchicine-binding site. Molecular docking studies confirmed this binding to occur via two and one hydrogen bonds between tubulin and CTR-20 and CTRiv 17, respectively. More interestingly, CTR compounds inhibit the proliferation of multi-drug resistant cell lines, which overexpress drug transporters involved in the efflux of clinically available microtubule targeting agents. In addition, the CTR compounds exhibit a synergistic relationship with paclitaxel in causing cytotoxicity to a P-glycoprotein overexpressing cell line. Therefore, these novel chalcone derivatives not only possess cancer-specific cell killing property but also the ability to exhibit similar cytotoxicity to both the multi-drug sensitive and resistant cells. Hence, CTR compounds possess substantial potential as safe and effective anticancer drugs.
Viral and bacterial sepsis: identification and characterization of cytokine profiles and cell death pathways
(2016-03-23) Michael, Paul
Sepsis and septic shock are the leading causes of death in intensive care units in North America. Approximately 800,000 cases of sepsis are reported every year in the USA and 215,000 will succumb to the disease despite aggressive antibiotic and supportive care [1-7]. The incidence of sepsis is increasing at a rate of 9% per year even with advances in the understanding of the pathophysiology of the disease [1]. Sepsis is commonly induced by bacterial and viral infections. Influenza A is the virus that causes the flu. The most recent pandemic caused by Influenza A (H1N1) demonstrated the potential of this virus to cause severe complications (viral induced sepsis characterized by pneumonia, acute respiratory distress and acute lung injury). This study investigated the inflammatory mediators of sepsis in H1N1 infected adults and from adults with bacterial induced sepsis. For H1N1 infections lung tissue and sera samples were characterized. Lung tissue showed hemorrhage, and interstitial congestion. Cytokine staining of these tissues revealed the presence of TNF-α, IFN-γ and IL-1β primarily from the recruited leukocytes. In addition TUNEL assay revealed apoptosis occurring in these same cell populations. ELISA quantification of sera from H1N1 infected adults also demonstrated high levels of TNF-α, IFN-γ and IL-1β at 113 pg/mL, 49 pg/mL and 26 pg/mL respectively. The sera also were able to induce apoptosis in cultured fibroblasts that was mediated through STAT1 signalling. The sera from bacterial induced sepsis were characterized for the presence of other cytokines and chemokines. TNF-α was consistently detected in all septic samples. Chemokine expression was more variable across the septic samples. The identification of different mediators of inflammation during sepsis will not only increase the basic knowledge about the progression of sepsis but would allow for the development of biomarkers that could better predict the progression of sepsis to more severe or fatal forms and allow for earlier intervention and treatment of these cases.
The use of surface plasmon resonance spectroscopy in characterizing small molecule-based nerve growth factor inhibitors
(2016-11-09) Kennedy, Allison E.
Neurotrophins are well known for their effects on neuronal survival and growth. Over the past two decades, considerable evidence has accumulated from both human and animal models that one neurotrophin, nerve growth factor (NGF), is a peripheral pain mediator, particularly in inflammatory pain states. NGF is upregulated in a wide variety of inflammatory conditions, and NGF-neutralizing molecules are effective analgesic agents in many models of persistent pain. Such molecules are now being evaluated in clinical trials and although seemingly effective in early clinical studies, many deteriorating side effects are now being brought to light. Thus, a need still remains for novel NGF-quenching therapeutics. In the past two decades, a handful of small molecule NGF-inhibitors have been described, however, their effects are still quite minimal compared to other inhibitory agents. The central aim of this thesis was to use novel screening strategies to identify small molecule NGF-inhibitors capable of modulating NGF signalling with greater efficiency than previously reported compounds for the purpose of therapeutic development. Using surface plasmon resonance (SPR) spectroscopy, established NGF-inhibitors and a series of novel compounds were analyzed for their specificity to NGF and their inhibitory properties of NGF binding to the TrkA receptor. In vitro techniques confirmed the high nanomolar inhibitory properties of a novel compound, BVNP-0197. Molecular modeling techniques also described a putative binding domain for BVNP-0197 to NGF at the loop II/IV cleft, an alternative domain than previously described by small molecule compounds. This binding domain describes an area with higher specificity for TrkA over binding for p75NTR. Serum albumin was also described as having a role in the binding events that occur between small molecule compounds to NGF in vitro. SPR kinetic analysis established that the addition of serum albumin into solution with small molecule compounds increased their specificity for NGF, as well as decreased their potential toxicity. The findings presented in this thesis will contribute to the future development of small molecule NGF-inhibitors.
Molecular analysis of Betula papyrifera populations from a mining reclaimed region: genetic and transcriptome characterization of metal resistant and susceptible genotypes
(2017-03-14) Theriault, Gabriel
The objectives of the present study were to; 1) determine if there’s an association between plant population diversity and genetic variation in white birch (Betula papyrifera) populations with soil metal contamination in the Greater Sudbury region (GSR), 2) assess if metal contamination and soil liming has an effect on global DNA methylation, 3) develop and characterize the transcriptome of B. papyrifera under nickel stress and, 4) assess gene expression dynamics in white birch in response to nickel stress. No association between plant population diversity and genetic variation with metal contamination was found. Liming increases plant population diversity but has no effect on genetic variation in the studied white birch populations. There was a decrease in root cytosine methylation in metal-contaminated sites compared to references. Treatment with the dose corresponding to total level of Ni or Cu (1,600 mg/kg Ni, 1,312 mg/kg Cu or combined) in sites of the GSR generated different responses within segregating populations analyzed. The main Ni resistance mechanism of white birch was associated with the prevention of translocation of Ni from root to shoot. We also observed lower ZAT11 and glutathione reductase expression in resistant genotypes compared to susceptible. The transcriptome of B. papyrifera was developed for the first time using Next Generation Sequencing. RNA from Ni resistant, moderately-susceptible, susceptible and water controls treatment was sequenced. A total of 209,802 trinity genes were identified and were assembled to 278,264 total trinity transcripts. In total, 215,700 transcripts were annotated and compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation and cell growth, and upregulation in genes involved in the plasma membrane. Seven candidate genes associated to nickel resistance were identified. They include Glutathione S-transferase, Thioredoxin, Putative transmembrane protein, Nramp transporter, TonB-like family protein and TonB-like dependent receptor. This TonB receptor was found to be exclusive to the Betula genus.
The effects of low-level electromagnetic fields and ultraweak photon emission on biological systems.
(2017-03-17) Karbowski, Lukasz M.
The functionality of biological organisms is a product of minute-invisible forces and structures, which together form a cohesive ensemble that initiate the processes of life. The electromagnetic and photonic nature of biological life and its basic unit the cell may be considered a viable option for communication, survival and senescence. The basis of this investigation considers the dynamic nature of low-level electromagnetic fields and ultraweak photon emission as a mechanism for intracellular and intercellular interactions, which work under the premise of only demanding small quanta of energy. Metastatic cancerous cells have been observed to emit specific frequencies of photon emission compared to healthy cells and increase this emission in unfavorable conditions. The application of a specific sequence of patterns (MuKarb) at a specific intensity, observed in the dissolution of planarian worms, when applied to cancerous cells produce complete death of the exposed metastatic cells but not healthy cells. When such patterns of light at specific wavelengths are pulsed into the aforementioned cells, the emitted light from the cells in the pulsed pattern is observed relative to the duration of the light exposure. These effects become critical to the specificity of the appropriate pattern and intensity of the field, which has been linked to the importance of appropriate equipment configuration. The smallest iv changes in the current/voltage flow within the circuitry of the digital to analogue converter and electromagnetic field devices can make drastic changes with respect to the elimination of cancerous cells being observed or not. This phenomenon reflects the Aharanov-Bohm phase shifts in voltage within the electromagnetic field equipment and its importance in producing effects as a result of low-level electromagnetic fields. The application of specificity within low-level electromagnetic generating equipment has also been shown through the process of non-locality (entanglement) where appropriate and tuned apparatus are required to produce specific non-local effects. Successful non-local effects were observed through observing decreases in growth of melanoma cells through nontangible means, comparable to the manipulate local melanoma cells. These results converge on the premise and importance of properly tuned equipment for successful low-level electromagnetic field and photon exposures. Furthermore the interaction between the use of low-level electromagnetic fields and photons at specific pattern has shown the importance of interfering with the propagation of metastatic cells.
The emission and application of patterned electromagnetic energy on biological systems.
(2017-03-31) Murugan, Nirosha J.
From the assembly of intricate biomolecules to the construction of tissues and organs from homogenous embryonic cells, patterns permeate throughout biological systems. Whereas molecules govern the multiform signalling pathways necessary to direct anatomy and physiology, biophysical correlates are inextricably paired to each and every chemical reaction – yielding a constant interplay between matter and energy. Electromagnetic energies represented as propagating photons or electromagnetic fields have shown to contain complex information that is specific to their paired molecular events. The central aim of this thesis was to determine whether these biophysical signatures or patterns can be obtained from biomolecules and subsequently be used in lieu of the chemical itself within a molecular cascade to elicit desired effects within biological systems. The findings presented here show that using a novel bioinformatics tool, namely the Cosic Resonant Recognition Model (RRM), biomolecules (proteins) can recognize their particular targets and vice versa by dynamic electromagnetic resonance. We also show using fundamental units of energies that this dynamic electromagnetic resonance is within the visible spectrum and can be used to define molecular pathways such as the ERK-MAP pathway, or distinctive viral proteins that mark certain pathogens such as Zika or Ebola viruses. Further findings presented herein show that these electromagnetic patterns derived from biomolecules can be detected using modern technologies such as photomultiplier tubes, and as every signature is unique to that system, can be used to identify insidious systems such as cancers from healthy populations. Furthermore, it is now possible to capture these unique electromagnetic signatures of biomolecules, parse the signals from the noise, and re-apply these patterns back onto systems to elicit effects such as altered proliferation rates of cancers or regenerative systems. The series of theoretical models and investigations outlined here clearly profiles the predominant electronic nature of the living matrix and its constituents, which lays the groundwork for reshaping our knowledge of cellular mechanisms that ultimately drive physiology, medicine and the development of effective diagnostic, preventative or therapeutic tools.
Biochemical and molecular characterization of microbial communities from a metal contaminated and reclaimed region.
(2017-04-18) Narendrula, Ramya
Metal contamination in the Greater Sudbury Region (GSR) resulted in severe environmental degradation. Soil liming and tree planting have been the main approaches to restoring the damaged ecosystem. The specific objective of the present study was to assess the effects of soil metal contamination and liming on 1) microbial biomass and abundance, 2) bacterial and fungal diversity, and 3) enzymatic activities and soil respiration. Phospholipid fatty acid (PLFA) analysis and 454 pyrosequencing were used to address these research objectives. Total biomasses for bacteria, arbuscular fungi (AM fungi), other fungi and eukaryotes were significantly lower in metal contaminated compared to uncontaminated reference areas. Analysis of bacterial communities revealed Chao1 index values of 232 and 273 for metal contaminated and reference soils, respectively. For fungi, the Chao index values were 23 for metal contaminated and 45 for reference sites. There was a significant increase of total microbial biomass in limed sites (342.15 ng/g) compared to unlimed areas (149.89 ng/g). Chao1 estimates followed the same trend. But the total number of OTUs (Operational Taxonomic Units) in limed (463 OTUs) and unlimed (473 OTUs) soil samples for bacteria were similar. For fungi, OTUs were 96 and 81 for limed and unlimed soil samples, respectively. Bacterial and fungal groups that were specific to either limed or unlimed sites were identified. Bradyrhizobiaceae family with 12 genera including the nitrogen fixing Bradirhizobium genus was more abundant in limed sites compared to unlimed areas. For fungi, Ascomycota was the most predominant phylum in unlimed soils (46.00%) while Basidiomycota phylum represented 85.74% of all fungi in the limed areas. Detailed analysis of the data showed that although soil liming increases significantly the amount of microbial biomass, the level of species diversity remained statistically unchanged. Soil respiration rates were higher in limed soils (65 ppm) compared to unlimed soils (35 ppm). They were significantly lower in metal contaminated sites (55 ppm) compared to reference sites (90 ppm). β-glucosidase (BG), cellobiohydrolase (CBH), β-N-acetylglucosaminidase (NAGase), aryl sulfatase (AS), acid phosphatase (AP), alkaline phosphatase (AlP), glycine aminopeptidase (GAP), and leucine aminopeptidase (LAP) activites were significantly higher in limed compared to unlimed sites. Metal contamination significantly reduced the activities of these enzymes with the exception of LAP. An opposite trend was observed for peroxidase (PER) enzyme activity that was higher in unlimed and metal contaminated sites compared to limed and reference areas.
Characterization of the relationship between two RBM5 family members
(2017-07-31) Loiselle, Julie Jennifer
RNA binding proteins (RBPs) control all aspects of RNA metabolism, and a single RBP can have numerous downstream effects. Alterations to their expression and/or function can, therefore, have remarkable consequences. For instance, decreased levels of the RNA binding motif domain (RBM) protein RBM5 are associated with increased risk of a number of cancer types, and RBM10 mutations can be lethal. Although these consequences are quite severe, little is known regarding the range of processes and events influenced by these two homologous RBPs. In fact, previous RBM5 and RBM10 functional studies were largely focused only on their abilities to promote two processes; apoptosis and cell cycle arrest. Potentially by control of these processes, RBM5 and RBM10 were shown to influence one event: differentiation. The objectives of this study were to identify all cellular processes and events enriched by changes in RBM5 and/or RBM10 expression in a particular cultured cell line, and to determine the extent of functional overlap for RBM5 and RBM10 in these cells. Towards these goals, a list of RBM5 and RBM10 mRNA targets and differentially expressed genes was determined using next generation sequencing techniques. Our data suggest that RBM5 and RBM10 do influence a wide range of cellular processes and events. Although there is overlap in RBM5 and RBM10 mRNA targets and differentially expressed genes, these RBPs can have antagonistic functions; for example our data suggest that RBM5 prevents the transformed state, whereas RBM10 actually promotes it in an RBM5-null environment. Furthermore, we present a working model by which RBM5 may regulate RBM10’s protransformatory function. Finally, we demonstrate a relationship between RBM5 and RBM10 in non-transformed cells. The results presented herein provide insight not only into the roles and regulation of RBM5 and RBM10, but of RBPs in general. Taken together, the results presented in the four papers included in this thesis expand the knowledge base of RBM5 and RBM10, which provides insight into the disease states associated with their disrupted expression or function. Our findings are thus relevant to a wide range of scientific fields including molecular, developmental and cancer biology.
Structures and functions of the post-mortem brain: an experimental evaluation of the residual properties of fixed neural tissues
(2017-08-18) Rouleau, Nicolas
Does brain function irreversibly cease after death? Billions of years of evolution and hundreds of thousands of years of human development have inculcated within us an intuition that death is a deep pit from which thoughts and behaviour cannot emerge. This dissertation serves to challenge the assumption of neurofunctional loss after death by employing modern technology to observe alternative mechanisms by which information can be processed by fixed, post-mortem neural tissues. The central aim of the thesis was to measure periodic, electric potential differences (μV) characteristic of the psychological definition of “response” within neuroanatomical loci while the fixed tissues were exposed to patterned current, complex electromagnetic fields, chemical probes, and other experimental conditions. The findings presented here show that fixed post-mortem tissues express regional, electrical anisotropies which can be modulated by various applications of electrochemical energy and that the areas surrounding the hippocampus are most responsive. We show that neuropathology secondary to repeated and protracted seizure activity can be detected in post-mortem rat brains with coupled depressions of low-frequency signal periodicities. Our findings demonstrate that injections of current into coronal sections of fixed human brain tissue are most potent when patterned to simulate neuronal spike-trains and the dominant frequency of the equivalent living tissue subsection. We also show that fixed, post-mortem brain tissues act as electromagnetic filters, expressing signals non-randomly and preferentially within the right cerebral hemisphere. Further findings indicate that receptor agonist-antagonist probes (e.g., glutamate and ketamine) as well as other chemical applications can induce regional electrical responses as well as habituation-type phenomena over repeated exposure. iv These responses are paired and can be inversely related to photon emission from the tissue proper as inferred by photomultiplier tube measurements. The bases of the electrochemical responses are thought to be due to phenomena associated with pH and ionic gradients in general as inferred by our experiments with post-mortem rat brains. The aforementioned experimental results are then synthesized to produce a working hypothesis upon which further research can be based. We conclude that the brain’s structure-function relationship is sufficient to elicit post-mortem responses characteristic of a composite material of otherwise unknown potential.
The effect of doxorubicin administration on skeletal muscle
(2017-08-29) Fabris, Sergio
Skeletal muscle (SM) is the largest organ in the human body and represents approximately 40% of the total body weight. Maintenance of SM mass and integrity is dependent on the delivery and removal of essential metabolic products as well as the dynamic balance between protein synthesis and degradation. The health and maintenance of skeletal muscle in cancer patients is of particular importance, as the significant loss of muscular mass is an indication of cachexia, a serious life-threatening condition. Doxorubicin (DOX) is a broad-spectrum anti-cancer chemotherapeutic and remains one of the most widely used chemotherapeutic agents for the treatment of solid tumors and hematological malignancies. The clinical use of DOX is limited by a well described dosedependent and cumulative cardiotoxic side effect. The majority of DOX-related research remains focused on reducing cardiotoxicity while little is known of the effect of the drug on SM. Therefore, the purpose of the thesis was to study the effects of DOX chemotherapy on SM. Study 1. The purpose of Study 1 was to examine the accumulation of doxorubicin (DOX) and its metabolite doxorubicinol (DOXol) in skeletal muscle of the rat up to 8 days after the administration of a 1.5 or 4.5 mg/kg i.p. dose. Subsequent to either dose, DOX and DOXol were observed in skeletal muscle throughout the length of the experiment. Interestingly an efflux of DOX was observed after 96 hrs, followed by an apparent reuptake of the drug which coincided with a spike and rapid decrease of plasma DOX concentrations. The interstitial space within the muscle did not appear to play a significant rate limiting compartment for the uptake or release of DOX or DOXol from the tissue to the circulation. Furthermore, there was no evidence that DOX preferentially accumulated in a specific muscle group with either dose. Study 2. Study 2 examined intracellular and interstitial nitric oxide (NO) concentrations in the SM following the administration of DOX. A single dose of 1.5 or 4.5 mg/kg was administered intraperitoneally to male Sprague-Dawley rats and interstitial (IS) and intracellular (IC) NO was quantified every 24 up to 192 hrs. post injection. There was no significant difference in IC NO following the injection of 1.5 mg/kg DOX when compared to control, however the administration of 4.5 mg/kg DOX resulted in lower (P<0.05) concentrations of NO in the IC. Interestingly, a consistently higher (P<0.05) concentration of NO in the IS was established following the administration of 1.5 mg/kg compared to control while no significant changes in IS NO resulted from the administration of the 4.5 mg/kg dose. The fluctuation of IS and IC NO were not a result of substrate availability as arginine concentrations remained stable throughout the experiment. Study 3. The purpose of Study 3 was to examine the effect of DOX administration on IC, IS and vascular concentrations of amino acids (AA) in SM of the rat up to 8 days after the administration of a 1.5 or 4.5 mg/kg i.p. dose. Intracellular total amino acids (TAA), essential amino acids (EAA) and branched-chain amino acids (BCAA) were significantly increased in each muscle group analyzed, following the 1.5 and 4.5 mg/kg doses compared to control. In the plasma, TAA were significantly increased compared to control where greater (P>0.05) concentrations were observed following the 1.5 mg/kg dose compared to the 4.5 mg/kg dose. Compared to control, the 1.5 mg/kg dose resulted in an increase (P<0.05) in interstitial TAA whereas the 4.5 mg/kg resulted in a sustained decrease (P<0.05). These data represent the first concurrent investigation into the accumulation of DOX in the IC, IS and vascular spaces as well as the resulting effects on NO and AA concentrations in these communicating compartments. Overall, SM plays an important factor in the availability and metabolism of DOX and the effect of DOX on SM may play a more significant role in the therapeutic impact of DOX than previously considered. This thesis provides a substantial foundation for future studies focused on reducing DOX-induced skeletal muscle damage.
Role of ammonium ions on the modulation of apoptosis in glutamine-starved cells
(2017-11-24) Abusneina, Abdelmuhsen
Highly proliferating cells reprogram their metabolism in order to fulfill their requirements in biosynthetic precursors. One consequence of this metabolic adaptation is glutamine (Gln) addiction, a phenomenon whereby cells become dependent on Gln for their survival. How cells sense Gln levels and link this information to the survival machinery remains to be fully understood. Our laboratory previously showed that when Sp2/0- Ag14 (Sp2/0) hybridoma cells were deprived of Gln, apoptosis is triggered within minutes. In this study, I used the Sp2/0 cell line to characterize the role which ammonium ions, a product of glutaminolysis, play in the modulation of cell survival. In a first series of experiments, I demonstrated that, when supplemented with ammonium salts, Gln-deprived Sp2/0 cell cultures showed a significant increase in viability. This effect of ammonium salts was independent of the cells’ ability to synthesize Gln, and was not affected by co-treatment with -ketoglutarate. In agreement with their effect on cell viability, ammonium salts caused a significant reduction in the number of Glnstarved Sp2/0 cells with apoptotic nuclear condensation and fragmentation. Unexpectedly, supplementing Gln-starved Sp2/0 cells with ammonium salts did not alter cytosolic cytochrome c release, caspase-3 activation, DNA fragmentation or nuclear lamin A/C cleavage, indicating that the mechanisms triggering the apoptotic machinery remained intact. In a second set of experiments, I used the Gln antagonists 6-Diazo-5- oxo-L-Norleucine (DON) and azaserine (AZA) to interfere with Gln metabolism in lieu of Gln deprivation. Both antagonists were toxic to Sp2/0 cells, an effect which was attenuated by supplementation with ammonium salts. This indicated that Gln metabolism was required for cell viability, and that the effect of ammonium salts did not require metabolic pathways utilizing Gln. Unexpectedly, the supplementation of DON or AZA to Gln-starved cultured led to a small but significant increase in cell viability. Moreover, the supplementation with ammonium salts of Gln-deprived Sp2/0 cultures treated with DON or AZA caused a marked increase in cell viability. More importantly, the combination of ammonium salts and Gln antagonists resulted in a significant reduction in caspase-3 activation in Gln-starved Sp2/0 cells, indicating that they interfered with the apoptotic process. Altogether, our data demonstrate that ammonium ions trigger a pro-survival cellular response in Gln-deprived Sp2/0 cells.
Metabolic engineering aimed at the production of keto acids from glycerol : an industrial by-product
(2018-01-26) Alhasawi, Azhar Ahmed
Worldwide, energy consumption is at an all-time high and projected to increasingly grow in the upcoming years. Thus, it is critical to uncover alternative sources of energy that are independent of fossil fuels and environmentally neutral. The transformation of biomass into various energy-rich chemicals is an important strategy that is being pursued globally. Biodiesel can be an interesting substitute to fossil fuels. However, this process generates excessive amounts of glycerol, a byproduct that needs to be converted into valuable products if the biodiesel industry is to be sustainable. The principle objective of this thesis is to study how glycerol can be used as a raw material by microbial systems to produce valuable products. The soil microbe, Pseudomonas fluorescens widely utilized in the numerous biotechnological applications due to its nutritional versatility is an obvious choice to tailor into a glycerol-transforming nanofactory. The abiotic modulators namely hydrogen peroxide (H2O2) and manganese (Mn) afforded uniquely facile means of triggering metabolic reprogramming aimed at the enhanced formation of pyruvate and α-ketoglutarate (KG). Under the influence of H2O2, P. fluorescens engineers an intricate metabolic network to synthesize ATP and pyruvate. As oxidative phosphorylation is severely impeded, the microbe invokes substrate level phosphorylation to generate energy. This is accomplished via the increased activities of various enzymes including pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPC) that were analyzed by blue-native polyacrylamide gel electrophoresis (BN-PAGE) and high performance liquid chromatography (HPLC). The high-energy phosphoenolpyruvate (PEP) is then converted into ATP and pyruvate, a process mediated by pyruvate phosphate dikinase (PPDK), phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK). Supplementation with a micro-nutrient such as Mn, a divalent metal involved in a variety of enzymes results in the reprogramming of the metabolic networks aimed at the accumulation of KG. The increased activities of isocitrate dehydrogenase (ICDH)- (NAD)P dependent and aminotransaminases aided the exocellular secretion of KG. The overexpression of pyruvate carboxylase (PC) that is evident in the Mn-treated cells provides oxaloacetate, an important precursor to the synthesis of citrate, a key ingredient in the synthesis of KG. Isocitrate lyase (ICL), fumarate reductase (FUMR), succinate semialdehyde dehydrogenase (SSADH), α-ketoglutarate decarboxylase (KDC) and γ-aminobutyric acid transaminases (GABAT) work in concert to produce KG. 13C-NMR helped identify the metabolites participating in the metabolic networks. Immunoblot experiments confirmed the presence of overexpressed enzymes. These disparate metabolic pathways that promote the overproduction of the keto-acids in P. fluorescens have the potential of converting glycerol to value-added products commercially. As the process utilized is devoid of any genetic manipulation, it can be readily implemented in an industrial setting. In conclusion, both H2O2 and Mn can orchestrate metabolic changes in P. fluorescens inducing the production of pyruvate and KG from glycerol respectively. These chemical manipulations may also be applied to other microbial systems.
The role of bile acids in overcoming resistance to chemotherapy
(2018-02-01) Chewchuk, Simon
In the context of cancer therapy, resistance to chemotherapy agents is a serious threat to patient welfare. In these circumstances, patients can either present with cancers that are naturally resistant to conventional therapy, referred to as innate resistance, or with cancers that become resistant following treatment, referred to as acquired resistance. In this thesis, we address the phenomenon of acquired drug resistance, involving cell lines selected for resistance to the anthracycline, doxorubicin. In the first study, we examined the role of the aldo-keto reductases AKR1C3 and AKR1B10 in doxorubicin resistance, enzymes that can hydroxylate doxorubicin to a less toxic form (doxorubicinol). Additionally, these enzymes can function to promote estrogen biosynthesis from estrone, which can have significant effects on cell growth and survival. We demonstrated in the first study that AKR1C3 and AKR1B10 are expressed at higher levels in doxorubicin resistant MCF-7 cells than their isogenic control counterparts. This change in expression correlated very well with increased estrogen synthesis. siRNA-mediated reduction in AKR1C3 and/or AKR1B10 transcript expression had no major effect on doxorubicin resistance, suggesting that these enzymes are not sufficient to mediate the doxorubicin resistance phenotype and that other mechanisms of doxorubicin resistance exist in these cells. We did, however, note that a pharmacological inhibitor of AKR enzymes (a bile acid termed β-cholanic acid) was effective in reversing doxorubicin resistance in doxorubicin-selected cell lines. This prompted a second study to investigate the mechanism for this reversal. We observed that β-cholanic acid strongly reduced doxorubicin resistance in cell lines that express the ABC transporter ABCC1, including doxorubicin-resistant MCF-7 breast tumour cells and H-69 lung cancer cells. Reversal of doxorubicin resistance was also observed in HEK293 cells transfected with ABCC1 expression vectors. Subsequent experiments confirmed that β-cholanic acid and another bile acid that does not inhibit the aldo-keto reductases was able to inhibit ABCC1-mediate doxorubicin efflux from tumour cells, thereby providing a mechanism for the reversal of doxorubicin resistance. Bile acids thus represent an important new class of compounds that could prove useful in improving the effectiveness of doxorubicin chemotherapy in cancer patients, specifically in recurrent tumours overexpressing the ABCC1 transporter.
Cancer cell behaviour in response to chemotherapeutics - a study of docetaxel induced inflammatory cytokine production and the effect of lipopolysaccharides
(2018-05-17) Edwardson, Derek William
The study of cancer is an ever‐evolving discipline and since the 1950's it has been driven by fundamental scientific research using cultured human tumour cell lines isolated from human cancer patients. Over the years it has become evident that patients with cancer of the breast, ovaries, and several other tissues, often respond well to initial chemotherapy treatment, only to be left with tumours that have become resistant to the cytotoxic effects of chemotherapy. This has prompted decades of cellular and mouse‐based studies to characterize the many biomolecular processes by which tumour cells in their microenvironments survive and reproduce in the presence of chemotherapy drugs. This dissertation discusses the role of cytokine production in chemotherapy drug efficacy both in the laboratory and the clinic. Cytokines are naturally released by healthy epithelial, endothelial, and immune cells to convey important messages to other cells and tissues of the body, driving immune responses upon recognition of pathogens or cellular damage. Cytokines have become increasingly considered for their roles in stalling or accelerating cancer progression as well as improving or limiting drug efficacy. In this thesis, we present primary research results that provide novel insight into the mechanism by which chemotherapy drugs induce inflammatory cytokine production and release from human tumour cells. We show that the semi‐synthetic taxane derivative docetaxel, as well as other structurally distinct chemotherapy drugs, induce the release of the inflammatory cytokine TNF‐α from breast and ovarian tumour cell lines. Constitutively increased production and release of TNF‐α and CXCL1 from breast and ovarian tumour cells was also observed upon their selection for survival in increasing concentrations of docetaxel. Docetaxel‐resistant cells were less responsive to acute treatment with docetaxel than their drug‐naive parental cell lines. These cells exhibited increased expression of the plasma membrane‐bound drug‐export protein, P‐glycoprotein, which promotes the efflux of docetaxel and other drugs from tumour cells. Interestingly, restoration of drug into the docetaxel‐ resistant cells not only restored the drugs' cytotoxic effect but also the ability of the cells to respond to drug with increased TNF‐α release. Current paradigms suggest that this response occurs through activation of the pathogen recognition receptor Toll‐like receptor 4 (TLR4), involving direct interaction with docetaxel at the cell surface. This model appears inconsistent with our results showing that cellular drug accumulation is necessary for the response of increased TNF‐α release to occur. We also show that the TLR4 agonist, lipopolysaccharides (LPS), causes increased production of TNF‐ in the presence of docetaxel and increased docetaxel cytotoxicity for both wildtype and docetaxel‐resistant MCF‐7 tumour cells, representing a potential novel strategy to restore chemoresponsiveness in chemoresistant tumours.
Antibacterial activity of freshwater green microalgae isolated from water bodies near abandoned mine sites in Ontario, Canada
(2018-07-06) Senhorinho, Gerusa Neyla Andrade
Progress of modern medicine relies on the discovery of new antibiotics. The increasing threat of antibiotic resistant bacteria, leading to an increase in morbidity and mortality of patients previously considered low risk, has highlighted the serious need for the expansion of antibiotic research and development. Historically, natural products have been the most successful source of antibiotics as they have complex and unique chemical structures and modes of action. Since most available antibiotics are originally a result of the secondary metabolism of bacteria and fungi, microorganisms from diverse environments capable of producing secondary metabolites have been and currently are being investigated for antibacterial production. Green microalgae are eukaryotic microorganisms that can be found in a very wide range of habitats, including extreme environments. These microorganisms are known to produce a series of commercially valuable compounds as a result of their secondary metabolism. The central aim of this thesis was to determine the potential of green microalgae as antibiotic producers collected (bioprospected) from water bodies near abandoned mine sites in Ontario, Canada. These water bodies exhibited a variety of chemical profiles, including high metal concentrations and low pH. Forty species of green microalgae were subsequently isolated and their extracts tested against various bacteria. The findings showed that 37.5% of these microalgae produced antibacterial compounds that seem to specifically inhibit the growth of Gram-positive bacteria, in particular the opportunistic pathogen Staphylococcus aureus. This was a higher success rate than any previous study on green microalgae. In addition, the evaluation of crude extracts of Chlamydomonas sp., the most common isolated species, demonstrated variation in antibacterial activity during cell growth. The highest antibacterial activity from this species was found in the exponential phase. Furthermore, green microalgal extracts exhibiting antibacterial activity also decreased the cell viability of malignant cells, particularly the rapidly dividing human ovarian carcinoma A2780 cells. However, the extracts did not decrease the cell viability of non-malignant cells. Taken together, the results of this thesis reveal that freshwater green microalgae from water bodies near abandoned mine sites are potential sources of antibacterial compounds against Grampositive bacteria and should be further investigated against rapidly dividing malignant cells.

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