Doctoral Theses

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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.
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.
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.
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.
Cellular effects of Ferula Assafoetida on breast cancer cells and inflammatory responses in cultured monocytes
(2021-12-16) Alharbi, Albatul
In traditional medicine, Ferula assafoetida (F. assafoetida), has been used as an antiseptic, anti-diabetic, anti-inflammatory, and anti-cancer agent. In recent years its anti-cancer and antiinflammatory activities have become a focus in drug research. We investigated the in vitro cytotoxicity and anti-inflammatory effects of ethanolic extracts of F. assafoetida and five known components (ferulic acid, vanillic acid, quercetin, ellagic acid, and p-coumaric acid) on a group of malignant and non-malignant breast cell lines and the THP-1 monocyte-like cell line. Our results showed that treatment with the ethanolic extract of F. assafoetida, and the components, had a significant effect on cell viability and apoptosis induction for the human MCF-7, MDA-MB-231, and murine 4T1 breast cancer cell lines compared to the non-malignant human HBL-100 breast cells. This research also showed that THP-1 peripheral blood monocytic leukemia cells, differentiated into macrophages, could be further polarized into the M1 inflammatory phenotype by treatment with extracts of F. assafoetida and the components. There was a significant increase in the expression of CD80, a marker associated with the M1 macrophage subtype, but no increase in expression of the M2 subtype marker, CD163, in treated cells. Further, this polarization of the THP-1-dependent macrophages showed an increased ability to damage MCF-7 or MDA-MB-231 cell monolayers in co-culture experiments. Therefore, treatment with F. assafoetida extracts can also indirectly cause the death of cancer cells via activation of immune cells. These results confirm that F. assafoetida is a potential source of anti-cancer and immune modulatory compounds and that further investigation is needed to reveal the mechanisms of F. assafoetida’s effects on apoptosis and immunomodulation.
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.
Characterization of the novel 4-chloro-1-piperidin-1ylmethyl-1H-indole-2,3-dione compound (Raja 42) for its antibacterial activity against Escherichia coli, Clostridium difficile, Staphylococcus aureus and Helicobacter pylori
(2020-11-18) Fong, Alexis
According to the World Health Organization (WHO), drug-resistant bacteria are prevalent in 83.3% of the regions where WHO conducts surveillance. Furthermore, the number of antibiotic resistant bacterial strains increases every year, necessitating the development of new classes of antibacterial agents. Toward developing a novel class of antibacterial agents, we have created a chemical library using chloroquine as the basic scaffold. We screened our chemical library of 211 compounds to identify antibacterial activity. Twenty-seven were effective on drug-sensitive E. coli strains as well as on those resistant to ampicillin, kanamycin or NDM-1. In addition, they were also effective against Staphylococcus aureus and methicillin-resistant S. aureus. Since all of them contain an isatin moiety, they are classified as the γ-lactam class of antibiotics. Although similarities can be seen in the spectrum of activities of γ-lactam-based and β-lactam-based antibiotics, there are marked differences in the activity against antibiotic resistant bacterial strains. One of the new compounds, Raja 42 (4-chloro-1-piperidin-1ylmethyl-1H-indole-2,3-dione), displayed a lowered MIC value and, therefore, was chosen for further studies. In addition to its excellent activity against E. coli, Raja 42 is also notably effective against Helicobacter pylori and Clostridium difficile isolates from patients. I set out to unravel the molecular mechanism by which Raja 42 exhibits its antibacterial effects. Data from cellular and fluorescent microscopic assays showed that bacteria were killed rapidly in the presence of Raja 42. A time-kill and membrane depolarization assays confirmed the rapid cell killing by Raja 42, suggesting that the mode of killing by the compound is likely due to the disruption of bacterial cell membrane. To further investigate this possibility, I carried out protein 2-D gel electrophoresis in an attempt to identify proteins involved in the Raja 42-mediated cell killing. In the process, those proteins differentially expressed in response to Raja 42 were isolated and their identities were determined by peptide fingerprinting using mass spectrometry. The resultant data revealed that several proteins involved in the reactive oxygen species (ROS) pathway are upregulated in the Raja 42-treated samples. In parallel, ten clones resistant to Raja 42 were generated, and their nucleotide sequences were determined. A 27 bp deletion upstream of the promoter region of ghrA, a necessary catalytic converter of glyoxylate to glycolate in the glyoxylate shunt pathway, was found to be present in all of the Raja 42-resistant clones. This data suggests that the ablation of ghrA is directly related to the Raja 42 resistant phenotype. To determine the quantitative gene expression of bacteria in response to Raja 42 treatment, QPCR analysis was carried out. To solidify the mechanism of Raja 42 further, rescue experiments were performed to determine the importance of ghrA. Taken all the data together, Raja 42 appears to kill bacteria by upregulating the level of cellular ROS through rapidly redirecting the metabolic pathways.
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.
Characterizing the structural Influence of electromagnetic field application geometry on biological systems
(2020-01-14) Carniello, Trevor N.
There is growing literature that describes the effects that exposures to different forms of magnetic and electromagnetic fields have on biological systems. Some robust effects have been reported when the temporal structure of the electromagnetic field is patterned after what has been observed biologically. However, there has been little effort devoted to ascertaining the role for that physical application geometry, the structure through that current is presented, plays in the bio-effectivity of patterned EMF. Here we devised a series of investigations that compared 4 unique geometric organizations of copper wire based application devices to generate patterned EMFs in order to discern if application geometry has any impact on biological responses from cell systems treated with exposure to EMF. Furthermore, we examined the structural pattern of a burst-firing EMF in order to characterize that parameters are important in optimizing the proportion of cells that can be induced to bear plasma membrane extensions in a cell model of induced neuritogenesis. Results of the experiments conducted within this thesis show that the pattern of the EMF applied to PC-12 cells is the most important factor to promote neurite outgrowth. Other parameters such as: the intensity of the applied field, the timing of the field, exposure duration, and whether or not the pattern in constantly or intermittently (i.e., rotated) exposed to PC-12 cells treated with forskolin do not appreciably impact the growth of neurites. Investigations using different magnetic geometries (e.g., structures around that copper wire is wound) were able to show that the physical structure of the EMF-generating device contribute to the efficacy of neurogenesis of PC-12 cells exposed to burst-firing pattern. Furthermore, unique EMF-generating devices influenced spectral profiles of ultra-weak photons emitted from B16-BL6 cells. The differences obtained between magnetic field generating devices suggest that the more heterogeneous the patterned EMF the more impactful it is on the structural and functional aspects of the biological system under investigation. Rigorous physical experimentation examining the features associated with unique structures around that copper wire is wound, showed that if the EMF-generating device approaches the structure of a dome, it has the capacity to reduce background magnetic field intensity and may provide insight (e.g., a mechanism) as to the efficaciousness of observed effects when a patterned EMF is generated through this device.
Comparative molecular analyses between red maple (Acer rubrum) and trembling aspen (Populus tremuloides) exposed to soil metal contamination: metal translocation, gene expression, and DNA methylation.
(2018-12-13) Kalubi, Kersey N.
he main objectives of the present study were to 1) compare the physiological responses and gene expression in red maple (Acer rubrum) and trembling aspen (Populus tremuloides) exposed to metal contamination, 2) determine if epigenetic events are associated with metal resistance in A. rubrum, and 3) assess global gene expression in A. rubrum exposed to different doses of nickel. Metal analyses of soil and plant tissues revealed that P. tremuloides is an accumulator of Mg, Zn, and Ni while A. rubrum does not accumulate these metals in the leaves as it shows avoidance as the main mechanism of coping with soil metal accumulation. Comparative analysis of gene expression revealed that the four genes tested (Nramps4, Nas3, At2G, and MRP4) were more upregulated in P. tremuloides compared to A. rubrum in a field study. AT2G and MRP4 genes were significantly down regulated in A. rubrum from the targeted metal contaminated sites compared to those from uncontaminated areas but environmental factors driving this differential gene expression couldn’t be established. The growth chamber experiment, showed differential gene expression based on p values when the effects of nickel doses were compared. There were more upregulated than down regulated genes in resistant genotypes compared to susceptible genotypes. Most of these genes are associated with coping with abiotic stressors and involves tolerance and detoxification mechanisms. There was a significant variation in the level of cytosine methylation among the metal-contaminated sites, with significant negative correlations between bioavailable nickel / copper content and cytosine methylation being observed. In Conclusion, the present study reveals that A. rubrum exhibits the avoidance strategy as the main mechanism of coping with soil metal accumulation. However, a more informative and indepth analysis of this mechanism would be very beneficial in deciding whether it is strictly physiological or genetic. The role of epigenetics can be further understood by determining the distribution of DNA methylation in both A. rubrum and P. tremuloides.
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.
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.
Effect of prenatal exposure to glucocorticoids and ionizing radiation on programming of adaptive behaviour and neural genetic dysregulation in adult offspring
(2020-07-23) Lalonde, Christine Nancy
Early life exposure to stress can lead to physiological and behavioural adaptations in offspring. Adaptive changes do not always benefit the organism, as it may result in adult diseases such as hypertension and diabetes in response to prenatal nutritional deficiencies. This thesis investigates the behavioural and genetic profiles of offspring exposed to two pathways of oxidative stress and DNA methylation: synthetic glucocorticoids and ionizing radiation. Synthetic glucocorticoids are able to bypass the placental enzymatic barrier and directly interfere with fetal gene expression by binding to glucocorticoid binding elements to either promote or inhibit expression as well as inducing changes in methylation of CpG islands. Ionizing radiation induces reactive oxygen species that will initiate DNA damage and oxidative stress leading to epigenetic modifications of gene regulation. The exposure to synthetic glucocorticoids induced adaptive phenotypical changes in Wistar-Kyoto offspring, inducing a stress-coping strategy and increased exploratory activity in combination with gene dysregulation in the prefrontal cortices. Exposure to ionizing radiation in C57Bl/6J mice did not induce significant behavioural changes; however, did elicit a few changes in gene expression in the prefrontal cortices, cerebral cortices, hippocampi, and cerebella that were sexually dimorphic. In contrast, the same radiation exposure study replicated in BALB/c mice induced extra-activity in offspring when faced with stress, arguably an adaptive response that may pose a risk to the animal. Significant gene dysregulation of oxidative stress and neuronal proliferation pathways was discovered in the prefrontal cortices, cerebral cortices, and cerebella of the BALB/c offspring. In consideration of the literature and the results of these studies, fetal programming of adult behavioural profiles may be accomplished through stress-induced genetic modifications.
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.
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.
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.
Immune - modulatory effects of sidr honey: implications for anti-proliferative effects on cancer cells
(2020-10-23) Almnayan, Danah
Honey has become popular as a potential treatment for several ailments, including many cancers. Being a natural product, honey is often considered to be a safe and inexpensive adjunct or sometimes even an alternative to the currently available cancer treatments (including chemotherapy and radiotherapy) that have adverse side effects. Honeys from different parts of the world have shown anti-proliferative, immune-modulatory, and anti-inflammatory actions. Yemeni Sidr Honey (YSH) is a world-renowned honey whose anti-inflammatory activity suggests the possibility of underlying anti-cancer and/or immune-modulatory actions. Our studies have shown that treatment with 1% YSH is able to inhibit proliferation, and induce apoptosis in breast cancer cell lines (MDA-MB-231 and MCF-7) and cervical cancer cell lines (Hela). We also showed that THP-1 monocyte-like cells differentiated by treatment with phorbol ester and then treated with YSH affected their polarization into M1 or M2 macrophages: treatment with YSH for 24 h, enhanced the expression of the M1 phenotype while treatment of the macrophages with LPS and YSH for 48 h increased the level of M2 markers of differentiation. Further, co-culture of the M1 differentiated macrophages with breast cancer cells showed that treatment of the macrophages with YSH decreased tumour cell growth and increased apoptosis. These results suggest treatment with YSH is able to impact cancer via two separate mechanisms: direct impacts on cancer cell survival and activation of anti-tumour immune system (monocyte) activation.
Impairment in mitochondrial oxidative phosphorylation alters clock gene expression
(2020-10-21) Baxter, Beverly Joan
Epidemiological studies provide evidence that workers who perform chronic night shift work are at significantly higher risk of a number of severe disease states including cancer. The perturbed activity/rest and feeding/fasting cycles, which occurs in persons performing shift work or who are subjected to jet lag, disrupt our normal 24- hour internal clock or circadian rhythm. The molecular mechanisms that link chronic circadian disruption to disease are not well understood. Since light exposure at night is known to decrease melatonin levels, some researchers have hypothesized that a reduction in the nocturnal levels of this pineal hormone predisposes individuals to disease. Animals that displayed atypical behaviours in their daily cycles, led to the identification of eight-key clock or circadian genes that are differentially expressed during the day and which determine normal internal timing. The expression of these genes was abnormal in a large number of human tumors including breast cancer. In this study, a temperature shift model was characterized and used as a means to synchronize the expression of these clock genes in a human breast cancer cell line. The model was compared to another cell synchronization protocol which utilizes serum shock and which showed differences in gene expression and cell cycle regulation between the two protocols. The temperature shift model was then used to study the impact of melatonin on clock gene expression and on the production of reactive oxygen species (ROS) in cells exposed to chemotherapeutic drugs. Melatonin influenced the cell cycle but did not cause significant differences in clock gene expression. Chemotherapeutic drugs differed in their effects on the production of intracellular ROS. A mitochondrial deficient (Rhø) cell line which exhibited impaired oxidative phosphorylation, was developed from MCF-7 cells. The profile of clock gene expression in Rhø cells that were also subjected to the temperature shift protocol was different than that of the parental MCF-7 line. This suggests that impairment of mitochondrial function disrupts clock gene expression and may be a link to the oncogenic transformation of cells.
The interaction among hydrogen sulfide, estrogen and insulin-like growth factor-1 in vascular smooth muscle cells
(2019-08-30) Shuang, Tian
The proliferation of vascular smooth muscle cells (SMCs) is regulated by an array of endogenous substances, including estrogen, insulin-like growth factor-1 (IGF-1), and hydrogen sulfide (H2S). Estrogen inhibits SMC proliferation via the activation of estrogen receptor-α (ER- α), but it stimulates the same in the absence of endogenous H2S. IGF-1, via its receptor (IGF- 1R), stimulates SMC proliferation and migration. ER-α and IGF-1R can form hybrid dimer with both ER and IGF-1 as the binding ligands. Furthermore, H2S produced by cystathionine-gamma lyase (CSE) inhibits SMC proliferation. It appears that the interaction and integration of the vascular effects of estrogen, IGF-1, and H2S determine the outcome of the proliferation of SMCs. In this thesis study, we found that plasma estrogen levels were significantly lower in female CSE knockout (KO) mice than in female wide-type (WT) mice. Estrogen treatment of atherogenic diet-fed mice attenuated hypercholesterolemia, oxidative stress, intracellular adhesion molecule-1 and NF-κB expression and increased H2S production in WT mice but not in CSE- KO mice. Not only estrogen and H2S affects each other’s production and function, H2S also interacts with IGF-1 to inhibit the stimulatory effect of IGF-1 on SMCs proliferation. This inhibitory effect of H2S was abolished by blocking IGF-1/IGF-1R signaling pathway. On the other hand, estrogen downregulated the protein expressions of IGF-1 and IGF-1R in mouse aortic tissues or aortic SMCs. Deficiency of IGF-1R expression or lower IGF-1R activity abolished the stimulatory effect of estrogen on the proliferation of CSE-KO SMCs. ER-α and IGF-1R were co-located on cell membrane and co-immunoprecipitated. The binding of estrogen to IGF-1R/ER-α hybrid catalyzed the stimulatory effect on SMC proliferation. Finally, H2S induced the S-sulfhydration of IGF-1R, but not ER-α, in mouse SMCs, which lead to the decreased formation of IGF-1R/ER-α hybrid. This decrease inhibited the phosphorylation of IGF-1R, and attenuated estrogen-induced SMC proliferation. It is concluded that the antiatherosclerotic effect of estrogen is mediated by CSEgenerated H2S. The absence of H2S favors the interaction of estrogen with IGF-1R/ER-α hybrid to stimulate SMC proliferation whereas the presence of H2S favors the interaction of estrogen with ER-α to inhibit SMC proliferation. Our studies demonstrate that H2S reverses the proproliferative effect of estrogen on SMCs and unmasks the dominative anti-atherosclerotic effect of estrogen. The appreciation of the critical role of H2S in the cardiovascular effects of estrogen and IGF-1 will help better understand the regulation of the complex vascular effects of estrogen and sex-related cardiovascular diseases

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