Biomolecular Sciences - Doctoral Theses

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On the radiobiology of anhydrobiotic saccharomyces cerevisiae : unraveling the role of intracellular water in the radiation response
(2024-03-26) Lapointe, Michel R.
Anhydrobiosis, denoting “Life without water” in Greek, is an adaptative characteristic held by few eukaryotic organisms. Given the integral role that intracellular water plays in the mechanisms of low linear energy transfer (LET) radiation-induced DNA damage, the question is raised as to what effect the absence of water may have on the radiation resistance of anhydrobiotic organisms. This project utilized anhydrobiotic Saccharomyces cerevisiae, which is a budding yeast and the same organism used in the fermentation of beer, wine, and bread. This yeast is extremely well studied. It has a radiation response on the genetic level, which is very similar to human cells. With its eukaryotic cell structure, numerous human gene homologs and paralleled molecular processes in the repair of ionizing radiation-induced DNA damage, it is regarded as an excellent biological model to study the foundational biological effects of radiation exposure. Furthermore, its anhydrobiotic capabilities make it an extremely low-maintenance model, excellent for running long-term experiments where laboratory access is acutely challenging. As such, its utility in the study of sub-natural background radiation (NBR) exposure has been investigated. Overall, the goal of this work was to decipher how anhydrobiosis influences radiation resistance, with a particular focus on the implications for sub-natural background radiation exposure. This work has shown that the radiation tolerance induced by desiccation is remarkably consistent through a wide range of radiation doses. Significant metabolic perturbations relating to sub-NBR exposure and potassium-isotope availability were observed. Anhydrobiosis perturbed the radiation response of S. cerevisiae in a predictable manner with low-LET radiation, and further investigation into the metabolic effects of sub-NBR radiation exposure is prudent to better understand the role of NBR in normal biological function and homeostasis.
Secretion and interaction of the heat-stable enterotoxin b (STb) with the gut hormone secreting STC-1 Cells
(2021-08-31) Kinkar, Eyad
Enterotoxigenic Escherichia coli (ETEC) employs a number of secretion systems for efficient translocation of a number of virulence factors during infection. Studies of the relationship between substrates and specific secretion pathways suggested that secretion substrates are identified and are targeted to their proper secretion pathway via inherent amino acid sequences, or secretion signals, within each substrate. ETEC utilizes the Sec-dependent pathway of the type two secretion system (T2SS) to secrete the Heat-Stable Enterotoxin B (STb), in addition to several other toxins, to the extracellular milieu. In this work, the nature of the nucleotide sequence of STb’s mRNA, rather than the amino acid sequence of its signal sequence, was investigated for its effect on STb secretion. Additionally, the interaction of STb with the gut hormone secreting cells (STC-1 cells) was investigated to elucidate the nature and consequences of such interaction. In the first study, the N-terminal signal sequence of premature STb (at the mRNA level) was subjected to various mutations (a number of accumulative silent mutations and a number of non-silent point mutations) to test whether or not its mRNA secondary structure affects its targeting for secretion. In the second study, the two main key secretion factors of Secdependant pathway, ffh and secb, were separately knocked out and overexpressed to uncover their potential involvement in pro-STb targeting and secretion processes. In the final study, an in vitro approach was devised to identify and characterize the STb cell surface receptor on STC-1 cells (mouse intestinal enteroendocrine cell line). The results indicated that the nucleotide sequence of the STb mRNA (within the N-terminal amino acid signal sequence of the toxin) can affect toxin secretion. Reducing the AU richness within the 5′ end of the STb mRNA (without altering the translated amino acid sequence of this region) significantly reduced toxin secretion. From the second study, deletion of FFh, the protein component of the signal recognition particle in E. coli, resulted in significant effects on targeting and translocation of pro-STb and STb to the cytoplasmic membrane and the extracellular milieu more so than SecB deletion. Overexpression of FFh, not SecB, was shown to enhance STb synthesis and secretion. Finally, the results of the third study confirm a direct disruption to the tight junction of STC-1 cells. The results strongly suggest that STb interaction with these cells is mediated, at least in part, by the tight junction associated protein occludin. This interaction ultimately led to Caspase 3-mediated apoptosis.
Unraveling the metabolic networks involved in the utilization of L-glutamine in Pseudomonas fluorescens exposed to nutritional stress
(2022-06-28) Legendre, Félix
Sulfur plays an essential role in oxidative homeostasis due to its participation in sulfhydryl groups (SH). A disruption of this vital nutrient is known to promote oxidative stress and activates a plethora of anti-oxidative strategies. Phosphate, a micronutrient that is part of adenosine triphosphate (ATP), the main molecule used as energy and other macromolecules in living cells. The stress response to sulfur and phosphate deficiency in Pseudomonas fluorescens was investigated with emphasis on ROS detoxification, and energy production. Metabolite profiling was performed by High Performance Liquid Chromatography (HPLC), enzymatic analysis was done using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and gene expression assessment of targeted genes was performed with SYBR-Green real-time PCR (qPCR). When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. The up-regulation of isocitrate dehydrogenase (ICDH)-NADP+ dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis. This reaction is aided by the concomitant increase in NAD kinase (NADK) activity. The latter converts NAD+ into NADP+ in the presence of ATP. Additionally, the microbe reprograms its metabolic pathways to produce KG and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KDC) work in partnership to synthesize KG. This process is further aided by the increased activity of the enzymes glutamate decarboxylase (GDC) and γ-amino-butyrate transaminases (GABA-T). Taken together, the data point to a metabolic network involving isocitrate, KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment. Finally, when cultured in low phosphate environments, the microbe can produce ATP via substrate level phosphorylation (SLP), in a mechanism involving the reductive isocitrate dehydrogenase (ICDH-NADH), isocitrate lyase (ICL), malate synthase (MS) as well as phosphoenol pyruvate carboxylase (PEPC), phosphoenol pyruvate synthase (PEPS) and pyruvate phosphate dikinase (PPDK). This metabolic reprogramming ensures the survival of the microbe and reveals the central role metabolism plays in cellular adaptation to abiotic stress.
Transcriptome and methylome analysis of trembling aspen (Populus tremuloides) under nickel stress.
(2022-04-28) Czajka, Karolina M.
Nickel is an essential micronutrient required at low concentrations for adequate plant growth and health. However, excessive amounts of bioavailable nickel ions in the surrounding soil can result in plant toxicity symptoms. Plants have evolved heavy metal tolerance mechanisms to adapt and cope with this abiotic stressor. The main objectives of the present research were to 1) further characterize the P. tremuloides transcriptome 2) compare gene expression dynamics between nickel-resistant and nickel-susceptible P. tremuloides genotypes with Whole Transcriptome (WT) sequencing, 3) determine the effects of different nickel concentrations on P. tremuloides gene expression and, 4) assess global methylation levels in P. tremuloides under nickel stress. Trembling aspen (Populus tremuloides) seedlings treated with varying concentrations of nickel nitrates (150 mg Ni / 1 kg of dry soil, 800 mg / kg, and 1, 600 mg / kg) showed phenotypic segregation of physical toxicity symptoms at the highest nickel dose of 1, 600 mg / kg. This study revealed that a metal transport protein (Potrs038704g29436 – ATOX1-related copper transport) was among the top upregulated genes in resistant genotypes when compared to susceptible plants. Other upregulated genes associated with abiotic stress were identified including a Dirigent Protein 10, GATA transcription factor, Zinc finger protein, Auxin response factor, Bidirectional sugar transporter, and thiamine thiazole synthase. Overall, an upregulation in ribosomal and translation activities was identified as the main response to Ni toxicity in the resistant plants. The results of the dosage analysis suggested that the 800 mg / kg nickel dose is the threshold at which an early abiotic stress response may be triggered as seen by the highly upregulated LEA protein and two calcium binding proteins when compared to water. The cluster of genes that had increased gene expression with increasing nickel dose also had multiple enriched GO terms related to heavy metal and abiotic stress including metal ion transport, antioxidant activity, photosynthesis, and ribosomal activity. Lastly, the initial screen for potential global methylation differences between nickel-resistant genotypes and water showed no significant difference in overall methylation levels. However, the potassium nitrate control for the 1,600 mg / kg dose did show significantl hypomethylation in comparison to the nickel-treated or water control samples. Future experiments could use targetspecific methylation and gene expression assays to investigate the biological significance of the heavy metal stress candidate genes identified in this top-down study in trembling aspen. Understanding the heavy metal tolerance mechanisms and responses used by hardy species like trembling aspen is important for environment bioremediation and maintenance of healthy ecosystems.
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.
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.
Unraveling the potential for the novel agent, VR23, and its use as an anti-inflammatory for both acute and chronic inflammatory conditions
(2021-06-29) Durkin, Amanda
Inflammatory conditions continue to be on the rise in Canada, due in part to the increase in aging population. Although effective in some cases, the anti-inflammatory drugs that are currently available have their own pitfalls, with toxic side effects and being non-selective in their mechanisms of action. In an attempt to develop an effective anti-inflammatory drug, I have characterized VR23, a novel 4-aminoquinoline derived sulfonyl hybrid compound. VR23 was initially developed in our laboratory as potentially an effective and safe anticancer agent. Previously, data obtained from an in vivo study for its anticancer effects raised a possibility that VR23 might also possess anti-inflammatory property. In a nutshell, data presented in this thesis confirm that the hypothesis is correct. In the study, I used both acute and chronic inflammatory models. In Chapter 1, I have shown that VR23 is able to effectively down-regulate proinflammatory cytokines comparably to dexamethasone, a well-known anti-inflammatory agent. Specifically, VR23 was able to down-regulate IL-6 with great sensitivity. In rheumatoid arthritis cell models of chronic inflammation, VR23 demonstrated superiority over the anti-rheumatic hydroxychloroquine in its ability to regulate pro-inflammatory cytokines. In Chapter 2, I demonstrated VR23’s anti-inflammatory mechanism is likely through its prevention of the phosphorylation of STAT3, leading to a decrease in the production of its down-stream targets, IL-6 and MCP-1. Lastly, in Chapter 3 I describe the discovery that VR23 is rapidly metabolized into CPQ and DK23. CPQ is not an active compound with respect to its anti-inflammatory activity, indicating that it is a by-product of the VR23 detoxification process. On the contrary, DK23 possesses active anti-inflammatory property, as potent as VR23 at their respective IC50 concentrations. Data from an acute lung injury model showed that the anti-inflammatory activity of VR23 is comparable to that of dexamethasone, a well-known corticosteroid. Data obtained from the rheumatoid arthritis study showed that VR23 is much more superior to hydroxychloroquine, a commonly used anti-rheumatic drug. Overall, this study demonstrates the potential for the novel compound, VR23, to be used as a non-toxic specific anti-inflammatory drug to treat IL-6 driven conditions such as rheumatoid arthritis.
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.
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.
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.
The role of reactive oxygen species and epigenetics in the fetal programming of hypertension by glucocorticoids
(2020-08-20) Lamothe, Jeremy D.
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
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.
Uncovering the therapeutic potential of Uncaria tomentosa using B16-BL6 mouse melanoma and 4T1 mouse breast cancer cells
(2019-03-14) Zari, Ali
Uncaria tomentosa (Rubiaceae), a medicinal plant native to Peru, has been traditionally used for centuries as a treatment for a wide variety of diseases, as well as to maintain health. It grows primarily in the Amazon rainforest and throughout South and Central America. U. tomentosa extracts have been demonstrated to have anti-oxidant, anti-apoptotic, anti-inflammatory and anti-cancer properties. The goal of this study was to examine Uncaria tomentosa in both in vitro and in vivo models in order to evaluate its potential anti-cancer activity using the B16-BL6 mouse melanoma and 4T1 mouse breast cancer cell lines. Both ethanol and PBS extracts of U. tomentosa were prepared and used to measure the effects on cell growth and survival using several different methodologies. Treatment of cells with ethanol extracts was much more effective at inhibiting cancer cell growth than treatment with PBS extracts in vitro, but no significant differences in the cancer inhibitory effects were observed in vivo. The in vitro experiments showed that treatment with the U. tomentosa extract significantly inhibited the growth of both B16- BL6 and 4T1 cell lines. It also inhibited the expression of the Ki-67 proliferation marker and promoted cell death as measured by increased DNA fragmentation using TUNEL assays in cancer cells. Treatment with the ethanol extract of U. tomentosa caused a significant increase in the fraction of apoptotic cells in flow cytometry (i.e. sub G1 peaks). Furthermore, two animal experiments were performed in order to evaluate the effect of U. tomentosa treatment on B16-BL6 cells in C57BL/6 mice. The results of the in vivo experiments concluded that treatment with U. tomentosa reduced tumour weight and tumour size. Histochemical analysis of the B16-BL6 tumours showed a strong reduction in the Ki-67 cell proliferation marker and a small but not significant increase in DNA fragmentation in U. tomentosa-treated mice compared to the control. Further, U. tomentosa extracts reduced staining for Factor VIII, a marker for endothelial cells, indicating a decrease in angiogenesis in treated mice. Since U. tomentosa has been shown to affect immune system function, the infiltration of several different immune cells into the tumour was examined. No significant differences in the number of infiltrating T cells (including T helper and cytotoxic T cells), B cells, or platelets were found between the treated groups and the control. Collectively, the results in this study concluded that U. tomentosa has potent anticancer activity that significantly inhibited cancer cell growth both in vitro and in vivo. The discovery of new medicinal plants that are effective against cancer cells may provide a strategy to develop cancer therapy and requires more attention.
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.
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.
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.
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.
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.

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