Chemical Sciences - Master's Theses

Permanent URI for this collectionhttps://laurentian.scholaris.ca/handle/10219/2073

Browse

Now showing 1 - 20 of 58

The impacts of sex and genetic background on the response of Drosophila melanogaster to essential and non-essential metal toxicity.
(2024-05-09) Slobodian, Mitchell Read
The following thesis investigates the impact of biological sex and genetic background on the response to copper and nickel toxicity in Drosophila melanogaster. Sex differences are historically understudied across biology despite significantly impacting metabolism, disease, and response to toxicants like toxic metals. Understanding the sexual dimorphism of metal toxicity is important for understanding the impact of metal pollution on the surrounding community and environment of mining communities around the globe. I predict that biological sex and genetic background will have a significant impact on the fly response to copper and nickel toxicity. I used the model organism D. melanogaster in multiple lethality assays to determine how four different isogenic lines and the two sexes respond to both copper and nickel toxicity. I performed multiple lethality assays using various concentrations of nickel and copper which concluded that sex and genetic background had a significant impact on the fly response to metal toxicity. The results of the lethality assays were supported by a subsequent starvation assay and measurement of ingested nickel content by inductively-coupled mass spectrometry (ICP-MS). The results of both the lethality assays and measurement of ingested nickel suggest that the common general metal response pathway which is responsible for maintaining metal homeostasis is significantly impacted by sex and genetic background.
Investigating the immune modulating effects of low dose ionizing radiation as a potential cancer therapy
(2023-05-30) Valiquette, Manon
Ionizing radiation is an established treatment modality for cancer. Unlike targeted high dose therapies, there is growing evidence that low dose radiation (LDR) can promote tumor reduction indirectly via stimulation of the immune system. Natural killer (NK) cells are one of the main immune cells that have been implicated in LDR induced anticancer effects. Despite this, the exact cellular and molecular mechanisms responsible for the modulation of the immune system following LDR, including the stimulation of NK cells and their cytotoxic properties, have not yet been identified. The goals of this study were twofold; to elucidate the cellular mechanisms involved in LDR immune stimulation and to investigate the dose response for NK cell cytotoxicity. Initially, a small animal model was used to comprehensively characterize the dose dependent effects on various immune cells. Mice were exposed to whole-body x-ray doses of 0.1, 0.25, 0.5 and 3 Gy and were sacrificed two days post-irradiation for isolation of their spleen, lymph nodes and blood. Flow cytometry was then used for immunophenotyping to identify potential shifts in the relative abundance of major immune cell populations. Data from this cohort of mice suggested that 2 days following an acute single exposure, LDR caused no significant changes in the numbers of the immune cell types tested, and that high dose radiation (HDR) caused a decrease in the cell populations of these cells in the irradiated mice. To further investigate the impact of LDR on the NK cells in-vitro, the NK-92 cell line was used. NK-92 cells were exposed to x-ray doses ranging from 0.1 to 1 Gy, and cell growth rates were measured postradiation. NK cell cytotoxicity was then quantified through the co-culturing of NK-92 cells with the tumorigenic K-562 cells, and the percent cytotoxicity was measured using flow cytometry. Lastly, transcriptional analysis was performed on the main genes involved in regulating NK cytolytic activity. Overall, data showed that LDR did not cause any significant increase in the growth, cytotoxicity and gene expression of NK-92 cells. To conclude, although no evidence of immune stimulation was found following LDR in both the mice model and NK-92 cell line, this study was successful in providing a good characterization of the baseline immune response to lower doses of radiation in healthy models.
The role of the Non-neuronal Cholinergic System in immune regulation
(2022-02-22) Roy, Danika
Activation of nicotinic acetylcholine receptors (nAChRs) by agonists like acetylcholine (ACh) can have anti-inflammatory properties via the regulation of cytokines. A recent novel class of nAChR specific molecules termed silent agonists are also thought to induce nAChRdependent metabotropic signaling cascades, while causing very little channel opening. These molecules may therefore be potent immune regulators. This study assessed the expression of cholinergic genes in human macrophages and gained insights on the anti-inflammatory properties of nAChR agonists, silent agonists and cholinergic inhibitors in human macrophages. For both aims, two human monocytic cell lines (THP-1 and U937) were cultured and differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) to obtain Mo macrophages and in some cases, further polarized into M1 (INF-γ) or M2 (IL-13 and IL-4). Expression of cholinergic genes were first determined via Western Blot and PCR. Secondly, experiments assessing expression of cell markers, cytokine expression, and viability were carried out following treatment with various conditions in the presence of LPS. It was hypothesized that macrophages would express cholinergic proteins and that nAChR agonists/silent agonists would result in anti-inflammatory responses whereas cholinergic inhibitors would result in proinflammatory responses. Overall, we found that differentiated macrophages expressed most cholinergic proteins and that the inhibition of cholinergic proteins and nAChR agonism modulated LPS-induced cytokine release. These experiments gave pertinent information regarding the presence of cholinergic genes and the anti-inflammatory properties of agonists/silent agonists in human macrophages.
Identification of radiation induced miRNA biomarkers using the CGL1 cell model system
(2022-06-01) Peterson, Jayden
The overall aim of this study was to identify novel radiation induced miRNA biomarkers for low dose radiation (LDR) exposures. MicroRNAs (miRNAs) have emerged as a potential class of biomolecules for diagnostic biomarker applications. miRNAs are small non-coding RNA molecules produced and released by cells in response to various stimuli. miRNAs also demonstrate remarkable stability in a wide range of biological fluids, in extreme pH fluctuations, and after multiple freeze-thaw cycles. Given these advantages, identification of microRNA-based biomarkers for radiation exposures can contribute to the development of reliable biological dosimetry methods, especially for LDR exposures. In this study, an miRNAome next-generation sequencing (NGS) approach was utilized to identify novel radiation induced miRNA gene changes within the CGL1 human cell line. Here, irradiations of 10, 100, and 1000 mGy were performed and samples collected 1, 6 and 24 h post-irradiation. Corroboration of the NGS results with RT-qPCR verification confirmed the identification of numerous radiation-induced miRNA expression changes at all doses assessed. Further evaluation of select radiation induced miRNAs including miR-1228-3p and miR-758-5p, as well as their downstream mRNA targets UBE2D2, PPP2R2D and ID2 demonstrated significantly dysregulated reciprocal expression patterns. In order to explore the miRNA profile in a sub-background ultra-low dose radiation (ULDR) environment, the CGL1 cells were grown in the underground SNOLAB laboratory in a specialized tissue culture incubator (STCI) designed to lower natural background radiation by a 27-fold difference relative to surface. Here, the cells were grown in the ULDR environment and natural background radiation (NBR) controls for a period of three months. Total RNA was periodically extracted to assess miRNA expression levels. Here, miR-502-3p was significantly upregulated in CGL1 cells cultured in the ULDR environment compared to the NBR control cells. Taken together, this study has identified novel radiation induced miRNA biomarkers at various radiation dose ranges, spanning from ULDR, LDR (10 and 100 mGy) and high dose (1 Gy) radiation exposures. Further evaluation is needed to determine whether the candidate miRNA biomarkers identified from this study can serve as suitable targets for radiation biodosimetry applications.
L-Carnitine Production in Pseudomonas fluorescens under phosphate starvation
(2021-09-30) MacLean, Alex
Glycerol is a by-product of the biodiesel industry and an important carbon source for bacterial growth. Phosphorus is an essential element in all living organisms and is utilized in numerous metabolic processes, such as the tricarboxylic acid (TCA) cycle and the electron transport chain. Without phosphorus, no organism can either grow or perform regular functions. In this study, we demonstrate that when the industrially-important microbe Pseudomonas fluorescens is deprived of phosphate, it elaborates a metabolic reconfiguration aimed at producing and secreting copious amounts of L-carnitine. To accomplish this biochemical adaptation, the organism bypassed the TCA cycle and utilized the glyoxylate shunt to generate a constant supply of L-carnitine through different metabolic networks. The upregulation of numerous enzymes including L-carnitine dehydrogenase (lcdH) and isocitrate lyase (aceA) mediated this process. The metabolic reprogramming triggered by phosphate may provide an effective means to transform an industrial waste into valuable L-carnitine.
The role of non-neuronal acetylcholine production in immune cells
(2021-10-01) Lachapelle, Maryse
The cholinergic system deals with the production, function, transport, and degradation of acetylcholine (ACh), a molecule commonly known as a neurotransmitter. This system has been shown to regulate inflammation via its resolution. Although increasing evidence suggests the importance of ACh in immune regulation, its role within specific immune cells remains inconclusive. Assessing the presence of cholinergic markers in immune cells in conjunction with their pharmacological inhibition will thus help to clarify the functional roles of the cholinergic system in the immune response. The aim of this study was to evaluate the role of non-neuronal ACh production in immune regulation. Expression studies using qPCR, western blots, mass spectrometry, and immunocytochemistry revealed the presence of ACh and choline acetyltransferase in immune tissues and within immune cells of such tissues. In addition, the effect of cholinergic inhibitors on immune function was examined on M1/M2 murine bone marrow derived macrophages (BMDMs) by examining their cytokine profile. Overall, inhibiting the synthesis of ACh seemed to significantly cause cell death in M1 BMDMs and non-significantly cause cell death in M2 BMDMs; yet other treatment conditions did not seem to cause cell death. However, due to pharmacological and statistical limitations, cytokine secretion profiles were generally inconclusive. These data further shed light on the role cholinergic system within immune cells, but further research would be necessary to validate these findings.
RNA disruption is a widespread phenomenon that is associated with cell death in tumor cells
(2021-10-19) Butler, Phillipe
A recent clinical trial found that chemotherapy-induced tumor rRNA degradation (RNA disruption) was associated with both pathological complete response (complete tumor destruction) and extended disease-free survival (DFS) in breast cancer patients treated with neoadjuvant chemotherapy. The RNA Disruption Assay (RDA) is a useful tool to quantify the magnitude of RNA disruption occurring within cells or tumors, expressed as the RNA Disruption Index (RDI), and is currently the subject of an international clinical trial. The purpose of this study was to investigate the phenomenon of RNA disruption in response to both chemotherapy agents and cellular stressors in the ovarian carcinoma A2780 cell line and the breast adenocarcinoma MDA-MB-231 cell line. Also, we assessed the relationship between RNA disruption and both cell death and RNase L expression using multiple cell viability assays and CRISPR-Cas9-mediated RNase L knockout clones, respectively. RNA disruption was induced in response to multiple chemotherapy agents and several cellular stressors, including oxidative stress, ER stress, protein translation inhibition and starvation. However, the induction and magnitude of RNA disruption was found to be both dose- and stressor-specific, and dependent on the cell line studied. Mycoplasma contaminations of host cell lines were also found to potentiate a markedly different RNA disruption banding pattern in response to chemotherapy. RNA disruption was also found to be associated with cell death, as significant increases in RDI values were observed for chemotherapy doses that induce partial or complete cell death, as measured by cell counting, and both cellular recovery and flow cytometry DNA content analysis. Finally, knockout of RNase L expression in the A2780 cell line did not affect RNA disruption, demonstrating that RNase L in not involved in the RNA disruption mechanism(s). Overall, these findings support the novel role of RDA as a tool for quantifying tumor cell death in vitro and in vivo.
The interaction of disulfiram and H2S metabolism in inhibition of aldehyde dehydrogenase activity and liver cancer cell growth
(2021-06-29) Read, Ethan
Disulfiram (DSF) is a sulphur-containing compound and has been used to treat chronic alcoholism and cancer for decades. DSF inactivates aldehyde dehydrogenase (ALDH) by modifying its cysteine residue(s). ALDH is recently identified as a cancer stem cell marker, facilitating cell self-renewal and tumour-initiating capacity. Hydrogen sulphide (H2S) as a new gasotransmitter regulates various cellular functions by the S-sulfhydration of cysteine residues in target proteins. H2S has also been shown to exhibit similar properties to DSF in the sensitization of cancer cells to chemotherapeutic agents. Here, the potential of DSF as a H2S-releasing donor under various conditions was investigated and the roles of H2S in the DSF-mediated inhibition of ALDH activity and decrease in cell viability in liver cancer cells were also examined. It was demonstrated that DSF facilitated H2S release from thiol-containing compounds, and DSF and H2S were both capable of regulating ALDH through inhibition of gene expression and enzymatic activity. The supplement of H2S sensitized human liver cancer cells (HepG2) to DSF induced reduction in cell viability. The expression of cystathionine gamma-lyase (a major H2S-generating enzyme) was lower but ALDH was higher in mouse liver cancer stem cells (Dt81Hepa1-6) in comparison with their parental cells (Hepa1-6), and H2S was able to inhibit liver cancer stem cell adhesion. In conclusion, these data provide some clues for combining with DSF and H2S for inhibition of cancer cell growth and tumour development by targeting ALDH.
Hydrogen sulfide (H₂S) attenuates lipotoxicity and cardiac cell senescence by regulating protein acetylation
(2021-07-26) Yu, Ruihuan
Hydrogen sulfide (H2S) is recently recognized as a novel gasotransmitter. H2S can be endogenously generated from cysteine in mammalian tissues, and cystathionine gamma-lyase (CSE) is a critical enzyme generating H2S in the cardiovascular system. Increasing evidence suggests that interference in H2S production is related to heart diseases. Obesity is a leading risk factor for heart dysfunctions by interrupting lipid metabolism. In the current work, the regulatory roles of the CSE/H2S system on lipid overload-induced lipotoxicity and cardiac senescence were explored. Here, it was found that incubation of H9C2 rat cardiomyocyte cells with a lipid mixture inhibited cell viability and promoted the cellular accumulation of lipids, formation of reactive oxygen species, mitochondrial dysfunctions, and lipid peroxidation; all of these could be reversed through incubation with the exogenously applied NaHS (the H2S donor). Further data revealed that H2S protected H9C2 cells from lipid overload-induced senescence by altering the expression of genes related to lipid metabolism and inhibiting both the production of acetyl-CoA and the level of protein acetylation. In vivo, knockout of the CSE gene strengthened cardiac lipid accumulation, protein acetylation, and cellular ageing in the mice fed a high-fat diet. Taken together, the CSE/H2S system is essential for maintaining lipid homeostasis and cellular senescence in heart cells under lipid overload. The CSE/H2S system would serve as a target for preventing and treating obesity and age-related heart diseases.
Glutamine synthetase expression and stress tolerance in Drosophila melanogaster
(2021-08-03) Daypuk, Jenna
This thesis explores glutamine synthetase in Drosophila melanogaster. L-glutamine, more than other amino acids, is strictly required for cell survival. Like many complex organisms, D. melanogaster has two forms of the enzyme glutamine synthetase (GS): GS1, located in the mitochondria, and GS2, located in the cytosol. Interestingly, how these two isozymes partition GS function is unknown. I validated a sample preparation protocol to separate the mitochondria and cytosol from Drosophila samples, and assay solutions for the measurement of GS transferase and GS biosynthetic activity. I created knockdowns of GS1 and GS2 and subjected these to heat shock and paraquat-induced stress. The GS1 and GS2 knockdowns displayed differences in their response to both stressors, indicating a potential functional differentiation of the two isozymes in stress tolerance. This project is a step in the use of Drosophila to explore the GS network and how glutamine modulates cell survival.
Modulation of antimicrobial activity by liposomal antibiotics for the treatment of Pseudomonas aeruginosa isolated from cystic fibrosis patients: potential role of the efflux pump inhibitor Phenylalanine Arginine-β-Naphthylamide
(2020-12-14) Gbian, Douweh Leyla
Cystic fibrosis (CF) is an autosomal recessive disease that stems from mutations in the CF transmembrane conductance regulator (CFTR) gene. Chronic pulmonary infections are the main cause of death observed in patients with CF, as they can bring about pulmonary exacerbations, inflammation and eventually lung and respiratory failure. Eradication of Pseudomonas aeruginosa, the principal pathogen found in CF patients, is extremely challenging due to its numerous resistance mechanisms, namely efflux pumps and reduced permeability. In the race against antibiotic resistance, there is a pressing need to develop new ways to revive existing antimicrobials and enhance their activity. Indeed, liposomal formulations of antibiotics were shown to revitalize the drugs by increasing their activity against pathogens and reducing associated toxicity. Similarly, phenylalanine arginine-β-naphthylamide (PABN) is a potent efflux pump inhibitor that demonstrated great activity against bacteria like P. aeruginosa by inhibiting drug efflux. In this study, liposomal gentamicin and erythromycin were prepared with the dehydrationrehydration vesicle method and their effects on biofilm formation, quorum sensing, virulence factors production and motility in the presence and absence of PABN were evaluated against clinical and laboratory isolates of P. aeruginosa. Liposomes and PABN combinations potentiated antibiotics and reduced the production of biofilms, virulence factors and motility in the bacteria. These results indicate that both liposomal gentamicin and erythromycin combined with PABN show good promise in keeping P. aeruginosa infections under control in CF lung infections.
Diet-induced insulin resistance & exercise in Drosophila melanogaster is highly influenced by genotype & sex
(2020-11-03) Eng, Michelle
Type 2 Diabetes, characterized by a combination of insulin resistance and impaired insulin secretion, is a serious metabolic disorder in humans that not only affects adults, but now young children. The model organism Drosophila melanogaster has emerged as an excellent model to study metabolic regulation. Using a high sucrose diet to induce insulin resistance, I used multiple fly lines to explore how genotype and sex interacts with the progression of insulin resistance and how exercise can influence this response. Flies were grouped and placed on one of four conditions: control diet (CD), high sucrose diet (HSD), control diet with exercise (CDEx), or high sucrose diet with exercise (HSDEx). Flies were exercised using a fly treadmill. Weight and various metabolites known to respond to diet and exercise were quantified, along with gene expression of various nodes in the insulin pathway. Twenty-five percent of fly lines on the HSD displayed symptoms of insulin resistance, such as hyperglycemia, glucose intolerance, and/or hyperinsulinemia, as well as patterns in insulin pathway gene expression that is indicative of insulin resistance. Flies experienced physiological changes in response to exercise, which changed 11-13% of the metabolome of each sex, and 20-29% of the metabolome of each line. Strikingly, across all experiments, fly response to diet and exercise was highly genotype and sex-dependent. This study demonstrates the complex nature of insulin resistance and most importantly, the importance of studying disease related states using multiple genetic backgrounds and both sexes.
Effect of doxorubicin and/or natural killer cells on RNA disruption in K562 chronic myeloid leukemia cells
(2020-09-22) Pascheto, Isabella
It has recently been observed that a variety of mechanistically distinct chemotherapy agents and cellular stressors induce ribosomal RNA (rRNA) degradation in tumour cells, a phenomenon termed RNA disruption. The RNA disruption assay (RDA) has been developed to quantify RNA disruption in a manner that can predict pathologic complete response and improved disease-free survival early in treatment. Immune checkpoint inhibitor (ICI) drugs are novel anti-cancer agents that can result in significant improvement in patient survival, but not all patients respond to these drugs. Thus, the RDA may be able to identify early in treatment ICI non-responders in order to avoid the continued costs and harmful side effects of this class of drugs and to consider alternate treatments. Since ICI drugs function by enhancing the efficacy of endogenous immune cells, this study assessed the ability of the RDA to quantify and monitor immune cell-mediated destruction of tumour cells in the absence or presence of the cytotoxic chemotherapy drug doxorubicin (DOX). This involved using the RDA to determine the RNA disruption index (RDI) for a particular sample via capillary gel electrophoresis and a proprietary algorithm. Loss of membrane integrity was also used to measure the cytotoxicity of immune cells towards tumour cells. In the K562 chronic myeloid leukemia cell line, RNA disruption was induced by a variety of chemotherapy agents, including DOX. Freshly isolated primary human natural killer (NK) cells also induced RNA disruption and loss of membrane integrity in K562 cells in a cell number-dependent manner. Pre-activation of NK cells with IL-2 augmented K562 cell RNA disruption and loss of membrane integrity in a dose-dependent manner. Preliminary studies suggested that pre-treatment with DOX may also augment NK cell-mediated RNA disruption in K562 cells. NK cell-mediated RNA disruption and loss of membrane integrity appear to correlate in a strong, positive manner in K562 cells. The pattern of rRNA degradation fragments induced by NK cells was very similar to that induced by chemotherapy agents. Taken together, these findings suggest that the RDA may have clinical utility in monitoring immune cell-mediated destruction of tumour cells induced by ICI therapies (prior to or after chemotherapy).
Analysis of tramadol and its metabolites in rat skeletal tissues following acute and repeated dose patterns using high performance liquid chromatography tandem mass spectrometry
(2020-10-15) Buckingham, Christian L.
The use of skeletal elements for the viable analysis of drugs of abuse has seen increased prevalence in the past 10 years. Advancements in the analytical methods used, including solid phase extraction and mass spectrometry, have allowed for increased sensitivity and selectivity. Previous studies have focused on the influence of dose-death interval, microclimate, differential patterns of exposure, and the influence of body position. In this work, the opioid analgesic tramadol was investigated for its pharmacological behaviour when administered as part of three dosage patterns to male Sprague Dawley rats. The three exposure patterns consisted of an acute low (n = 4, 1 doses, 30 mg/kg) group, a repeated high survived group (n = 5, 3 doses, 30 mg/kg) and a repeated high overdosed group (n = 11, 3 doses, 30 mg/kg). Drug free rats (n = 4) served as negative controls. Following euthanasia by CO2 asphyxiation, animals were decomposed to skeleton outdoors over the summer of 2019 in Sudbury, Ontario. Bones were sorted by animal and skeletal element (skull, vertebrae, ribs, pelvis, femur, tibia/fibula), then washed and ground to powder before undergoing dynamic methanolic extraction. Semi-quantitative analysis of tramadol and four of its metabolites – O-desmethyltramadol, N-desmethyltramadol, N,Odidesmethyltramadol and tramadol N-oxide – was conducted using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) in positive ion mode. Analyte levels were expressed as a mass-normalized response ratio (RR/m) in order to account for the exact mass of bone used. Method validation for the analysis of tramadol and its metabolites was investigated in accordance with the Scientific Working Group of Toxicologists (SWGTOX) standards of practice, with all criteria except for dilution integrity successfully met at a limit of detection and limit of quantitation of 1 ng/mL. The effect of exposure pattern on analyte level and analyte level ratio was assessed using the Kruskal-Wallis test for significant differences (P < 0.05). A total of 315 pairwise comparisons were performed to assess significant differences, with the ratio of tramadol to N-desmethyltramadol determined to be the metric most commonly able to identify these differences in 91% of tests. Additionally, the effect of skeletal element on analyte level and analyte level ratio was also assessed, with a total of 675 pairwise comparisons. Skeletal element was determined to be a significant factor in all cases. This data suggests that both skeletal element and dose pattern are important measures to evaluate with respect to the analysis of drugs of abuse in bone tissues. Furthermore, different metrics, including analyte level and analyte level ratios, may be useful for discriminating between these different dosing patterns.
Studies on the unfolding and zinc status of thermolysin and carboxypeptidase A, and the design of a fret-quenched peptide for assaying thermolysin-like proteases
(2020-09-16) Goulet, Danica
The chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) has previously been employed as a probe to determine structural transitions during protein (un)folding in the zincdependent anthrax lethal factor protease (LF). Whether PAR can be employed more widely as a zinc accessibility probe (ZAP) to study the unfolding of other zinc proteins remains to be established. Using a combination of intrinsic tryptophan fluorescence spectroscopy and chelator studies, the unfolding pathways and the influence of the protein fold on the metal status were investigated for the zinc proteases thermolysin (TL) and carboxypeptidase A (CPA). These studies revealed considerable differences in the unfolding pathways and the resistance to metal dissociation for these enzymes. In addition, the observations reported herein demonstrate that ZAPs might be of value in future investigations to gain further insight into the mechanisms underlying the metal-mediated misfolding of proteins involved in a variety of neurodegenerative diseases. Finally, this study describes a new fluorescence-quenched heptapeptide (Dabcyl-FKFLGKE-EDANS), which shows the largest specificity constant documented for any TL substrate.
The roles of H2S on hepatic acetyl-CoA and lipid metabolism
(2020-07-13) Ali, Amr
Hydrogen sulfide (H2S) is a novel gasotransmitter that is endogenously produced in the liver by cystathionine γ-lyase (CSE). The CSE/H2S system dysfunction has been linked to various liver diseases such as fatty liver diseases. Hepatic acetyl-CoA is a key intermediate from the metabolism of glucose, amino acid, and lipid in most species, but the roles of H2S in the regulation of hepatic acetyl-CoA and lipid metabolism have not been explored. Here, we found that incubation of human liver carcinoma (HepG2) cells with a mixture of free fatty acids (FFAs) or high glucose reduced CSE expression and H2S production and promoted intracellular accumulation of acetyl-CoA and lipid. Supply of exogenous NaHS (an H2S donor) or cysteine (an H2S precursor) reduced acetyl-CoA content and lipid accumulation, while blockage of CSE activity by DL-propargylglycine promoted intracellular lipid accumulation. Furthermore, H2S blocked FFAs-induced transcription of de novo lipogenesis, inflammation, and fibrosis-related genes. In vivo, knockout of CSE gene stimulated more hepatic acetyl-CoA and lipid accumulation in mice induced by high-fat choline-deficient diet. The expression of lipogenesis, inflammation, and fibrosis-related genes were significantly higher in liver tissue from CSE knockout mice when compared with wild-type mice. In conclusion, the CSE/H2S system is indispensable for maintaining the homeostasis of acetyl-CoA and lipid accumulation and protecting from the development of inflammation and fibrosis in liver under excessive caloric ingestion, and CSE/H2S system constitutes an interesting target for the prevention and treatment of fatty liver disease.
Development of a glycoliposomal delivery system to improve dynantin antagonism of the kappa opioid receptor
(2019-12-13) Boraman, Amanda
κ opioid receptors (KOP) play a role in the addictive properties of nicotine by opposing its rewarding effects. Using a KOP antagonist, we aim to modulate this relationship, and prevent the negative effects of withdrawal that causes individuals to relapse. A novel glycoliposomal delivery system was used to improve the stability of dynantin, a KOP antagonist peptide, in human plasma. Reverse phase high-performance liquid chromatography (RP-HPLC) was used to study the entrapment and stability of dynantin. Subsequently, the in vitro Parallel Receptor-ome Expression and Screening via Transcriptional Output–Transcriptional activation following arrestin translocation (PRESTO-TANGO) system was used to study the affinity and selectivity of dynantin at the KOP compared to the δ and μ opioid receptors (DOP and MOP). Results showed that dynantin had good selectivity for KOP without activating or blocking DOP or MOP. The ratio of peptide: mannose lipid: cholesterol was modified to improve the stability of dynantin in human plasma. Dynantin was completely degraded in plasma after 24 hours with no cholesterol present, while a 1:5:2 ratio (peptide: mannose lipid: cholesterol), had 71±4% of dynantin remaining after 24 hours. Importantly, the delivery system had no effect on the affinity of dynantin at the KOP. Our delivery system therefore shows promise in its ability to increase the bioavailability of therapeutic peptides such as dynantin, for which we demonstrate here a promising ability to act selectively as an antagonist at the KOP.
An optimized and validated method for screening and quantification of commonly encountered stimulant drugs and selected metabolites in dried blood spot (DBS) samples by ultra performance liquid chromatography-quadrupole time of flight-high resolution mass spectroscopy (UPLC QTOF-HRMS) analysis
(2019-11-13) Unger, Kirk
Blood microsampling techniques, like Dried Blood Spots (DBS), have seen recent development as an alternative to wet blood samples and offer numerous advantages. Quantification of ng/mLrange concentrations using DBS microvolumes (10-20 µL) is made possible with cutting edge instrumentation. Worldwide establishment of per se limits for drug impaired driving requires timely methods of sampling blood for accurate drug concentration measurements and interpretation. The method validation for the analysis of stimulants and metabolites in DBS samples by Ultra Performance Liquid Chromatography-Quadrupole Time of Flight-High Resolution Mass Spectroscopy (UPLC-QTOF-HRMS) is presented. Limits of detection and quantitation to 10 ng/mL was achieved. Method validation criteria was satisfied for 10 of 14 analytes. DBS drug stability over 8 weeks varied by analyte. DBS samples may assist in overcoming challenges in blood sampling as they are less invasive, easily transported and stored, and accurate drug quantitation even at low concentrations in DBS samples is possible.
Nanoscale examinations on the sequestration of Cu by organic material in contaminated surface soils in Timmins, Ontario
(2018-07-24) Mantha, Haley
The deposition and alteration of particulate matter (PM) in soils is a world-wide health concern. This study investigates the fate of Copper in organic-rich surficial soil layers (0-5 cm) contaminated by mining related activity at the Timmins Kidd Creek metallurgical site, Canada. The sequestration and mobilization of Cu by organic material (OM) in the soil layer is investigated using focused ion beam (FIB) technology and transmission electron microscopy (TEM). Copper occurs as incidental metallic Cu and covellite (CuS) nanoparticles (NPs), formed via ion reduction by humic substances and magnetite. Additionally, TEM analysis of the colloidal fraction, extracted via ultracentrifugation from the surface soil layer, indicates the occurrence of incidental covellite NPs in close association with dissolved OM. This study shows for the first time that Cu is sequestered as incidental metallic Cu and covellite NPs within OM and magnetite in surficial organic-rich soil layers. Furthermore, the mobility of Cu is facilitated through the transport of covellite nanoparticles embedded within organic colloids.
Genetic background and environmental effects on single nucleotide polymorphisms in the NADPH pathway
(2018-12-13) Baath, Simran
A major focus in modern genomics is determining the connection between genotypes and quantifying phenotypes. In this connection, many factors come into play including different genetic backgrounds, genetic variation at a locus, and environmental conditions. Genetic variation in Drosophila melanogaster, and specifically the simple polymorphisms within Malic enzyme (Men), can provide insight into the pathways between genotypes and phenotypes. Globally, there are two polymorphic sites in the malic enzyme gene. On site is near the protein (MEN) active site and found at an allelic frequency of 50% glycine amino acid and 50% alanine amino acid. The second polymorphism is buried within the protein and found at an allelic frequency of 90% methionine amino acid and 10% leucine amino acid. To determine the complexity of the pathway between genotypes and phenotypes, multiple genetic backgrounds for each genotype, using multiple D. melanogaster lines, were included to explore and quantify genetic background effects, and paraquat was used to induce oxidative stress. The biochemical characteristics of the alleles varied significantly between the genotypes under benign conditions and both polymorphic sites effected some phenotypes. The first site played a role in the MEN Vmax and Km; the glycine allele had 14% higher Vmax activity than the alanine allele and the glycine allele had 8% higher Km than the alanine allele. The second site influenced the Km and Vmax/Km ratio (relative activity); the methionine allele had 34% higher malate Km than the leucine allele the leucine allele had 52% higher relative activity than the methionine allele. Interestingly, the protein product encoded by the rarer allele, leucine, had a higher relative activity and lower Km concentration, having a large impact on the enzymatic phenotype. These extreme phenotypes of that allele may be an indication of the why the allele is maintained at 10% across populations. Different lines with the same genotype had different biochemical phenotypes, indicating the importance of backgrounds effects influencing the final phenotype. Further, the flies’ phenotypes differed between benign and oxidative stress conditions. Flies exposed to paraquat had a decrease in MEN Vmax, and the MEN alleles did not significantly differ from each other. Overall, the findings from this study suggest that the final phenotype are strongly influenced by the polymorphisms found in MEN, the interactions between genetic background and environmental conditions.

Browse

Recent Submissions