MacEwan Open Journals (MacEwan University)
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Identification and Quantification of Microplastics Using Nile Red Staining
No full textPlastic is a very useful and versatile product, however extensive use and unchecked disposal has resulted in significant global impacts. Microplastic (0.1 µm–5 mm) is particularly problematic and is a widespread pollutant impacting aquatic ecosystems. The accumulation of microplastics produces negative repercussions such as aesthetic and economic impact and most importantly adverse biological and ecological effects. Existing identification and quantification techniques such as Raman spectroscopy, Pyrolysis-gas chromatography with mass spectrometry and FT-IR spectroscopy are time consuming and require expensive instruments. The aim of this research is to develop a rapid fluorescent staining procedure for microplastic quantification using a fluorescent dye, Nile Red. Developing a fluorescent staining procedure will provide a rapid way of differentiating microplastics from the other natural materials, enabling easier and more accurate quantification of microplastics. The first step would be the formation of microplastics from common materials such as freezer bags (polyethylene), bottle caps (polypropylene) or food containers (polystyrene). These will be used as standards for further tests including the selection of a suitable organic solvent that would not degrade or stain the filter paper but effectively stain the microplastics. Stained microplastics will be irradiated with blue or green light causing fluorescence, which can then be detected using red filter. This method will be compared to traditional methods such as Raman spectroscopy and brightfield microscopy and then be applied to the samples extracted from the North Saskatchewan River. The results from the study will help in efficient sample processing and understanding microplastic contamination in our environment.
Faculty Mentor: Dr. Matthew Ross
Discipline: Chemistr
The Blackfoot Confederacy in Canada
No full textOur digital story focuses on the Blackfoot in Canada before, during, and after colonization. Our digital story reveals the impacts that colonization had upon their health and well-being. Colonizers caused disease epidemics, implemented residential schools, severed the connection to their culture and traditional ways, and caused a near extinction of the buffalo which was the Blackfoot\u27s primary resource.
 
Understanding the Functional Role of the C-Terminal Domain of the Conserved Protein LepA
No full textA large number of biochemical reactions that enable and regulate life are mediated by enzymatic proteins, which work by lowering the activation barrier of a biochemical reaction to increase the rate at which the reaction occurs. During translation, the ribosome is assisted by multiple different proteins which efficiently catalyze the formation polypeptides, to achieve a functional protein with as little error as possible. Many of the ribosomal associated proteins are GTPases, such as the Elongation Factors (EF-Tu and EF-G), which act as molecular switches by catalyzing the hydrolysis of GTP into GDP and an inorganic phosphate. For example, EF-G catalyzes the translocation of peptidyl-tRNA from the aminoacyl site, to the peptidyl site, as well as the movement of mRNA by one codon.
There are many translational GTPases whose functions are still unknown, one of them being LepA. LepA is a highly conserved GTPase, present in bacteria and eukaryotic mitochondria and chloroplasts. It is the third most highly conserved protein in bacteria, falling behind EF-Tu and EF-G, implying that it is likely serves a significant function in the cell. LepA is very similar in structure EF-G, apart from it’s unique C-terminal domain (CTD). The role of the CTD and how it makes LepA different from EF-G in function are still unknown and is the basis for this study.
For my project, six variants of LepA were constructed to study the role of the CTD including C-terminal truncations and single point mutations. Initial data from our lab showed that several of these variants had altered GTP hydrolysis rates presumably because key functional amino acids in the CTD were removed or mutated. To ensure this was the case, several control experiments were carried out including measuring the binding affinity of LepA wild type and variants to nucleotides (GTP / GDP) were measured via rapid kinetics using a stopped flow. To do so, a fluorescent analog of GTP / GDP was used to monitor binding and unbinding to LepA. The results showed that all LepA variants tested were able to bind nucleotides with the same affinity, suggesting that altered nucleotide binding was not responsible for the change in GTP hydrolysis observed prior, and also that LepA has approximately 8 times higher affinity toward GDP when compared to GTP. Overall the work has contributed to the understanding of the CTD in LepA, and thereby helping to determine the functional role of LepA in protein synthesis.
*Indicates presente
Identification of Conserved Proteins in Nocardia brasiliensis and other Actinomycetoma causative agents using a Bioinformatic approach
No full textMycetoma, a neglected tropical disease, is caused by a multitude of different microorganisms. This condition may be caused by both bacterial and fungal species, with the bacterial species suggested to spread more quickly through the body. Often untreated due to lack of medical access, amputation is ultimately a commonly relied upon resolution to avoid further complications in patients. Nocardia brasiliensis, although a rare pathogen, is the main culprit of actinomycetoma (bacterial-caused mycetoma). This underreported disease has not been fully explored. Currently, actinomycetoma has no preventative measures. Given the difficult patient accessibility of medical care, an effective, practical, pre-emptive approach is worth exploring. One such measure is the implementation of a broad-spectrum vaccine aimed at most, even all, mycetoma causative bacterial agents by targeting a conserved motif. I hypothesize that at least one Nocardia brasiliensis encoded protein is conserved
amongst other actinomycetoma bacteria. Using a bioinformatical approach, identified Nocardia brasiliensis proteins were used to search for homologs in other potential mycetoma-causative agents. Three key DNA replication proteins have been identified as potential candidates. Interestingly, homologs were identified in other Nocardia species, as well as Rhodococcus, Actinobacteria, and Corynebacteriales species. Furthermore, nucleotide sequences encoded by the three most common causative agents of mycetoma were compared. From these analyses, the 16S ribosom was identified as partially conserved amongst them. These identified elements can now serve as a platform for future studies exploring them as a potential vaccine candidate
Light Pollution: An Aerial Perspective
No full textArtificial Sky glow is a growing obstacle for both the public to enjoy the night sky, and Astronomers to make accurate measurements of the universe around us. Given Calgary’s expanding urban geography and population, light pollution and resulting sky glow has continued make the stars less accessible. In this first aerial light pollution observation in Canada, this study will give quantifiable resolution to which areas of the SW quadrant of Calgary and area surrounding the Rothney Astrophysical Observatory create Sky Glow. Measuring Sky glow from above and below the plane using a sky quality meter, giving results in magnitudes per square arcseconds, and further instruments on the bottom giving photon counts from ground sources via digital CCD and a wide lens optics. Resulting in measurements of Sky Glow from above and tying them with sources from below to see if there is a correlation. Further using this data, we will explore the impact of urban development on our night sky.
*Indicates presente
Visualizing Riemannian Surfaces in 3D Using OpenGL
No full textUsing a hand-written C++ program, I will explore visualizations of Riemann surfaces in OpenGL. These surfaces will be plotted by visualizing discrete points in the complex plane. This will allow exploration of these surfaces in a first-person 3D environment
Investigation of myo-inositol Phosphates on Human Cells by Phenotypic Cell-Based Assays
No full textMyo-inositol phosphates (IPs) are a large family of compounds that are ubiquitous in eukaryotes. They are involved in many critical cellular functions, from signal transduction to DNA repair in eukaryotes. Myo-inositol hexakisphosphate (InsP6), commonly known as phytic acid, is the most abundant cellular IP and previous studies have suggested that exogenous InsP6 has anti-proliferative effects on multiple cancer cell lines. By contrast, derivatives of InsP6 with lower phosphate number, such as myo-inositol (1,2,4,5,6)-pentakisphosphate (InsP5), have not been studied due to their costly preparation. Here we investigated the effects of InsP6 and InsP5 on the human bone osteosarcoma cell line, U2OS, by phenotypic assays. After 48 h after treatment with either InsP6 and InsP5 at 15 or 50 µM, cells display a striking vacuole formation, as observed by light microscopy. By staining with acridine orange and observing by fluorescence microscopy, the vacuolated cells showed acidic vesicle accumulation, indicative of increased autophagy. By staining with the non-permeant fluorescent dye Lucifer yellow and fluorescence microscopy we observed that vacuolated cells showed higher levels of the fluorescent dye, indicative of endocytosis, than non-treated cells. Vacuoles observed by phase contrast microscopy are frequently stained with Lucifer yellow and acridine orange. These findings suggest that InsP5 and InsP6 affect endocytosis, which subsequently leads to increased lysosomal degradation the engulfed material. Understanding the effects of IPs on human cells is important in understanding inositol signalling pathways and may lead to the discovery of novel anticancer compounds.
*Indicates presente
Dilbit toxicity: Behaviour changes in freshwater amphipods.
No full textThe Alberta oil sands contain an estimated 167.2 billion barrels of bitumen, a heavy crude oil that comprises the majority of Canada’s crude oil production. Unrefined bitumen is extremely viscous compared to conventional oil, which is a fluid at atmospheric pressure and temperature, and therefore must be diluted with natural-gas condensates to create diluted bitumen or ‘dilbit’, so that it can flow through pipelines. New Canadian dilbit pipelines such as the Enbridge Northern Gateway Pipeline and the Kinder Morgan Trans Mountain Expansion Pipeline will come in close proximity to thousands of freshwater ecosystems. When dilbit is spilled, it will initially float in freshwater, but the added diluents will begin to evaporate and the remaining dilbit will combine with sediments, causing it to sink in freshwater Ongoing research is addressing the toxicity of sediment-bound dilbit to the freshwater amphipod Hyalella azteca. In this study, Hyalella azteca will be exposed to sediment bound dilbit for 96 hours and the adverse behavioural and physiological effects of dilbit will be assessed. To date, sediment bound dilbit has not been toxicologically studied. This research will address these questions and refine research techniques using dilbit and bitumen products in toxicity research using amphipods and other organisms. Not only that but addressing these issues will lead to better knowledge on how to best lead cleanup efforts in freshwater dilbit spills, saving money, time and possibly preventing environmental devastation as a result of pipeline spills
Characterization of Engineered Protein Switches Using Intramolecular Disulfide Bridges
No full textProtein engineering is the process of developing proteins that can perform functions that often are novel and do not occur in nature. Development of proteins with new properties is critical for a wide range of biotechnology applications including new metabolic pathways, biosensors, and molecular scaffolds. Engineered proteins are frequently used in the food industry, medicine, nanobiotechnology, and environmental applications. One of the limits of protein engineering is the lack of ability to rationally design molecular switches. Here we report on the development of a molecular switch which utilizes cysteine disulfide bridges.
Our model system for the development of a designer molecular switch is the translation elongation factor (EF) G. EF-G stimulates translocation of tRNA through the ribosome, a process that requires a conformational change in EF-G. We have developed and implemented a Molecular Dynamics simulation pipeline to design 3 EF-G variants (G163C-T650C, P121C-K675C, and T125C-S679C) with disulfide bridges that limit the conformational changes of EF-G to specific functional states. To assess correct folding of these conformationally restricted EF-G variants, we have used circular dichroism and determined key enzymatic properties such as nucleotide binding and GTP hydrolysis rates for these EF-G variants.
The reported data validates the ability of our Molecular Dynamics pipeline to rationally design novel molecular switches.
*Indicates presente
Development of a Cell-Free Synthetic Biology Platform
No full textCell-free systems allow for a reliable and consistent expression of recombinant proteins outside of a living cell, bypassing issues with genetic regulation and cellular noise (Hodgman and Jewett, 2012). Such systems are advantageous over cell-based synthetic biology due to the capability of tolerating toxins normally detrimental to the cell, increased freedom of design, reduced risk for biocontamination, and a rapid design-build-test cycle. Emerging as a new platform for synthetic biology, cell-free systems have shown potential for use in a variety of applications, including biofuel production, biomanufacturing, health and medicine. However, current cell-free systems are inaccessible due to their high cost or incredibly laborious lab work required to reproduce them. The goal of this work is to develop a completely customizable and accessible cell-free system composed of 38 proteins required for transcription and translation. Each protein is designed with a hexa-histidine tag on the N or C terminus to allow for easy nickel-sepharose purification. Protein overexpression and purification is verified using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS- PAGE), and all 38 proteins are reconstituted to form a function cell-free system. 8 proteins have currently been successfully overexpressed and confirmed by SDS-PAGE.
To simplify the process, multiple proteins are purified at once by combining cell pellets from multiple overexpressions and purifying them on a single nickel-sepharose column. The concentrations of purified proteins will be determined using mass spectroscopy. Preliminary results include successful multi-protein purification of four proteins (Release Factor 3, Histidine Synthetase, Tryptophan Synthetase and Ribosome Recycling Factor) on a single nickel-sepharose column, subsequently verified by SDS-PAGE. Future goals for this work include complete overexpression and purification of all 38 proteins, followed by functional validation. Ultimately, this work will provide a safe and customizable cell-free system for protein production.
REFERENCE
Hodgman, C.E. and M.C. Jewett, Cell-Free Synthetic Biology: Thinking Outside the Cell.
Metabolic Engineering, 2012. 14(3): p. 261-269