The Undergraduate Research in Science Conference of Alberta Proceedings (URSCA) provided access to abstracts and examples of scientific research conducted by undergraduate students from across Alberta from 2015-2018. Please note that this proceedings website has been discontinued.
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Glucose Tolerance is Unchanged During Acute Normobaric Hypoxia
Blood glucose regulation is critical to support metabolism, particularly in contexts of metabolic stressors (e.g., high altitude). Data regarding insulin sensitivity and glucose tolerance in hypoxia are scant and inconclusive. We aimed to characterize the interactive effects of acute normobaric hypoxia and glucose regulation following an oral glucose tolerance test (OGTT) compared to normoxia. Following 12 hours of fasting on two separate days, 28 healthy participants (21.8±0.3 yrs; BMI 22.8±0.48 kg/m2; 16 females) were randomly exposed to either normoxia (FIO2 0.21) or hypoxia (FIO2 0.148) in a normobaric hypoxia chamber. Blood glucose was tested from finger capillary samples via glucometer and sterile lancets. Following a 10-min baseline in normoxia or hypoxia, participants consumed an OGTT (75g, 300ml) and blood glucose was sampled every 10-min for 80-min. Peripheral oxygen saturation was lower at baseline in hypoxia (88.6±0.7 vs. 96.8±0.41%; P<0.0001), but fasted blood glucose was not different between trials (hypoxia=4.86±0.07 vs. normoxia=4.80±0.08 mmol/L; P=0.47). Blood glucose responses following OGTT were compared between oxygen conditions using a two-factor repeated measures ANOVA (2FRM-ANOVA; factors = oxygen condition x time) and area under the curve (AUC; paired t-test). Blood glucose responses were not different between trials (2FRM-ANOVA interaction, P=0.765; AUC normoxia vs. hypoxia, P=0.227). We conclude that glucose tolerance is unchanged with acute normobaric hypoxia, likely protecting the metabolic rate of organs that do not require insulin for uptake or storage (e.g., brain) during hypoxic stress. Funding Sources: NSERC Discovery, MRU Faculty of Science and Technology
Consequences of 1,3-butadiene DNA adducts – R,R-N6,N6-DHB-dA and R,S-1,N6-g-HMHP-dA – on DNA base pairing
DNA is the hereditary material in humans and almost all other organisms. Unfortunately, upon exposure to carcinogenic species and other compounds, our DNA can be damaged in a variety of ways. An example of DNA damage includes the formation of bulky nucleobase addition products (adducts), which may give rise to cancer. This poster focuses on adducts formed upon exposure to 1,3-butadiene – a human carcinogen that has been associated with an increased incidence of leukemia (cancer of the blood cells). Sources of 1,3-butadiene exposure include automobile exhaust and tobacco smoke, as well as polluted water and air at or near chemical, plastic or rubber facilities. In order to understand the consequences of DNA exposure to 1,2,3,4-diepoxybutane – a metabolite of 1,3-butadiene – this study examines the hydrogen-bonding properties of two adenine (dA) adducts formed upon addition of a bulky moiety to the N6 position, namely the R,R-N6,N6-DHB-dA and R,S-1,N6- g-HMHP-dA adducts. Previous studies have shown that human translesion DNA polymerases hand k preferentially incorporated A, C, G or T opposite R,R-N6,N6-DHB-dA, and A, G or T opposite R,S-1,N6-g-HMHP-dA. This study seeks to explain the observed mutagenicity of these adducts and is the first step in determining the effect of these adducts on the DNA duplex. Figure 1. 1,3-butadiene adducts R,R-N6,N6-DHB-dA (left) and R,S-1,N6-g-HMHP-dA (right)
Investigating the Interaction Between DDX17 and The Rift Valley Fever Virus 3’ Terminal Region Using a Nano-scale Structural Approach
Rift Valley Fever (RVF) is a currently an untreatable disease that affects both humans and livestock in sub-Saharan Africa. This disease is viral in origin and the Rift Valley Fever virus (RVFV) is transmitted primarily through mosquito vectors Aedes and Culex. RVFV is a part of the Bunyaviridae family, and the genus Phlebovirus. Recent studies have indicated that RVFV interacts with the human DEAD-box RNA helicase protein DDX17. There is minimal research on the specific interactions between this host protein and the viral RNA, although DDX17 has been shown to inhibit RVFV replication. The DDX17 helicase performs a critical role in viral replication, and we are trying to dissect the specific role by which it inhibits RVFV replication. In addition, we are also studying two regions of the RVFV which forms a stem-loop structure that is proposed to interact with DDX17. Using selective 2’-hydroxy acylation analyzed by primer extension (SHAPE) and small-angle x-ray scattering (SAXS) techniques we aim to gain both secondary and 3-dimensional structural information. To identify specific domains of DDX17 that interact with both viral constructs, we require a highly pure preparation of full-length DDX17 and its truncated domains. Thus, the primary objective of my project is to establish protocols for large scale preparation of full-length and individual domains of DDX17 to identify a specific domain of DDX17 that interacts with the RVFV RNA using Electrophoretic mobility shift assays (EMSA) and study interactions using microscale thermophoresis (MST). *Indicates presenter
Using 1H NMR Spectroscopy to Compare Urinary Metabolites of Stroke and Spinal Cord Injury Patients Before and After Neurorehabilitation
Background: Rehabilitation represents the primary approach to promote long-term functional recovery after neurological injury. There is an urgent need to improve the effectiveness of rehabilitation therapies to optimize the potential for recovery in an individual. Precision medicine approaches using modern ‘omics’ techniques represent vital prerequisites in stratifying individual patients with neurological injury to their optimal rehabilitation program. Metabolomics research from our laboratory has demonstrated that metabolite levels in urine detected by NMR spectroscopy serve as reliable prognostic markers for neurological injury. This study aims to determine if a proton NMR-based quantitative metabolic profiling approach can identify novel biomarkers in clinically accessible biofluids that are indicative of both the repair processes and treatment efficacy following neurorehabilitation from stroke and spinal cord injury. The main hypotheses of this study are (a) that each of the neurological conditions will yield changes in the metabolic profiles that can be correlated to the extent of recovery of a patient and (b) that biological pathway analysis will provide an insight into the mechanisms behind the repair process. These techniques will provide a greater understanding of the biochemical processes that mediate neural repair in the central nervous system for clinical application. Methods: Patients with stroke and spinal cord injury (n≥14 per group and sex) were recruited through the Foothills Medical Centre in Calgary, Alberta. Urine samples were collected from patients within 48-72 hours of injury and again at 6-months post-injury, following neurorehabilitation. A 700MHz Bruker Avance III HD NMR spectrometer located at the Canadian Centre for Behavioural Neuroscience was used to acquire the metabolic profiles of urine samples pre- and post-neurorehabilitation. Multivariate statistical and biomarker analysis tests were used to determine changes in the metabolic fingerprint which can potentially be linked to clinical outcomes. Impact: Within the rehabilitation field there is an urgent need to generate evidence-based therapies and validate existing ones. Metabolomic analysis will provide a time- and cost- effective method to identify optimal rehabilitation therapies based on an individual’s impairments, through translational biomarker discovery. This personalized medicine approach offers a new strategy to evaluate and improve the efficacy of neurorehabilitation strategies for stroke and spinal cord injury patients. *Indicates presenter
Eukaryotic Initiation factor 5B (eIF5B) provides glioblastoma multiforme with resistance towards apoptotic agents
Eukaryotic initiation factors (eIFs) are proteins involved in the general process of mRNA translation. However, under conditions of physiological stress, alternative methods of translation initiation are often activated and relied upon to continue translation of a subset of mRNAs. Specific eIFs such as eIF5B have been shown to use cap-independent mechanisms to promote the production of anti-apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP). In the present study, we have explored if eIF5B provides resistance to glioblastoma multiforme (GBM) cells towards apoptotic agents. Using siRNA, we depleted eIF5B from various GBM cells and treated them with agents that promote apoptosis. In order to activate intrinsic apoptosis pathway we treated these cells with genotoxic agents such as doxorubicin (DXR), and temozolomide (TMZ). Moreover, we treated GBM cells with tumor necrosis factor alpha (TNFα), and/or TNF-related apoptosis-inducing ligand (TRAIL) to activate extrinsic apoptosis pathway. The depletion of eIF5B did not sensitize GBM cells to DXR or TMZ. However, further viability assays showed that knockdown of eIF5B sensitized U343 cells to TRAIL or TRAIL + TNFα. Further, the combination of TRAIL + SMAC- mimetic compounds (SMC) showed statistically significant decreases in cell viability when eIF5B was depleted. So far, six different glioblastoma cell lines have been assessed for this effect, and further studies are underway to observe the effects of eIF5B depletion on cell viability of various cancer and non-cancer cells. Our preliminary data suggest that eIF5B enhances cell survival of GBM cells and could represent a potentially important therapeutic target. *Indicates presenter
Potential sulfate sources for the Paskapoo aquifer of Alberta
The Paskapoo aquifer of Alberta is one of the most significant sources of groundwater for domestic and industrial use in the entire province. However, there are pronounced levels of sulfate in the southern part of the aquifer between Red Deer and Calgary, above the 500 mg/L drinking safety limit advised by Environment Canada. The focus of this study is to examine the potential origins of this sulfate from three primary sources: (1) self-sourced from the Paskapoo Formation, deposited ~60 million years ago; (2) overlying Cordilleran till; and (3) and overlying Laurentian till. Oxidation of sulphide minerals from Laurentide till is the primary source of sulfate based on findings from published research. The Cordilleran till has not been examined as a sulfate source to the same extent. This study used three methods to determine whether Cordilleran till had any potential to add sulfate to the Paskapoo aquifer. A geochemical test utilizing X-Ray Fluorescence (XRF) determined sulfur content in rock samples from several potential source formations of Cordilleran till. Next, we immersed two grain-sizes of sediment (10 grams) from each formation into distilled water (40 ml) and reacted the sample with barium chloride to determine if and how much sulfate each formation could generate. Finally, a GIS map was constructed using flow directions of the Cordilleran and Laurentide Ice sheets, and the outcrop distribution of bedrock formations. This was used to determine whether each formation had the potential to be integrated as till material and subsequently deposited over the Paskapoo. This study found that several of the tested formations from the Cordillera had sizeable levels of sulfate and were capable of producing sulfate when immersed in water. Although sulfur did have the potential to be integrated into Cordilleran till, the flow direction of the Laurentide glacier ultimately restricted placement of Cordilleran till over the Paskapoo aquifer and may have limited the western input of sulfate into the aquifer. However, regional groundwater flow could potentially transport the sulfate to other aquifers due to the regional dip of Tertiary and uppermost Cretaceous strata. This study presents a multidisciplinary approach involving GIS, geochemistry, geology, and hydrogeology to determine potential sources of groundwater contamination. *Indicates presenter
Detecting chemical modifications of Ribonucleic Acid
Transfer Ribonucleic acid (tRNA) is one of the highly chemically modified RNA in the cell, and the isomerization of uridine to pseudouridine (ᴪ) is the most abundant RNA modification found in all domains of life. Although the function of RNA modification is not yet fully understood, the chemical changes in RNA have been implicated in cellular fitness and development of genetic diseases. For example, the bacterial proteins TruA and RluA are both pseudouridine synthases which catalyze the isomerization of uridine to pseudouridine. TruA is capable of modifying several nearby sites in tRNAs while RluA modifies different RNAs at positions with a shared sequence and structure. These modifications are conserved in all forms of life, but little is known about their function. The objective of this research project was to modify tRNA using modification enzymes and to establish a method to detect multiple RNA modifications at once using High Performance Liquid Chromatography (HPLC). Using a reverse phase chromatography column will allow to separate samples based on hydrophobicity. On one side, tRNA was successfully in vitro transcribed, purified and digested into nucleosides, which were then separated using the HPLC column. On the other side, TruA and RluA wild-type proteins were successfully overexpressed and purified, in order to prepare modified tRNA. In the future, this system will enable us to detect and quantify the presence of pseudouridine as well as other chemical modifications in any type of RNA. This tool will thereby greatly advance research in the Kothe lab to understand the mechanisms and functions of RNA modifications. *Indicates presenter
Synergistic Effects of Ancestral Stress and Aging on Anxiety-like Behaviours
Exposure to adverse environments such as prenatal stress early in life is associated with anxiety-like behaviours in adulthood, which is potentially further exacerbated by aging. Recent studies have indicated that ancestral prenatal stress can propagate across generations to alter emotional wellbeing of unexposed offspring. Here we investigated if exposure to prenatal stress in the great-grandmother (transgenerational stress), or exposure across four consecutive generations (multigenerational stress) can alter anxiety-like behaviours in male and female fourth (F4) generation offspring. Anxiety-like behaviours were evaluated by means of the elevated plus maze in both males and females across three different groups: transgenerational stress (SNNN), multigenerational stress (SSSS), and non-stressed controls (NNNN). Both sexes were evaluated at the age of 12 months (middle age) and 18 months (old age). Our results demonstrate that aging and ancestral stress synergistically heightened anxiety-like behaviour, especially in males. Interestingly, the highest levels of anxiety-like behaviours were observed in transgenerationally stressed offspring of both ages. Overall, these results indicate that males are more sensitive to ancestral stress and more likely to respond by developing anxiety-like behaviours. Thus, ancestral stress and aging may synergistically alter mental health outcomes particularly in males. *Indicates presenter
Implementation of a new method to identify Verticillium isolates affecting potato cultivars in Alberta
Potato (Solanum tuberosum L.) is the most important non-cereal crop in the world, with 300 million tons produced every year. Despite its economic importance, potato plants are susceptible to numerous diseases caused by fungi, bacteria, and viruses, that lead to tremendous financial losses for the industry. A common potato disease called Verticillium wilt, or potato early dying, is caused by fungi from the genus Verticillium. The two species that affect potato cultivars in Alberta are V. dahliae and V. albo-atrum. These soil-borne fungi affect xylem tissues preventing water intake causing dehydration in plants. This results in necrosis of the tissues and thus, leads to early plant mortality. Unfortunately, once a crop has developed disease symptoms, there is little that can be done to prevent harvest losses that year. This is why a fast and reliable method for early detection of the fungi is necessary. Traditionally, fungal identification is performed by observing morphological characteristics of mycelia on solid media with a microscope; however, it is time consuming, requires specialized skill set, and species identification can only be achieved by genome sequencing. We are currently implementing a new method developed by Inderbitzin et al. (2013) to identify the species of Verticillium isolates by detecting the presence of species-specific nucleotide sequences1. After extracting fungal DNA, a polymerization chain reaction (PCR) is conducted with species-specific primers for both V. dahlia and V. albo-atrum. By visualizing the presence of an amplified fragment on agarose gel, it is possible to determine the Verticillium species. We are testing and optimizing this method on eight non-identified pure Verticillium cultures isolated from fields in Alberta to determine their species. We are also working on the isolation of new Vertcillium cultures from seven potato plant samples collected during the past growing season across Southern Alberta that showed symptoms of Verticillium wilt. This will allow us to build a collection of Verticillium isolates for future genetic analysis. Overall, this new method will be a very powerful tool to detect the presence of Verticillium fungi in soil and plants before the emergence of any disease symptoms, which allows potato grower to control this disease in a timely manner preventing harvest losses. 1Inderbitzin P, Davis RM, Bostock RM, Subbarao KV (2013) Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays. PLoS ONE 8:e65990 *Indicates presenter
Sprouting behavior of potato seed tubers (cv. Russet Burbank) after the application of different plant growth regulators
The yield of a potato crop is strongly influenced by the physiological age of potato tubers at planting. Physiological age can be defined as the stage of development of a seed tuber, which changes progressively by increasing chronological age and is modified by growth history and storage conditions (Struik & Wiersema, 1999). It is often characterized as the sprouting potential of seed. It is hard to overstate the importance of physiological age of seed potatoes to the potato industry: it strongly affects emergence of sprouts, number of stems per plant, number of tubers per stem, tuber-size distribution and tuber yield of the progeny crop (Van der Zaag & Van Loon, 1987; Van Ittersum, 1992; Struik & Wiersema, 1999; Struik, 2007). Thus, understanding the mechanisms of physiological aging and the ability to control it is crucial to maximizing crop yield and quality. Previous studies on physiological age of seed potato have been focused on environmental factors, such as relative humidity, temperature, photoperiod, and diffuse light (Struik & Wiersema, 1999; Struik et al., 2006). Tuber respiration appears to be a pacemaker of aging: the longer the respiration rate remains high, the older the seed tubers will be (Blauer et al., 2013). In addition, research has shown that plant growth regulators such as abscisic acid (ABA), ethylene, indole-3-acetic acid (IAA), cytokinins and gibberellins play a role in the control of tuber dormancy and subsequent sprout growth (Suttle, 2007). Up to date, there is still a strong need for further research to better understand their role and the effect on the physiological age (Suttle, 2004; Suttle, 2007; Kloostermann & Bachem, 2014; Wohleb et al., 2014). In this study, different growth regulators such as Gibberellic Acid, ABA, Auxin, Cytokinin and 2.4-D were applied to the meristematic tissue of the seed tuber at three different concentrations (2.5, 5, 10 ppm) and stored at two different temperatures (5, 12°C). Afterwards, the break of dormancy and the sprouting behaviour were observed. At the time of writing, the study was not completed and thus no results can be presented at this time. The findings of this study will give indications for further research to develop new methods to control physiological age. Key words: seed potato, growth regulator, dormancy, sprouting, physiological age, Solanum tuberosum L. *Indicates presenter
As part of the Campus Ecology Project at the University of Lethbridge, a twenty question online survey was administered to 265 students in Psychology 1000 or select Psychology 2000 level courses, who attend the campus. The students answered anonymously, and provided insight into their interactions with different plant and animal species that exist on the University of Lethbridge campus. This 10-20 minute survey gathered information about how landscapes and ecology affect and improve an individual’s experience on campus. The survey also attempted to gain insight about how individuals feel about allocating funds towards ecology on campus, and the protection of potentially endangered species. Lastly, students were given the opportunity to indicate any ways in which they felt that the University of Lethbridge campus could better promote wellness for it’s visitors.
Identification of Conserved Proteins in Nocardia brasiliensis and other Actinomycetoma causative agents using a Bioinformatic approach
Mycetoma, 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.
Maternal Preconception Nicotine and Enriched Housing
A mother’s environment can affect her offspring even before conception. Both positive factors, such as an enriched environment, and negative factors, such as drug use, have the potential to shape offspring development. The influence of psychoactive drugs is especially concerning because of the role they play in modulating important survival instincts, such as anxiety; therefore, strategies to counteract the changes induced by drug use are of particular interest. One possible way to counteract the transgenerational effects of drugs may be through enriching the maternal environment, which has been previously shown to attenuate the effects of nicotine. Enriched environments have huge beneficial effects on individuals, and hopefully these benefits can be passed down to offspring as a protective factor. In this experiment, female rats received moderate levels of nicotine in their drinking water (15mg/L sweetened with 1% sucralose) while being housed in either standard lab conditions or an enriched environment. The rats undergoing enriched housing were placed in a larger cage with multiple levels, more cage mates, and a rotation of stimulating objects. Control females received water sweetened with 1% sucralose while living in either environment. The offspring underwent behavioural testing in adolescence and adulthood to look for changes in elevated plus maze behaviours, a test that measure anxiety-like behaviours. Cortical thickness and thalamic size were also examined to look for effects on brain morphology. The results showed significant effects of preconception nicotine and enriched housing in the anxiety-like behaviours of the offspring in both adolescence and adulthood, dependent on sex. Results suggest that maternal preconception nicotine use lowers the amount of anxiety present in offspring. When the mothers are kept in enriched housing, however, the anxiety levels in the nicotine cohort return to levels near that of the control group. These results have important implications in the ability of the offspring to survive their environment, as differences in anxiety levels affect the survival strategies used by the offspring. Additionally, the return to control levels in the offspring whose mothers lived in an enriched environment does in fact suggest that maternal enrichment acts as a protective factor against preconception nicotine use. These results are not surprising as nicotinic acetylcholine receptors play a major role in the modulation of anxiety pathways, evidenced by both increases and decreases in anxiety due to nicotine use.
Investigation of myo-inositol Phosphates on Human Cells by Phenotypic Cell-Based Assays
Myo-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 presenter
Artificial 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 presenter
Over the past decades, polar regions such as the Canadian Arctic Archipelago have experienced pronounced changes associated with climate warming (e.g., decreased sea ice extent and thickness). Such relatively recent environmental shifts have motivated research regarding past climate variability to understand how polar marine environments respond to changing conditions, for example glacial to interglacial transitions. This presentation outlines ongoing research that is part of a larger project (funded by ArcticNet and NSERC) focussed on the palaeoclimatology, palaeoceanography and deglacial histories of the Canadian Arctic Archipelago. In particular, the use of foraminifera (benthics and planktonics) is highlighted as a proxy for reconstructing past environmental conditions via assemblage and geochemical (δ13C and δ18O) analyses, including water temperatures, sea ice conditions, and ecosystem productivity, in the eastern (Lancaster Sound/Baffin Bay) and western (M’Clure Strait) entrances to the historical Northwest Passage. To allow for accurate interpretations of past environments, ongoing work also focusses on foraminiferal distribution in surface (modern) sediments in relation to measured oceanographic parameters (temperature, salinity, sea ice) as a baseline for paleo-interpretations. Preliminary results from foraminiferal surface sediment calibrations, along with the taxonomic issues associated with some important indicator taxa will be discussed. *Indicates presenter
Analysis of Potential Anti-Cancer Effects of Cannabinol and Cannabidiol using HCC1806 and HEK293 Cell Lines
Humans produce endocannabinoids that act as neuromodulators in the endocannabinoid system. They bind to Gαi protein-coupled cannabinoid receptors to control the release of many neurotransmitters. Cannabinoids receptor 1 (CB1) mediates psychoactive effects through its location mostly in the central nervous system while Cannabinoid receptor 2 (CB2) regulates various immune responses through its location in peripheral tissues. The endocannabinoid system has been used as a molecular target by research to treat diseases such as multiple sclerosis, cardiovascular disorders, obesity and inflammatory pain. Thus, the endocannabinoid system is a potential molecular target to treat cancer. With the proposed legalization of recreational marijuana and with growing number of patients using cannabis for medicinal purpose, there is an urgent need to provide data on potential medicinal value of cannabis and cannabinoids. The Cannabis Sativa plant naturally synthesizes numerous different cannabinoids of which (CBN) and cannabidiol (CBD) have promising properties in cancer treatment. CBD is a phytocannabinoid known for its anticonvulsant and anti-nausea properties. Previous research suggests that CBD can target breast cancer cells while preserving normal cells. CBN is another phyotocannabinoid with anti-inflammatory properties that can potentially aid to reduce inflammation resulting from cancer. This study aims to determine if CBN and CBD have an effect on cancer cells and normal cells. We hypothesize that we may observe an increase in apoptosis of cancer cells treated with the two compounds but no effect or perhaps even a slight increase in normal cell growth. Preliminary data in lab suggests that these compounds have anti-cancer properties and we want to solidify this evidence through repetition of the experiment. Various concentrations of CBN and CBD were administered separately to HCC1806 breast cancer cells and HEK293 normal human kidney cells for 24 hours before conducting apoptosis and cell cycle assays to determine if the cannabinoids had induced cell death or affected proliferation. *Indicates presenter
Quantification of Tissue Specific Expression of ABC type B transporters in Eschscholzia californica
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are a diverse superfamily of transmembrane proteins found in all eukaryotes. These proteins couple the hydrolysis of ATP to the transport of a wide variety of substrates across cellular membranes. In plants, ABC type B (ABCB) transporters have been shown to be involved in the transport of both growth regulators (auxin) and secondary metabolites. Plant secondary metabolism refers to the vast array of small molecules being produced, but not directly required for plant growth and development. However, secondary metabolites have been shown to play important roles in plant defense, such as in deterring herbivory or resistance to pathogens. It has been shown that transport between cell types is necessary in the biosynthesis of many of these metabolites and that ABC transporters play a necessary role in this trafficking. Eschscholzia californica, or the California poppy, accumulates alkaloids within specialized cells within roots. Our lab previously identified EcABCB1, a transporter involved in the transport of these alkaloids. In this project, using the recently-released draft genome of E. californica, we have identified seven additional putative ABCB transporters (ABCB2-7). Using quantitative polymerase chain reaction (qPCR), we will quantify their expression profiles in a tissue-specific manner. These results will then be used to draw inferences of the function of each candidate gene, based on comparative analysis of expression profiles of ABCB homologs in Arabidopsis thaliana. Preliminary data shows that EcABCB1 is expressed in both roots and stems, whereas EcABCB2 is expressed ubiquitously across all tissues. This may indicate that EcABCB2 may be involved in the transport a growth factor such as auxin, which is a metabolite necessary for growth across all organs. In order to obtain a better understanding of the biochemistry of plants and the synthesis of their alkaloids, the transporters required for this synthesis must be characterized. *Indicates presenter
The Effect of Varying Doses of UVC Radiation on the Viability of Human Cancer Cells
UVC radiation has the ability to damage DNA in cells. This causes the cell to either arrest division or undergo apoptosis. In this study we observed the effects of increasing doses of UVC radiation on the division rates and viability of cells from the human cancer cell line MCF7. We hypothesized that at a certain dose the cells would no longer be able to repair DNA and apoptosis would be induced. Furthermore, we hypothesized that the division rate would decrease as the dose of UVC increased. Cells were irradiated at either, 0 mJ/cm2 (negative control), 0.31 mJ/cm2, 1.55 mJ/cm2, 4.65 mJ/cm2, 9.3 mJ/cm2, 18.6 mJ/cm2, 37.2 mJ/cm2, or 74.4 mJ/cm2. 48 hours after irradiation, a Trypan Blue viability assay was performed to determine the percentage of dead cells at each dose. To compare division rates, the total number of cells in each treatment was taken as a percentage of the total number of non- treated cells. The IC50 for apoptosis was found to be 21.25 mJ/cm2. Our results showed that the total number of cells decreased significantly between 0 mJ/cm2, and 4.65 mJ/cm2. At concentrations above 4.65 mJ/cm2, the total number of cells remained constant. Suggesting that this is the point in which cells may have stopped growing. In contrast, the percentage of dead cells did not differ between the nontreated cells and those treated with UVC doses up to 9.3 mJ/cm2, but was significantly higher at doses above 18.6 mJ/cm2. The point at which the percentage of dead cells begins to increase significantly and where division rates plateau is relatively close, thus, we infer that this the point in which cells stopped trying to repair their DNA and instead began to undergo apoptosis. As we hypothesized, at a high enough dose, the DNA in cells would become irreparable and the cells would die. Because all cells above this dose show significant death, the observation is consistent with our hypothesis. *Indicates presenter
Identification and Alignment of Human and Mouse SNPs in the DMD Gene using a Bioinfomatical Approach
Muscular dystrophy (MD) is a fatal, muscle wasting disease affecting individuals who have acquired a mutation in their dystrophin (DMD) gene. Some of these mutations are a single nucleotide alteration. Dystrophin is a vital part of a protein complex in skeletal and cardiac muscle tissue. The gene is the largest in the human genome, spanning 79 exons on the X chromosome. MD is X linked dominant, targeting mostly males that are affected by the one of the two most common forms, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Both DMD and BMD result from single nucleotide polymorphisms (SNPs). To date, there is no cure for the disease. Of the identified SNP mutations, the phenotype for many has not yet been characterized. To this end, identifying conserved regions between humans and mice would allow for further characterization of these SNPs, as dystrophinin in mice is functionally analogous. Therefore, I hypothesize that some of the human SNPs in DMD are conserved in mouse DMD. Using a bioinformatical approach, I collected various SNP mutations, and using Ensemble, I then aligned the genes. Of the exons examined, 212 SNPs were identified, 17 of which were on exon 57. Furthermore, many of the human SNPs align with mouse DMD gene SNPs. This study will allow for further characterization of the DMD SNPs, as well as serve as a platform for future in vivo studies, opening opportunities to possible gene therapies for individuals with muscular dystrophy.
Single-dose UV-C irradiation induces the formation of gamma-H2AX foci in the human cell line (MCF7) that are not resolved after 2 and 24 hours of recovery as determined by immunofluorescence microscopy
The integrity of the genome is of utmost importance for proper functioning existence of all living systems. Genotoxic insults, such as UV-C radiation, may exert adverse effects on genomic integrity. Cells have developed a number of highly conserved repair mechanism to counteract the variety of mutagenic and cytotoxic DNA lesions induced by genotoxic agents. Phosphorylation of gamma-histone H2AX is a known DNA-repair signaling response to double- stranded breaks (DSBs); however, the formation of DSB by UV-C radiation is reported to be rare. We sought to determine whether a single dose of UV-C radiation (0.31 J/cm2, 254nm) can induce the formation of gamma-H2AX foci in the human cell line (MCF7) and determine the amount of DNA damage present after a recovery period of two and 24 hours by immunofluorescence microscopy. We also exposed cells to 50 nM of camptothecin (CPT), a DNA damaging agent, as a positive control. We found that similar to CPT, human cells exposed to UV-C radiation (0.31 J/cm2) showed upregulated gamma-H2AX foci at both 2 h and 24 h after the genotoxic insult. Cells exposed to this single dose showed significantly higher gamma-H2AX from non- treated cells and the extent of DNA damage remained the same between 2 and 24 h. Our results indicate that UV-C radiation is capable of inducing the formation of the DSB signaling foci gamma-H2AX and that cells are unable to repair the genotoxic insult within 24 h of recovery. This further indicates that human cell line (MCF7) may require longer than 24 hours to noticeably repair the extent of DNA damage induced by (0.31 J/cm2) of UV-C light.
Modifying the Stripe Rust Resistance Gene Yr10 in Wheat (Triticum aestivum L.) by PCR Mutagenesis
Stripe rust is a disease in cereal crops caused by the fungal pathogen Puccinia striiformis which can have devastating effects on crop yields. Cereal plants have unique resistance (R) genes which code for resistance (R) proteins with coiled coil (CC), nucleotide binding site (NBS), and leucine rich repeat (LRR) domains. The CC and LRR domains are thought to be particularly important for protein function/interaction in the resistance response. The Yr10 gene is a stripe rust resistance gene in Triticum aestivum that codes for a unique CC-NBS-LRR sequence but no longer confers resistance to some races of rust. Previous studies indicate that by mutagenizing the DNA sequences that code for the CC and LRR domains it is possible to change the R protein’s sequence, and possibly the interaction during the resistance response3. Thus, the defeated R genes can confer resistance once again when mutagenized in these regions. This study used PCR mutagenesis (by overlap extension) to mutagenize the Yr10 gene in the regions encoding the CC and LRR domains. Primers were designed to amplify Yr10 such that products contained mutated sequence as extensions, available for overlap. The amplifications were the expected size, indicted primers were successful. The PCR products containing the mutagenized sequences of Yr10 were then assembled with the pANIC 6D vector to be used in further experiments involving the transformation of wheat crops to test resistance. *Indicates presenter
Mathematical Modelling of a Measles Outbreak in Pre-vaccine England and Wales
We present a spatial variant of the time series susceptible-infectious-recovered (TSIR) stochastic population-based model to capture the spatial transmission dynamics of a measles outbreak across the landscape of England and Wales during the pre-vaccine era. Specifically, we explore how the basic dynamical features of a measles outbreak with a seasonal forcing of transmission acts as a major driver of a long-term epidemic behavior. We use a 20-year pre-vaccination era biweekly time series data (births by year and incidence of measles for the years 1944-1966) from 60 towns and cities in England and Wales to capture the spatial spread of measles. In England and Wales prior to vaccination, measles was endemic in large cities, but in smaller cities disease fadeout occurred. Reappearance of the disease would then occur only after a case was imported from a surrounding city where measles was endemic. To capture spatio-temporal dynamics, multi-city models must be developed, but these models can become very large requiring more memory and processing power than a single computer can deliver. Rather than represent the population as a linked set of cities, we represent the population as a gridded map. Each grid cell can transmit infectious disease to its neighbors, with probabilities that decline exponentially with distance. We present a stochastic spatial model with six compartments. We call this the kids-susceptible- infectious-recovered-adults-dead (KSIRAD) model. From the simulation, we recover spatiotemporal maps of the incidence of the infection. We compare simulated time-series graphs with real data compiled by Grenfell and others. Our future work includes testing of our spatial model for measles outbreaks reported in the modern era, for example, in conflict affected areas of the Republic of the Niger in Western Africa in 2016. Socioeconomic disparities in a country like Niger presents significant challenges to reporting and real-time tracking of human infectious diseases. *Indicates presenter
Development of a Cell-Free Synthetic Biology Platform
Cell-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.
An aerial inventory of rock glaciers in the Southern Alberta Rocky Mountains
Rock glaciers are large lobes of rocky debris commonly found within alpine permafrost in the Alberta Rocky Mountains. Alpine permafrost is of significant scientific interest as it encompasses a large area and exhibits unique responses to climate change. Rock glacier falls from these increasingly unstable mountain slopes can represent significant geohazards, which makes mapping the spatial distribution of these features and classifying their morphology important. They can also be used to identify and understand past climatic change in these alpine regions, with relict (fossilized) rock glaciers indicating a past climate formerly able to create and support these features. Although a common feature within the Alberta Rocky Mountains, information on rock glacier characteristics and distribution within this region is rather limited. The inventory is based on the aerial classification of rock glaciers using high- resolution satellite imagery available through Google Earth and in ArcMap, a geospatial processing program. Geospatial mapping and remote sensing techniques were used for the quantitative and spatial analysis of these features to create an inventory that classifies, quantifies, and characterizes the rock glaciers within this region. Classification of talus-derived and glacier-derived rock glaciers was done using morphological characteristics as the elemental method for identification. Digital Elevation Models (DEM) and optical satellite imagery were used to quantify various attributes such as shape, state, slope, aspect, and elevation. A digital database with the findings from this research will be made publically available for future research through the Alberta Geological Survey. *Indicates presenter
Metabolomic Profiling of Gestational Diabetes Mellitus
Introduction: Gestational diabetes mellitus (GDM) has been shown to increase the occurrence of pregnancy complications, including preeclampsia, preterm births, caesarean sections, intrauterine growth retardation, and neonatal intensive care unit admissions. Current tests for diagnosing GDM and pre-diabetic states in pregnancy are time-consuming and often unreliable. Our main objective was to characterize any metabolomic differences in the urine of women with GDM and healthy pregnant controls to identify diagnostic and predictive biomarkers. Methods: Urine samples from the second trimester of pregnancy from 33 women with GDM and 34 age-, income-, and education-matched healthy controls were obtained from the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort. Proton nuclear magnetic resonance (1H-NMR) data was acquired using a 700 MHz spectrometer and subsequently binned for data analysis. Group separation was evaluated using principle component analysis (PCA) and partial least square discriminant analysis (PLS-DA). Variable importance analysis based on random variable combination (VIAVC) and Mann-Whitney testing were used to determine which metabolites were significantly altered across the comparison groups. Lastly, pathway analysis was carried out to determine which biological pathways were potentially altered by these metabolites. Results: The analyses revealed group separation between the metabolomic profiles of pregnant women diagnosed with GDM and health pregnant controls. Several significantly altered metabolites, along with some potentially altered biological pathways, were identified across the comparison groups. Conclusions: These results demonstrate the potential for 1H-NMR and metabolomics to diagnose pregnant woman with, or assess their risk for developing, GDM. Furthermore, the subset of metabolites identified as significantly altered provides potential biomarkers that can be correlated with pregnancy outcomes in order to identify women at high risk of negative pregnancy outcomes. *Indicates presenter
Modification of the Stripe Rust Resistance Gene Yr10 in Triticum aestivum
Stripe rust is a disease in Triticum aestivum (bread wheat) that is caused by the fungal pathogen Puccinia striiformis. The pathogen has evolved to defeat an R gene in T. aestivum known as the Yr10 gene. The Yr10 gene was found to encode an evolutionary-conserved sequence known as the CC-NBS-LRR. This conserved sequence was found to be involved in producing resistance to various pathogens. Within this sequence the coiled coil (CC) and leucine rich repeat (LRR) domains are thought to be important to the protein’s function. This project made use of PCR overlap-extension mutagenesis to mutagenize the CC and LRR domains in an attempt to create modified constructs of the Yr10 gene. PCR reactions gave fragments of the expected sizes which were then assembled into the pANIC6D vector to be used in transformation. Transformation of the vector with inserted sequences into Escherichia coli will be done to confirm the successful insertion of the fragments. There is hope that in the future the modified constructs could be transformed into wheat. *Indicates presenter
Development and Characterization of Calcium Sensors for In vivo Neuronal Activity Imaging
Genetically-Encoded Fluorescent Calcium Indicators for Optical Imaging (GECOs), that modulate their fluorescence intensity in response to changes in calcium ion concentration, are powerful tools for the investigation of cell biology. These fluorescent indicators are vital when it comes to cellular imaging because they allow the non-invasive study of cells, tissues, and sub-cellular structures at a detail that was previously not possible. The focus of this project is engineering a new GECO that exhibits favourable characteristics, such as increased brightness and a higher fold change. To develop this new GECO, we started from mNeonGreen, the brightest monomeric fluorescent protein currently available. An initial prototype construct was made using rational design, following the precedent of the GCaMP series of indicators. This construct was further improved using directed protein evolution with colony-based screening of libraries of randomly generated variants. We observe the brightness of promising new variants and perform tests to see how new mutations have affected the brightness and the fold change of the variant. After many rounds of screening, our latest variant of mNeonGreen-GECO exhibits a Ca2+-dependent change of eight. When compared to the original construct, this indicator appears significantly brighter with higher contrast between Ca2+-bound and Ca2+-free states. Directed evolution is ongoing and we expect to produce a fluorescent indicator that will be used for in vivo imaging of intracellular Ca2+ dynamics. We will further image its calcium dynamics directly in Zebrafish and compare our variants with mNeonGreen fluorescent protein and the GCaMP series. Using mNeonGreen, we hope to create a calcium indicator that researchers can use to investigate the physiological activity of organs, understand the signaling patterns within tissues, and use to study the various disease states which may lead to the development of new therapies.
Using cortical thickness and elevated plus maze as a means to assess brain and behaviour in adolescent rodents
The effect of maternal experience on the prenatal environment is well researched, however, comprehensive examination of father experience is lacking. This project aims to gain insight into paternal nicotine exposure and complex housing on offspring brain and behaviour. In this study, four experimental groups were used; nicotine enriched, nicotine pair housed, control enriched and control pair housed. Male Long Evans rats were either kept in pair houses or enriched in complex housing at P45 for 96 days and administered the appropriate solution (nicotine/control) during the last 48 days. Rats were then immediately mated with female Long Evan rats following cessation of enrichment and treatment administration. Offspring were then born to the mothers, never coming into contact with the fathers. A subset of the offspring were perfused on P21 for anatomical analysis while the remaining underwent behavioral testing on P36 and were subsequently perfused. Cortical thickness measurements were completed on the animals perfused at P21 and elevated plus maze (EPM) behavioral task - a measure of anxiety - was conducted on the remaining animals at P36. An increased amount of time spent in the closed arms of the EPM is used as a measure of anxiety in these rodents. Nicotine pair housed males as well as control enriched males were found to have significantly thicker cortexes. However, when these treatments were combined in the nicotine enrichment males, this observed thickness in the cortex seemed to be nullified as the cortex thickness was now comparable to the control pair housed males. Male and female offspring of enriched fathers and male offspring of nicotine exposed fathers spent significantly more time in the closed arms of the EPM, thus being considered more anxious than the controls. *Indicates presenter