Testing novel therapeutics to overcome T-ALL chemotherapy resistance in an in vivo mouse model.

Abdus Anwar, Biochemistry with Dr. Marjorie Brand

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by aggressive hematological tumors, 40% – 60% of which express the TAL1 gene which can serve a potential therapeutic target. The clinical prognosis for this subtype of T-ALL is that less than 50% of patients survive after five years of treatment. This is because chemotherapy does not target tumors specifically and as a result fails to eliminate all the T-ALL cells causing them to become resistant. A drug screen was performed in the TAL1+ cell line Jurkat/TR and several drugs were identified that target various transcription factors involved in epigenetic modifications such as histone deacetylases and methylases. Comprehensive dose response experiments were then performed to determine optimal drug concentrations to target TAL1+ T-ALL. 25uM of the drugs identified in the screen significantly decreased the proliferation of the T-ALL cells after 36 hours. A similar dose response was also performed for the standard chemotherapeutic drugs and a similar decrease in T-ALL proliferation was observed. All combinations of the epigenetic and chemotherapeutic drugs were used at the GI50 concentrations obtained to understand their combinatorial effect on TAL1+ T-ALL proliferation. Drug combinations that were found to have the greatest combinatorial negative effect on T-ALL proliferation will be tested in a mouse model to determine their in vivo efficacy. This research is critical to identifying and determining the efficacy of potential therapeutics.

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Analysis of Black Carbon in Canada's Arctic

Marianne Lahaie Luna, Earth and Environmental Science

 

Black carbon is a significant greenhouse gas, which contributes to climate change and in high concentrations it can cause detrimental effects to human health. The purpose of our study is to determine the dominant source of black carbon in Canada’s Arctic air in order to better understand its origins and to aid in the creation of policies to better regulate its emissions. Samples of black carbon from Canada’s Arctic were collected using glass fibre filters attached to high volume air samplers. The 14C content of the samples was evaluated to determine whether the source of carbon is anthropogenic or natural, 14C is produced in the atmosphere and incorporated into plant material. During natural combustions of biomass the organic compounds that are released will contain 14C. However 14C is not present in the combustion products of petrochemical substances. The filter papers were combusted at different temperatures, their gasses collected and then the carbon was graphitized in order to determine the isotopic signature of the components of particulate matter on the filters using accelerator mass spectrometry.

 

Resurrecting ancestral proteins to help us make better drugs.

Jean-Paul Salameh, Biochemistry with Dr. Corrie DaCosta

Proteins, life’s essential molecular machines, are linear polymers made from combinations of 20 naturally occurring amino acids. Since the unique amino acid sequence of proteins dictates their function, our goal is to identify the amino acid determinants of protein function. Most proteins are hundreds of amino acids long, meaning that the number of possible amino acid sequences is essentially infinite. Even for a relatively small protein with 200 amino acids, there are 20E200 possible amino acid sequences. This number of sequences is much greater than the number of atoms in the observable universe! This number makes it nearly impossible to experimentally test the amino acid determinants of protein function. The solution has been given to us through the evolutionary analysis of these proteins: evolution has been exploring the sequences of proteins for billions of years, and selected only those amino acid sequences giving rise to proteins whose functions are capable of supporting life. Growing databases of protein sequences, bioinformatics algorithms, as well as advances in molecular evolution, allow us to reconstruct the sequences of extinct ancestral proteins, and trace the amino acid changes that have accrued throughout evolution, giving rise to modern protein function. Potency of the resulting protein can be estimated through real-time monitoring of ligand-gated ion channels using direct electrical measurement with a patch-clamp microchip. We are using this approach to map and resurrect strategic ancestors of proteins implicated in disease to decipher how they work, so that their function can be better controlled through the design of more targeted pharmaceuticals.

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Polyphosphorylation: a novel post translational modification.

Jimmy Tseng, Cellular molecular medicine with Dr. Michael Downey

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Post translational modifications (PTMs) play a crucial rule in many cellular processes such as cell cycle regulation, protein degradation, and cell signaling. Recently, a new type of PTM termed polyphosphorylation has been characterized. Polyphosphate is a chain of phosphates that generally serves as an energy and phosphate reservoir. Through the process of polyphosphorylation, these polyphosphate chains are added on lysine residues covalently and non enzymatically. Before our study, only two proteins were identified to be polyphosphorylated, which prevented a complete understanding of the function of this modification. Moreover, it is completely unknown how this modification is regulated within the cell. To gain more insight about this modification, the Downey lab carried out a screen of ~100 potential polyphosphorylated candidates and uncovered 18 novel targets. We analyzed these proteins in yeast strains that are known to have less polyphosphate content and observed a reduction in polyphosphorylation. This suggests a connection between polyphosphorylation and the cellular polyphosphate content. Since polyphosphate can serve as an energy and phosphate reservoir, we next hypothesized that polyphosphorylation can be regulated by altered metabolic states. In support of this hypothesis, a decrease in polyphosphorylation was observed in cultures grown to saturation or after treatment with rapamycin, a drug mimicking cellular starvation. We propose that the reduction in polyphosphorylation under cellular starvation and saturation suggests a connection between polyphosphorylation and cell growth.

 

 

Enhancing the viability of microencapsulated probiotics.

Paul Wambo & Alan Cruz, Microbiology with Dr. Rudy Jones

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Probiotics are living bacteria that are good for your health and in particular your digestive system. Recent studies have shown that viability of probiotics from many commercial products (yogurt kefir, etc.) is compromised during gastro-intestinal transit due to the acidic nature of the digestive system – thus limiting the efficacy. Microencapsulation using calcium-alginate beads is one of a series of recent methods that has been identified as being a potential candidate to provide a solution to this problem. Probiotic cultures were prepared and encapsulated using an extrusion process for bead production. The probiotics, encapsulated within the beads were exposed to a simulated stomach acid solution, and timed samples were collected to measure the viability as a function of exposure time to stomach acid. Viability was assessed through the use of standard plate count techniques. Based on our study, this calcium-alginate encapsulation technique demonstrated to be an effective and simple method of probiotic delivery into the intestinal flora.

 

 

CENP-B factor in stabilizing nucleosomes in neocentromeres.

Tamjeed Elahee, Bioinformatics (Computational Biology) with Dr. Sergey Hosid

 

My project looks at the relationship between the location of the CENP-B factor and the stability of the CENP-A nucleosomes in neocentromeres, through the use of computational biology. As well my project looks into the periodicity of CENP-A nucleosome regions in neocentromeres and its role in the formation and stability of the neocentromere. The main hypothesis is that the closer the CENP-B factor is to the CENP-A nucleosome on the DNA strand the more stable the CENP-A nucleosome complex becomes. As well, that the CENP-A nucleosome rich regions are periodical in their occurrence, as a result, the CENP-A nucleosome help stabilise the neocentromere. The methodology consists of two main parts. The first, being to investigate the relationship between the location of the CENP-B factor and the stability of the H3 nucleosomes as a control. This is used as a reference to suggest the significance of any relationship found between the location of the CENP-B factor and the stability of the CENP-A nucleosomes, which was done afterwards. In this section we also attempted to find the preferential location of the CENP-B factor on CENP-A nucleosomes. The second part looked at the periodicity of the CENP-A nucleosomes rich regions in experimentally found stable neocentromeres. The results of the first part have been found and analysed. For the H3 neucleosomes, there was no preferential location of the CENP-B factor and there was no significant correlation between CENP-B location and the stability of the H3 nucleosome. For the CENP-A nucleosome, it was found that CENP-B factor was preferentially between 15-20 base pairs from the nucleosome. As well there was a negative correlation found between the stability of the CENP-A nucleosome and the distance of the CENP-B factor from the nucleosome. The results for the second results have not been fully analysed as of yet.
 

 

PRMT6 as a regulator of stress granule formation and chemoresistance.

Sorush Rokui, Medicine with Dr. Jocelyn Côté

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Protein arginine N-methyltransferases (PRMTs) are a family of proteins responsible for the posttranslational methylation of arginine residues. The members of this family are classified based on their pattern of methylation: type I PRMTs symmetrically dimethylate arginine (ie. SDMA), type II PRMTs asymmetrically dimethylate arginine (ie. ADMA), and type III PRMTs can only monomethylate arginine (ie.MMA). This family of proteins has been previously shown to play a role in cell signaling, transcriptional regulation and cancer invasiveness and proliferation. Specifically, PRMT1, 5, 6, 7 and CARM1 (PRMT4) have all been associated with poor breast cancer prognosis. Further, stress granules are cytoplasmic bodies composed mainly of untranslated mRNA, translational machinery and signaling proteins. The formation of SGs is upregulated in response to such cellular stresses as UV radiation, hypoxia and proteasome inhibitors (eg. bortezomib) and allow for increased cell survival upon exposure to acute stresses; thus, they are directly linked to chemoresistance. Our lab has previously demonstrated a link between PRMT6 expression and SG formation in that PRMT6 knockdown via shRNA reduces cellular capacity for SG formation in HeLa (cervical cancer) cells. Further, rescue of PRMT6 in knockdown HeLa species via flag-PRMT6 transfection allowed for partial recovery of stress granule formation. However, overexpression of PRMT6 in HeLa cells did not allow for more prolific stress granule formation.
Our current research has focused on the mechanistic nature of this link. GFP-expressing cell lines have been established in an effort to conduct a mass spectrometry experiment that will surely conclude in fruitful data concerning the interactions PRMT6 experiences throughout its role in stress granules regulation and associated chemoresistance.
 

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Optimal incubation parameters for silver nanoparticle production using Neochloris oleoabundance.

Sam Delisle, Biotechnology with Dr. Christopher Lan

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Silver nanoparticles (AgNPs) have attracted attention from researchers all over the world due to their interesting properties resulting from their large surface:volume ratios. Their miniature size makes them more versatile, allowing them to have applications in various fields such as chemistry (Hemantha & Sureshbabu, 2011), physics (Nie & Emory, 1997), and medicine (Cho et al., 2008). For example, AgNPs are widely used as antibacterial agents (Prabhu & Poulose, 2012) as well as catalysts (Yan et al., 2006), solar energy converters (Kamat, 2007), and electrical productivity enhancers (Ma et al., 2008). However, conventional ways of producing AgNPs involve high costs and environmental pollution (Yin et al., 2004). As a result, the biosynthesis of AgNPs emerged as a topic for scientific research. Microalga’s fast reproduction, easy cultivation and non-toxic properties make it one of the most promising bio-producers of AgNPs able to operate at an industrial scale. It has been discovered that the addition of AgNO3 to Neochloris oleoabundans cell extract, followed by incubation for 24 hours results in the production of AgNPs. However, in order to optimize this process, the optimal incubation parameters such as AgNO3 concentration, pH, light intensity, and temperature must be discovered. This study was mainly focused on testing various values for each of the incubation parameters mentioned previously in an attempt to combine the optimal ones. In general, a large number of monodispersed nanoparticles is desired, making this our main factor in determining the optimal conditions. Since the presence of AgNPs results in an absorbance peak in the UV-vis spectrum around 410nm, the intensity and shape of this peak were used to determine the dispersity and concentration of AgNPs. As far as we know, these results are the first to provide future researchers with a starting point for determining incubation parameters from which adjustments can be made.

 

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Are pituitary hormone transcripts altered in secretogranin II knockout zebrafish?

Brendan Kelly, Biology with Dr. Vance Trudeau

 

Secretoneurin (SN) is a peptide composed of 33-34 amino acids derived from its secretogranin (SgII) precursor. In teleost fish there are two paralogues of SgII referred to as SgIIa and SgIIb that are processed by prohormone convertases to produce the bioactive peptides SNa and SNb. Previous studies have shown that SN is present in certain hypothalamic neurons in the brain and in prolactin cells in the anterior pituitary. Additionally, SN acts through a paracrine pathway to stimulate the release of luteinizing hormone (LH) from the gonadotrophs. In contrast, almost nothing is known about these peptides role in organismal development. Our study will focus on the role of SgII in regulating the development and functioning of the pituitary using the zebrafish (Danio rerio) as a model organism. SgIIa and SgIIb are present during early zebrafish larval development (18 and 5 hours post-fertilization, respectively), and their expression increases thereafter. It is hypothesized that gonadotropic hormone subunits ( lhβ fshβ and gsuα) and neurohypophysial peptides (oxt, avp) are present during early larval development. It is also hypothesized that inhibition of SgII expression via specific gene knockout (KO) will alter the expression of these gonadotropins and peptides, which may lead to defects in sexual function later in life. In this study embryos from four genetically-modified lines (WT, SgIIa-KO, SgIIb-KO, and SgII(a+b)-KO) will be collected at key time points post-fertilization. RNA will then be extracted from these samples using the Trizol method, after which cDNA will be synthesized. Samples will then be run using real-time quantitative PCR in order to quantify the transcript levels of the genes listed above. This data will augment the current knowledge of the regulation of reproduction in zebrafish as well as providing support that SgII is a regulator of this system.