Browsing by Author "Bernstein, Nina"
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Item9-(2-Phosphonyl-methoxyethyl)-adenine promotes erythrocytic differentiation and disrupts cell replication in chronic myelogenous leukemia K562 cells(2021) Wiseman, Brittany; Harcombe, Kimberley; Bernstein, NinaDisruption during cellular differentiation can cause hematopoietic stem cells to proliferate uncontrollably, resulting in the development of cancer. Differentiation therapies are being investigated as a type of cancer treatment which involve inducing agents that promote the differentiation of cancer cells into those with similar properties to normal blood cells. These cells can then undergo apoptosis at an accelerated and controlled rate compared to cancer cells, making this a potential therapeutic technique. In this study, the ability of human chronic myelogenous leukemia K562 cells to undergo cellular differentiation in response to the inducing agent 9-(2-Phosphonyl-methoxy ethyl)-adenine (PMEA) is investigated. PMEA has previously been shown to disrupt cell replication, and promote erythrocytic differentiation in K562 cells. In order to further test the effectiveness of this inducer, cell proliferation was measured with a cell growth curve, hemoglobin presence was measured with benzidine staining, and gamma-globin expression (a protein subunit of fetal hemoglobin) was measured in both induced and uninduced K562 cell cultures via RT-qPCR and western blotting. The results indicate that PMEA slows cell replication, and promotes hemoglobin (and subsequently gamma-globin) expression in treated cells. In summary, the findings support the conclusion that PMEA is able to promote erythrocytic differentiation in K562 cells, and provides information that supports differentiation therapies as a method for cancer treatment. ItemBiochemical characterization of DNA repair enzyme inhibitors, molecules with possible applications to improving cancer treatment(2020) Hamel, Jolie; Bernstein, NinaPolynucleotide kinase-phosphatase (PNKP) is a critical DNA repair enzyme responsible for processing DNA damage caused by radiation. A loss of function in this enzyme results in increased cell susceptibility to radiation-induced DNA damage and subsequent cell death. As radiation therapy is commonly used in cancer treatment, targeted inhibition of PNKP has been proposed to increase the effectiveness of radiation therapy at lower doses. We characterized two previously identified PNKP inhibitors, Candesartan and S4, by their effects on the kinase activity, kinase substrate binding, and phosphatase substrate binding of Caenorhabditis elegans and mouse PNKP. The binding assays were conducted using electromobility shift assays (EMSA), while in vitro kinase assays were performed to assess kinase activity. Both inhibitors had an effect on both domains of PNKP, but were more effective at displacing the phosphatase substrate than the kinase substrate. Comparisons of kinase activity inhibition by new and older samples of inhibitors showed that both Candesartan and S4 degrade over a span of 3-4 months and lose their effectiveness. These inhibitors show promise for applications in cancer treatment, but further research is needed. ItemBiochemical characterization of the kinase activity of DNA repair enzyme, PNKP from C. elegans(2015) Oladogba, Oluwatosin; Bernstein, NinaDNA damage by genotoxic agents such as ionizing radiation or reactive oxygen species is likely to occur in the DNA of all living organisms. Therefore the cells of living organisms have developed complex protein networks overtime to help discover and repair DNA damage (Bernstein et al. 2005). Polynucleotide Kinase/Phosphatase (PNKP) is an enzyme that plays a crucial role in repairing a type of DNA damage known as DNA strand breaks (Bernstein et al. 2005). This enzyme has 3 domains, a kinase domain at the C-terminal, a phosphatase domain at the center, and an FHA domain at the N-terminal (Figure 1) (Bernstein et al. 2008). The kinase and phosphatase domains are responsible for directly repairing DNA strand breaks while the FHA domain is responsible for binding PNKP to other DNA repair enzymes (Bernstein et al. 2008). The general objective of this study is to analyze the kinase activity of PNKP derived from C. elegans (CePNKP) in comparison to PNKP derived from humans (hPNKP) by conducting kinase assays. A long term goal for this research is to characterize useful orthologs of PNKP for structural studies of an inhibitor binding to this enzyme. Results from this research showed that the kinase activity of CePNKP is more selective for the recessed 5’ terminus compared to the kinase activity of hPNKP, and this suggests that it might possibly be a good model for hPNKP. ItemCloning and purification of a glycerol-specific alditol oxidase for biosensor construction(2022) Barroma, Chrissa; Kryjak, Amanda; Bernstein, NinaWine production is dependent on ethanol, but also on optimal glycerol concentrations, both of which are produced by S. cerevisiae fermentation. Wine characteristics like sweetness levels are influenced by glycerol concentrations. Additionally, elevated glycerol levels can be an indication of abnormal blood sugar levels. In both situations, close observations of glycerol levels are essential. One proposed method of measuring glycerol concentrations is through enzymatic oxidation with a glycerol biosensor. Alditol oxidase (AldO) is a recently discovered carbohydrate oxidase in S. coelicolor. Despite specificity for longer-chained polyols, studies have proposed that AldO can be used as a glycerol oxidase. Using random point mutations, an AldO mutant was isolated and had increased specificity for glycerol. These results suggest that potential for AldO with glycerol biosensor development. This project aimed to produce a glycerol specific alditol oxidase to be used as a biosensor. A synthetic alditol oxidase (AldOG) gene was used to produce AldOG via cloning methods. We plan to overexpress and purify the AldOG protein to use in construction of a glycerol biosensor in collaboration with Dr. Samuel Mugo (MacEwan University). ItemFlexible electrochemical aptasensor for cortisol detection in human sweat(2021) Mugo, Samuel; Alberkant, Jonathan; Bernstein, Nina; Zenkina, Olena V.This communication demonstrates an electrochemical DNA aptasensor for the detection of cortisol in human sweat. The aptasensor was fabricated via layer-by-layer assembly on stretchable polydimethylsiloxane (PDMS) coated with conductive nanoporous carbon nanotube-cellulose nanocrystals (CNC/CNT) film using a linker to a cortisol specific DNA aptamer. The flexible cortisol aptasensor had a dynamic range of 2.5–35 ng mL−1. The aptasensor precision was determined to be 2.7% relative standard deviation (%RSD) across the concentration dynamic range. The aptasensor was determined to have a limit of detection (LOD) of ∼ 1.8 ng mL−1. The aptasensor was demonstrated to have high selectivity to cortisol and was unresponsive to interfering species including glucose, sodium lactate, and β-estradiol. The aptasensor was successfully evaluated for the detection of cortisol in human sweat indicative of its high specificity. ItemMolecular modelling and kinase assay for CePNKP binding to DNA(2017) Manary, Brandon; Bernstein, Nina; Llano, JorgeComputational techniques of homology modelling, enzyme–substrate docking, and molecular dynamics were applied to elucidate the structure and substrate binding properties of the DNA repair enzyme polynucleotide kinase/phosphatase from the nematode C. elegans (CePNKP). PNKP is involved in the repair of DNA strand breaks, a form of DNA damage caused by reactive oxygen species, ionizing radiation and certain chemical mutagens. Mutations in human PNKP have been associated with the neurological disorders Microcephaly with Intractable Seizures (MCSZ) and Ataxia Oculomotor Apraxia 4 (AOA4). In addition, human PNKP has been identified as a potential drug target for the development of chemo- and radiosensitizing agents for cancer treatment. CePNKP is a useful model system for studying the human enzyme. The substrate preference for both human and C. elegans PNKP has been investigated, and found to be similar, with higher selectivity for recessed over blunt DNA ends. However, CePNKP exhibits a more exclusive preference for recessed DNA ends than human PNKP. To elucidate the reason for the unique substrate specificity of CePNKP, the structure of CePNKP in complex with its DNA substrate must be determined in atomistic detail by molecular modelling. The generated structural model is compared with our experimental results of kinase activity assays of wild-type CePNKP. ItemUsing anticipated learning outcomes for backward design of a molecular cell biology course-based undergraduate research experience(2020) Hills, Melissa; Harcombe, Kimberley; Bernstein, NinaAnticipated learning outcomes (LOs) were defined and used for the backward design of a Course-based Undergraduate Research Experience (CURE). These LOs reflect the inquiry-based nature of CUREs and capture key knowledge and skills inherent to scientific practice and essential in research. The LOs were used to plan a formative and summative assessment strategy to support and evaluate student achievement. A research question was identified that aligned with the learning goals of the course, provided an opportunity for discovery and iteration, and introduced a variety of molecular, cellular, and biochemical techniques. The course is offered to students in the final year of their degree and delivered over a 12-week period with two 3-hr labs each week. These LOs, and the rigorous assessment strategy used to support them, could be adapted to different projects. Likewise, the laboratory exercises are presented as a series of modules highlighting opportunities for adaptation to a variety of schedules.