Browsing Graduate Studies by Department "Biochemistry and Molecular Biology"
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- ItemOpen Access1α, 25-dihydroxyvitamin D3 induced osteogenesis in murine embryonic stem cells: the role of Wnt5a(2006) Davis, Lesley Anne Margaret; Rancourt, Derrick
- ItemOpen AccessA novel cdc2 homologue in brain: identification and biomechanical characterization(1993) Lew, G. John; Wang, Jerry H.
- ItemOpen AccessA search for binding partners of Golgin-67: potential interaction with guanine nucleotide dissociation inhibitor I(2003) Swancott, Abigail Jane Mary; Fujita, Donald J.
- ItemOpen AccessA study of genes in spermatogenesis: the potential functions of tgap and oaz3(2010) Ruan, Yibing; van der Hoorn, Frans A.
- ItemOpen AccessA Study of the Therapeutic Value of the Mashkiki Bacteriophage: A lambda display and subunit vaccine system(2009) Thomas, Bradley Scott; Rancourt, Derrick
- ItemOpen AccessA Tale of a ‘Tail’ – Understanding the Role of Ku80 C-terminal Region in Non-Homologous End Joining(2015-12-14) Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan; Cobb, Jennifer; MacDonald, JustinNon-homologous end joining (NHEJ) is the major DNA double strand (DSB) break repair pathway in mammalian cells. The first step in NHEJ is recognition of DSBs by the Ku heterodimer and subsequent recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a serine/threonine protein kinase, to form the DNA-PK complex. This complex aligns DSB ends, activates Artemis endonuclease activity and finally recruits XRCC4-DNA ligase IV, which ligates DNA ends. The Ku heterodimer consists of 70 and 80 kDa subunits and is conserved throughout evolution. It has been suggested that the extreme C-terminal 14 amino acids of Ku80 is required for DNA-PKcs recruitment and activation. However, another study demonstrated that deletion of the Ku80 C-terminal region (CTR) does not abolish DNA-PKcs activation. Thus, there is considerable ambiguity regarding the role of the Ku80 CTR in DNA-PKcs recruitment and activation. The aim of this study is to understand the role of Ku80 CTR in NHEJ with focus on its ability to recruit and activate DNA-PKcs kinase activity. Using clonogenic cell survival assays, I confirmed that hamster cells expressing Ku80 CTR deletions are radiosensitive and also showed sensitivity to other DSB inducing agents such as doxorubicin and neocarzinostatin. I then generated Ku80 C-terminal deletions (Ku80 residues 1-718 and 1-569), cloned them into baculovirus vectors and expressed and purified the corresponding Ku heterodimers from insect cells. In vitro autophosphorylation reactions, in presence of calf-thymus DNA, using purified proteins showed that Ku heterodimer with Ku80 residues 1-718 showed only a slight defect in DNA-PKcs autophosphorylation, whereas heterodimer with Ku80 residues 1-569 had significant defects in multiple DNA-PKcs autophosphorylation sites. Surprising results were observed when defined DNA structures such as 25 base pair (bp) blunt ended double stranded (ds) DNA was used. Deletion of the entire Ku80 CTR (residues 570-732) lead to abrogation of DNA-PKcs kinase activity and inability to interact with DNA-PKcs protein. On the other hand, deletion of extreme C-terminal 14 amino acids of Ku80 did not affect DNA-PKcs kinase activity but showed defects in its ability to interact with DNA-PKcs. These defects may underlie the radiation and chemosensitivity of Ku80 CTR deletion mutants.
- ItemOpen AccessADP-ribosylation of neuronal tissue specific protein B-50(1995) Philibert, Keith D.; Zwiers, Henk
- ItemOpen AccessAdvances in Hydrogen Exchange Mass Spectrometry to Study Microtubules and MAPs(2015-05-27) Burns, Kyle; Schriemer, DavidMicrotubules are a fundamental component to the cellular cytoskeleton and are responsible for cell structure, motility, intracellular transport, and formation of the mitotic spindle. The ability to serve the diverse functions requires constant flux between polymerization and depolymerization. The regulation of microtubule dynamics is accomplished by microtubule associated proteins that can interact with microtubules, or its tubulin dimer, to induce polymerization or depolymerization. The depolymerization mechanism induced by mitotic centromere-associated kinesin (MCAK) was studied in detail. Studying a large protein system such as the regulation of microtubule dynamics at the molecular level requires an integrative structural biology approach. Hydrogen exchange mass spectrometry (HX-MS) is a vital technique for studying the protein dynamics and was optimized for the analysis of large protein complexes. Two HX-MS platforms consisting of a FT-MS and a high-resolution QTOF mass spectrometer were evaluated by comparing the figures-of-merit for a typical bottom-up HX-MS experiment: peptide identification, deuterium measurement accuracy, and deuterium measurement precision. The Orbitrap Velos identified 64% more peptides than the TripleTOF 5600, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the TripleTOF, depending on the Orbitrap resolution setting; however, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate. The findings presented support the use of the TripleTOF 5600 for further development of hydrogen exchange methods. A data-independent acquisition approach was developed that combines peptide fragmentation data and a new peptide scoring algorithm (WUF, Weighted Unique Fragment) to provide MS/MS data for HX measurements while reducing manual validation. The scoring incorporates elements of the validation process and preserves high peptide identification accuracy. When compared to a conventional Mascot-driven HX-MS method, HX-MS2 produces two-fold higher tubulin sequence depth at a peptide utilization rate of 74%. The HX-MS2 method was applied to study the microtubule depolymerization process induced by MCAK. In the described model, the N terminus is responsible for the lateral separation in conjunction with the outward curvature induced by the motor domain. The C terminus is responsible for regulating the microtubule interactions.
- ItemOpen AccessAlternative splicing of NCX1 affects the sensitivity of its Ca2+ sensors(2012-10-01) Torry, Lisa; Lytton, JonathanThe Na+/Ca2+ exchanger, NCX1, is important for Ca2+ homeostasis in many different tissues. Exchange function is regulated by Ca2+ binding to two cytoplasmic domains, CBD1 and CBD2, that are subject to tissue-specific alternative splicing. To test if alternative splicing modulates Ca2+ binding to the CBDs, the isolated domains CBD1, CBD2, and the tandem construct CBD12 from different NCX1 spliced isoforms were produced, purified and analyzed for Ca2+ binding in a competition binding assay using fluorescent Ca2+ indicators. NCX1.4-CBD2 bound two Ca2+ ions while NCX1.3-CBD2 did not bind Ca2+. CBD1 bound four Ca2+ ions, and some of the CBD1 Ca2+ binding sites in the CBD12 tandem construct had isoform-specific affinities significantly higher than CBD1 alone. My results confirm that alternative splicing of NCX1 affects regulatory Ca2+ binding in both CBDs, and thus tailors NCX1 function to suit tissue-specific requirements for Ca2+ homeostasis.
- ItemOpen AccessAn in vitro evaluation of two novel oncolytic viruses for the treatment of esophageal cancer(2012-10-01) Fratiloiu, Andreea; Johnston, RandalAbstract Novel therapeutic regimens are needed to improve the dismal outcomes of patients with esophageal cancer as it is the 9th most common malignancy and the 6th most frequent cause of cancer related deaths worldwide. The major goal of anti-cancer therapies is to destroy the tumor cells with little damage to the surrounding normal cells. Oncolytic viruses are experimental agents that are intended to target and kill various types of cancer cells, leaving normal cells healthy. Two novel oncolytic viruses, Reovirus and Coxsackievirus, were investigated for the possible treatment of esophageal cancer by determining whether these two agents can replicate and kill esophageal cancer cells in vitro by apoptosis. Both viruses meet this goal and Reovirus appears to be the most effective.
- ItemOpen AccessAn investigation of nutrition-dependent mRNA translation in Drosophila larvae(2015-01-28) Nagarajan, Sabarish; Grewal, SavrajDietary nutrients promote organismal growth via activation of cellular pathways that are necessary for cellular growth and division. However, upon conditions of nutrient scarcity, organisms ranging from yeast to mammals alter their metabolism to conserve nutrients and energy. One of the key cellular events required for sustained growth downstream of nutrition is mRNA translation. In order to coordinate cellular and organismal growth with nutrient availability, organisms have devised several control checkpoints to limit the energy consuming process of translation under starvation. Work on mammalian cell lines have shown that mRNA translation is inhibited in response to a multitude of stresses including nutritional stress, DNA damage, viral stress, hypoxia and oxidative stress. However, it is unclear as to how an organism in general responds to starvation in terms of mRNA translation. I have used polysome gradient analysis in developing Drosophila larvae to show that starvation led to a rapid decrease in translation, seen as early as 30 minutes after removal of larvae from food. A maximal decrease in translation was observed after 6-18 hours of starvation. Using qRT-PCR I have looked at the translation profile of 18 individual genes and observed that starvation led to a general decrease in translation, irrespective of the starvation-mediated change in total transcript levels. Sugars and amino acids have been shown to be important regulators of translation in animal cells. I have determined that neither sugars nor amino acids were sufficient to maintain translation in larvae removed from food. However, a complex diet such as yeast was sufficient to maintain translation in larvae removed from food. The majority of work in the field of translation suggest three main signaling pathways functioning downstream of nutrition to regulate translation - insulin signaling,TOR signaling and eIF2α signaling. I have shown that TOR signaling is required for translation in fed conditions. On the other hand, genetic activation of TOR and/or insulin signaling was not sufficient to prevent the starvation-mediated inhibition of translation. I have also shown that the eiF2α kinases - GCN2 and PERK were not required for starvation-mediated suppression of translation.
- ItemOpen AccessAnalysis of GTP variability and the erythrocytic GTP concentration determining trait (Gtpc) in the mouse(1997) Wiebe, Glenis Jane; Snyder, Floyd F.
- ItemOpen AccessAnalysis of the role of the Pnk1 in DNA damage repair in fission yeast(2006) Qi, Tao; Young, Dallan B.
- ItemOpen AccessAnalysis of transcription factor DNA interactions utilising energy filtered transmission electron microscopy(2001) Brown, Mark Lesley; Bazett-Jones, David P.
- ItemOpen AccessAssociation of kinesin light chain 3 with outer dense fibers(2003) Zhang, Ying; van der Hoorn, Frans A.
- ItemOpen AccessBiochemical and molecular studies on the candidate tumor suppressor ING1 during apoptosis and senescence(2003) Vieyra, Diego; Riabowol, Karl T.
- ItemOpen AccessCalcium-independent smooth muscle contraction: a focus on zipper-interacting protein kinase (ZIPK)(2011) Moffat, Lori Dawn; Walsh, Michael P.; MacDonald, Justin A.