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MUSA - Alberta Pharmacy Students' Association

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University of <strong>Alberta</strong> Summer Students’ Research Day<br />

In 2011, over 200 undergraduate students participated in the Faculty of Medicine & Dentistry Summer Student<br />

Research Program. On October 15, 175 students presented posters at the 44th Annual Summer Students’ Research<br />

Day. Listed below are the 14 finalists from the poster competition. We congratulate the finalists and all participants.<br />

From these 14 finalists, two students were selected to represent the<br />

University of <strong>Alberta</strong> at the annual National Students’ Research<br />

Forum in Galveston, Texas. Their abstracts are presented below.<br />

Uncovering the role of topoisomerase II-beta<br />

binding protein 1 in dna replication stress response<br />

Mark Assmus, Charles Leung, Mark Glover<br />

DNA replication stress can lead to genomic instability which has<br />

been shown to be one of the primary hallmarks of cancer. TopBP1 is a<br />

crucial mediator protein found within the replication stress response<br />

in mammalian cells. TopBP1 activates Ataxia telangiectasia mutated<br />

related (ATR) kinase which phosphorylates many of the downstream<br />

substrates to initiate this response. The replication stress response<br />

involves specific interactions between the nine BRCA1 C terminus<br />

(BRCT) domains of TopBP1 and various proteins. More specifically,<br />

TopBP1 has been shown to provide an essential role in interacting<br />

with both ATR-interacting protein (ATRIP), Rad9-Rad1-Hus1 (9-1-1)<br />

complex as well as Mediator of DNA damage checkpoint protein 1<br />

(MDC1) which are all essential components of the response pathway.<br />

The crystal structure of TopBP1 BRCT 4/5 in complex with MDC1 was<br />

previously solved in our lab. The structure shows a unique mode of<br />

TopBP1 binding to MDC1 that involves the dimerization of two BRCT<br />

4/5 molecules. In an effort to further examine this interaction, I used<br />

a fluorescence polarization (FP) binding assay involving an MDC1<br />

FITC labelled di-phospho-peptide. I was able to express and purify<br />

GST fusion proteins of TopBP1 BRCT 4/5 and TopBP1 BRCT 5, as well<br />

as TopBP1 BRCT 5 alone, which were used for further FP studies.<br />

The results of the FP assays indicated that it is the BRCT 5 binding<br />

pocket which is primarily responsible for the interaction with MDC1<br />

and that the dimerization induced by GST allows for tighter binding.<br />

Additionally, mutant constructs of the putative BRCT 5 binding<br />

pocket were designed, successfully over-expressed and purified.<br />

The FP assays showed decreases in binding affinity associated with<br />

mutation of key conserved residues in the binding pocket. FP was<br />

also used to confirm that the phosphorylation of the MDC1 peptide<br />

is essential for TopBP1 BRCT 4/5 recognition. Taken together, these<br />

FP results further support the unique dimerization-based binding<br />

mechanism suggested by the crystal structure.<br />

PaX3 expression in melanoma<br />

Zachary Tan and D. Alan Underhill<br />

The transcription factor PAX3 is critical for development of neural<br />

crest lineages including melanocytes. Prior to birth, PAX3 is<br />

required for the proliferation of melanocyte precursors and it is<br />

thought to maintain an ‘undifferentiated plastic state’ in epidermal<br />

melanocytes after birth, as well in melanocyte stem cells. In<br />

addition, PAX3 is expressed throughout melanoma progression,<br />

from nevi to metastatic disease. Nevertheless, little is known about<br />

how PAX3 carries out these diverse roles. PAX3 is reported to be<br />

phosphorylated by Glycogen Synthase Kinase 3ß (GSK3ß). In the<br />

present study, the potential role of this kinase in modulating PAX3<br />

activity in B16F10 melanoma cells was examined using chemical<br />

inhibitors. Fluorescence Activated Cell Sorting (FACS) was used<br />

to assess cell cycle distribution and PAX3 levels were monitored<br />

by immunoblotting. Treatment of cells with the GSK3ß inhibitors<br />

lithium chloride (LiCl) or BIO caused decreased cell proliferation<br />

(P=0.05) and G2/M accumulation (P=0.05), and was associated<br />

with increased PAX3 expression (P=0.05). In contrast, knockdown<br />

of PAX3 using siRNA resulted in G1 accumulation (P=0.05).<br />

Immunofluorescence techniques for exogenous BrdU incorporation<br />

and endogenous PS10H3 allowed for direct microscopic visualization<br />

and quantification of cells in S and G2/M phase respectively. Upon<br />

PAX3 knockdown, there was significantly less BrdU incorporation<br />

and PS10H3 staining (P=0.05). Lastly, cell motility assays were<br />

conducted using live-cell Differential Interference Contrast (DIC)<br />

microscopy and analyzed using T-Scratch software. Interestingly,<br />

inhibition of GSK3ß as well as PAX3 knockdown was associated<br />

with markedly decreased cellular motility and proliferation. These<br />

investigations identify GSK3ß and as an important modulator of<br />

PAX3 levels in melanoma cells, and also suggest broader roles for<br />

PAX3 in regulating the G1 to S-phase transition in melanoma.<br />

Student Poster Title Supervisor<br />

Mark Assmus Uncovering the role of topoisomerase II-beta<br />

binding protein 1 in DNA replication stress<br />

response<br />

Christopher<br />

Beavington<br />

Department/<br />

Division<br />

Dr. Mark Glover Biochemistry<br />

University of <strong>Alberta</strong> Health Sciences Journal • April 2012 • Volume 7 • Issue 1 3<br />

Alanna<br />

Chomyn<br />

The structural studies of bacterial lactoferrin<br />

binding protein B from Neisseria meningitides<br />

Isolation of trkA expressing and IB4-binding<br />

sensory neurons through the use of saporin<br />

Nicholas Chua A model system for complex redox enzyme<br />

maturation<br />

Alexandru<br />

Cojocaru<br />

Michelina<br />

Kierzek<br />

Using inhibition of protein N-myristoylation<br />

towards the design of a synthetically lethal<br />

treatment of B-cell lymphomas<br />

Elucidating the molecular mechanisms<br />

of heart disease-linked mutations of<br />

phospholamban<br />

Stephanie Mah Capase 1 Inhibition in inflammatory bowel<br />

disease reduces epithelial cell extrusion<br />

Scott Meyer Investigating the quinone binding site of<br />

Escherichia coli fumarate reductase<br />

Robyn Millott The novel interaction between<br />

N-myristoyltransferase 1 and calnexin<br />

Kian Parseyan Proposed improvements for intraspinal<br />

microstimulation array fabrication and<br />

insertion<br />

Amit Persad Expression of ST8Sia family in developing<br />

chick retina and their role in AP2deltamediated<br />

axonal generation<br />

Raheem<br />

Suleman<br />

Does long life come from mom? Isolation of<br />

a longevity-conferring mitochondrial DNA<br />

mutation in Caenorhabditis elegans<br />

Dr. Joanne Lemieux Biochemistry<br />

Dr. Christine Webber Anatomy<br />

Dr. Joel H. Weiner Biochemistry<br />

Dr. Luc G.<br />

Berthiaume<br />

Cell Biology<br />

Dr. Howard S. Young Biochemistry<br />

Dr. Julia Liu Medicine/<br />

Gastroenterology<br />

Dr. Joel H. Weiner Biochemistry<br />

Dr. Marek Michalak Biochemistry<br />

Dr. Vivian K.<br />

Mushahwar<br />

Cell Biology<br />

Dr. Roseline Godbout Oncology<br />

Dr. Bernard D.<br />

Lemire<br />

Biochemistry<br />

Zachary Tan PAX3 expression in melanoma Dr. Alan Underhill Oncology<br />

Terri Waller Partial deficiency of adipose trigylceride<br />

lipase (ATGL) does not protect against<br />

diabetes-induced cardiac dysfunction<br />

Dr. Jason R.B. Dyck Pediatrics<br />

RESEARCH

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