11th ANNUALOHRI RESEARCH DAY

PROGRAMThursday, November 10th, 2011

7:30 A.M. – 5:00 P.M.

St. Elias Centre750 Ridgewood Ave.

Ottawa, ON

SPONSORS

THIS EVENT IS GENEROUSLY SUPPORTED BY THE FOLLOWING SPONSORS:

COMMITTEE

The Ottawa Hospital Research Institute (OHRI) would like to express its appreciation to members of the OHRI Research Day Committee for their dedication and hard work in organizing this event. Dr. Duncan Stewart Dr. Jim Dimitroulakos Dr. Luc Sabourin Dr. Jay Baltz Dr. Dean Fergusson Dr. Fraser Scott (Chair) Dr. Angela Crawley Dr. Cathy Tsilfidis Ms. Melanie Genereaux Ms. Jennifer Paterson Ms. Melissa Bowerman Ms. Alessandra Pasut

VOLUNTEERS Mr. Greg Canham Ms. Jane Caniff Ms. JoAnn MacDonald

MESSAGE FROM THE CEO & SCIENTIFIC DIRECTOR Welcome to the 11th Annual OHRI Research Day! As in previous years, we have a full-day program designed to promote scientific interaction and highlight the outstanding work of our students and fellows. Our young researchers are a vital part of the OHRI, providing the insight, enthusiasm and dedication that are crucial to our success. Whether you are formally involved in the judging or not, I strongly encourage you to ask questions of our trainees during the poster sessions and oral presentations. This is an important training exercise, and also a fantastic opportunity to learn about all the exciting research projects going on at our Institute. I would also like to draw your attention to the OHRI IMPACT (Identification of Marketable Products, Applications and Commercializable Technologies) Award, which is designed to encourage trainees to identify technologies and innovations stemming from their research and think about how their research could lead to innovations. This award is part of a larger effort at OHRI to foster the translation of our research findings into benefits for Canadians, and based on the interest in this award, I’m sure we will be continuing it in future years. The keynote Dr. J. David Grimes Lecture is another highlight of OHRI Research Day, and this year I am delighted that our lecturer is Dr. Jennifer Van Eyk of Johns Hopkins University. Dr. Van Eyk is one of the most promising young Canadian scientists in the cardiovascular field, and she has been uniquely successful in both basic laboratory research and translational science. I am sure that her lecture will be enlightening and inspiring for everyone. On behalf of everyone at OHRI, I would like to extend thanks to our guest speaker, our presenters, our judges, our planning committee and our volunteers for their contributions to this important day. I would also like to thank the sponsors for making it possible.

Duncan Stewart, MD, FRCPC

CEO & Scientific Director and Senior Scientist in the Regenerative Medicine Program and Evelyne and Rowell Laishley Chair, Ottawa Hospital Research Institute

Vice President, Research, The Ottawa Hospital Professor, Department of Medicine, Faculty of Medicine, University of Ottawa

J. David Grimes, MD, FRSPC

This annual lecture is named in honour of Dr. J. David Grimes, founder of the Loeb Research Institute (predecessor of the OHRI at the Civic Campus). Dr. Grimes served as the Institute’s CEO & Scientific Director until he retired in 1997. He recruited and mentored many of Ottawa’s leading health researchers. He also practiced neurology for over 25 years, specializing in Parkinson’s disease. After a long and courageous battle with lung disease, Dr. Grimes passed away on May 9th, 2001. A man of great vision and compassion, Dr. Grimes is missed by everyone who has known him. His memory lives on at OHRI in many ways, including in this annual lecture and in the OHRI’s Dr. J. David Grimes Research Career Achievement Award and the Dr. J. David Grimes Research Chair at the University of Ottawa.

DR. J. DAVID GRIMES LECTURE

2011 J. DAVID GRIMES LECTURER

Dr. Jennifer Van Eyk, Ph.D. is an Associate Professor of Medicine, Biological Chemistry and Biomedical Engineering at Johns Hopkins University, Baltimore and Director of the Hopkins NHLBI Proteomics Innovation Center on Heart failure, the JHU Bayview Proteomics Center that has the mandate to facilitate application of proteomics in medicine. Most recently, she has become director of the JHU Clinical Translational Science Award Biomarker Development Center that works to bring biomarkers to clinical use through the application of innovative technologies. Her research laboratory studies the underlying molecular mechanism of heart and vascular disease using a large number of proteomic methodologies focusing on the role of disease induced post-translational modifications. In particular, the site-specific identification, quantification and specific functional role of protein modifications ranging from cell surface proteins to those constituting the myofilament and mitochondria. The philosophy of the group is that key clinical questions must drive the development and application of innovative technologies - for protein and biochemical understanding - in order to better define new therapeutic intervention and robust biomarkers for the diagnosis, prognosis and risk stratification. Dr. Van Eyk has over 160 peer reviewed manuscripts, reviews, book chapters and has co-edited two books entitled “Proteomics and Genomic Analysis of Cardiovascular Disease” and “Clinical Proteomics: Insights into Disease”. Dr. Van Eyk is on the editorial boards or a consulting editor for Circulation Research, Circulation: Genetics, J. Molecular and Cellular Cardiology and is senior editor for Proteomics and Proteomics: Clinical Applications.

RESEARCH TRAINEE SALARY AWARDS

Melissa Bowerman Justin Boyer Marie Brier Andre Bussieres Christine Cabrera Annabelle Caron Wing Yean Chang Heather Colquhoun Mary-Ann Doyle Mehdi Eshraghi Feras Ghazawi Vanessa Illing Ji Kim Bao Kong John Paul Michalski Andre Molgat Lyndsay Murray Ryan O’Meara Scott Ryan Kenny Schlosser Rany Shamloul Amanda Smith Scott Sugden Brianne Thrush Qi Wang Hang Yin

GOODMAN COHEN SUMMER STUDENT AWARDS

Charlie Foster Jovian Tsang

PROGRAM OUTLINE

7:30 a.m. Registration / Poster Setup / Continental Breakfast 8:15 a.m. Opening Remarks (Dr. Fraser Scott, Dr. Duncan Stewart, Dr. Bernard Jasmin) 8:30 a.m. Neuromuscular Development and Disease

Moderator: Dr. Hang Yin Wnt7a – a potential new therapeutic for muscle wasting (Dr. J. von Maltzahn, Rudnicki

group) A prkl-1 suppressor screen to identify new planar cell polarity components involved in

neuronal polarity (David Carr, Colavita group) Delayed skeletal muscle development and intrinsic muscle weakness in mouse models

of spinal muscular atrophy (Justin Boyer, Kothary group)

9:15 a.m. Cancer Cell Regulation Moderator: Janet Manias PKCiota depletion induces p21-dependent senescence in glioblastoma (Ian Restall,

Lorimer group) SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and

efficient cell migration (Jennifer Quizi, Sabourin group) The epigenetic cofactor UTX is involved in TAL1-mediated transcription deregulation in T

cell acute lymphoblastic leukemia (Fengtao Dai, Brand group) 10:00 a.m. Refreshment Break (Sponsored by Illumina) 10:15 a.m. Poster Viewing

Including judging of posters prepared by postdoctoral fellows, clinical fellows, research associates and residents

11:00 a.m. Consequences of Chronic Disease and Aging Moderator: Dr. Chenxi Zhou

The Nox1/4 inhibitor GKT136901 reduces oxidative stress and prevents development of renal injury in a model of type II diabetes (Dr. M. Sedeek, Touyz group)

Deficiency of ornithine decarboxylase contributes to oocyte aneuploidy in mice (Dr. Yong Tao, Liu group)

Pro-inflammatory activation of J774A.1 macrophages reduces their ability to prevent 3T3-L1 preadipocyte apoptosis (André Molgat, Sorisky group)

11:45: a.m. Advances in Medical Practice Moderator: Dr. Lee-Hwa Tai

Practice patterns and variations in spine x-ray ordering among chiropractors enlisted with provider Networks offering complementary care in the United State (André Bussières, Grimshaw group)

Systematic review and meta-analysis of mesenchymal stromal cells in preclinical models of sepsis (Dr. Manoj Lalu, McIntyre and Stewart groups)

Anticholinergic medication for bladder dysfunction in multiple sclerosis can adversely affect cognitive functioning (Dr. Roxana Cruce, Freedman group)

12:30 p.m. Lunch 1:15 p.m. Poster Viewing

Including judging of posters prepared by MSc, PhD, honours, and co-op students and IMPACT award posters

2:00 p.m. The Dr. J. David Grimes Keynote Lecture Moderator: Dr. Duncan Stewart

Clinical proteomics: tackling the unknowns in heart disease presented by Dr. Jennifer Van Eyk, Johns Hopkins University

3:00 p.m. Advances in Stem Cell Research Moderator : Andrea Bravi

Adult brain and spinal cord progenitors respond differently to differentiation factors following proliferation in vitro (M. Coyle, Tsai group)

Modeling Hutchinson-Gilford Progeria Syndrome Using Patient Derived Induced-Pluripotent Stem Cells (Dr. Wing Chang, Stanford group)

Paradoxical increase in BMP kinome activity in blood-derived endothelial cells from Bmpr2 mutation carriers with hereditary pulmonary arterial hypertension (Jessie Lavoie, Stewart group)

3:45 p.m. Refreshment Break 4:00 p.m. The Science Behind the Headlines (moderated by Jennifer Paterson)

“Canadian-made virus shows promise as cancer treatment” (Dr. John Bell) “Longer CPR not necessarily beneficial: study” (Dr. Ian Stiell) “Supreme Court ruling opens doors to drug injection clinics across Canada” (Dr. Mark

Tyndall) 4:45 p.m. Poster and Oral Presentation Awards and Closing Remarks (Dr. Fraser Scott and Dr.

Duncan Stewart) 5:00 p.m. Reception with Cash Bar

ORAL PRESENTATIONS

Neuromuscular Development and Disease (8:30 to 9:15)

Moderator: Hang Ying

1. Wnt7a – a potential new therapeutic for muscle wasting

J. von Maltzahn, ( PhD Candidate ) C. F. Bentzinger, J.M. Renaud and M.A. Rudnicki OHRI Regenerative Medicine Program

Loss of skeletal muscle is a consequence of many chronic diseases, e.g. Duchenne muscular dystrophy (DMD) or cachexia, as well as of aging (sarcopenia). So far, the muscle loss in these chronic diseases remains without adequate treatment. Wnt signalling plays an important role in homeostasis of adult tissues. In skeletal muscle Wnt7a drives the expansion of the satellite stem cell pool through the PCP signalling cascade thereby facilitating regeneration. Furthermore, we discovered that Wnt7a activates the Akt/mTOR anabolic pathway in differentiated myofibers thereby inducing hypertrophy in concert with its receptor Fzd7. Notably the activation of the Akt/mTOR pathway through Wnt7a is independent of IRS-1 and IGF-receptor. Treatment of dystrophic mice with Wnt7a resulted in increased numbers of satellite cells and pronounced hypertrophy. Wnt7a treatment not only increased muscle size significantly but also tetanic force in normal and dystrophic mice thereby providing additional physiological relevance. Wnt7a also benefited mice suffering from cachexia, treated muscles displayed less atrophy than control muscles. This dual function of Wnt7a in adult skeletal muscle provides us with a promising candidate for ameliorative treatment of muscle wasting diseases like DMD or cachexia.

2. A prkl-1 suppressor screen to identify new planar cell polarity components involved in neuronal polarity

David Carr (MSc. Student) Jiravat Visanuvimol Leticia Sanchez-Alvarez Antonio Colavita OHRI Neuroscience Program The planar cell polarity (PCP) pathway is responsible for organizing cell polarity in the plane of the epithelium. We have recently shown that the core PCP pathway components Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1) are required in a subset of peripheral motor neurons (VC neurons) to restrict neurite emergence to a specific tissue axis. In loss-of-function mutants, VC4 and VC5 neurons display ectopic neurites that extend abnormally along the anteroposterior (AP) body axis. The unc-4 promoter is expressed in the VA, DA and VC motor neurons. unc-4 driven over-expression of PRKL-1 in the VC neurons is sufficient to suppress neurite growth. These transgenic animals also displayed backwards locomotion impairment; a movement controlled by the VA and DA neurons. This inability to move backwards could be suppressed by vang-1 null mutants, which are known to interact with prkl-1 in the PCP pathway. We used these observations as the basis of a genetic screen to identify other suppressors that can restore backwards locomotion. Our preliminary findings include the identification of several suppressor mutants including at least 8 new alleles of vang-1 and at least one allele of fntb-1, the beta subunit of farnesyltransferase. Farnesyltransferases post translationally modify proteins by attaching a 15-carbon isoprenoid lipid, farnesyl, to the cysteine residue of a C-terminal CAAX motif. The addition of a farnesyl group facilitates membrane association of the protein due to the hydrophobic nature of the lipid. We believe that FNTB-1 prenylates PRKL-1 at its C-terminal CAAX motif to promote membrane association and interaction in the PCP pathway. We will be describing the characterisation of fntb-1 and genetic interactions with prkl-1 as well as further findings from the genetic screen.

3. Delayed skeletal muscle development and intrinsic muscle weakness in mouse models of spinal muscular atrophy

Justin G. Boyer1 (PhD Candidate),2, Kyle Scott2 , Yves De Repentigny1, Céline Boudreau-Larivière3, Jean Michaud4 Jean-Marc Renaud2 and Rashmi Kothary1,2,5 1Ottawa Hospital Research Institute, Regenerative Medicine Program, Ottawa, ON, Canada K1H 8L6 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 3School of Human Kinetics, Laurentian University, Sudbury, ON, Canada, P3E 2C6 4Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 5Department of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5 OHRI Regenerative Medicine Program Background The disruption of the survival motor neuron (SMN1) gene, either by deletion, rearrangement, or mutation leads to the neurodegenerative disease spinal muscular atrophy (SMA). SMA is characterized by degeneration of α-motor neurons and associated skeletal muscle atrophy. Although SMA is primarily a motor neuron disease, the involvement of muscle in its pathophysiology has not been entirely ruled out. Objectives/Methods The purpose of this study was to elucidate intrinsic muscle defects in SMA mouse models. To do so we took advantage of two different SMA mouse models; the most severe model, Smn-/-;SMN2, and a second less severe model, the Smn2B/-model. Results We report that tibialis anterior muscles of Smn-/-;SMN2 mice generate 40% less maximal force than control muscles independent of aberrant motor neuron signal transmission. Moreover, we demonstrate presymptomatic muscle weakness greater than 60% of control values in both Smn-/-;SMN2 and Smn2B/- mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant expression of the Ca2+ pump sarcoplasmic reticulum Ca2+ ATPase, and the skeletal muscle sodium channel protein Nav 1.4. Despite decreased force production, muscles from SMA mice showed intact sarcomere

organization albeit with an increased number of immature myofibers. The expression of molecular markers of myogenesis including, Pax7, MyoD, myogenin and MRF4, was delayed suggesting that Smn is crucial for early muscle development. Conclusions The findings from this study highlight the presence of muscle defects independent of motor neuron degeneration in SMA mice. Thus, targeting skeletal muscle should not be overlooked in development of SMA therapeutics.

Cancer Cell Regulation (9:15 to 10:00)

Moderator: Janet Manias 1. PKCiota depletion induces p21-dependent senescence in glioblastoma

Restall, IJ (PhD Candidate)1,2; Simard, MA 1; Lavictoire, SJ 1; Parolin, DA 1; Lorimer, IAJ 1-3 1Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa 3Department of Medicine, University of Ottawa, OHRI Cancer Therapeutics Program Cellular senescence is a defense mechanism against the progression of tumours that can be triggered by oncogenes and other cellular stresses. Senescence must be circumvented for tumourigenesis to occur and there is therefore an exciting opportunity to reactivate senescence in cancer cells as a therapeutic strategy to treat human cancers. The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in tumours either by mutations in the PI3K catalytic domain, loss of the PTEN tumour suppressor, or by activation through other regulatory proteins. Protein kinase C iota (PKCiota) is a member of the atypical protein kinase C family of the AGC kinases and is a downstream mediator of the PI3K pathway. In glioblastoma cell lines, where PKCiota is activated by the loss of PTEN, depletion of PKCiota increased the number of senescent cells. The induction of senescence occurred in the absence of a detectable DNA damage response (DDR) but was dependent on the presence of p21, a known substrate of PKCiota. The aurora kinase inhibitor VX-680 further increased the senescence response in the absence of a detectable DDR, suggesting that senescence is induced by an inability to successfully progress through mitosis. We suggest that PKCiota is activated in cancer cells to avert the induction of senescence and that targeting PKCiota could be an effective method to reactivate senescence in cancer.

2. SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and efficient cell migration Jennifer L. Quizi ( PhD Candidate), Paul O’Reilly* and Luc A. Sabourin* *Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H8M5. Ottawa Hospital Research Institute, 725 Parkdale Ave., Ottawa, Ontario, Canada K1Y 4E9. OHRI Cancer Therapeutics Program The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at serine 250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle and subsequent ubquitylation in migrating cells. Interestingly, an accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

3. The epigenetic cofactor UTX is involved in TAL1-mediated transcription deregulation in T cell acute lymphoblastic leukemia Fengtao Dai (PhD Candidate) OHRI Regenerative Medicine Program The transcription factor TAL1 is a potent oncogene whose aberrant expression in the T-cell lineage has a causative role in T cell acute lymphoblastic leukemia (T-ALL), an aggressive blood cancer (Condorelli et al., 1996; Kelliher et al., 1996). Furthermore, TAL1 is aberrantly expressed in over 60% of T-ALL patients. Our group (Palii et al., 2011) and others (Ferrando et al., 2002; Kusy et al., 2010) have previously shown that TAL1 is involved in inhibiting T-cell differentiation and apoptosis while also activating cell proliferation in T-ALL. In addition, using ChIP-sequencing and gene expression profiling upon TAL1 knockdown in leukemic cells, we have recently identified a number of TAL1 target genes that mediate its leukemogenic effect (Palii et al., 2011) . However the molecular mechanism through which TAL1 regulates the expression of these genes remains unclear. To get insight into the mechanism of TAL1-regulated transcription in a leukemic context, we performed an extensive proteomic study to identify cofactors that interact with TAL1. Here, we report for the first time that TAL1 associates with the coactivator complex UTX, which is normally involved in regulating the expression of developmentally regulated genes through removal of the repressive histone H3 lysine 27 trimethyl

(H3K27me3) modification. Furthermore, we demonstrate that TAL1 recruits the epigenetic regulator UTX to activate specific genes that display anti-apoptotic and pro-proliferative function via demethylation of H3K27me3. Consistent with these findings, we also found that in the absence of UTX, T-ALL cells exhibits severe inhibition of cell growth, resulting from both increased apoptosis and decreased cell proliferation. Together, these results demonstrate that the anti-apoptotic and pro-proliferative function of TAL1 in T-ALL is mediated at least partially through the recruitment of the epigenetic factor UTX.

Consequences of Chronic Disease and Aging (11:00 to 11:45)

Moderator: Chenxi Zhou 1. The Nox1/4 inhibitor GKT136901 reduces oxidative stress and prevents development of renal injury in a model of type II

diabetes. M Sedeek1 ( Postdoctoral Fellow), A Gutsol1, Y He1, A Nguyen Dinh Cat1, A Yogi1, D Burger1, A Montezano1, F Heitz2, C Szyndralewiez2, P Page2, C Kennedy1, K Burns1, R Hebert1 and R Touyz1. 1 Kidney Research Center, Ottawa Hospital Research Institute, Ottawa, ON, Canada and 2 GenKyoTex, GenKyoTex, Geneva, Switzerland. OHRI Chronic Disease Program BACKGROUND AND OBJECTIVES: Diabetes is a major cause of end stage kidney disease. Oxidative stress has been implicated in this process. Data from our laboratory showed that higher levels of albuminuria in db/db mice are associated with increased expression of renal NADPH oxidase 4 isoform (Nox4), but not Nox1, and increased oxidative stress, implicating a role for Nox4 in renal injury in diabetes. Here we investigated effects of Nox1/4 inhibitor, GKT136901, on oxidative stress, antixoidants and kidney injury in a model of type 2 diabetes. METHODS: Male db/m and db/db mice on BLKS background, aged 8 weeks were used. Mice were divided into 6 groups, db/db and db/m control group received normal chow, db/db and db/m treated with GKT136901, at low dose, 30 mg/kg/day or with high dose, 90 mg/kg/day, for 16 weeks. The drug was administered orally mixed with food. 24 h urine collections were performed every 4 weeks for measurement of urinary malondialdehyde (MDA) and urinary albumin. Kidney tissue was collected at the end of the study for morphometirc, WB and PCR analysis. RESULTS: db/db mice showed increased urinary MDA excretion compared to db/m mice (141 ±10 vs. 17± 1 nmol MDA/ 24 h urine, n=14-19, P < 0.001). Compared with untreated db/db mice, MDA excretion was significantly reduced in mice treated with high dose (107 ±17 nmol MDA/ 24 hrs urine, n=14, P < 0.05). Expression of superoxide dismutase (SOD-1) was significantly lower in db/db mice compared to db/m (P < 0.05). Treatment with GKT136901 normalized SOD-1 expression at both doses in db/db mice. Reduction of oxidative stress was associated with improved survival of diabetic mice: db/db mice survival is about 70% compared to 95% survival of db/db mice on low dose of GKT136901 and to 100% survival in db/db mice on high dose of GKT136901, P < 0.05 db/db vs. db/db on high dose. Renal morphometric analysis showed a 30% increase in glomerular surface area in untreated db/db mice versus db/m mice. This glomerular hypertrophy was partly reduced to a 14% increase in db/db mice treated with high dose GKT136901 CONCLUSIONS: Chronic treatment of db/db mice with GKT136901, a specific inhibitor of Nox1/4, resulted in a reduction of systemic oxidative stress, increased SOD-1 expression, and improved survival of diabetic mice. Development of nephropathy and renal injury were ameliorated by treatment. Our data suggest that the Nox1/4 inhibitor may have clinical utility in preventing diabetic nephropathy.

2. Deficiency of ornithine decarboxylase contributes to oocyte aneuploidy in mice

Yong Tao (Postdoctoral Fellow), Johne Liu Ottawa Hospital Research Institute OHRI Chronic Disease Program An increasing number of women struggle with fertility problems in their early 40’s, 10-15 years before they reach menopause. Clinical studies suggest that egg aneuploidy, particularly those resulted from premature separation of sister chromatids in meiosis I, is a key factor in pre-menopausal infertility. Using several mouse models, we demonstrated that inhibition of or-nithine decarboxylase (ODC), either during oocyte maturation in vitro or during ovulation in vivo, caused a significant increase in the number of aneuploid eggs, especially those with single sister chromatids. Oocytes from older mice (8 months) contained significantly reduced levels of ODC mRNA, compared to oocytes from young mice (2 months). Most remarkably, while these older mice exhibited 16.1 % egg aneuploidy (again mainly the presence of single sister chromat-ids), this was reduced to 4.7 % by putrescine supplementation. These results reveal a novel link between ODC deficiency and increase of oocyte aneuploidy and provide the rationale for a poten-tially effective intervention for reducing the risk of aneuploid pregnancy in pre-menopausal women.

3. Pro-inflammatory activation of J774A.1 macrophages reduces their ability to prevent 3T3-L1 preadipocyte apoptosis.

André Molgat (PhD Candidate), AnneMarie Gagnon, Alexander Sorisky Chronic Disease Program, Ottawa Hospital Research Institute; Departments of Medicine and of Biochemistry, Microbiology & Immunology, University of Ottawa Background: Obesity is associated with macrophage accumulation in the adipose tissue of mice and humans. We have previously reported that macrophage-conditioned medium (MacCM) prevents preadipocyte apoptosis in a PDGF-dependent manner. Adipose tissue macrophages have been reported to exist in various states of activation. Objective: Here, we have investigated the effect of pro-inflammatory macrophage activation on the pro-survival activity of MacCM

and on MacCM-stimulated preadipocyte PDGFR signaling. Methods: MacCM was generated by treating J774A.1 macrophages with 100ng/ml lipopolysaccharide (LPS) or vehicle control for 0.5-6h, and then placing them in serum-free medium for 24h. Preadipocytes (3T3-L1) were placed in serum-free control medium, MacCM (vehicle control), or LPS-activated MacCM, and their survival was assessed. Results: Preadipocyte cell number decreased by 48% upon serum-withdrawal (p<0.001, 6h control medium without vs with serum); MacCM completely prevented this cell loss. LPS activation of macrophages impaired the pro-survival effect of MacCM, resulting in a 25% reduction in cell number (p<0.05, 6h LPS- vs vehicle- treated MacCM). The attenuation of pro-survival activity observed with LPS-treated MacCM was not associated with a reduction in pro-survival PDGF signaling. We have previously reported that PDGFR, Akt and ERK phosphorylation are necessary for MacCM-dependent preadipocyte survival, and these three responses were the same for both vehicle- and LPS-treated MacCM. Untreated J774A.1 macrophages expressed PDGFA and PDGFB mRNA; PDGFB message was 14-fold more abundant. A transient 2-fold upregulation in macrophage PDGFB expression was observed 2h post LPS treatment, whereas PDGFA expression was reduced by 50% following LPS treatment. Conclusions: Our data suggest that macrophage activation with LPS significantly interferes with the pro-survival activity of MacCM, without altering MacCM-stimulated PDGFR-signaling. Further studies will be needed to identify the mechanism by which LPS-activation hinders the pro-survival activity of MacCM.

Advances in Medical Practice (11:45 to 12:30)

Moderator: Lee-Hwa Tai

1. Practice patterns and variations in spine x-ray ordering among chiropractors enlisted with a provider Networks offering

complementary care in the United States. André E Bussières (DC, MSc, PhD (Candidate))1 Jeremy M Grimshaw, MBChB, PhD, FRCGP2,7 Timothy Ramsay, MSc, PhD3 Steven Hilles DC, MBA5 for the Canada PRIme Plus Team6 1Population Health Program, University of Ottawa, Ottawa Hospital Research Institute, Université du Québec à Trois-Rivières, Quebec, Canada. 2Clinical Epidemiology Program, Ottawa Hospital Research Institute – Civic Campus, Ottawa, Ontario, Canada. Faculty of Medicine, University of Ottawa, Smyth Road, Ottawa, Ontario, Canada. 3Associate Scientist, Ottawa Health Research Institute, Ottawa Hospital, General Campus, Scientific Director, The Ottawa Methods Centre, Ontario, Canada. 4Director, Health Service Quality Management, American Specialty Health Network, Inc. California, United States. 5Canada PRIme Plus is a collaboration of international researchers (Canada, United Kingdom). OHRI Clinical Epidemiology Program Background: Common back pain has been associated with high x-ray utilization rate, yet current evidence suggests routine imaging of the spine is unnecessary. Knowledge of practice characteristics and variation in x-ray ordering behaviour can help set goals and develop quality improvement strategies to improve process of care. However, such background information is critically lacking in the chiropractic profession. Objectives: This study sets to determine the baseline rates and variations of spine x-rays among chiropractors (DCs) enlisted with ASH, a large network of providers in the US, and to establish professional and practice-related determinants of inclination to order spine x-rays for spine disorders. Methods: A cross-sectional analysis of administrative claims data from a sample of 10,052 chiropractors enlisted within ASH was carried out. Routinely collected data on claims paid for spine x-rays and patients characteristics aggregated at the provider level were linked with survey data containing demographic and enrolment information. We first performed a descriptive analysis on survey data chiropractors completed prior to registering with the ASH, and determined baseline rates and variations of spine x-rays across the US for contracted chiropractors for the year 2010. Binomial regression analyses were performed to identify significant predictors of x-ray utilization and to estimate the associated incidence risk ratio with 95% confidence interval (CI) for two spine x-ray utilization rates: 1) claims paid per patients, and 2) claims paid within 5 days of initial visit per new patient exams. Results: Complete data for 6,946 DCs and 249,193 patients aged 18 to 65 were available for analyses. Claims were paid for a total of 90,652 new patient exams and 23,369 spine x-rays, of which 17,511 were ordered within 5 days of initial patient exam. Rate of spine x-rays was 125 per 1000 patients. In contrast, rate of spine x-rays within 5 days of initial patient visit was 204 per 1000 new patient exams, with rates in the states of Georgia and Ohio 2 to 3 times higher than California. Significant predictors of higher x-ray ordering rates included having onsite imaging, provider gender, lower tier levels, practicing in an urban setting in the East North Central, South Atlantic, and Mountain areas, chiropractic school attended, and number of patients treated per hour. Conclusion: Although large regional variation persists, spines x-ray ordering practice among ASH chiropractors was lower than previously reported figures for non-Network providers. Having onsite imaging services was the most influential predictor for ordering spine x-rays.

2. Systematic review and meta-analysis of mesenchymal stromal cells in preclinical models of sepsis

Manoj Lalu, (Resident,) Department of Anesthesiology, The Ottawa Hospital ; Lauralyn Mcintyre (The Ottawa Hospital); Jupinder Bains (University of Ottawa); Francois Lamontagne (Université de Sherbrooke); Dean Fergusson (The Ottawa Hospital Research Institute); Shirley Mei (The Ottawa Hospital Research Institute); Duncan Stewart (The Ottawa Hospital Research Institute) OHRI Neuroscience Program Background: Mesenchymal stromal cells (MSCs) are powerful immunomodulatory cells. Pre-clinical evidence suggests that MSCs modulate the malignant inflammatory response and may enhance pathogen clearance in septic shock. Prior to designing a translational clinical trial we performed a systematic review and meta-analysis of all preclinical studies of MSC therapy for sepsis. Objective: To systematically review the effect of MSC therapy on death, organ dysfunction, inflammation, and pathogen clearance in preclinical septic shock models. Methods: A systematic search of MEDLINE, EMBASE (1948- July 2011), and a manual search of bibliographies was conducted to identify all preclinical studies of septic shock treated with MSCs as compared to a control group.. A priori efficacy outcomes measured at pre-specified time points post experimental sepsis induction included death, organ dysfunction, inflammatory cytokines, and pathogen clearance. Results were pooled using the ratio of means method with inverse variance random effects modeling and expressed as odds ratios (ORs) and 95 percent confidence intervals (95% CI). Results: Eight studies were included; three used a cecal ligation and puncture model and five used a severe endotoxemia model. Treatment with MSCs as compared to controls decreased death at all observation time points:< 2 d (OR 0.24, 95% CI 0.10-0.61), 2-4 d (OR 0.19, 95% CI 0.07-0.53), and > 4 d (OR 0.27, 95% CI 0.08-0.91). At > 24 h post-sepsis induction pulmonary neutrophil infiltration (OR 0.59, 95% CI 0.37-0.96, and creatinine concentration were decreased (OR 0.66, 95% CI 0.50-0.0.89), and there was a trend toward a reduction in aspartate aminotransferase (OR 0.81, 95% CI 0.64-1.02).; TNF-α was significantly decreased < 6 h post-sepsis induction (OR 0.68, 95% CI 0.47-0.97). IL-1β at 6-24 h (OR 0.51, 95% CI 0.42-0.62), and IFN-γ at < 6 h (OR 0.53, 95% CI 0.34-0.83) and 6-24 h (OR 0.44, 95% CI 0.31-0.60). Bacterial colony forming units were decreased in blood (OR 0.37, 95% CI 0.25-0.54), the spleen (OR 0.37, 95% CI 0.22-0.63), and the peritoneum (OR 0.69, 95% CI 0.63-0.75). Conclusions: MSC therapy in preclinical models of septic shock reduced early and late mortality, reduced markers of organ dysfunction, reduced the inflammatory response, and increased pathogen clearance.

3. Anticholinergic medication for bladder dysfunction in multiple sclerosis can adversely affect cognitive functioning

Roxana Cruce (Clinical and Research Fellow), Reza Vosoughi, Mark S. Freedman - The Ottawa Hospital Research Institute, The Ottawa Hospital, University of Ottawa Background: Cognitive impairment in Multiple Sclerosis (MS) occurs even at the earliest stages of the disease. Some studies have suggested that agents facilitating cholinergic input improve cognition in MS, similar to other dementias. However, there has been little attention paid to drugs with anticholinergic properties, such as those used to treat bladder symptoms. Anticholinergic drugs (ACD) may worsen cognition in dementia, but it is unknown what effect - if any - they may have on cognition in MS. Objective: To examine whether the use of ACD for the treatment of bladder symptoms has an impact on cognitive functioning in MS. Methods: Study patients were selected and consented from the MS clinic who were age 18-65, taking ACD steadily for ≥6 months and excluded if they were receiving additional agents with high/moderate - level anticholinergic properties or if they had major psychiatric co-morbidities. 42 patients on bladder ACD were compared to 46 patients not receiving ACD, in terms of their scores on a single Symbol Digit Modality Test (SDMT) and Selective Reminding Test (SRT). Also administered were tests of fatigue (Modified Fatigue Impact Scale - MFIS) and depression (Beck Depression Inventory - Fast Screen - BDI-FS) in order to examine for possible confounders of cognitive testing. Results: Patients using ACD showed significantly lower SDMT and SRT scores compared to those not using ACD (p < 0.001; t-test). The association of cognitive impairment with ACD usage was robust, even when other variables were considered. Conclusion: Given the widespread use of ACD for the treatment of bladder symptomatology in MS, these results importantly suggest that their chronic use can have a negative impact on cognitive functioning. Whether or not more selective ACD would have a similar effect requires further investigation.

Advances in Stem Cell Research (3:00 to 3:45M)

Moderator: Andrea Bravi

1. Adult brain and spinal cord progenitors respond differently to differentiation factors following proliferation in vitro.

M. COYLE ( PhD Candidate) 1, U. SHANMUGALINGAM 1, H. WESTWICK 1, X. CAO 2, E. C. TSAI 1; 1 Ottawa Hosp. Res. Inst., Ottawa, ON, Canada; 2 Univ. of Ottawa, Ottawa, ON, Canada Background/Objective: Neural stem/progenitor cells (NSPCs) have the potential to repair the brain and spinal cord after injury. We evaluated adult spinal cord (SC) and subventricular zone (SVZ) NSPC differentiation into neurons, oligodendrocytes and astrocytes with retinoic acid (RA), platelet derived growth factor (PDGF) and bone morphogenic protein-4 (BMP-4), respectively. Methods: Adult SC and SVZ NSPCs from Sprague Dawley rats (~150g) were cultured for a period of seven days with epidermal growth factor and basic fibroblast growth factor. Primary neurospheres were then removed from the mitogens, washed and plated in concentrations of RA, PDGF and BMP-4 ranging from 0-500 ng/ml. Seven days post factor exposure NSPCs were fixed and stained for O4, glial fibrillary acidic protein (GFAP), β-III-tubulin

(βIIIT), brain lipid binding protein (BLBP), Nestin, BrdU (pulsed 24hr before fixation) and TUNEL. Cells were counterstained with Hoechst and imaged with fluorescent microscopy. Percentage of positive cell staining out of the total number of cells plated with differentiating factor were obtained and compared to the control conditions (differentiating factor absent). Results: Our results showed that the number of βIIIT positive SVZ NSPCs increased with RA concentrations greater than 125ng/ml (p<0.001). In contrast, there was no significant increase in βIIIT positive SC NSPCs with RA concentrations as high as 500 ng/ml. However, with RA concentrations ≥250 ng/ml an increase in the proportion of BLBP positive cells was seen in both SVZ and SC cells compared to control (p<0.01). PDGF increased the number of O4 positive SVZ NSPCs when it was >250 ng/ml (p<0.01). SC NSPCs did not show a significant change in the number of O4 positive cells with PDGF concentrations as high as 500ng/ml. With PDGF concentrations ≥250 ng/ml, there was an increase in the number of BrdU positive SVZ NSPCs (p<0.001), but not with SC NSPCs. While an increase in the proportion of GFAP positive SVZ NSPCs was seen with BMP-4 concentrations ≥ 250 ng/ml (p<0.001), BMP-4 at concentrations ≥ 125 ng/ml reduced the number of GFAP positive SC NSPCs (p<0.001). Conclusion: Therefore we conclude that it is likely that factors inducing differentiation for SVZ NSPCs may not produce the same result with SC derived NSPCs. Further work identifying the appropriate differentiation cues will be required before NSPCs can be appropriately applied therapeutically.

2. Modeling Hutchinson-Gilford progeria syndrome using patient derived induced-pluripotent stem cells

Wing Y. Chang1 (Postdoctoral Fellow), Alton Etheridge2, Ji-Hoon Cho2, Kai Wang2, Anna Omelyanenko1, Vicki Ling1, Sarah Y. Kwon1, Leslie Chen2, Abner Huang2, Peter Pasceri3, Paul Bradshaw3, Manuel Alvarez1, Paul A. Cassar1, Kamal Garcha1, Hanna Song1, Mark Ungrin1, Akitsu Hotta3, Milica Radisic1, Stephen Meyn3, Peter W. Zandstra1,4, James Ellis3,5, David J. Galas2,6, and William L. Stanford1,2,4,5 1Institute of Biomaterials and Biomedical Engineering; University of Toronto, Toronto, Ontario, Canada; 2Institute for Systems Biology, Seattle, Washington, USA; 3Hospital for Sick Children, Toronto, Ontario, Canada; 4Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada; 5Ontario Human Induced Pluripotent Stem Cell Facility, Toronto, Ontario, Canada; 6Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg. OHRI Regenerative Medicine Program Hutchinson-Gilford Progeroid Syndrome (HGPS) patients exhibit traits of accelerated aging, where numerous cell-types and tissues deteriorate. Vascular smooth muscle cells (VSMCs) are a affected in HGPS, leading to vascular dysfunction and death. Lack of appropriate disease models impedes the search for new therapeutics to prevent or slow disease progression. Here, we describe the generation and analysis, including global expression of mRNA and miRNA and epigenetic markers, of HGPS-induced pluripotent stem cells (iPSCs) to model the disease. Reprogramming reset aberrant gene expression and factors perturbed in HGPS such as H3K4me3, H3K27me3, HP1γ, and Lamin B1, are normal in early passage (young) HGPS-iPSC derived VSMCs. The normal signature of H3K4me3 and H3K27me3 is reflected on the chromatin of HGPS-iPSCs as ChIP-seq analyses show a similar profile to hESCs. Lastly, prolonged culture of VSMCs exhibit Progerin accumulation, increased gamma-H2Ax foci, and nuclear blebbing. Taken together, we have developed a framework to monitor the progression of HGPS and draft the HGPS disease regulatory network.

3. Paradoxical increase in BMP kinome activity in blood-derived endothelial cells from Bmpr2 mutation carriers with hereditary pulmonary arterial hypertension Jessie R. Lavoie (PhD Candidate), Mark Ormiston c, Carol Perez-Iratxeta a, David W. Courtman ab, Nicholas W. Morrell c, Duncan J. Stewart ab aOttawa Hospital Research Institute, Sprott Stem Cell Centre and Regenerative Medicine Program b Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa c Department of Medicine, University of Cambridge School of Clinical Medicine, Hills Road, Cambridge, UK OHRI Regenerative Medicine Program Background: Severe pulmonary arterial hypertension (PAH) is a rare but lethal disease, which is characterized by profound vascular remodeling which is limited to the lung, characterized by excessive proliferation of pulmonary endothelial cells. Hereditary PAH (HPAH) is usually caused by heterozygous “loss of function” mutations in bone morphogenetic protein type II receptor (Bmpr2). However, the precise mechanism by which mutation of this gene causes PAH is unclear. Objective: To elucidate the effects of Bmpr2 mutations on the activity of proteins in the BMP signaling pathway in endothelial cells using an unbiased proteomic approach. Methods: Blood-derived, late outgrowth endothelial cells (EC) were expanded ex vivo from peripheral blood samples of four PAH patients with Bmpr2 mutations and four healthy control subjects. Proteins were isolated from quiescent cells with and without exposure to BMP-9 (1 ng/ml, 8 hours) and subjected to high-resolution 2D electrophoresis following by multiplex-staining for proteins (Sypro Ruby) and phosphoproteins (Pro-Q Diamond), and differential protein expression and phosphorylation was determined using PDQuest software. Results: Rigorous statistical analysis revealed a systematic increase in the baseline phospho-protein profile of cells derived from PAH patients carrying Bmpr2 mutations compared to cells from healthy controls. In all but one instance, the basal level phosphorylation in HPAH cells in the absence of BMP-9 stimulation was at, or above, the phosphorylation level induced by BMP-9 in the healthy control cells. Furthermore, the response to BMP-9 was lost entirely in the patient-derived cells. The statistical analysis of the total protein profile indicated that these changes were not due to changes in protein abundance in the patient cells, since there was an equal number of proteins that were up and down-regulated in HPAH vs. healthy cells. These proteins have been harvested and are being subjected to mass spectrometry analysis (LC-MS/MS) to allow protein identification. Conclusions: These data suggest that while Bmpr2 mutations generally resulted in the loss of responsiveness to the BMP ligand, they were associated with a profound basal increase in phosphorylation of signaling proteins, independent of ligand availability. This increase in BMP kinome activity could explain how a single copy of a mutant Bmpr2 gene can have such an important effect on EC biology.

POSTER ABSTRACTS

Cancer Therapeutics Program

1. Enhancing the Oncolytic Efficiency of Vaccinia Virus by Mutagenic Augmentation of EEV Production Aimée N. Laporte (MSc student, 1,2), Chris Storbeck (Post-doc,2), Laura Evgin (PhD student, 1,2), Fabrice Le Boeuf (Post-doc,2), John C. Bell (Senior scientist, 1,2) 1Department of Biochemistry, Microbiology and Immunology, University of Ottawa; 2Centre for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI), Ottawa, Ontario, K1H 8L6.

Background: Oncolytic viruses are currently under investigation as anti-cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti-tumour effect include inefficient viral spread and immune-mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic candidate vaccinia virus (VV). Naturally, a small proportion (<1%) of VV virions are released from infected cells with a cell-derived membrane and become known as extra-cellular enveloped virus (EEV). Due to this additional membrane, EEV is shielded from many anti-virus immune factors allowing it to travel further and largely avoid host-mediated neutralization. This form of VV is important for long range virus dissemination and sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release in influenced by Abl tyrosine kinase function. Objectives: Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. Further EEV enhancing modifications are under investigation with various VV strains. This augmentation of EEV production is expected to enhance the oncolytic potential of VV in vivo through enhanced spread and immune evasion. Methods: Engineer each specific mutation into three different VV strains, namely WR, Copenhagen (Cop) and Wyeth (Wy), in order to determine if one specific backbone and one mutation combination is superior in EEV production. Engineer and assess combinations of the mutations in one backbone in order to investigate further enhancement potential. Develop in vitro assays for the investigation of enhanced EEV production as a result of the specific mutations. Develop in vivo models for the investigation of enhanced viral spread and improved anti-cancer effects as a result of the specific mutations. Results: Cop A34R and Cop B5R demonstrate enhanced viral spread in vitro and in a lung met mouse model. Conclusions: Enhanced spread can be achieved in vivo by mutagenic modifications to the envelope proteins of vaccinia virus.

2. RhoB controls endothelial cell vessel formation in part via negative regulation of RhoA

Grant A. Howe (PhD Student) and Christina L. Addison Angiogenesis is required for tumor growth beyond 1mm3, and as such, studies on angiogenesis are crucial to understanding cancer progression. Recent studies have suggested a role for the small GTPase RhoB in the control of processes that are required for angiogenesis, but mechanisms remain poorly understood. Using human umbilical vein endothelial cells, we have shown that stimulation with vascular endothelial growth factor (VEGF), the major growth factor governing angiogenic processes, results in increased expression of RhoB protein. Additionally, we observed that RhoB is required for cell migration, vessel sprouting, and capillary morphogenesis in these cells. Depletion of RhoB in HUVEC using siRNA treatment resulted in increased RhoA activation in response to VEGF stimulation. Conversely, activity levels of RhoC are lowered in RhoB-depleted cells. Increased RhoA activation contributed to the cellular defects observed when RhoB was depleted, as inhibition of RhoA activity led to a partial restoration of capillary morphogenesis in the absence of RhoB. Increased RhoA signaling through Rho-dependent kinases ROCK I and II lead to reduced capillary morphogenesis as treatment with two inhibitors of ROCK restored some vessel formation in RhoB-depleted cells. Thus our data suggest that RhoB regulates the activity of RhoA and exerts control over the RhoA/ROCK pathway whose activation levels must be appropriately regulated for angiogenesis to occur. Additionally, RhoB appears to affect the activity levels of RhoC, and while the importance of this interaction remains to be determined, these results indicate an important role for RhoB in angiogenesis centered on the regulation of activation of other Rho proteins.

3. Old drugs, new use: Microtubule-targeting viral sensitizers for oncolytic virotherapy Diallo JS (Postdoctoral Fellow), Arulanandam R (Postdoctoral Fellow), Batenchuk C (Ph.D candidate), Julie Cox (Masters student), Vanessa Garcia (Co-op student), Anisha Sinha (Co-op student), Theresa Falls, Markus Vaha-Koskela (Postdoctoral Fellow), Le Boeuf F (Research Associate), Bell JC (Senior Scientist) Oncolytic viruses (OVs) are promising anti-cancer agents but like other cancer monotherapies, the genetic heterogeneity of human malignancies can lead to treatment resistance. We previously used a virus/cell based assay to screen diverse chemical libraries that identified several small molecules that could act in synergy with OVs to destroy tumour cells that resist viral infection. While some of these are completely novel molecules a large proportion are known microtubule-targeting drugs that have been previously evaluated for various disease indications including autoimmune disorders and cancer. We found that these drugs can potently enhance the activity of a prototypical oncolytic rhabdovirus named VSV (vesicular stomatitis virus) in vitro, and lead to improved therapeutic efficacy in vivo. Importantly, the tumor-specificitiy of oncolytic VSV is retained using these viral sensitizer drugs. Evidence suggests that the microtubule-targeting viral

sensitizers work through a previously unknown effect of these compounds, which is to decrease virus-mediated type I interferon (IFN) secretion. Decreased IFN secretion induced by microtubule-targeting viral sensitizers coincided with decreased Type-I IFN-stimulated gene expression as assessed by gene expression microarrays and gene set enrichment analyses. Altogether, these effects led to a robust increase in viral spread through resistant tumor cells but were insufficient to sensitize normal cells to VSV. We believe that this supports further testing of oncolytic rhabdoviruses in combination with microtubule-targeting agents in future clinical trials. Furthermore, we believe this highlights the power of our “pharmaco-viral” screening approach for identifying enhancers of viral oncolysis, which in addition to improving OV therapy, have led us to uncover new aspects of virus/host biology and drug mechanisms of action.

4. Mechanism of oncolytic virus targeting of tumor-associated vasculature Rozanne Arulanandam (Postdoctoral Fellow), Caroline Brietbach, Carolina Ilkow, Cory Batenchuk, Naomi De Silva, Christina Addison, John Bell OHRI, University of Ottawa, Ottawa, Ontario While efforts to selectively disrupt established tumor vasculature through VEGF/VEGFR inhibitors have resulted in transient clinical benefit, the upregulation of alternate pro-angiogenic pathways and the development of ‘evasive resistance’ through increased tumor invasiveness and spread have shown to hinder chances of long term survival in patients. New therapies which target tumor blood supply are needed. It has been demonstrated that a number of different virotherapeutic platforms, designed to selectively infect and lyse cancer cells may also impact the tumor vasculature. Indeed, intravenous administration of two different OVs, Vesicular Stomatitis Virus and vaccinia virus, can lead to decreased tumor perfusion leading to acute vascular shutdown in murine tumor models. In patients with advanced, solid tumors, oncolytic vaccinia can infect tumor-associated endothelial cells, thus having a direct antiangiogenic effect, while while normal vessels remain unharmed. Recent studies have attributed a role for VEGF in stimulating the delivery and efficacy of OVs through an increase in vascular permeability and immune-mediated destruction of the tumor endothelium. We believe that the selective targeting of tumor vasculature by OVs may be due to a VEGF-mediated suppression of interferon (IFN) and other IFN-regulated cytokines, characteristic of the innate antiviral response. To examine this possibility, human umbilical vein (HUVEC) and dermal (HMVEC) endothelial cells were subject to treatment with VEGF165 and other pro-angiogenic growth factors, followed by OV infection. Viral titers, as well as IFN production through qRT-PCR or ELISA, were compared over time in the presence or absence of VEGF. Our results reveal that OV replication and transgene expression in actively proliferating, human endothelial cells could be enhanced ~10 fold by both VEGF165 and FGF-2, but not FGF-1. Confluent monolayers of HUVEC were resistant to OV infection demonstrating a selectivity towards dividing endothelial cells, characteristic of the angiogenic phenotype. Given that the tumor endothelium is known to be rich in growth factors such as VEGF165 and FGF-2, these findings elucidate a mechanism explaining the preferential infection of tumor vasculature by OVs. The fact that the stimulation of at least two different angiogenic pathways may sensitize HUVECs to OV infection further validates their therapeutic potential in the context of VEGFR-sensitive and resistant tumors. Furthermore, while the role of IFN in suppressing angiogenesis is fairly well understood, our results identify VEGF165 as a novel inhibitor of IFN production, pointing towards a potential bi-modality between the two signalling molecules.

5. E-selectin produced by endotoxic endothelial injured cells augments ECFCs homing and is further increased by SDF-1

binding to CXCR4 Jie Sun, (Postdoctoral Fellow) Yuhua Li, David Allan Background: Endothelial colony-forming cells (ECFCs) are highly proliferative endothelial precursors that can be mobilized into peripheral blood and facilitate vascular repair. The mechanisms that govern the circulating ECFCs’ homing to injured endothelial area are lacking. In this study, we developed an in vitro model of endotoxic endothelial injury to explore the factors that released from injured endothelial cells which might attract circulating ECFCs and find e-selectin plays a critical role in ECFCs’ homing. We also address a synergistic effect on this homing process mediated by SDF-1α which was known can mobilize ECFCs from bone marrow. Methods: ECFCs were expanded from mononuclear cells isolated from human umbilical cord blood samples and expanded in EGM2 (Lonza). Endotoxic injury was induced by treating human umbilical vein endothelial cells (HUVECs) with 0.1ug/ml LPS. HUVECs were also exposed to hypoxic injury (1% O2) and radiation injury (8Gy). Adhesion assay and migration assay were used to evaluate homing ability of ECFCs. Expression levels of particular genes were quantified by PCR. Western blot and flow cytometry were used to evaluate protein expression of e-selectin ligands (CD44 and CD162) on ECFCs. Results: We observed exogenous SDF-1α can enhance ECFCs’ adhesion to fibronectin and this enhance can be attenuated by its antagonist: plerixafor. However SDF-1α mRNA expression was not changed in HUVECs but up-regulated in MRC5 (human pulmonary fibroblasts) following endotoxic injury. e-selectin mRNA and protein level was dramatically increased in injured HUVEC cells with a peak level on 3h after injury. Both endogenous and exogenous e-selectin increased adhesion and migration of ECFCs to fibronectin, this increase can be blocked by anti-CD44 antibody. After pre-treated with SDF-1a for 2h, ECFCs have more homing ability following e-selectin induction, which can be attenuated by plerixafor. This might be caused by an increasing of CD44 and CD162 expression on ECFCs after SDF-1α treatment while a decrease after blocking with plerixafor. Conclusion: Our study provides a new sight on the microenvironment between circulating ECFCs and injured endothelial cells, where SDF-1α produced by adjacent injured fibroblasts mobilizes ECFCs from bone marrow to peripheral blood and up-regulates e-selectin ligands on ECFC cell surface. Then e-selectin produced by endotoxic injured endothelial cells allows ECFCs to adhere and migrate to injured endothelial area through its ligands on ECFCs. SDF-1α and e-selectin may play synthetically to complete a self-renew process mediated by endothelial progenitor cells. Our findings encourage new drug applications based on endothelial cell released factors for patients with severe endothelial injury which often leads death and currently has no effective treatment.

6. Virally-encoded carboxylesterase potentiates the combination of oncolytic Vaccinia virus and irinotecan Michelle Becker (MSc Student), Chris Storbeck, Madison Foster, Judith Paget, Harold Atkins and John Bell Background Oncolytic viruses are capable of infecting and replicating in cancerous cells, resulting in their destruction, yet leaving healthy cells relatively unaffected. Oncolytic Vaccinia virus (VV) selectively infects, replicates and expresses transgenes in tumour tissue following intravenous delivery in patients. Although anti-tumour activity was shown, there is still room for improvement. This work intends to combines VV with irinotecan (CPT-11) for the treatment of colon cancer. CPT-11 is a colon cancer chemotherapeutic that is infused intravenously and converted to the more active chemotherapeutic SN-38 by carboxylesterase (CE) enzymes, high levels of which are found in the liver and intestine. SN-38 is a topoisomerase I inhibitor that eventually induces DNA double strand breaks, leading to growth arrest and apoptosis. However, intestinal CE produces SN-38 in the colon, leading to gastrointestinal damage and resulting in diarrhea, a dose-limiting toxicity. In addition to simply combining the oncolytic virus with the chemotherapeutic, we have engineered VV recombinants to express a CE enzyme, which should increase the conversion of CPT-11 to SN-38. Moreover, the CE used is derived from human liver CE, but contains mutations that render it 100 – 1000 times more effective. Since the CE-expressing viruses are tumour tropic, the enhanced conversion of CPT-11 should only be occurring in the tumour. Objective Our objectives are to examine the combination of parental VV and CPT-11, confirm that the recombinants are functional and determine if they increase efficacy when combined with CPT-11. We hypothesize that virally-encoded CE will potentiate the combination of VV and CPT-11. Methods / Results / Conclusions Here we show, in vitro, that CPT-11 does not hinder parental virus replication in four human colon cancer cell lines. SN-38, however, at very high concentrations does. Nevertheless, SN-38’s cytotoxic effects may outweigh its dampening of viral replication and depending on how much SN-38 is made in vivo, this may not even be a problem. Also, we show that the recombinant viruses express functional protein that greatly enhances human colon cancer cell death in the presence of CPT-11, compared to their non-CE expressing counterpart, and that this is mediated through increased DNA double strand breaks. There are two major potential implications; the first is that virally-encoded enzyme may improve the safety of CPT-11 as lower doses can be administered yet its activity should remain high in the tumour only. And secondly, virally-encoded CE may improve the oncolytic virus’ efficacy as it may kill residual tumour cells.

7. Calpain-mediated processing of p53-associated, parkin-like cytoplasmic protein (PARC) promotes p53 subcellular trafficking and chemosensitivity in human ovarian cancer cells 1,2,3Michael G. Woo (PhD Candidate), 1,3,4Kai Xue, 4Jiayin Liu, 2Heidi McBride and 1,2,3,5Benjamin K. Tsang 1Reproductive Biology Unit and Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Ottawa 2Department of Cellular and Molecular Medicine, University of Ottawa 3Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1Y 4E9 4Clinical Reproductive Medicine Centre, Nanjing Medical University, Nanjing, Peoples’ Republic of China 5World Class University (WCU) Biomodulation Major, Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea Background: Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Objective: To present evidence that CDDP promotes calpain-mediated Parc down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Methods: Chemosensitive and resistant OVCA cells were treated with RNA interference for PARC and p53, dominant-negative Akt (DN) or API-2, inhibitors of calpain, caspases and the ubiquitin proteasome pathway, ionomycin, and CDDP. Subcellular p53 was determined by confocal microscopy; assessment of apoptosis by Hoechst 33258 nuclear staining and PARP cleavage; Calpain mediated PARC processing was examined using in vitro cleavage assays and pharmacological inhibition with Calpeptin. Results: Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. Conclusions: The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

8. Investigating the role of tumour suppressor lethal giant larvae in glioblastoma Alexander Gont (MSc Student), Dr. Ian Lorimer (Supervisor) Background: Glioblastoma is an aggressive, invasive and deadly type of neurological cancer. In glioblastoma there is increased activation of protein kinase C iota (PKCι) through enhanced PI3K signalling predominantly as a result of PTEN loss. Specific knockdown of PKCι leads to increased induction of cellular senescence, increased sensitivity to chemotherapeutics, and decreased cell motility. Lethal Giant Larvae (LGL), one of the earliest tumour suppressors discovered, is predominantly phosphorylated and inactivated by PKCι in glioblastoma multiforme cells. In mammalian cells LGL has effects on cellular proliferation and motility. Its expression is decreased or lost in multiple human cancers and its knockout leads to mouse neuronal tissue overgrowth. LGL interacts with non-muscle myosin IIa, a major mediator of cell motility. Objective: The objective is to elucidate the functions of LGL in glioblastoma multiforme related to its phosphorylation by PKCι Methods: Stably selected inducible lentiviral vectors were used for the expression of Flag-tagged LGL proteins. Fluorescent imaging was done by confocal microscopy. Viability assays were done using trypan blue exclusion and induction of cellular senescence was measured by an increase in senescence-associated beta galactosidase staining. Results and Conclusions: LGL protein levels are detectable as early as six hours after induction without any expression in the absence of doxycycline. A mutant form of LGL, LGL-3SA, which is non-phosphorylatable by PKCι was used to determine the effects of PKCι phosphorylation on the functions of LGL. LGL-3SA localized predominantly to the cell membrane while wild-type LGL remained mainly cytosolic. This membranous localization is a marker of active LGL. LGL-3SA co-localized with non-muscle myosin IIa more abundantly than with the wild-type. This co-localization was principally around the perimeter of the cell rather than having a distinct front suggesting improper leading edge formation. Expression of the LGL-3SA led to a reduction in cell proliferation and an increase in senescent cells while expression of wild-type LGL gave no discernible change in phenotype. In glioblastoma LGL is an important substrate for PKCι. Inactivation of LGL by PKCι appears to be a critical step in gliomagenesis.

9. Ontario Tumour Bank initiative at The Ottawa Hospital H. Sekhon (MD, Dept. of Pathology, TOH), J. Werier (MD, Dept. of Surgery, TOH), C.A. Jodouin (Coordinator of Research Operations, TOH), E. Pitre (Ontario Tumour Bank Research Technician, OHRI), M. Sienkiewicz (Ontario Tumour Bank Research Technician, OHRI), J. Bartlett (Transformative Pathology Director, OICR), M. Albert (Ontario Tumour Bank Manager, OICR) S.Kodeeswaran (Ontario Tumour Bank Director, OICR) The Ontario Tumour Bank is a province-wide biorepository and data bank focused on collection of tumour-related human biospecimens. It provides academic and industry cancer researchers with a diverse selection of high quality tumour-related specimens and data obtained directly by dedicated tumour bank staff, who follow a stringent set of procedures and ethical guidelines. The biospecimens and clinical data are an important resource for scientists engaged in translational research who are developing better diagnostic tools and new drug therapies. Researchers depend on the Ontario Tumour Bank to provide research biospecimens of high quality, diversity, and integrity. Operating at state-of-the-art hospitals and cancer centres across Ontario, including The Ottawa Hospital since 2005. The Ontario Tumour Bank coordinates the collection, storage, analysis, annotation, and distribution of tumour and peripheral blood samples. Working in collaboration with local pathologists, medical oncologists, surgeons and other hospital personnel, specially trained staff obtain patient consent, collect tissues and assemble comprehensive clinical information about each donor and the corresponding samples. The Ontario Tumour Bank is a program of the Ontario Institute for Cancer Research (OICR). Funded by the Government of Ontario, OICR is a not-for-profit corporation that supports research on the prevention, early detection, diagnosis, treatment and control of cancer.

10. A multicentre study assessing 12-weekly intravenous bisphosphonate therapy in women with low risk bone metastases from breast cancer using bone resorption markers J.F.Hilton( Clinical Fellow),I.Kuchuk, N. Bouganim, L. Vandermeer, S. Hopkins, D. Robbins, E. Amir, S. Dent, C. Milano, O. C. Freedman, R. A. Dent, G. Dranitsaris, M. J. Clemons; McGill University and Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada; NCIC Clinical Trials Group, Kingston, ON, Canada; Ottawa Hospital Cancer Centre, Ottawa, ON, Canada; The Ottawa Hospital Cancer Centre, Ottawa, ON, Canada; Division of Medical Oncology and Hematology, Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada; Durham Regional Cancer Centre, Oshawa, ON, Canada; Sunnybrook Health Sciences Centre, Toronto, ON, Canada; Health Economics and Biostatistics Consultant, Toronto, ON, Canada Abstract: Background: Metastatic bone disease is a major cause of morbidity and mortality for breast cancer patients (pts). Bisphosphonates (BP) have been shown to significantly delay the onset and frequency of skeletal related events (SREs), improve pain control and overall quality of life. Most pts receive intravenous BP every 3-4 weeks regardless of their individual risk for a SRE. This “one size fits all” strategy exposes those pts at a relatively low risk of SREs to the financial and quality of life burden of multiple visits to the cancer centre for treatments as well as an increased risk of drug toxicity. This study aimed to assess whether IV BP can be safely given at reduced frequency. Methods: The primary objective of this study is to demonstrate in women with biochemically defined low-risk bone metastases that the administration of IV BP every 12 weeks is sufficient to maintain biochemical stability for one year. Eligibility criteria include; bone metastases from breast cancer, have received at least three months of regular 3-4 weekly IV BP, satisfactory renal function, adequate dental health, no systemic treatment change or SRE within 4 weeks of study entry. Low risk disease will be defined as serum CTx levels <600 ng/L Biochemical failure defined as CTx levels >600 ng/L measured at predefined time points. Secondary objectives are to evaluate the palliative benefit of 12-weekly IV BP therapy as reflected by occurrence of SREs, analgesic use, self-reported pain using the validated BPI and FACT-BP questionnaires. Sample size was calculated at 68 pts. Given the small sample size, nonparametric Bootstrapping will be

employed to calculate point estimates, standard deviations and 95% confidence intervals. An exploratory multivariable analysis will also be undertaken to determine baseline factors that were associated with pt’s maintaining their telopeptide levels in the low risk range. Conclusion: TRIUMPH was planned as multicenter study, but due to circumstances beyond our control the other two centres did not open – so we actually performed a multi-centre trial at a single centre with high accrual rate-full accrual (n=68) within 1 year. This trial has the potential to allow lower risk women to receive less frequent dosing of BP, thus improving their quality of life with less cancer center visits and reducing their chance of drug induced adverse events.

11. Identifying Patients at High Risk for Acute and Delayed Chemotherapy Induced Nausea and Vomiting (CINV): The Validation of a Prediction Tool (Emesis 1) N. Bouganim( Clinical Fellow),I. Kuchuk, G. Dranitsaris, L. Vandermeer, S. Hopkins, S. Dent, P. Wheatley-Price, S. Verreault, C. Young, M. J. Clemons; McGill University and Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada; Health Economics and Biostatistics Consultant, Toronto, ON, Canada; Ottawa Hospital Cancer Centre, Ottawa, ON, Canada; The Ottawa Hospital Cancer Centre, Ottawa, ON, Canada; Division of Medical Oncology, The Ottawa Hospital Cancer Centre, Department of Medicine, University of Ottawa, Ottawa, ON, Canada Abstract: Background: Despite the use of standardized anti-emetic guidelines, up to 20% of cancer patients (pts) suffer moderate to severe CINV (≥ grade 2). Prediction of pts at high risk for CINV, through the use of validated mathematical models, is important in developing management strategies. We previously developed cycle-based prediction models for acute & delayed CINV. As part of the validation process, we prospectively evaluated the ability of the scoring systems to accurately identify pts deemed to be high risk for ≥ grade 2 CINV. Methods: Pts receiving chemotherapy were provided with symptom diaries. Compliance to these was enhanced via patient phone calls 24 hours and 5 days post-chemotherapy (CT) after every cycle. All pts received standard anti-emetic prophylaxis. Prior to each cycle of CT, acute and delayed CINV scoring systems were applied to stratify pts into low and high risk groups. The external validity of each system was assessed via a receiver operating characteristic curve (AUROC) analysis. Logistic regression modeling was applied to compare the risk of ≥ grade 2 CINV between pts considered to be high vs. low risk. Results: CINV outcomes data were collected from 95 pts following 181 cycles of CT. Incidence of ≥ grade 2 acute & delayed CINV was 17.7% and 18.2% respectively. Major predictors for ≥ grade 2 CINV included; younger patient age, platinum or anthracyline-based chemotherapy, low alcohol consumption, earlier cycles of CT, previous history of morning sickness and prior emetic episodes with CT. Both the acute & delayed scoring systems had good predictive accuracy when applied to the external validation sample (AUROC = 0.69, 95%CI: 0.59-0.79 and AUROC = 0.70, 95%CI: 0.60-0.80). Pts identified to be at high risk by the scoring systems were 2.8 (p = 0.025) and 3.1 (p = 0.001) times more likely to developed ≥ grade 2 CINV. Conclusions: This study demonstrates that our scoring systems are able to accurately identify pts at high risk for acute and delayed CINV. Their application and planned continued refinement will be an important source of patient-specific risk information that will allow the optimization of anti-emetic therapy.

12. Insect cell carriers for systemic delivery of oncolytic viruses Dominic Roy(PhD Student)1,2, Anthony Power1,2, Fabrice LeBoeuf1,2, Theresa Falls1, John Bell1,2. 1Cancer Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 2Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada. Background: Replication-competent oncolytic viruses are tremendously potent therapeutic agents when applied to tumour cells in vitro or by direct injection in vivo; however their capacity for systemic delivery is severely limited due to a host immune system that is well-trained to eliminate foreign pathogens from the circulation. In recent pre-clinical investigations, a novel approach using cells as carriers for oncolytic viruses has shown promise in overcoming such obstacles. A number of candidate cell types have been proposed ranging from immune cells to stem cells to transformed cell lines. However the clinical utility of carrier cell types investigated to date is limited by one or more of the following features: cumbersome isolation and/or manufacturing protocols, limited viral productivity (in the case of non-tumour cells), off-target tissue homing tropisms, and safety concerns (in the case of tumourigenic cell lines). Objective: In efforts to improve the clinical feasibility of the carrier cell concept, we have investigated the potential of insect cells for systemic delivery of oncolytic viruses. We have identified insect cell lines that are efficiently infected with both rhabdo- and poxviral oncolytic agents. My objective is to investigate the ability of insect cell carriers to deliver oncolytic viruses to tumour cells both in vitro and in vivo. Results/Conclusion: When systemically administered to tumour-bearing mice, virus-loaded insect cells were able to deliver both classes of oncolytic agent to distally-localized tumour beds in immune-competent animals. Importantly, systemically administered insect cell carriers were well tolerated and circulated extensively with little off-target tissue homing. Uniquely, the insect cell culture system offers the capacity for large-scale biotherapeutic production without the tumourigenicity that limits the safety of continuous mammalian cell lines. Therefore the insect cell system described here may help to fuel the launch of cell-borne oncolytic viruses into clinical trials.

13. Preventing perioperative cancer metastases formation by enhancing Natural Killer cell activity Lee-Hwa Tai1 (PhD, Postdoctoral Fellow), Rashmi Seth1,2(MD, MSc), Christiano Tanese Souza1(Research Technician), Rebecca Auer1,2(MD, Associate Scientist) 1 Centre for Innovate Cancer Research, Ottawa Hospital Research Institutre, Ottawa, Ontario, Canada 2 Department of Surgery, Division of General Surgery, University of Ottawa, Ottawa, Ontario, Canada Background: Surgical resection is the mainstay of therapy for patients with localized solid malignancies. Even with complete resection, many patients develop metastatic recurrence and ultimately die of their disease. The perioperative period is a uniquely susceptible time for the formation of metastases. A number of changes that occur following surgery

promote the formation of metastases, including dissemination of tumour cells, a pro-mitotic, pro-angiogenic cytokine milieu and suppression of the immune system. Natural Killer (NK) cells are a key component of the innate immune response against cancer, in particular the elimination of micrometastases. NK cell suppression and dysfunction have been linked to the development of metastatic disease in animal models and in cancer patients. Objective: We hypothesized that surgical stress enhances the development of postoperative metastases by impairing NK clearance of tumor cells. Specifically, we like to 1) define the mechanisms involved in NK cell suppression following surgery and 2) explore novel anti-cancer therapies to modulate postoperative NK cell suppression and inhibit metastases. Methods: A mouse model of surgical stress and tumor metastases was used to address these questions. In this model, NK cell functions were assessed using immunological functional assays following tumor challenge, surgical stress and novel perioperative anti-cancer therapy. Results and Conclusions: We observed enhanced lung metastases in tumor-bearing, surgically stressed mice depleted of NK cells compared to intact mice suggesting that NK cells play a crucial role in mediating tumor clearance. Accordingly, in vivo CFSE labeled splenocyte rejection and ex vivo chromium labeled target cell killing by NK cells were significantly reduced in surgically stressed mice compared to untouched mice. A significant reduction in spleen NK cell numbers and expression of NKG2D and KLRG1 NK cell surface receptors were also observed in surgically stressed mice. These data suggest that surgical stress impairs global NK cell function. For the therapeutic arm of my project, we have demonstrated that using the perioperative administration of the non-specific NK cell activator poly I:C can reverse NK cell suppression following surgery and that this correlates with a reduction in the postoperative formation of metastases. We have also evaluated the oncolytic vaccinia virus, tkVV(GMCSF), and observed a similar attenuation of metastases and recovery of NK cell cytotoxicity following perioperative administration. Cancer therapies aimed at targeting the mechanisms responsible for the prometastatic effects of surgery will reduce recurrences and improve survival in surgical cancer patients when used in the perioperative period.

14. The role of LMO4 in ErbB2-driven mammary tumorigenesis Kyla Garbuio (PhD Student)1,2; Chris Storbeck 1; and Luc Sabourin 1 1 Cancer Therapeutics Program, Ottawa Hospital Research Institute 2 Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa BACKGROUND: This project investigates factors mediating the metastatic ability of breast cancer cells and specifically, the Ste20-like kinase (SLK), the role of its interaction with the LIM-only domain 4 (LMO4) protein and the role of this complex in regulating cell migration and invasion. ErbB2/HER2/Neu, a member of the epidermal growth factor (EGF) receptor tyrosine kinase (RTK) family, over-expressed in 25 – 30% of breast cancers, confers an invasive phenotype to ErbB2 positive tumors. Our lab has shown that SLK is activated downstream of ErbB2/HER2/Neu and SLK activity is required for cell adhesion turnover and migration. The LIM binding domain proteins Ldb1 and Ldb2 regulate SLK catalytic activity. Both over-expression and knock-down of Ldb1/2 causes an increase in SLK activity suggesting that SLK regulation involves complex stoeichiometry. Knock-down of Ldb1/2 leads to increased cell motility. Ldb1/2 are known binding partners of LMO4, which is over-expressed in 56% of breast tumors and 62% of ErbB2-positive cases. In mice, overexpression of LMO4-induced mammary cell proliferation and tumour cell invasion. HYPOTHESIS: Up-regulation of SLK activity through the interaction with LMO4 contributes to ErbB2-mediated breast cancer cell invasiveness and metastasis. METHODS: Conditional LMO4 (flox/flox) mice have been crossed with mice overexpressing (MMTV-driven) a bicistronic Cre recombinase + activated ErbB2 (NIC) for immunohistological, survival and biochemical analysis of the effect of loss of expression of LMO4 on ErbB2 tumor development and progression. The role of LMO4 in SLK-dependent cell migration and adhesion turnover will be investigated using LMO4 knock-down approaches and over-expression studies in fibroblast, mammary epithelial and ErbB2-postive cell lines. The interaction of LMO4 with SLK and its activity following scratch wounding and ErbB2 stimulation will be assessed. Finally, an investigation into the role of the LMO4/SLK complex in migration and metastasis will include the evaluation of the invasive and metastatic potential of tumors and epithelium isolated from NIC x LMO4 (-/-) mice using grafting experiments. RESULTS: Preliminary data show that LMO4 expression is increased in ErbB2-derived murine mammary tumors. LMO4 directly binds to Ldb1, Ldb2, and SLK. Over-expression as well as knock-down of LMO4 resulted in an increase in SLK activity. Knock-down of LMO4 expression inhibited cell migration. LMO4 is present in the cytosol in a pattern similar to SLK, localizing to the membrane ruffle. CONCLUSION: Thus far, we believe that our results suggest that up-regulation of SLK activity through direct interaction with LMO4 contributes to breast cancer cell invasiveness and metastasis.

15. Characterization of putative stem cells in fallopian tubes as an origin of ovarian cancer Kholoud Alwosaibai (PhD Student) Background: Fallopian tubes are the part of the reproductive tract that is closest to the ovary. It has been shown in ovarian cancer patients that the tubal fimbriae, the most distal part of fallopian tubes adjacent to the ovary, contain carcinomas. Preneoplastic epithelial lesions have been reported in fallopian tubes based on loss of Pax2 and induction of p53 expression. These observations raise the possibility that the fallopian tubes may be the site of origin for some ovarian cancers. Objective: We hypothesize that TGF-beta in the ovary, responding to peri-ovulatory inflammation, may flow with the follicular fluid into the fallopian tube at ovulation. In the presence of TGF-beta, oviductal epithelial cells (OVE) will become more mesenchymal, undergoing an epithelial to mesenchymal transition (EMT). EMT has been shown in mammary epithelium to increase stem cell features, and we therefore aim to show that induction of EMT in OVE cells increases characteristics associated with stem cells and susceptibility to cancer. Methods: Oviducts were isolated from female mice and OVE cells were extracted and clonal cell lines were developed. OVE cells were treated with 10 ng/ml TGF-beta for 6 days and collected for counting. Cells were also collected after 3 days for RNA extraction, reverse transcription and qPCR, and for western blot analysis. Prepubertal (25 days old) mice were superovulated with PMSG and hCG and oviducts were isolated at 8 and 14 hours after hCG, which is 4 hours before and 2 hours after ovulation. Isolated oviducts were fixed, embedded and sectioned for immunohistochemistry. Results: OVE cells treated with TGF-beta

acquire EMT characteristics. Morphologically, TGF-beta-treated cells develop a spindle shape and their rate of proliferation is reduced. mRNA encoding vimentin and snail are up-regulated, whereas E-cadherin mRNA is down-regulated. Initial investigation of stem cell marker expression by qPCR has shown that TGF-beta-treated OVE cells up-regulate CD44, Sca-1 and CD133. TGF-beta also suppresses expression of the marker of oviductal epithelium, Pax2. Conclusions: TGF-beta inhibits the proliferation of oviductal epithelial cells, promotes an epithelial-mesenchymal transition, and enhances the expression of mRNA encoding for stem cell markers. These results, in combination with the data showing that TGF-beta suppresses Pax2 expression, reminiscent of preneoplastic epithelial lesions, suggest that induction of EMT in oviductal epithelium promotes cellular responses associated with susceptibility to transformation.

16. Reproductive steroid hormones alter ovarian cancer progression in mouse models Kendra Hodgkinson (PhD Student)1,4, Laura Laviolette1,4, Carolina Perez-Iratxeta3, Barbara C. Vanderhyden1,2,4 Departments of Cellular and Molecular Medicine1 and Obstetrics and Gynecology2, University of Ottawa; Ontario Genomics Innovation Centre3; Centre for Cancer Therapeutics, Ottawa Hospital Research Institute4 Background Epithelial ovarian cancer is often asymptomatic during its initial stages and due to a lack of effective early screening methods, the disease is frequently diagnosed at advanced stages. Identification of causative factors is valuable for the development of preventative measures and early treatment options. Epidemiological studies have demonstrated that women who take hormone replacement drugs after menopause have an increased risk of developing ovarian cancer. Objective This study investigates the actions of estrogen and progesterone on ovarian cancer in two mouse models Methods This study uses two mouse models of ovarian cancer to evaluate the impact of steroid hormones on ovarian cancer progression, particularly after menopause, when the majority of ovarian cancer is diagnosed. A xenograft model will examine the effects of 17-β estradiol (E2) treatment on ovarian cancer progression. To model the development of ovarian cancer in a menopausal ovary, menopause will be induced in the tgLS-CAG-TAg transgenic mouse model of ovarian cancer by vinylcyclohexene diepoxide (VCD) treatment prior to oncogene activation. Results E2 treatment in vivo decreased the length of survival in ovariectomized SCID mice xenografted with murine ovarian cancer cells. Treatment with E2 in vitro did not affect proliferation of the cancer cells or mouse ovarian surface epithelial cells. Microarray analysis of the tumours has revealed significant upregulation of 196 genes and downregulation of 55 genes in animals treated with exogenous E2, including genes involved in cell differentiation, proliferation and migration. Preliminary results suggest that the menopausal mice tend to have prolonged survival and altered tumour histology relative to non-menopausal mice. Conclusions Current experiments are exploring the impact of E2 and progesterone treatment on disease progression in this model. Elucidation of the molecular pathways altered by E2 will improve our knowledge of the etiology of ovarian cancer and may lead to advances in the prevention, early detection or treatment of the disease.

17. Mir-105 influences proliferation and anchorage-independent growth in prostate cancer cells D.R. Honeywell (MSc Student) and C.L. Addison Program for Cancer Therapeutics, OHRI and Departments of Biochemistry, Microbiology and Immunology and 1Medicine, University of Ottawa Background: MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression. Many have demonstrated roles in controlling aspects of cancer such as cell proliferation, epithelial to mesenchymal transition, invasion and metastasis. As the tools for miRNA analysis become more readily available, the involvement of many previously unassociated miRNAs in cancers are being identified. Objectives: 1) To determine what miRNAs are differentially expressed between normal and cancerous prostate cells. 2) To evaluate what role these miRNAs play in cancer phenotypes. 3) To discover what pathways the miRNAs alter to modulate cancer phenotypes. Methods: Microarray screening of miRNAs expressed in prostate cancer cell lines compared to normal prostate epithelial cells revealed a number of differentially expressed miRNAs which were validated using TaqMan quantitative RT-PCR. One of these, miRNA 105 (miR-105) was chosen for further study to evaluate its role in the modulation of the tumorigenic phenotype in prostate cancer. miR-105 was over-expressed in tumor cells following transfection of control (cel-miR-67) or miR-105 synthetic miRNA duplexes (Dharmacon), and its effects were examined on cell proliferation, anchorage independent growth in soft agarose and invasion through Matrigel coated transwells. Microarray screening of miR-105 over-expressing compared to control miR transfected PC3 cells was performed to identify putative targets regulated by miR-105, which were then validated by qRT-PCR and western blot analysis. Results: Microarray screens showed that miR-105 was down-regulated in prostate tumor cells compared to normal prostate epithelium, thus we hypothesized that it may be an important negative regulator in prostate cancer growth and metastasis. In support of our hypothesis, we found that that over-expression of miR-105 led to suppression of proliferation and anchorage-independent growth in PC3 and DU145 prostate tumor cells. Over-expression of miR-105 in tumor cells also appears to inhibit cell invasion. Results of differential gene expression studies suggested that miR-105 can target and inhibit Cdk6. We confirmed that over-expression of miR-105 resulted in reduced Cdk6 protein and message levels in tumor cells, thus supporting its role in inhibiting tumor cell proliferation. Based on our initial results we are now investigating additional gene targets of miR-105 and their contribution to its role in inhibiting tumorigenesis. We will also evaluate effects of over-expression of miR-105 in a mouse model of prostate tumor growth and metastasis. Conclusion: We have identified a miRNA with apparent novel tumor suppressor functions in prostate cancer. In our future work, we hope to further elucidate what pathways miR-105 targets to control these changes in prostate cancer and what relevance this has to human prostate cancer progression.

18. VV-mE3L as a novel oncolytic virus candidate Komar M.H.1,2 (MSc Student), Rintoul J.L.1,2, Laporte A.N.1,2, Falls T.2 and Bell J.C.1,2 1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario Canada 2 Centre for Cancer Therapeutics, Ottawa Hospital Research Institute (OHRI), Ottawa, Ontario, Canada

Background Selectivity of Oncolytic viruses (OVs) to infect cells is in part due to the interferon (IFN) signaling pathway, involved in antiviral innate immunity. Many cancer cells have IFN signaling defects and therefore may leave themselves vulnerable to viral infection and as a result, cell death. Over the last few decades several oncolytic poxviruses, such as vaccinia virus (VV), have emerged as potential cancer therapies. Wild-type (wt) poxviruses naturally display broad cell tropism. Research on poxviruses as cancer therapy candidates has progressed, with many strategies available to attenuate or better target these viruses to the tumour. The deletion of VV thymidine kinase and/or vaccinia growth factor genes are two common strategies used, while attenuation by increasing the IFN sensitivity of the virus has not been examined extensively. VV-mE3L is a novel oncolytic candidate. It was generated from the wt Western Reserve (WR) strain that has been mutated in the E3L locus. The E3L gene encodes a viral inhibitor of the PKR, an IFN-induced kinase. VV E3L mutants are extremely attenuated, with severe replication defects. Therefore, to compensate for the replication defect, VV E3L was instead replaced with a homologue of E3L from the parapoxvirus Orf virus. Previous studies have shown that this virus, designated VV-mE3L, maintains its ability to infect cells in vitro, but is attenuated compared to its wt counterpart in vivo. Objectives This study was designed therefore to determine if VV-mE3L is safer than its parental virus, WR, in vitro and in vivo and to determine if oncolytic VV-mE3L occupies a different oncolytic niche than a current clinical OV candidate. Results We screened VV-mE3L and WR in vitro using normal human fibroblast cells and found that VV-mE3L viral replication was abrogated in the presence of human IFN-beta while in WR-infected cells viral replication was still present showing that VV-mE3L is sensitive to the effects of IFN in these cells. Viral replication of VV-mE3L and WR in human cancer cells was comparable. Safety, as determined through maximum tolerated dose (MTD) of VV-mE3L in vivo was 10-fold less pathogenic than WR in C57Bl/6 mice. Lastly, using an HT-29 in vivo mouse model, tumor bearing mice treated with VV-mE3L showed in stability of disease. Conclusions These data indicate that VV-mE3L is a potential OV candidate. Current poxvirus OVs were designed with different targeting mechanisms and VV-mE3L may target cancer cells that are resistant to therapy by other poxvirus OVs.

19. ORFV: A novel oncolytic and immune stimulating parapoxvirus therapeutic Julia L. Rintoul1 (PhD Student), Chantal G. Lemay1, Lee-Hwa Tai1, Theresa J. Falls1, Christiano Tanese de Souza1, Byram W. Bridle2, Marianne M. Stanford1, Pamela S. Ohashi3, Yonghong Wan2, Brian D. Lichty2, Andrew A. Mercer4, Rebecca C. Auer1, Harold L. Atkins1, and John C. Bell1 1Dept. of Biochemistry, Microbiology & Immunology, Faculty of Medicine, University of Ottawa, Ottawa Hospital Research Institute, Centre for Innovative Cancer Research, 501 Smyth Rd. Box 926 Ottawa, Canada, K1H 8L6. 2Centre for Gene Therapeutics, Dept. of Pathology and Molecular Medicine, McMaster University, Faculty of Health Sciences, 1280 Main St. W. Hamilton, Canada, L8S 4K1. 3The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, 620 University Avenue, 10-1030, Toronto, Canada, M5G 2M9. 4Department of Microbiology and Immunology, University of Otago, Dunedin PO Box 56, New Zealand 9016. Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (parapoxvirus ovis, or Orf virus) is the prototypic species of the parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here we show for the first time that live replicating ORFV induces an anti-tumour immune response in 2 syngeneic mouse models of cancer which is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal Natural Killer cells. We have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis and replication including efficacy in an A549 xenograft model of cancer.

20. Harnessing oOncolytic virus mediated anti-tumour immunity with an infected cell vaccine Chantal G Lemay1,2 (PhD Student), Julia Rintoul1,2, Agnieszka Kus1, Vanessa Garcia1,2, Theresa Falls2 Kelley Parato2, John C Bell 1,2. 1 University of Ottawa 2 Ottawa Hospital Research Institute Owing to high genetic instability, tumours are often highly heterogeneous; vaccines that are multi-valent and multi-modal will lead to the most well rounded and efficient anti-tumour assault. VSV-D51 leads to a robust anti-tumour T cell response, on which in vivo efficacy is dependent. However, many tumours are not susceptible to VSV-D51 due to an intact type 1 IFN response. In an attempt to harness the adjuvant properties of VSV-D51, we have developed a powerful oncolytic vaccine platform that presents the full breadth of the patient’s unique tumour antigens in the context of an ex vivo VSV-D51 infection. This infected cell vaccine (ICV) is made from cells lines, CT26.wt and B16-F10, that are resistant to VSV-D51 in vivo infection. We demonstrate that prophylactic VSV-ICV immunization in both models protected mice from future live tumour challenge and that the addition of GM-CSF into the viral genome (VSVgm-ICV) greatly increased efficacy. Examination of the early immune phenotype following immunization with the VSVgm-ICV in the B16-F10 model revealed a marked increase in dendritic cell and natural killer (NK) call maturation. The challenge tumour is rapidly infiltrated by a very large number of IFNg-producing T cells and NK cells. Finally, we demonstrate that this approach is robust enough to significantly impact tumour growth in the therapeutic setting. This therapy should be broadly applicable as it does not depend on the ability of VSV to replicate in the cancer cells, is personalized to the patient’s unique tumour antigens, and is capable of eliciting both T cell and NK cell responses

21. Exploiting the tumour microenvironment to enhance anticancer effect of oncolytic viruses Naomi De Silva(PhD Student), Caroline Breitbach, Theresa Davidson-Falls, Usaf Aladl, Laura Evgin, Dominic Roy, Manijeh Daneshamand, Aaron Fenster, Harold Atkins, John Bell Department of Biochemistry, Microbiology, and Immunology. University of Ottawa, Ottawa, Ontario, Canada Robarts Imaging Institute, University of Western Ontario, London, Ontario, Canada. Background: Oncolytic viruses (OVs) were designed to kill cancer cells by direct infection and lysis. OVs replicate within, and kill, certain cancer cell lines extensively in vitro; however, when these same cell lines form tumours in mice, viral replication during treatment is limited. Despite this limited infection, it has been observed that OVs exhibit a secondary killing activity of uninfected cells. This "Bystander Effect", where uninfected cancer cells are rapidly killed, is due to the virus’ ability to specifically target tumour vasculature which results in a loss of tumour blood flow. Targeting tumour vasculature has major advantages over only targeting tumour tissue; these include an ease of targeting with IV administration as well as a presumably greater genetic stability of tumour endothelial cells that should decrease the likelihood of any evolved resistance to therapeutics. Interestingly, oncolytic Vaccinia is able to induce a similar loss of tumour blood flow in patients enrolled in clinical trials for the virus. Objectives: To investigate how oncolytic virus is able to target tumour vasculature; characterize the tumour rim that remains after OV therapy; and to identify components of the tumour microenvironment that protect the tumour rim from viral oncolysis. Methods: To understand how OVs target tumour vasculature, histological studies of tumour vascularity, virus distribution, and cellular proliferation were carried out on tumours treated with oncolytic VSV and Vaccinia virus. 3D models of VSV infection and tumour perfusion were generated from serial histological sections of immunohistochemical staining for VSV and microspheres respectively. To investigate why the rim remains viable and resistant to direct infection, live tumour slices were infected ex vivo. Results/Conclusions: VSV is capable of targeting tumour vasculature by directly infecting tumour vessels, and recruiting neutrophils which mediate blood clot formation within tumour vasculature. A rapid loss of perfusion is observed coincident with apoptosis and necrosis of the tumour core, leaving a viable rim. Similar observations were made for Vaccinia virus where tumour endothelium was infected in a variety of mouse models and patients enrolled in clinical trials. 3D models of infection and perfusion show that infection occurs in small pockets within the tumour rim and perfusion is absent in the tumour core. Tissue slice experiments revealed that in the absence of physical barriers to virus delivery, the majority of the tumour rim continues to be resistant to direct infection, indicating that tumour vasculature is not the sole barrier to OV delivery.

22. Stem Cell Antigen-1 expression marks a population of mouse ovarian surface epithelial cells with enhanced stem/progenitor cell characteristics Lisa F Turchet (PhD Student), Olga Collins, Barbara C Vanderhyden. Department of Cellular and Molecular Medicine, University of Ottawa; Centre for Cancer Therapeutics, Ottawa Hospital Research Institute; Ottawa, Ontario, Canada. Introduction: Stem cell antigen-1 (Sca-1) is the most common marker used to enrich for murine hematopoietic stem cells and with the realisation that many stem cell types share conserved biochemical pathways, Sca-1 has become an attractive stem cell marker candidate for identifying a variety of tissue specific stem/progenitor cells (referred to as stem cells). The mouse ovarian surface epithelium (OSE) is a single layer of poorly differentiated cells that covers the surface of the ovary and is ruptured during ovulation. The remaining OSE cells surrounding the ovulatory wound then proliferate rapidly to regenerate the surface layer of cells. Since rapid healing also occurs in other epithelial tissues where stem cells are the source of proliferating cells, we propose that the OSE contains a Sca-1+ stem cell population which gives rise to the rapidly proliferating cells that close ovulatory wounds. Methods and Results: Using flow cytometry we have shown that the OSE contains a side population of cells (SP, 3% of all OSE) that rapidly effluxes Hoechst 33342, a characteristic that is frequently found in stem cells in other tissues. Since SP analysis typically identifies a heterogeneous population of cells we used quantitative RT-PCR to compare expression levels of specific stem cell markers in SP versus non-SP cells. We have determined that OSE SP cells express 5-fold higher levels of Sca-1 mRNA and 3-fold higher levels of the stem cell marker CD117 (KIT) mRNA when compared to non-SP cells. These results indicate that both Sca-1 and CD117 are potential candidate stem cell markers in the OSE. To compare growth potential and the ability of the Sca-1+/- and CD117+/- OSE cells to self-renew in vitro, spheroid generating efficiency was assessed. Using magnet assisted cell sorting, we divided OSE cells into Sca-1+ and Sca-1- fractions (0.89% and 99.1% respectively) and CD117+ and CD117- fractions (2.3% and 97.7% respectively) and compared sphere generating efficiency in methylcellulose. The Sca-1+ fraction yielded significantly more spheres (p<0.001) compared to all other fractions (% sphere generating efficiency after 28 days: Sca-1+ 0.37; Sca-1- 0.020; CD117+ 0.028; CD117- 0.013) suggesting that it is a good marker for OSE stem cell enrichment. The sphere generating efficiencies of the CD117+ and CD117-OSE fractions (0.028% vs. 0.013%) was not statistically different indicating that CD117 may not be a useful marker for isolating a population of OSE cells with enhanced stem cell characteristics. The incidence of sphere formation can be increased by at least two factors highly expressed at ovulation (TGF-beta and LIF) indicating that ovulation may influence the OSE stem cell population. Currently, we are performing in vivo experiments to determine if Sca-1+ OSE have the ability to repopulate the surface of the ovary. Preliminary results show that only the Sca-1+ OSE are able to attach to the surface of the ovary and survive for at least 5 weeks which indicates that this fraction may be enriched for OSE stem cells. Conclusion: These results indicate that the OSE contains a population of Sca-1+ cells with enhanced stem cell properties (inclusion in SP, enhanced sphere generating efficiency, ability to attach and survive on the surface of the ovary). Interestingly, we have also found that factors highly expressed in follicular fluid at the time of ovulation may influence OSE stem cell activity thereby driving ovulatory wound healing.

23. Identifying patients at high risk for acute and delayed chemotherapy induced nausea and vomiting (CINV): The validation of a prediction tool Iryna Kuchuk(Clinical Fellow), George Dranitsaris, Nathaniel Bouganim, Carolyn Milano, Lisa Vandermeer, Susan Dent, Paul Wheatley-Price, Jenny Laporte, Karen-Ann Oxborough, Mark Clemons Abstract: Background: Despite the use of standardized anti-emetic guidelines, up to 20% of cancer patients (pts) suffer moderate to severe CINV (≥ grade 2). Prediction of pts at high risk for CINV, through the use of validated mathematical models, is important in developing management strategies. We previously developed cycle-based prediction models for acute & delayed CINV. As part of the validation process, we prospectively evaluated the ability of the scoring systems to accurately identify pts deemed to be high risk for ≥ grade 2 CINV. Methods: Pts receiving chemotherapy were provided with symptom diaries. Compliance to these was enhanced via patient phone calls within 5 days post-chemotherapy (CT) after every cycle. All pts received standard anti-emetic prophylaxis. Prior to each cycle of CT, acute and delayed CINV scoring systems were applied to stratify pts into low and high risk groups. The external validity of each system was assessed via a receiver operating characteristic curve (AUROC) analysis. Logistic regression modeling was applied to compare the risk of ≥ grade 2 CINV between pts considered to be high vs. low risk. Results: CINV outcomes data were collected from 97 pts following 401 cycles of CT. Incidence of ≥ grade 2 acute & delayed CINV was 13.5% and 21.4% respectively. Major predictors for ≥ grade 2 CINV included; younger patient age, platinum or anthracyline-based chemotherapy, low alcohol consumption, earlier cycles of CT, previous history of morning sickness and prior emetic episodes with CT. Both the acute & delayed scoring systems had good predictive accuracy when applied to the external validation sample (AUROC = 0.70, 95%CI: 0.62-0.77 and AUROC = 0.75, 95%CI: 0.69-0.80). Pts identified to be at high risk by the scoring systems were 3.1 (p = 0.006) and 4.2 (p < 0.001) times more likely to developed ≥ grade 2 CINV. Conclusions: This study demonstrates that our scoring systems are able to accurately identify pts at high risk for acute and delayed CINV. The application of these prediction tools will be an important source of real-time risk information for the practicing oncologist and can enhance patient care by optimizing preventative therapies in a proactive manner.

24. Modeling the interactions of oncolytic Vaccinia virus in the blood to achieve efficient systemic delivery Laura Evgin (PhD Student), Chantal Lemay,Theresa Falls, Harold Atkins, John Bell Systemic delivery of oncolytic viruses is the ideal mode of treatment for metastatic disease because metastatic beds can be dispersed or their extent undiscovered. There are however, many barriers to the systemic delivery of oncolytic Vaccinia virus that are presented by the innate and acquired immune systems. Most notably, complement and neutralizing antibody, the latter of which is specifically present in the demographic of patients vaccinated against smallpox, act together to neutralize the virus and induce activation of the immune system. We have identified that complement and antibody in human blood together can neutralize up to 99% of the virus in vitro. The importance of these clearance mechanisms is then amplified when patients are treated with multiple doses and the amount of antibody produced significantly increases. Additionally, we have discovered that the virus partitioned preferentially to the cellular compartment over the plasma, as demonstrated in vitro as well as in blood samples from patients treated intravenously with oncolytic Vaccinia virus as part of a clinical trial. We have identified that the virus preferentially associates with B cells and phagocytic cells such as neutrophils and monocytes, however the red blood cell compartment also represents an important reservoir of virus due to its size. We are currently evaluating the mouse as a surrogate model for the delivery of oncolytic Vaccinia virus to tumours in the face of all the important clearance mechanisms. Through modeling the interactions between the virus and blood components both in vitro, in vivo, and in samples from previous and upcoming clinical trials, we will identify the interactions that are most important in lowering the effective dose of delivery, and undertake the corollary questions; which mechanisms can we manipulate in order to abrogate neutralization, and which ones can we capitalize on to increase delivery.

25. UNAVAILABLE

26. Perioperative sepsis and potentially hypothermia, but not hemorrhagic shock promotes the development of cancer metastases in a murine model. A. Ananth1,2* (MSc Student), R. Seth1,3*, L. Tai1, T. Lam1, T. Falls1, C. Souza1, J. Bell1, H. Atkins1, R. Boushey3, R. Auer1,3. 1Center for Innovative Cancer Research, The Ottawa Hospital Research Institute, Ottawa, ON 2Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON 3Department of Surgery, Division of General Surgery, The Ottawa Hospital, Ottawa, ON. *co-authors Background In cancer patients, surgery is known to promote tumour metastases. However, the effects of perioperative complications such as sepsis, blood loss, and hypothermia on cancer metastases have not been adequately assessed. We hypothesize that sepsis, blood loss, and hypothermia will further enhance the prometastatic effect of surgery. We sought to establish a murine model of surgical stress to evaluate the effect of each perioperative factor on postoperative tumour metastases. Methods Prior to surgery, pulmonary metastases were established by intravenous challenge of CT26LacZ colon cancer cells in Balb/c mice or B16LacZ melanoma cells in C57Bl/6 mice. Surgical stress was generated through partial hepatectomy (PH) or left nephrectomy (LN). Sepsis was induced by puncturing the cecum with an 18G needle and expressing stool into the abdomen. Hemorrhagic shock was induced by removal of 30% of total blood volume via saphenous vein. Hypothermia was induced by removing the heating apparatus during surgery and lowering core body temperatures to 30°C. Lung tumour burden was quantified 3 days post surgery. Results In both murine models, surgical stress induced by either PH or LN resulted in a 2-fold increase in metastases compared to non-surgery mice. Mice that

received anaesthesia only had a lung tumor burden equivalent to untouched controls. The prometastatic effect of surgery was amplified in mice subjected to perioperative sepsis resulting in a further 2-fold increase in metastases. In contrast, surgically stressed mice subjected to Stage 3 hemorrhagic shock did not show an additional increase in their lung tumor burden. Currently, we are optimizing the murine model of surgical stress and hypothermia, however, initial experiments comparing hypothermia and normothermia in non-surgically stressed mice have shown that hypothermia significantly increases lung tumor burden. Conclusions Overall, sepsis and potentially hypothermia, but not hemorrhagic shock results in further augmentation of cancer metastases. Additional studies aimed at exploring immune suppression as a key mechanism are being actively pursued and will be important in the future development of perioperative immunomodulation strategies aimed at attenuating metastatic disease in the setting of sepsis, hypothermia, and blood loss.

27. Role of the tumour microenvironment during oncolytic virus replication Carolina Ilkow (Postdoctoral Fellow), Rozanne Arulanandam, Cory Batenchuk, John Bell Background: Oncolytic virotherapy has emerged as a novel therapeutic for the treatment of cancer. Up to date, several viruses have been successfully engineered to selectively replicate and kill cancer cells. However, the tumour microenvironment is a growing target for consideration of cancer therapeutics; only recently research in oncolytic virotheraphy began to focus on the tumour microenvironment as a separate cancer associated entity that may be targeted by oncolytic viruses (OVs). The tumour microenvironment consists of a complex interplay of molecular signals that control a wide range of biological functions including tumour growth, progression, neo-vascularization and metastasis. Hypothesis: We hypothesized that these signals may also play an important role in modulating replication and spread of OVs within different compartments of the tumour. Methods: In order to begin investigating the impact of tumour stroma-secreted molecules on OVs replication and spread, a screen measuring the impact of various growth factors and cytokines on oncolytic vesicular stomatitis virus (VSV51) and oncolytic Vaccinia virus (VV) replication was performed. Results: We found that the fibroblast growth factor-2 (FGF-2) exclusively enhances VSV51 and VV replication not only in several cancer cell lines but also in activated fibroblasts. Ongoing studies are aimed at revealing the molecular mechanism by which FGF-2 specifically enhances OVs replication in cancer-associated fibroblasts. Conclusion: The approach of uncovering signalling pathways that enhance OVs replication within different compartments of the tumour will undoubtedly contribute to a better design of oncolytic viruses.

28. Regulation of Ste20-like kinase in cell motility is dependent on FAK and PI3K signalling Lilia Antonova (Postdoctoral Fellow), OHRI Center for Cancer Therapeutics Luc Sabourin, OHRI Center for Cancer Therapeutics Background: Ste20-Like Kinase (SLK) is a ubiquitously expressed kinase whose main function appears to be in cell migration, where it is involved in induction of actin reorganization and focal adhesion turnover. Due to the key role of SLK in cell migration we are interested in identifying the pathways regulating SLK in this process. We have previously found that one such pathway is initiated by the receptor tyrosine kinase, ErbB2, a protein overexpressed in a large number of breast cancer cases, and that this pathway is dependent on Focal Adhesion Kinase (FAK) activity. We are now working on delineating the signals downstream of FAK which lead to SLK upregulation. Results: FAK is activated by autophosphorylation on its 397 tyrosine residue, subsequent recruitment of regulatory kinases to that residue, and further phosphorylation on tyrosines associated with downstream signalling. Phospohorylation of FAK tyrosines 861 and 925, specifically, has been correlated with the regulation of cell migration. In order to identify signalling pathways leading from FAK to SLK activation, we studied the importance of FAK pTyr-397, 861 and 925 in SLK activation and localization to the leading edge of migrating cells. We found that mutation of each of these tyrosine residues results in loss of SLK kinase activity and disrupts SLK localization during migration. Further, both the FAK pTyr-397 binding PI3K, and the pTyr-925 associated MAPK were shown to play a role in SLK regulation. An important downstream target of PI3K signalling is the Phosphoinositide Dependent Protein Kinase (PDK1). In order to elucidate PI3K signalling to SLK we examined the possibility that PDK1 may act as an intermediary in this signalling pathway. With the use of kinase assays employing a recombinant PDK1 protein we found that PDK1 directly phosphorylates both recombinant SLK protein and SLK immunoprecipitated from cells. Furthermore, PDK1 presence acted to increase SLK kinase activity both in vitro and in MEF cells transfected with a PDK1 expression vector. Direct association of SLK and PDK1 was observed in PDK1-overexpressing cells. Interestingly, these effects were seen both in the presence or absence of PDK1 kinase activity, suggesting that direct phosphorylation may not be the only mechanism through which PDK1 regulates SLK. In addition to its effect on SLK activity, PDK1 was also found to be necessary for SLK localization. Cells treated with the PDK1 inhibitor BX912 showed a decrease in the localization of SLK to the leading edge of migrating cells, a process previously shown to be necessary for cell migration. Finally, we confirmed that PDK1 acts downstream of PI3K in regulating SLK, as addition of PDK1 to cells was able to rescue the negative effect of PI3K inhibition on SLK kinase activity. Future Directions: The findings described here represent novel molecular mechanisms of SLK regulation and contribute to the understanding of the process of cell migration. Further studies will focus on isolating the mechanisms of PDK1 association with SLK and the importance of this association to cell migration. This work is supported by Canadian Breast Cancer Foundation, and Canadian Institute of Health Research grants.

29. Monitoring and identification of sepsis development through a composite measure of heart rate variability Andrea Bravi (PhD Student) - Department of Cellular and Molecular Medicine, University of Ottawa André Longtin - Department of Physics, University of Ottawa Andrew JE Seely - Department of Cellular and Molecular Medicine, University of Ottawa Background: Severe sepsis and septic shock are the most common causes of mortality in critically ill patients, with a mortality of approximately 50% and an average annual cost of $16.7 billion in the USA. Furthermore, a retrospective study on 2,731 subjects showed that each hour of delay in the initiation of effective antimicrobial therapy is associated with a mean decrease in survival of 7.6%. Therefore, tracking the development of sepsis is a challenge of particular interest. Being both sensitive to its presence and severity, heart rate variability holds the appropriate information to develop a monitoring tool specific to sepsis. Objective: The creation of a unique measure tracking sepsis development and of an alarm system to provide physicians with an indication of the probability a patient is developing sepsis at a given time. Methods: The studied dataset included 17 adults who underwent bone marrow transplant. Of them, 14 developed sepsis. The analysis of their heart rate variability was pursued extracting 101 measures of variability. Several measures were used with the aim to better characterize the heart rate variability. Those measures underwent filtering and two steps of data reduction. In the first one, Spearman’s nonlinear correlation was used to identify the measures showing a decrease in variability correlated with the development of sepsis. In the second one, the measures were projected into a new reference system and reduced to a unique measure, which we called a composite measure of variability. The projection and reduction was performed using the Principal Component Analysis. Then, the sum of the energy at very low frequency of the composite measure was used as the unique independent variable of a logistic regression classifier, which provided the probability of developing sepsis at a given time. Results: The described procedure created a unique measure which was capable of tracking the development of sepsis. The composite measure showed a positive increasing trend for patients who did not develop sepsis, and a negative decreasing trend for patients who developed sepsis. The alarm system developed from the composite measure was capable to detect sepsis development 60 hours before of sepsis diagnosis (median value). Conclusions/Significance: A signal processing module based on the multivariate study of heart rate variability was proposed. This module was capable to identify the presence, and track the severity of sepsis, making its application in clinical settings potentially beneficial in sepsis treatment.

Chronic Disease Program

30. The essential role of IL-7 signalling in CD8+ T-cell activities

Crawley AM1,2 ( Research Associate)., Vranjkovic A1., McGuinty M2., Faller E1., MacPherson P1,2,3., Angel JB1,2,3. 1. Ottawa Health Research Institute, Ottawa, Canada. 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada 3 Division of Infectious Diseases, Ottawa Hospital-General Campus, Ottawa, Canada. Plain language summary: Anti-viral CD8+ T-cell responses become impaired in HIV infection in part due to decreased T-cell survival, function and memory cell development; aspects mediated largely by interleukin-7 (IL-7). In progressive HIV infection, decreased T-cell expression of the IL-7 receptor alpha (CD127) and impaired IL-7 signalling, despite increased IL-7 production, may contribute to failing T-cell activity. Here we examine the effects of IL-7 on a panel of signalling pathways and investigate how IL-7 signalling pathways are associated with IL-7-related activities in CD8+ T-cells. Background: Anti-viral CD8+ T-cell responses become impaired in HIV infection in part due to decreased T-cell survival, function and memory cell development; aspects mediated largely by the cytokine IL-7. In progressive HIV infection, decreased T-cell expression of the IL-7 receptor alpha (CD127) and impaired IL-7 signalling, despite increased IL-7 production, may contribute to failing T-cell activity. This study examines the effects of IL-7 on a panel of signalling pathways and investigates how IL-7 signalling pathways are associated with IL-7-related activities in CD8+ T-cells. Methods: Activation of intracellular signalling pathways (eg. Jak-STAT, PI3K, MAPK) in isolated CD8+ T-cells cultured with increasing concentrations of IL-7 was measured. In addition, IL-7-related activities (Bcl-2 production, proliferation, glucose uptake, perforin release) were evaluated. The effect of inhibiting IL-7-induced signalling pathways on IL-7 activities was assessed. Results: Low concentrations of IL-7 (10 pg/ml) are capable of inducing maximum activation of the Jak-STAT and PI3K signalling pathways, while higher concentrations (500-1000 pg/ml) were required to induce Bcl-2 production and glucose uptake. Even higher concentrations of IL-7 (10,000 pg/ml) were needed to induce cell proliferation and perforin release. Inhibition of Jak activation reduced IL-7-induced Bcl-2, and perforin production, while inhibition of either Jak/STAT or PI3K pathways reduced glucose uptake and proliferation. Conclusions: The activation of intracellular signalling pathways by IL-7 in human CD8+ T-cells has now been comprehensively described by this research. These pathways are associated with IL-7-induced functions. Furthermore, the kinetic and optimal concentrations of IL-7 required for the induction of these functional outcomes suggest a complex control of IL-7-associated functions. Future work will investigate which specific signalling pathways and associated functions are impaired in HIV infection, providing insight into the use of IL-7 as a therapy to improve the immune status in HIV+ individuals.

31. Cholesterol trafficking regulation in pathological conditions Eid, W. M. (PhD Biochemistry, 2nd year) The mTOR pathway is a key regulator of cell growth and proliferation; it integrates different signals to regulate many processes, such as lipid synthesis. mTORC1 activates lipid synthesis through activation of SREBP, a transcription factor that controls the expression of lipogenic genes. However, the molecular mechanism that link them both is yet unknown.

mTORC1 also inhibits the catabolic process of autophagy. It was found that autophagy is involved in regulating intracellular lipid stores through the hydrolysis of lipid droplets. The cholestryl ester hydrolysis is recently shown to depend on autophagy. We hypothesize that mTORC1 activates SREBP through the CE cycle. To establish the relationship between autophagy and the CE cycle, we analyzed lipids intracellular localization & mobilization of lipid droplets. Our data support autophagy involvement in the CE cycle. we obtained evidence that ABCA1 promotes both autophagy and the CE cycle to establish the relationship between autophagy and ABCA1 function.

32. Janus kinase 2 mediate embryonic responses to hyperosmotic stress by regulating Na+/H+ exchanger isoform 1 activation in mouse preimplantation embryos Chenxi Zhou1,2 (Research Associate), Jay M. Baltz1,2,3 1Ottawa Hospital Research Institute, Ottawa, Ontario K1Y 4E9, Canada; 2Department of Obstetrics and Gynaecology (Division of Reproductive Medicine), 3Department of Cellular and Molecular Medicine University of Ottawa Faculty of Medicine, Ottawa, Ontario K1H 8M5, Canada Background: Preimplantation embryos are sensitive to the environment in which they develop and are especially sensitive to increased osmolarity. Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitous electroneutral membrane transporter that can be activated by hypertonicity in many cell types to counter cell volume decreases. Despite its potential importance, neither the involvement of NHE1 in cell volume regulation in mammalian preimplantation embryos nor the relevant signalling mechanisms by which hypertonicity activates NHE1 have been investigated. Objective: The present study was conducted to assess whether preimplantation embryos respond to increased osmolarity by activating NHE1, and to screen for important signalling pathways required for NHE1 activation following exposure of preimplantation mouse embryos to hyperosmotic environments. Major methods: Mouse embryo NHE1 activity was measured by monitoring intracellular pH using a fluorescence imaging system. Signaling pathways were dissected using inhibitors, functional manipulation of Jak2 by mRNAs (wide-type or dominant negative form) microinjected into embryos, and immunostaining and Western blots to assess Jak2 activity and localization. Results and conclusion: Hypertonicity that induced a cell volume decrease rapidly activated NHE1 in preimplantation mouse embryos in an osmolarity-dependent manner at each stage of development, with highest activity at the 2-cell stage. Our results indicated that protein tyrosine phosphorylation plays a substantial role in this NHE1 activation, since genistein (20µM), a potent broad-spectrum tyrosine kinase inhibitor, totally inhibited NHE1 activity elicited by cell shrinkage in mouse 2-cell embryos. By using more selective inhibitors, we further found evidence that Janus kinase 2 (Jak2) and calmodulin (CaM) were essential for hypertonicity-induced NHE1 activation. In contrast, inhibitors of PKA, PKC, p38 MAPK, ERK1/2, JNK, Src, PI3K and MLCK had no significant effects. The involvement of a Jak2 was further confirmed by overexpression of dominant negative Jak2 mutant in mouse embryos, which markedly suppressed the hypertonicity-induced NHE1 activity. Western blots also clearly demonstrated that hypertonic treatment rapidly stimulated tyrosine phosphorylation in embryos, likely of Jak2 at Tyr1007/1008, critical sites that must be phosphorylated for kinase activation. Consistent with NHE1 activity data, we also found that the abundance of hypertonically-induced tyrosine phosphorylation in preimplantation mouse embryos was highest amount at 2-cell stage. We have determined the subcellular distribution of Jak2 protein in mouse embryos by immunostaining and the expression of a YFP-tagged Jak2 construct. We found that Jak2-YFP was present throughout the cell with some cytoplasmic localization, and was concentrated at the cell surface, but was absent from the nucleus. Using selective inhibitors, we demonstrated that PLC and IP3 were also required for NHE1 activation in mouse embryos upon cell shrinkage. These outcomes further our knowledge of how culture environments can affect embryo development. This research was supported by Canadian Institutes of Health Research operating grants MOP74515 and MOP62730 to JMB.

33. Study of inhibitory effect of epididymal CRES on PC4/PCSK4 activity Priyambada Mishra ( Research Technician), Qin Qiu, Majambu Mbikay, Ajoy Basak Chronic Disease Program Ottawa Hospital Research Institute Loeb, Civic site PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in M range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

34. Mice deficient in transient receptor potential melastatin 7 (TRPM7) cation channel are hypertensive with altered vascular signaling to angiotensin II. Antunes TT (Postdoctoral Fellow) , Yogi A, He Y, Callera GE, Montezano AC, Ryazanov AG, Ryazanova LV. Touyz RM.

Background: Transient receptor potential melastatin 7 (TRPM7) cation channel, a unique chanzyme, regulates transmembrane Mg2+ transport through its channel domain and intracellular signalling through its kinase domain. We demonstrated aberrant TRPM7 signaling in experimental hypertension. Whether TRPM7 plays a pathophysiological role in hypertension is unknown. Objective: We tested the hypothesis that decreased TRPM7 activity amplifies pressor responses to Ang II, an effect associated with vascular dysfunction and arterial remodeling. Results: TRPM7-deficient mice with deletion of the kinase domain (TRPM7+/-) were studied. Baseline blood pressure (BP) was increased in TRPM7+/- mice vs wild type (WT) controls (121±3.3 vs 108±1.6 mmHg, p<0.05). This was associated with increased sensitivity to acetylcholine (WT vs TRPM7+/-; pD2=6.4±0.1 vs 6.9±0.1; p<0.05). Ang II infusion (400 ng/kg/min, 14 days) increased BP in WT (148±4.5mmHg) and TRPM7+/- mice (165±2.9mmHg). Ang II-infused TRPM7+/- mice exhibited increased sensitivity to acetylcholine compared to vehicle treated TRPM7+/- (untreated vs TRPM7+/-Ang II; pD2=6.9±0.1 vs 7.8±0.4; p<0.05); with similar Emax values (93±4 vs 86±6%). Media-lumen ratio was similar in Ang II-infused WT (0.28±0.02) and TRPM7+/- mice (0.23±0.02). To investigate in greater detail the role of TRPM7 kinase in vascular signaling, vascular smooth muscle cells (VSMC) from TRPM7+/- and WT mice were cultured. Ang II increased Mg2+ influx in both strains (2.5-fold for both; p<0.001). Ang II increased phosphorylation of JNK, p38MAPK and ERK1/2 in VSMC from WT mice by 10; 4; ;and 6.4-fold; respectively. MAPK responses were blunted in TRPM7-deficient VSMC. Conclusion: Our findings identify TRPM7 kinase as a novel signaling system in the regulation of endothelial function, vascular signaling and BP control.

35. The role and regulation of Checkpoint kinase 1 in ovarian cancer cells Ahmed Y. Ali (PhD candidate), Mohammad R. Abedini, Benjamin K. Tsang The Ottawa Hospital Research Institute The University of Ottawa Cisplatin (CDDP) resistance is a major hurdle in the treatment of human ovarian cancer (OVCA). A better understanding of the mechanisms of CDDP resistance can greatly improve therapeutic outcome for patients. p53, a determinant of CDDP sensitivity in ovarian cancer, is activated by Chk1 in response to DNA damage. Although the oncogenic phosphatase Protein Phosphatase Magnesium-dependent 1 (PPM1D) can deactivate both p53 and Chk1 through site-specific dephosphorylation, whether PPM1D plays a role in CDDP resistance is unknown. Here, using pair-matched wt-p53 CDDP-sensitive (OV2008) and –resistant (C13*) cells, and p53-compromised CDDP-resistant cells (A2780cp, OCC-1, OVCAR-3, and SKOV3), we have demonstrated (i) the existence of site-specific differences in phospho-Ser-Checkpoint kinase 1 (Chk1) content between sensitive and resistant cells in response to CDDP; (ii) PPM1D, but not ataxia telangiectasia and Rad3-related kinase (ATR), is important in the regulation of CDDP-induced Chk1 activation and ovarian cancer cell chemosensitivity; (iii) PPM1D down-regulation sensitizes resistant cells to CDDP primarily by activating Chk1 and p53. Our findings establish for the first time that PPM1D confers CDDP resistance in ovarian cancer cells through attenuating CDDP-induced, Chk1-mediated, p53-dependent apoptosis. These findings extend the current knowledge on the molecular and cellular basis of cisplatin resistance and offer the rationale for PPMID as a potential target for treatment of chemoresistant ovarian cancer.

36. Role and regulation of NR4A1,NR5A1 and NR5A2 in mechanisms of polycystic ovary syndrome (PCOS) Kai Xue(PhD Student)1 2,Jiayin Liu 2, Bruce Murphy 3 , Benjamin K. Tsang 1 1 Ottawa Hospital Research Institute 2 Nanjing Medical University 3 Centre for Animal Reproduction Research, University of Montreal Background: Excessive androgen is associated with ovarian pathology. The role of androgens in the regulation of ovarian follicle development remains controversial. We (present studies) and others have shown that preantral follicles treated with DHT in vivo and in vitro exhibited enhanced growth compared to non-treated controls. However, in chronically androgenized rats and in human polycystic ovary syndrome (PCOS) subjects with hyperandrogenism, follicles growth is arrested in early-antral stage. These studies raise the possibility that the role of androgen in the regulation of follicular growth may be follicular stage-specific. NR4A1, NR5A1 and NR5A2, members of nuclear receptor superfamily, are important regulators of ovarian steroidogenesis and follicle development. Their expression is follicular cell-specific and is regulated by DHT and gonadotropin. PCOS is a common female endocrine disorder and a leading cause of anovulatory infertility.Understanding the molecular regulation of follicular growth by androgen may provide important insight for novel therapy for this syndrome. We hypothesize that androgen promotes preantral follicle growth while suppresses early-antral follicle development though regulation of orphan receptor expression and actions. Excessive androgen exposure at early antral stage results in PCOS phenotype. Our Objectives are: (1) To compare the influence of androgen (DHT) on pre- and early-antral follicle growth in vitro and in vivo; (2) To investigate the expression pattern of nuclear receptors during follicular development and in PCOS models; (3) To examine whether nuclear receptors could differentially regulate pre- and early-antral follicle growth and determine their target genes involved in such regulation. Experimental Strategies: Our laboratory has recently developed a chronically androgenized rat "PCOS" model which exhibits many phenotypes of the human PCOS, we plan to: (1) Compare the relative growth dynamics in cultures of pre-antral and early antral follicles isolated from the PCOS and CTL rats, including follicle growth (follicle volume), cell proliferation [Ki67 IF, cell number (alamar blue assay)]; (2) Assess the relative ovarian expression pattern and follicular sites of expression of NR4A1, NR5A1 and NR5A2 in PCOS and CTL rats (Western blot, qPCR, ISH and IHC). Moreover, protein and mRNA levels of these receptors in PCOS and non-PCOS human ovaries will be compared (IHC and ISH). (3) Examine the influence of follicular cell-specific up- and down-regulation of the nuclear receptors (with adenovirus sense cDNA or siRNA) in theca (NR4A1, NR5A1) and granulosa cells (NR5A1 and NR5A2) in follicles cultured in the presence or absence of DHT. Follicular growth and cell proliferation will be assessed in cultured preantral and early antral follicles. Responses of isolated granulosa cells (cell cycle progression) to these manipulation will be assessed by flow cytometry; and (4) Identify the target genes possibly involved in the regulation follicular cell proliferation and differentiation by the nuclear receptors

(ChIP assays). We will then: a) confirm the regulation of these genes with forced expression or knockdown of specific nuclear receptors, b) determine if knockdown of these specific genes (siRNA) would abrogate DHT- and orphan nuclear receptor-mediated follicular growth, cell proliferation and differentiation.

37. IL-7 induces rapid internalization of CD127 by clathrin mediated endocytosis, leading to subsequent degradation by the proteasome. Elliott Faller (Postdoctoral Fellow), Feras Ghazawi and Paul MacPherson Summary/Background: IL-7 signaling is important for CD8 T-cell homeostasis and function and we have previously shown decreased expression of the IL-7R alpha-chain (CD127) on CD8 T-cells in HIV+ patients. Suppression of CD127 is mediated by both the HIV Tat protein and IL-7, both of which are elevated during HIV infection. We show here by flow cytometry, western blot analysis and confocal microscopy that IL-7 induces the rapid internalization of CD127 by endocytosis and subsequent degradation by the proteasome. Methods: CD8 T-cells from healthy donors were incubated with IL-7 at concentrations ranging from 10-10 000 pg/ml. Surface CD127 expression was determined by flow cytometry and total protein was determined by western blot. Intracellular CD127, RAB7, Clathrin, LAMP1, MCP20 and EEA1 expression were visualized by confocal microscopy. Results: IL-7 decreases surface CD127 expression on CD8 T-cells in a dose- and time-dependent manner with initial suppression as early as 20 minutes and maximal suppression at 12 hours. This decrease at the cell surface is followed by loss of total CD127 protein within the cell as demonstrated by western blot. IL-7 induces internalization of surface CD127 through endocytic vesicles. Both Filipin, which disrupts lipid rafts, and Dynasore, which prevents fission of vesicles from the cell membrane, prevent IL-7 induced down regulation of CD127 at the cell membrane. Consistent with this we have shown by confocal microscopy that in resting CD8 T-cells CD127 is distributed evenly throughout the cell. After treatment with IL-7, CD127 forms multiple intracellular punctae with increased co-localization with clathrin by 5 minutes followed by co-localization with the early endosomal marker EEA1 after 30 minutes. By 2 hours CD127 staining associates with the late endosome marker RAB7 and the proteasomal 20S subunit, while at the same time showing decreased colocalization with the lysosomal marker LAMP1. Interestingly, the proteasome inhibitors lactacystin and MG132 both blocked IL-7’s ability to down regulate CD127 confirming IL-7 directs the receptor to the proteasome for degradation. Conclusions: IL-7, a potent immunomodulatory cytokine, is currently being investigated as a potential therapy and vaccine adjuvant in the treatment of HIV infection. In order to maximize these therapeutic strategies, we must first understand how the IL-7 receptor is regulated. We show here that IL-7 down regulates CD127 protein on the surface of CD8 T-cells through a mechanism independent of transcriptional suppression. IL-7 binding to its receptor induces aggregation of CD127 into lipid rafts, internalization through endosomes, and ultimately degradation via the proteasome.

38. The effects of HIV-1-Tat and HIV-1-Vpr on the maturation and function of monocyte derived dendritic cells. Peter Fairman (PhD Student)Dept. Biochemistry, Microbiology, and Immunology University of Ottawa, Ottawa, Ontario; Ottawa Hospital Research Institute, Ottawa, Ontario) Jonathan B. Angel (M.D., F.R.C.P.C., Senior Scientist, Chronic Disease, Ottawa Hospital Research Institute, Infectious Disease Specialist, Associate Professor, Faculty of Medicine, University of Ottawa) Dept. Biochemistry, Microbiology, and Immunology University of Ottawa, Ottawa, Ontario; Ottawa Hospital Research Institute, Ottawa, Ontario; Division of Infectious Disease, Ottawa Hospital-General Campus, Ottawa, Ontario. Background: Dendritic cells (DC) are mediators of the adaptive immune response responsible for the presentation of antigens to naïve T-cells in secondary lymph organs. During HIV-1 infection, DC maturation and function are compromised in spite of low infection rates. Therefore the effects of HIV-1 on DCs in the absence of productive DC infection are important. Two HIV-derived proteins Tat and Vpr found in the plasma have been found to have effects on dendritic cell maturation and function. Objective: The objective of this study was to evaluate the effects of the HIV-1 accessory proteins Tat and Vpr on the maturation and function of monocyte derived dendritic cells (MDDCs) in vitro. Surface molecule expression, phagocytic activities, and antigen presentation over the course of DC maturation will be measured. Methods: Monocytes isolated from peripheral blood mononuclear cells were differentiated into immature MDDCs (iMDDC) according to established methods. Cells were first incubated with HIV-Tat or Vpr, and then with or without a maturation inducing inflammatory cytokine cocktail (IL-1β, IL-6 and PGE2), and then examined by flow cytometry for expression of CD14, DC-SIGN, CD80, CD83, CD86, CD40, CCR7, MHC I and MHC II. To measure endocytosis, immature MDDCs were incubated with HIV-Tat or HIV-Vpr followed by a 1 hour incubation with saturating concentrations of FITC-conjugated dextran and then examined by flow cytometry. Antigen presentation was measured by incubating immature MDDCs with HIV-Tat or Vpr and then co-culturing with CFSE stained autologous PBMCs for 7 days. Co-cultures were then stained with PC5-CD8 and cellular proliferation was measured by flow cytometric analysis. Results: Neither incubation with HIV-Tat or HIV-Vpr induced any differences in the expression of any of the examined surface molecules either in the presence or absence of the maturation inducing cytokines. The endocytosis of FITC-dextran was observed to be decreased after incubation with both HIV-Tat and HIV-Vpr. Antigen presentation was inhibited affected by both HIV-Tat and HIV-Vpr. Conclusion: In vitro the incubation with HIV-1 accessory proteins Tat and Vpr inhibit the ability of iMDDCs to endocytose carbohydrate antigens without inducing changes in cell surface molecule markers typically associated with maturation. HIV-Tat and HIV-Vpr also inhibited antigen presentation to autologous T-cells. Understanding the mechanisms of dendritic cell dysfunction in HIV infection will provide further insight into HIV immune pathogenesis.

39. Development of cell volume-regulatory mechanisms during oocyte growth Samantha Richard (MSc Student) Dr. Jay Baltz (research supervisor/PI) Chronic Disease Program, OHRI, Depts of OB/GYN & Cellular and Molecular Medicine, University of Ottawa.

Background Pre-implantation embryos are sensitive to changes in osmolarity and utilize volume regulatory mechanisms to maintain normal cell volume. Prior to ovulation, GV oocytes depend on the strong adhesion to the rigid zona pellucida (ZP) to determine cell size. Following ovulation, detachment of the oocyte-ZP adhesion results in decreased cell volume and initiation of independent cell volume regulation through the intracellular accumulation of organic osmolytes, primarily glycine. Glycine accumulation is mediated by the GLYT1 transporter, a mechanism unique to eggs and early embryos. Objective: Determine what mechanism is responsible for the maintenance of GLYT1 quiescence in the follicle. Hypotheses: 1)The mural granulosa and/or follicular fluid components are required for the maintenance of GLYT1 quiescence. 2)Gap junctional communication from the granulosa cells is required to transport the GLYT1 inhibitor components to the oocyte. 3)Detachment from the ZP is required for GLYT1 activation. Methods: I will use intact antral follicles, cumulus-oocyte complexes (COCs), and denuded GV oocytes maintained in culture. GLYT1 activity will be assessed by measuring the rate of [3H]glycine uptake. GLYT1 is fully activated by 4 hours after ovulation or removal from the follicle; Therefore, GLYT1 activity will be determined at 4 hours post-isolation. To address hypothesis 1, GLYT1 activity will be measured after 4 hours. Oocytes that were cultured denuded, in COCs, or in antral follicles will be compared. If GLYT1 remains quiescent in antral follicles, to address hypothesis 2, antral follicles will be incubated with and without gap junction inhibitors (18-α-glycyrrhetinic acid, octanol, lindane) and GLYT1 activity determined after 4 hours. To address hypothesis 3, oocyte-ZP adhesion release will be delayed using protease inhibitors (my preliminary data), and the timecourse of GLYT1 activation measured vs. control. Results/Conclusion Using oocytes at various stages of development, preliminary data indicates that growing oocytes gain the ability to activate GLYT1-mediated glycine transport when they reach 70-75µm in diameter. This coincides with the acquired ability of the oocyte to undergo oocyte-ZP detachment and be released from prophase of meiosis I arrest. Preliminary data showing a lack of GLYT1 activation following immediate oocyte removal from antral follicles suggests that a factor from the follicle is necessary for the maintenance of GLYT1 quiescence. Understanding the mechanisms involved in this method of volume regulation is important in providing optimal conditions for oocyte and embryo culture to avoid failure of infertility treatments, and damage that could result in dysregulation in fetal development, and disease in the offspring.

40. Angiotensin Ii-induced aldosterone synthesis in adipocytes is mediated via calcineurin/Nfat-dependent pathways Aurelie NGUYEN DINH CAT (PhD, Postdoc Fellow)1, Ana M. BRIONES 1, Glaucia E. CALLERA 1, Alvaro YOGI 1, Dylan BURGER 1, Ying HE 1, Augusto C. MONTEZANO 1, Anne Marie GAGNON 2, Celso E. GOMEZ-SANCHEZ 3, Elise P. GOMEZ-SANCHEZ 3, Alexander SORISKY 2, Kevin D. BURNS 1, Rhian M. TOUYZ 1. 1 Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Canada; 2 Chronic Disease Program, Ottawa Hospital Research Institute, University of Ottawa, Ottawa; 3 Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, Jackson; Adipocytes possess a local renin-angiotensin-aldosterone system. We previously reported that 3T3-L1-differentiated adipocytes and mouse mature adipocytes express aldosterone synthase (CYP11B2 gene and protein) and produce aldosterone, which is enhanced by angiotensin II (Ang II). In adrenal glands, aldosterone production is mediated via calcineurin-induced upregulation of CYP11B2 gene. Objective: In the present study, we evaluated if calcineurin and the nuclear factor of activated T cells (NFAT) play a role in Ang II-induced aldosterone production by 3T3-L1 adipocytes. Methods: To address our hypothesis, differentiated 3T3-L1 adipocytes were stimulated with Ang II for 24 hours. Nuclear translocation and phosphorylation of the NFATc4 were evaluated. Aldosterone concentration was evaluated by ELISA in medium from 3T3-L1 adipocytes in the presence or absence of the inhibitors of calcineurin, cyclosporine A (CsA, 10-7M) and tacrolimus (FK506, 10-8M) and the specific NFAT inhibitor (VIVIT, 10-6M). Results: Ang II induced an increase in NFATc4 translocation into the nucleus (212%, p<0.01), which was blocked by candesartan, an AT1R antagonist (10-6M). This response was followed by a decrease in the phosphorylation of NFATc4 at 24 hrs of stimulation with Ang II (Ctrl vs Ang II, 1 ± 0.07 vs 0.64 ± 0.08, p<0.05), which was inhibited by CsA and FK506 (respectively, 1.34 ± 0.25 and 1.28 ± 0.18, p<0.05). Moreover, Ang II-induced increase in aldosterone production (Ctrl vs Ang II, 3.4 ± 0.3 vs 11.5 ± 2.7, n=6, p<0.001) was inhibited by CsA (3.5 ± 0.6, p<0.001), FK506 (2.9 ± 0.3, p<0.001) and VIVIT (4.2 ± 0.5, p<0.001). Conclusion: Our findings suggest that in calcineurin and NFAT play a role in adipocyte-derived Ang II-induced aldosterone synthesis.

41. Antimicrobial peptide, LL-37, as a potential vaginal contraceptive with anti-sexually transmitted disease (STD) property Nopparat Srakaew1,(PhD Candidate) Hongbin Xu1, Riccardo di Brisco1,2, Duriya Fongmoon1, Greanggrai Hommalai1, Krista Quesnel1, Charlene Young1,6, Wattana Weerachatyanukul3, Luigi Panza4, Fiamma Ronchetti2, and Nongnuj Tanphaichitr1,5,6 1Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; 2Dipartimeto di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Milano, Italy; 3Department of Anatomy, Faculty of Science, Mahidol University, Bangkok; 4Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale, Novara, Italy; 5Department of Obsterics/Gynecology, and 6Department of Biochemistry/Microbiology/Immunology, Faculty of Medicine, University of Ottawa, Ontario, Canada Background: Currently, there is a strong need for effective contraceptives that also have anti-sexually transmitted disease (STD) property. The use of natural compounds, e.g., antimicrobial peptides (AMPs), which are present in the reproductive system and have broad-spectrum of microbicidal activities, is promising. hCAP-18 found in the semen is a propeptide of the AMP, LL-37, with anti-microbial/HIV-1 properties. Processing of hCAP-18 to LL-37 occurs in the vagina 2-6 h post-coitus, by which time sperm have entered the uterus. LL-37 therefore protects the vagina from pathogenic attacks. However, it was unclear whether LL-37 had any effects on sperm. Since sperm contain anionic sulfogalactosylglycerolipid (SGG), LL-37 may interact with the sperm surface in the same manner as its interaction with an anionic bacterial membrane lipid, phosphatidylglycerol. It is possible that the LL-37-sperm interaction may lead to sperm dysfunctions.

Objective: To determine whether LL-37 can be used to induce sperm dysfunctions. Methods: LL-37-SGG binding was determined by ELISA. Binding of exogenous LL-37 to live mouse/human sperm was detected by immunoblotting and immunofluorescence. Sperm fertilizing abilities were measured from various parameters: motility, viability, acrosomal status and in vitro fertilization. Cytotoxic effects of LL-37 on vaginal/cervical epithelial cell lines were assessed by MTT assay and Sytox Green staining. Results: LL-37 decreased mouse and human sperm viability, motility and mouse sperm fertilizing ability in a dose dependent manner. At 3.6 µM LL-37, , motility and fertilizing ability of LL-37 treated mouse sperm were zero, whereas sperm viability was 25% of the control value. The loss of sperm fertilizing ability was due to premature acrosomal exocytosis; ~80% of mouse sperm treated with 3.6 µM LL-37 became acrosome reacted. The effect of LL-37 on the sperm was likely due to SGG affinity (Kd = 455.7 ± 59.1 nM). LL-37 at 3.6 µM was not toxic to the vaginal/cervical epithelial cell lines. Conclusions: LL-37 markedly inhibited sperm fertilizing ability without cytotoxicity to vaginal/cervical epithelial cells. The selective adverse effects of LL-37 on sperm may be due to its interaction with SGG, which may lead to acrosomal exocytosis. Our results suggest that LL-37 can be potentially developed into a vaginal contraceptive with anti-STD actions.

42. The role of gelsolin in regulating chemoresistance in ovarian cancer Bao Kong (Ph.D Student), OHRI&University of Ottawa; Heidi M. McBride (Associate Professor), University of Ottawa Heart Institute; Benjamin K. Tsang (Professor), OHRI and University of Ottawa Background: Chemoresistance is an important reason for failure of treatment of ovarian cancer (OVCA). Gelsolin was found to be involved in the regulation of apoptosis and chemoresistance of OVCA cells. Drp1 is known to be a mediator of mitochondrial fragmentation. Objective: To better understand the role of Gelsolin in regulating apoptosis and chemoresistance in OVCA. Methods: Gelsolin was knocked-down using SiRNA in chemosensitive OVCA cells with or without CDDP treatment. Apoptosis was examined with Hoechst assay. The levels of Gelsolin and Drp1 in mitochondria fraction were also checked when chemosensitive OVCA cells were treated with CDDP. Possible binding of Gelsolin and Drp1 was explored in chemosensitive OVCA cells with IP. Results: Down-regulation of Gelsolin attenuated CDDP induced apoptosis in chemosensitive OVCA cells. CDDP caused accumulation of Gelsolin and Drp1 in mitochondria and mitochondrial fragmentation. Drp1 was found to bind to Gelsolin in chemosensitive OVCA cells. Conclusion: Gelsolin possibly binds to Drp1 and facilitates mitochondrial fragmentation in chemosensitive OVCA cells when treated with CDDP.

43. Characterization of dysregulated ovarian follicular development in rat model for polycystic ovarian syndrome Kim JY (Postdoctoral fellow), Wang Q, Xue K, Leader A and Tsang BK Background: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder, which is characterized by anovulatory infertility, hyperandrogenism and insulin resistance. Also, it is associated with suppressed granulosa cell proliferation, differentiation and ovarian follicular growth arrest at the early antral stage of development. Objective: To characterize the dysregulated ovarian follicular development in rat PCOS model Methods: Immature 21 day-old rats were implanted with a silastic capsule with daily release of 83 µg Dihydrotestosterone (DHT) to mimic the hyperandrogenic state in women with PCOS. After implanting for 12 weeks, animals were sacrificed and ovaries collected from control (CTL) and DHT-treated rats were fixed with formalin, embedded with paraffin, and sectioned for morphological (HE staining and PAS staining) and immunohistochemical analysis. Expression of cleaved casapse-3, aromatase, calpain, its substrates (vimentine, fodrin and b-tubulin), p-AKT and PTEN was analyzed by immunofluorescence. In order to study whether hyperandrogen induces granulosa cells apoptosis, apoptosis-related molecules (caspase-3, PARP, XIAP and p-AKT) in granulosa cells cultured with DHT were examined by Western blot in vitro. Results: DHT treatment resulted in ovary structural changes, which is associated with decreased number of follicles in preantral to preovulatory stages and absence of corpus luteum but increased number of condensed atypical follicles. Atypical follicles in DHT-rats constitute predominantly theca cells. Follicle exhibited higher calpain expression in DHT-rats compared to CTL. This response is inversely correlated with down-regulation of cytoskeleton proteins (vimentin, fodrin and β-tubulin) which are known substrates of calpain. Granulosa cell aromatase expression is down-regulated and apoptosis (active-caspase-3 and DNA fragmentation) increased in follicles of DHT-rats. Survival protein phospho-AKT (Ser473) is decreased in granulosa cells in DHT-rats. However, PTEN level is considerably higher, suggesting that down-regulation of phospho-AKT may be induced by PTEN in vivo. DHT induced granulosa cells apoptosis and downregulation of XAIP, PARP and phospho-AKT contents in a dose-dependent manner in vitro. Conclusion: The dysregulated follicular development in PCOS rat ovary, which is characterized as condensed ovarian structure, decreased number of late stage follicles, absence of corpus luteum and increased atypical follicles. Atypical follicle constitutes predominantly theca cells, which is associated with calpain activation and granulosa cell apoptosis by casapase-3. All these results suggest that the cell death modulators such as caspase-3 and calpain may contribute to the dysregulated follicular development in rat PCOS model.

44. Involvement of CyclinD2 and HuR in FSH-stimulated granulosa cell proliferation Yingying Han (PhD Student), Guoliang Xia and Benjamin K. Tsang Background: Follicle stimulating hormone (FSH) is the main hormone to drive granulosa cell proliferation and follicular growth. And Cyclin D2 is required for FSH-stimulated granulosa cell proliferation. However, the cellular mechanism by which FSH stimulates granulosa cell proliferation is not fully understood. HuR (human antigen R) is a ubiquitous RNA-binding protein affecting mRNA stability during cellular proliferation process. It has been proposed that the mRNA-stabilizing influence of HuR requires its translocation to the cytoplasm. However, the function and regulation mechanism of HuR in Follicle-Stimulating Hormone (FSH) regulated follicle development process is poorly understand. Here we propose the hypothesis that FSH regulate granulosa cell proliferation though increase cyclin D2 half-life at the early culture stage, which is due to HuR translocation from nucleus to cytoplasm through PKA signal pathway. Objective: To

examine whether HuR involved in FSH regulates granulose cell proliferation with stabilization of cyclin D2 mRNA. Methods: Granulosa cells in the present experiment were obtained from DES-injected rat ovaries. Granulosa cell proliferation was examined by both alamar blue assay and flow cytometry. EdU staining was also used to check the DNA synthesis condition. Cyclin D2 mRAN and protein expression were checked at different culture time (2h, 6h, 12h and 24h). And cyclin D2 mRNA half-life will be detect with adding actinnomycin D into the culture medium. The subcellular location of HuR was firstly analyzed by immunofuorescence in rat both ovary and granulosa cells. HuR knockdown and overexpress will be done in the future, and cyclin D2 expression and granulosa cell proliferation will be checked at the same time. Result: FSH significantly increase alamar blue reduction and cells percentage in G2/M phase at 24h of in vitro culture. Interesting, FSH stimulated EdU absorption and cells percentage in S phase happened very quickly (2h), and disappeared at the lateral culture stage (12h and 24h). At the same time, we proved FSH increase cyclin D2 mRNA and protein expression only at the early culture stage (2h and 6h) but not the lateral culture stage (12h and 24h). We proposed that the quick cyclin D2 mRNA increase due to posttranscriptional regulation, which stabilize cyclin D2 mRNA at 2h. In the present study, HuR is widely expressed in rat ovary, predominantly (>90%) localized in the nuclei of granulosa cells. Our next work is to check whether FSH increase HuR stability and regulate HuR translocation in granulosa cells at 2h of in vitro culture.

45. Induction of heme oxygenase-1 inhibits development of type 1 diabetes in BBdp rats Mahmoud Husseini (MSc Student) Jennifer A. Crookshank (Senior Research Technician) Dr. Gen-Sheng Wang (Research Associate) Dr. Fraser W. Scott (Senior Scientist) Background: Type 1 diabetes (T1D) is an autoimmune disease involving loss of immune tolerance to insulin-producing β-cells in the pancreas. There is increasing evidence to suggest a role for the cytoprotective enzyme heme oxygenase-1 (HO-1) in controlling components of the innate and adaptive immune systems. Induction of HO-1 by cobalt protoporphyrin (CoPP) protects NOD mice from developing T1D. Through the breakdown of toxic heme, HO-1 produces potent antioxidants and suppresses inflammatory immune responses. Previous studies by our group and others indicated that animals and humans with T1D display abnormal pro-inflammatory responses in the gut immune system where the majority of immune cells in the body reside. This raised the possibility of immune dysregulation in the gut. Our preliminary data indicate that BioBreeding diabetes-prone (BBdp) rats have fewer gut-resident M2 CD163+ macrophages compared with control rats. Hypothesis/Rationale: Because HO-1 is the rate-limiting enzyme involved in the CD163 macrophage-dependent heme degradation pathway, we hypothesized that it could also be an important factor in T1D. Our aim was to investigate the effects of HO-1 upregulation on T1D development. Methods: Beginning at 30 d, BBdp rats were injected i.p. with CoPP (6.5 mg/kg, n=17) or saline (n=15) twice per week for three weeks to induce HO-1. Animals were sacrificed at ~51 d or monitored for development of T1D until 114 d. Results: T1D incidence was inhibited in CoPP-treated rats (CoPP, 27% vs. saline 65%, p=0.01). CoPP-treated animals (51 d, n=8/gp) showed a marked increase in HO-1 expression predominantly by CD68+ macrophages in lamina propria, pancreatic lymph nodes and pancreatic interstitial space. Confocal analyses of pancreas using anti-insulin and anti-HO-1 antibodies showed no HO-1+ cells in BBc (control) islets in contrast with BBdp islets which displayed abundant HO-1+ non-β-cells which were likely CD68+ macrophages. Initial flow cytometry analyses showed that isolated lamina propria mononuclear cells from BBdp rats displayed impaired upregulation of HO-1 in response to lipopolysaccharide and wheat gluten peptides compared with control animals. Conclusion: These data demonstrate a strong inhibitory effect of HO-1 induction on development of T1D possibly through modulation of the gut immune system and upregulation of cytoprotective processes in the pancreas.

46. Proteomic aAnalysis of sperm anterior head plasma membrane: unfolding the molecular components underlying sperm-egg interaction Kongmanas, K.1,2 (PhD Student), Sugeng, C. 1,2, Souda, P.4, Faull, K.F. 4, Whitelegge, J. 4, Tanphaichitr, N.1,2,3 1Chronic Diseases, Ottawa Hospital Research Institute, Ottawa, ON, Canada; 2Department of Biochemistry/Microbiology/Immunology, University of Ottawa, ON, Canada; 3Department of Obstetrics and Gynecology, University of Ottawa, ON, Canada; 4Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, USA Testicular sperm do not yet have the fertilizing competency including binding ability to zona pellucida (ZP), extracellular matrix glycoproteins surrounding the egg, and ability to fertilize eggs. To gain this ability, they have to undergo a maturation process called capacitation which involves reorganization and modification of sperm plasma membrane components. It is known that anterior sperm head plasma membrane (APM) is the site for ZP binding. Therefore, capacitation-related changes at this site would lead to acquisition of sperm full fertilizing ability. To understand the molecular basis underlying this process, we have performed quantitative mass spectrometry (MS) based proteomic analyses of APM vesicles isolated from non-capacitated and capacitated sperm using nitrogen cavitation. As expected, number of proteins, including egg-binding molecules (i.e., SED-1, ADAMs 1, 2 and 3) and chaperones (i.e., Hsp70), were present with higher amounts in capacitated APM. Blue native gel electrophoresis also revealed increased levels of high-molecular-weight (HMW) protein complexes in the capacitated APM samples. Moreover, far western blotting analysis using biotinylated ZP showed that the HMW protein complexes in the capacitated APM possessed the affinity for ZP. Altogether, the results from this study provide compelling evidence that multimeric protein complexes with ZP affinity were formed on the APM after capacitation to mediate sperm-egg interaction and fertilization.

47. Chemerin, a novel regulator in follicular steroidogenesis and its possible involvement in the pathogenesis of Polycystic Ovarian Syndrome (PCOS) Qi Wang (PhD Student), Jiyoung Kim, Kai Xue, Mingju Cao, Jia-Yin Liu, Arthur Leader and Benjamin K Tsang Departments of Cellular & Molecular Medicine and Obstetrics & Gynecology, University of Ottawa; Chronic Disease Program, Ottawa Hospital Research Institute; Ottawa Fertility Centre, Ottawa, Ontario, Canada; Centre for Clinical

Reproductive Medicine Nanjing Medical University, Nanjing, Peoples’ Republic of China Background: Polycystic ovarian syndrome (PCOS) is a heterogeneous syndrome and accounts for 75% of anovulatory infertility. It is typically associated with follicle growth arrest, minimal granulosa cell proliferation, dysregulated sex hormone profile, hyperthecosis and insulin resistance. Using a rodent model for PCOS which recapitulates the reproductive and metabolic phenotypes of the human conditions, we have begun to examine molecular and cellular mechanisms underlying the dysregulated reproductive and metabolic functions of PCOS. Chemerin is a novel adipokine and associated with obesity and metabolic syndrome. Elevated plasma chemerin level has been shown in obese women and PCOS patients. However, whether and how chemerin is involved in the regulation of follicular growth/steroidogenesis and pathogenesis of PCOS is unknown. Objective: To better understand the complex regulatory mechanisms involved in the control of ovarian follicular growth and steroidogenesis and gain insights in their dysregulation in the pathogenesis of PCOS. Methods: Immature rats (Day 21) were implanted with a silastic capsule with daily release of 83µg DHT to mimic the hyperandrogenic state in women with PCOS. After implanting for 12 weeks, ovaries were obtained for morphological analysis (IHC). Granulosa cells were collected by follicle puncture and cultured with FSH (0-100ng/ml, 48h)± testosterone (0-0.5µM). Estradiol and progesterone secretion (EIA) and steroidogenic enzymes expression (WB) were determined. In addition, granulosa cells and preantral follicles from immature rats were treated with chemerin (0-100ng/ml) ± FSH. The effects of chemerin on follicle growth (diameter), steroids production (EIA) and FSH-induced signaling were examined. Results: 1) PCOS rats exhibited increased body weight gain, decreased ovarian weight, disruption of estrus cyclicity, decreased insulin sensitivity and aberrant expression of the adipokine chemerin. 2) Increased serum and ovarian chemerin levels were associated with decreased progesterone/estradiol secretion and down-regulated p450scc and aromatase expression in granulosa cells from DHT-rats. 3) Chemerin and CMKLR were expressed in all ovarian cells and down-regulated by gonadotropin in vivo. 4) Chemerin suppressed FSH-induced preantral follicular growth and progesterone/estradiol secretion in preantral follicles and granulosa cells. Supported by CIHR, CIHR-QTNPR graduate and fellow scholarship (to QW and JYK) and CIHR-STIHRS Postdoctoral Fellowship (to MJC)

48. In vitro HIV infection reduces IL-17 expression by human Th17 cells Jason Fernandes (Postdoctoral Fellow) Ottawa Health Research Institute, Ottawa, Ontario Dr. Jonathan Angel (MD FRCPC, Supervisor, Department of Biochemistry Microbiology and Immunology, University of Ottawa, Ottawa, ON, Ottawa Health Research Institute, Ottawa, Ontario, Division of Infectious Disease, Ottawa Hospital - General Campus, Ottawa, ON) Background: T cell dysfunction persists in HIV+ individuals despite restoration of CD4+ T cell counts to near normal levels with HAART. Selective loss and deficiency of Th17 cells in the gut was recently described and may contribute to microbial translocation and persistent inflammation observed in HIV infection. It is unclear whether this deficiency results from HIV inhibiting Th17 cell differentiation or function. The known interaction of HIV regulatory proteins with intracellular signaling pathways related to differentiation and cytokine production may explain the ongoing deficiency in Th17 cells in HIV patients even after HAART. We hypothesize that Th17 differentiation and function is impaired in HIV infection due to altered intracellular signaling. Objectives: The objective of this study is to identify the effects of HIV on intracellular signalling pathways related to Th17 differentiation and function. Methods: CD4+ T cells were isolated from peripheral blood mononuclear cells and treated with antibodies against CD3 and CD28 in the presence of IL-1β, IL-6, and IL-23 for 7 days to generate Th17 cells as described previously. The resulting Th17 cells were infected with a dual tropic HIV strain (HIVCS204) for 24 hours. Following infection these cells were re-stimulated with PMA and ionomycin to stimulate cytokine production. Expression of Th17 cytokines was assessed by flow cytometry. Results: CD161 expression of cultured cells increased following activation in culture, consistent with literature describing in vitro Th17 differentiation. Re-stimulation of uninfected Th17 cells induced the expression of IL-17, a distinctive Th17 cytokine. IL-17 induction was inhibited when Th17 cells were infected in vitro with HIVCS204 prior to re-stimulation with PMA and ionomycin. Conclusion: In vitro HIV infection inhibits production of IL-17 by in vitro differentiated Th17 cells suggesting that HIV may play a role in altering Th17 responses through inhibition of upstream intracellular signaling pathways. Understanding the mechanism of this inhibition may provide insight into therapies that will correct the gut-associated immunopathogenesis in HIV infection.

49. Aldosterone-induces vascular pro-inflammatory and fibrotic effects through Nox1-dependent mechanisms independently of blood pressure changes. Montezano AC( Research Associate) , He Y, Ceravolo GS, Callera GE, Carter A, Yabe-Nishimura C, Touyz RM. Kidney Research Centre, Ottawa Hospital Research Institute, Ottawa, Canada. Aldosterone (Aldo) plays an important role in the pathogenesis of vascular remodelling, a condition where reactive oxygen species (ROS) formation is exacerbated. The vasculature possesses functionally active ROS-producing NADPH oxidase (Nox) enzymes, of which Nox1 may be important in remodelling. We hypothesized that Aldo-induced ROS-dependent vascular damage is mediated by activation of Nox 1. To address our hypothesis, we treated C57B6 (WT) and Nox1 knockout (KO) mice with Aldo (400 ug/Kg/day) or saline (Ctl) in osmotic minipumps for 4 weeks. Blood pressure was measured by tail-cuff. At the end of treatment, the mesentery was harvested and small arteries were collected for expression of MAP kinases and, pro-inflammatory (PAI-1), pro-fibrotic (fibronectin, pro-collagen I) and senescence (pSchp66 activation) markers (immunoblotting). Plasma was collected for analysis of potassium (K+), sodium (Na+), magnesium (Mg2+) and calcium (Ca2+) levels. Aldo did not increase blood pressure in WT and KO mice. Aldo decreased K+ plasma levels in both (p<0.05), WT (Ctl: 5 mmol/L; Aldo: 2.5 mmol/L) and KO (Ctl: 4.5 mmol/L; Aldo: 2.7 mmol/L) mice, with no changes in levels of Na+, Mg2+ or Ca2+. Aldo increased fibronectin (Ctl: 0.9 arbitrary units (AU), Aldo: 1.50 AU, p<0.05) and PAI-1 (Ctl: 0.7 AU, Aldo: 1 AU, p<0.05) expression, as well as JNK (Ctl: 0.6 AU, Aldo: 1.1 AU, p<0.05) and ERK1/2 (Ctl: 0.55 AU, Aldo: 1 AU, p<0.05) activation in WT mice. Aldo increased pShcp66 activation (Ctl: 0.75 AU, Aldo:

1.25 AU, p<0.05) in WT mice. No changes were observed in p38MAPK phosphorylation. Aldo did not induce any effect on proteins analyzed in mesenteric arteries from KO mice. In conclusion, our study suggests that Nox1 plays an important in Aldo-induced pro-fibrotic, inflammatory and senescence responses in vessel without modulating blood pressure.

50. Oxidative stress is involved in vascular smooth muscle cell differentiation to an osteogenic phenotype: role in hypertension. Yusuf H(BSc Student), Montezano AC(Research Associate), Zimmerman D(MD), Callera GE(Research Associate), Touyz RM(Supervisor). Vascular calcification, a process that involves vascular smooth muscle cells (VSMC) transformation to an osteoblast-like phenotype, is associated with hypertension, a condition where reactive oxygen species (ROS) production is increased. We previously demonstrated that magnesium (Mg2+) plays a protective role on VSMC calcification and also on oxidative stress and vascular dysfunction in hypertension. However, the role of ROS in vascular calcification is unclear. Here, we tested the hypothesis that oxidative stress induces VSMC mineralization. Cultured VSMCs from mesenteric arteries from WKY and SHRSP rats were exposed to calcification medium (CaM) (Ca2+ 1.8 mmol/L, PO4 2.0 mmol/L) for 10 days in the presence/absence of tempol (superoxide dismutase mimetic), diltiazem (L-type calcium channel blocker) and N-(p-amylcinnamoyl)anthranilic acid (ACA, TRPM2 inhibitor). Calcification was assessed by the expression of osteocalcin (OC) and BMP-2 (increase), and BMP-7 (decrease) by immunoblotting. p47 phox translocation (cytosol:membrane) was determined by immunoblotting. ROS generation was evaluated by chemioluminescence. ROS production (Ctl: 30 AU/ug protein; CaM: 60 AU, ug protein, p<0.05) and p47 translocation (Ctl: 0.7 AU; CaM: 1.1AU, p<0.05) were increased by the CaM in VSMCs from WKY. CaM-induced increase in OC and BMP-2 (p<0.05), followed by a decrease in BMP-7 (p<0.05), expression in VSMCs from WKY was inhibited by tempol, diltiazem and ACA. In SHRSP VSMCs, the increase in OC and BMP-2 expression (p<0.05) induced by the CaM was also blocked by tempol. TRPM2 expression (a redox-sensitive Ca2+ channel) was decreased by the CaM in WKY but not in SHRSP VSMCs (p<0.05). However, TRPM2-S expression, the short isoform and endogenous inhibitor of TRPM2, was decreased in WKY and SHRSP VSMCs (p<0.05). Inhibition of c-Src, a redox-sensitive non-receptor tyrosine kinase involved in osteoclast signalling, reduced the CaM-induced OC expression increase only in VSMCs from SHRSP. In conclusion, c-Src-regulated oxidative stress may play an important role in VSMC calcification, particularly in SHRSP.

51. Clusterin localization during mouse sperm capacitation: a marker for sperm capacitation? Suraj Kadunganattil (Postdoctoral Fellow) 123, Arpornrad Sae-wu 1, Kessiri Kongmanas 12, Lucy Nguyen 1, Steven Tshakatumba 1 and Nongnuj Tanphaichitr 123 1Chronic Diseases Program, OHRI, Ottawa, Canada 2Dept of Biochemistry, Microbiology and Immunology and 3Dept of Obstetrics/Gynecology, Faculty of Medicine, University of Ottawa, Ottawa, Canada Clusterin (CLU) is a sulfated glycoprotein having ubiquitous tissue distribution. It has been implicated in a plethora of biological processes such as sperm maturation, tissue differentiation and remodeling, membrane recycling, lipid transport, cell-cell interaction, cell proliferation, cell death and as a soluble chaperone. In the testis, CLU is one of the major secretory products of Sertoli cells. CLU is also secreted by the epididymal epithelial cells. CLU secreted by the male reproductive tract has been implicated in spermatogenesis, sperm protection, agglutination of abnormal spermatozoa and fertilization. In addition to the male reproductive tract secretions, CLU has also been detected on sperm surface. However, the function of sperm surface CLU has not been investigated. Mammalian sperm undergo membrane modification during maturation within the epididymis and in the female reproductive tract prior to fertilization. These maturational changes involve alterations in lipid and protein components of the sperm membrane. CLU has been shown to play a role in cholesterol efflux (lipid transport) from macrophage foam cells. Since cholesterol efflux is an important event accompanying sperm maturation our objective in the present study was to investigate the role of CLU in epididymal sperm maturation and in vitro capacitation of mouse sperm. Sperm isolated from caput and caudal epididymal regions showed different staining patterns for CLU. In caput sperm, only 50% showed CLU staining whereas majority of the caudal sperm stained for CLU. In the caudal epididymal sperm, immotile sperm showed bright CLU staining in the entire sperm head except the convex ridge. Motile caudal sperm showed less intense speckled staining in the entire head with absence of staining in the convex ridge. Following in vitro capacitation, caudal sperm showed strong CLU staining in the hook region of the head and this pattern represented the major percentage of stained cells. Thus, in vitro capacitation of sperm caused a re-localization of CLU. CLU re-localization on sperm was associated with cholesterol efflux as sperm with CLU staining in the hook region showed faint staining with filipin, an antibiotic which binds specifically to cholesterol. Our results suggest that clusterin re-localization during in vitro capacitation is an indicator of sperm fertilizing ability. We are currently investigating if changes in CLU distribution during in vitro capacitation play a role in facilitating sperm-egg interaction. These studies will thus help in defining a unique functional role for CLU in sperm.

52. Male subfertility: The incidence of lysosomal storage disorder in sertoli cells K. Kongmanas (PhD Student)1,2,*, S. Kadunganattil1,2,*, H. Xu1,2, C. E. Smith3, T. Rupar4, N. Goto-Inuoe6, L. Hermo3, K. Faull5, N. Tanphaichitr1,2 1Department of Biochemistry/Microbiology/Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada; 2Department of Chronic Diseases, Ottawa Hospital Research Institute, Ottawa, ON, Canada; 3Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada; 4Child and Parent Resource Institute, Health Research Institute and the Department of Paediatrics and Biochemistry, University of Western Ontario, London, ON, Canada; 5The Pasarow Mass Spectrometry Laboratory, University of California Los Angeles, CA, USA; 6Hamamatsu University, Hamamatsu, Shizuoka, Japan We have shown that arylsulfatase A (ASA) on the sperm head surface is involved in sperm-egg binding in vitro. In the

testis, ASA is localized to developing acrosomal granules of spermatogenic cells and lysosomes of Sertoli cells; ASA in Sertoli cells is enzymatically active, although its roles are unknown. In this report, we showed that ASA-/- mice up to 5-month old had normal sperm fertilizing ability and spermatogenesis. However, when the ASA-/- males became older these reproductive parameters as well as fecundity were significantly reduced. Concurrently, apoptotic germ cells (GCs) were highly accumulated (20x of wild type values) in the ASA-/- seminiferous tubules. Sulfogalactosylglycerolipid (SGG) exists specifically and substantially in male GCs; it plays roles in gamete binding and sperm lipid raft formation. Since ASA desulfates SGG in vitro, we quantified SGG in total testis lipids, testicular GCs and epididymal sperm in 8-month old ASA-/- and wild type mice by ESI- and imaging MS and flow cytometry. SGG levels were reduced to half of wild type values in all germ cells, but increased to 150% in total testes in ASA-/- mice. As a ZP binding molecule, the reduced SGG amounts in ASA-/- sperm explained their attenuated fertilizing ability. Since apoptotic GCs and/or their remnants (containing SGG) are normally uptaken by Sertoli cells for degradation, the increased SGG levels in ASA-/- mouse testes likely belonged to Sertoli cells due to ASA deficiency. This statement was supported by the preferential incorporation of SGG liposomes into cultured Sertoli cells as well as intracellular existence of SGG in Sertoli cells isolated from adult mice. Significantly, we observed lysosome swelling and lipid droplet accumulation, a typical lysosomal strorage disorder (LSD) feature, in 8-month old ASA-/- mice. Like sulfatide in brain cells of ASA deficient individuals, accumulated SGG in Sertoli cells likely exerted cytotoxicity, leading to dysfunctions of Sertoli cells in supporting spermatogenesis events; these included reduced SGG biosynthesis in germ cells, leading to the observed decreased sperm SGG levels in the ASA-/- mice. Our work reported here is the first evidence of LSD in Sertoli cells, which resulted in male subfertility.

53. IL-7 down regulates CD127 expression in human CD8 T cells through two independent mechanisms Feras Al Ghazawi(PhD Student), Elliott Faller, Scott Sugden, Juzer Kakal and Paul MacPherson Background: In view of the role interleukin (IL)-7 plays in T cell development, homeostasis, and activation it is no surprise expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. HIV Tat protein and IL-7 either alone or in synergy down regulate expression of CD127 on CD8 T cells. Here we show a detailed delineation of the suppressive effects of IL-7 on CD127 expression at both the level of transcription and surface protein in resting primary human CD8 T cells, and demonstrate for the first time that the loss of surface CD127 protein following stimulation with IL-7 is independent of transcriptional suppression. We also examine the signaling pathways activated by IL-7 and their involvement in down regulating both CD127 transcripts and surface protein. Methods: Primary human CD8 T cells from healthy HIV-negative volunteers were treated with IL-7 at concentrations ranging from 100 pg/ml-10 ng/ml for varying lengths of time and CD127 transcripts and surface protein were measured by qPCR and flow cytometry respectively. Levels of phospho-STAT3 and -STAT5 were measured in IL-7 stimulated cells and in untreated controls by flow cytometry. Inhibitors of protein translation, STAT5 phosphorylation, JAK and PI3K were used as described. CD127 transcripts and surface protein were similarly measured in a Jurkat cell line engineered to express the CD127 cDNA from the CMV promoter. Results: We show that IL-7 down-regulates the expression of CD127 transcripts and surface protein in resting human CD8 T cells in a time- and dose-dependent manner, and that constant and high levels of IL-7 (10 ng/ml) are required to maintain suppression. The maximal suppression of transcripts and surface protein occurs at 3 and 12 hours respectively. We also show that the early IL-7 mediated down regulation of surface CD127 protein occurs within 20 minutes and is independent of the transcriptional suppression which is delayed by 40-60 minutes. Further, we show in the Jurkat cell line IL-7 down regulates the expression of surface CD127 protein but not transcripts, confirming IL-7 binding results in the direct loss of CD127 from the cell surface. We also show that IL-7-mediated down regulation of CD127 transcripts in CD8 T cells is dependent on JAK/STAT5 signaling while the early down regulation of surface CD127 protein is JAK/STAT5-independent. Conclusions: IL-7 down regulates expression of the IL-7R alpha-chain by two independent mechanisms, by inducing the rapid removal of CD127 from the cell surface and by suppressing CD127 gene transcription.

54. Gene expression profiling of the gut in the LEW.1AR1-iddm rat fed type 1 diabetes-promoting or protective diets Ariana Noel (MSc Student), Christopher Patrick (PhD Student), Jennifer A. Crookshank (Senior Research Technician), Fraser W. Scott (Senior Scientist). Chronic Disease Program, Ottawa Hospital Research Institute. Background: Type 1 diabetes (T1D) is an autoimmune disorder in which beta cells of the endocrine pancreas are destroyed resulting in impaired glucoregulation. There is evidence that diet, increased gut permeability and inflammation play significant roles in the development of T1D in NOD mice, BBdp rats and humans. The gut epithelium is the major interface between the diet and immune system. We hypothesize that defects in the gut barrier and associated immune system also play a pivotal role in pathogenesis of T1D in the LEW.1AR1-iddm (LEW-DP) model. Objective: To investigate gene expression differences between the gut of LEW-DP and LEW-control (LEW-C) animals and identify effects of cereal and hydrolyzed casein (HC) diets on gene expression in the jejunum of 45 day LEW-DP rats. Methods: LEW-C and LEW-DP animals were fed a diabetes-promoting cereal diet or a protective HC diet. At end-of-study, the diabetes incidence of the LEW-DP on the HC diet was 38% versus 62% on the cereal diet (p=0.03). At 45 days, rats were euthanized and jejunum samples were snap frozen in liquid nitrogen. Total RNA was extracted from LEW-C cereal-fed (n=5), LEW-DP cereal-fed (n=6) and LEW-DP HC-fed animals (n=7) using the RNA II kit (Macherey-Nagel). RNA integrity was verified and concentration was quantified using a Nano Labchip with an Agilent 2100 Bioanalyzer. Rat Gene 1.0 ST microarrays were used to evaluate gene expression followed by identification of molecules, networks, and pathways. Microarray results were interrogated using bioinformatics tools such as Cytoscape with the Reactome plug in. cDNA was synthesized for the PCR array using the RT2 First Strand kit (SA Biosciences). Innate and Adaptive Immune Response PCR Arrays (SA Biosciences, PARN-052) were used to investigate differential expression of inflammation associated genes followed by analyses using SA Biosciences web-based software. Results: The results of the PCR array analyses demonstrated a bias towards pro-inflammatory conditions in the gut of LEW-DP rats. Strain: In the LEW-DP cereal-fed (n=4) versus the LEW-C cereal-fed (n=4) animals, fibronectin 1 (p=0.01) was downregulated possibly indicating dysfunction in regulatory T-

cell development in the LEW-DP rat. Diet: Genes promoting the inflammatory response were upregulated in the LEW-DP cereal-fed (n=4) when compared to LEW-DP HC-fed (n=4) rats. Tissue necrosis factor receptor superfamily 1a (p=0.0006) was upregulated in cereal-fed rats. Interleukin-1 family member 10 (p=0.04) and NF-kappaB inhibitor alpha (p=0.03) were both downregulated in cereal-fed rats. Interleukin-1 family member 10 shares similar structure and function to Interleukin-1 receptor antagonist. In general, the results of the microarray analysis were consistent with the gene expression changes observed both diet and strain analyses. Conclusion: The gene expression data from the PCR array analyses suggest that as in other animal models and some human patients, the LEW-DP gut is characterized by pro-inflammatory conditions that are reduced by a low antigen protective diet.

55. Decreased intestinal immune regulatory cells in autoimmune diabetes-prone LEW.1AR1-iddm rats – Modification by diet Christopher Patrick (PhD student), Jennifer A. Crookshank (Senior Research Technician), Dr. Gen-Sheng Wang (Research Associate), Ariana Noel (MSc student), Ariel Hendin (summmer student), Dr. Fraser W. Scott (Senior Scientist); Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; Dept. Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada Background: Gastrointestinal abnormalities are present in animal models and humans with type 1 diabetes (T1D), possibly contributing to T1D pathogenesis. T1D in LEW.1AR1-iddm (LEW-DP) rats is inhibited when animals are fed a low-antigen hydrolyzed casein (HC) diet compared with a standard cereal diet. Objective: Determine whether the small intestine of pre-diabetic LEW-DP rats displays altered immune cell populations compared with control LEW (LEW-C) rats and whether T1D-modifying diets alter inflammatory status. Methods: Jejunum samples from pre-diabetic 45 d cereal-fed LEW-C rats and cereal- or HC-fed LEW-DP rats were analyzed to evaluate immune cell distribution in the epithelium and lamina propria using immunohistochemistry and morphometry. RNA from jejunum was isolated to evaluate gene expression using qRT-PCR. Results: Strain: Pre-diabetic 45 d LEW-DP rats displayed fewer tolerogenic CD163+ M2 macrophages and Foxp3+ cells, as well as a decreased Foxp3+/CD3+ T cell ratio in lamina propria compared with control LEW-C rats. qRT-PCR analyses revealed decreased expression of immunoregulatory factors Foxp3 and CTLA-4 in LEW-DP compared with LEW-C rats. Diet: The Foxp3+/CD3+ T cell ratio was increased in HC-fed LEW-DP rats compared with cereal-fed LEW-DP rats. CD3+ lamina propria lymphocytes (LPL), CD3+ intra-epithelial lymphocytes (IEL) and CD8α+ IEL were decreased in HC-fed LEW-DP rats compared with cereal-fed LEW-DP rats. HC-fed LEW-DP rats displayed a decreased IFN-γ/IL-4 cytokine mRNA ratio compared with cereal-fed rats, suggesting amelioration of a Th1-biased inflammatory profile. Interestingly, the intestinal Foxp3+/CD3+ T cell ratio decreased significantly with age between 45 d and 100 d in cereal-fed LEW-DP rats but did not change in HC-fed LEW-DP rats nor in cereal-fed LEW-C rats. Preliminary microarray analyses of pooled intestinal RNA samples from these animals using Affymetrix Rat chips were consistent with these findings. Conclusion: Compared with control LEW-C rats, young LEW-DP rats exhibit decreased markers of intestinal immune regulation in conjunction with pro-inflammatory features before T1D onset. Inflammation was partly attenuated in LEW-DP rats by feeding a low-antigen HC diet.

Clinical Epidemiology Program

56. PRISMA-P: preferred reporting items for systematic reviews and meta-analyses David Moher (Senior Scientist), Larissa Shamseer (Research Coordinator, OHRI), Mike Clarke (Professor, University of Belfast), Davina Ghersi (Coordinator, International Clinical Trials Registry Platform, WHO) , Alessandro Liberati (Professor, University of Modena), Mark Petticrew (Professor, London School of Hygiene and Tropical Medicine), Paul Shekelle(Director, Southern California Evidence-Based Practice Center, RAND Corporation), Lesley Stewart (Director, Centre for REviews and Dissemination, University of York) Background: Systematic review protocols are seldom published (with some exceptions, such as the protocols for Cochrane reviews). Furthermore, for systematic reviewers interested in publishing their protocols, there is limited guidance currently. Where protocols are accessible, they show what was – and was not -planned for the review which might not be clearly understandable in published reports. Objectives: To develop a guideline to aid authors when preparing and reporting protocols. This will extend the PRISMA (Preferred Items for Reporting Systematic Reviews and Meta-Analyses (PRISMA) Statement, to provide guidance for protocols (PRISMAP). Methods: Development of PRISMAP will follow the process for reporting guideline development designed by the EQUATOR (Enhancing the QUAlity and Transparency Of health Research) group. Potential checklist items were identified through the Delphi process and extensive consensus-building during the development of PROSPERO – an international register for systematic review protocols. The PROSPERO items will be debated and further refined into a checklist for the reporting of systematic review protocols during a June 2011 meeting of international experts, facilitated by the developers of PROSPERO and experts in the development of reporting guidelines. The draft guideline with accompanying checklist will be shared at the 2011 Cochrane Colloquium. Potential Impact: The availability of a tool to help Cochrane and other systematic reviewers create and report protocols will hopefully improve the quality of both protocols and the subsequent reviews. PRISMAP might also make it easier for readers and peer reviewers to identify selective reporting biases in systematic reviews

57. The Influence of CONSORT on the quality of reporting on RCTs Lucy Turner (Research coordinator), Larissa Shamseer (Research coordinator), Doug G. Altman (Director of Center of Statistics and Medicine), Laura Weeks (Senior research associate), Jodi Peters (Research Coordinator), Ken Schulz (Director of FHI), David Moher (Senior Scientist)

Background: The Consolidated Standards of Reporting Trials (CONSORT) Statement was developed in response to concerns about the quality of reporting of randomized controlled trials (RCTs). It is an evidence-based minimum set of recommendations for reporting RCTs, intended to facilitate complete and transparent reporting and aid in critical appraisal and interpretation. A 2006 systematic review examining the effectiveness of CONSORT for improving the reporting of RCTs in endorsing journals (i.e. those which, at minimum, recommend that authors use CONSORT), found CONSORT endorsement to be associated with better quality of reporting, despite poor methodology of some included studies. Five years on from the publication of that review, an update is needed. Objective: To update the systematic review of CONSORT effectiveness by Plint et al. Methods: Conventional systematic review methods employed in the original review were followed. The search for new comparative studies evaluating the quality of reporting of RCTs spanned August 2005 – March 2010. Two reviewers independently screened studies for eligibility; data extraction and study validity assessments were conducted by a single reviewer and verified by a second reviewer. Results: In the five year period since publication of the original review, 41 new eligible studies were identified in addition to the eight included in the original review. When comparing endorsing and non-endorsing journals, items such as sequence generation, allocation concealment and participant flow were reported better in those endorsing CONSORT. Further details of the comparison between endorsers and non-endorsers as well as between trials published before and after CONSORT publication (both 1996 and 2001) will be presented. Impact: This review will provide further evidence on whether CONSORT is effective at improving the reporting of RCTs. This information will be helpful to authors, peer-reviewers and journal editors in helping to decide whether to endorse CONSORT.

58. Improving patient care: thoracic surgery quality monitoring information management and clinical documentation (TSQIC) Jelena Ivanovic, (MSc Student);2* Zeb Khan, MSc;1* Tim Ramsay, PhD;2 Donna E Maziak, MDCM, MSc, FRCSC;1,2 James Patrick Villeneuve, MD, PhD;1 Sebastian Gilbert, MD, FRCSC;1 Farid M Shamji, MBBS, FRCSC;1 R Sudhir Sundaresan, MD, FRCSC;1 Andrew JE Seely, MD, PhD, FRCSC.1,2 *Indicates presenting authors 1Department of Surgery, Division of Thoracic Surgery, University of Ottawa, the Ottawa Hospital – General Campus, 501 Smyth Road, K1H 8L6, Ottawa, Canada 2Clinical Epidemiology Program, Ottawa Hospital Research Institute, 501 Smyth Road, K1H 8L6, Ottawa, Canada Background: In the Ottawa Hospital’s (TOH) Thoracic Cancer Assessment Clinic, a paper-based, voice-dictation documentation system is currently in use; disadvantages of this type of system include length of time spent on documenting, delays in and significant costs of transcription, and variable standards and quality of documentation. Health information and communication technologies, such as electronic record keeping systems, are transforming the healthcare industry and improving efficiency of care. Building upon existing initiatives to standardize and improve surgical quality, such as the Ottawa Thoracic Morbidity and Mortality classification system, the Ottawa Division of Thoracic Surgery has created a web-based, iPad-optimized, point-of-care software application to perform Quality monitoring, Information management and Clinical documentation (TSQIC). Objective: The objective of this multi-year project is to scientifically evaluate the impact of TSQIC on documentation accuracy and completeness, efficiency of communication, as well as feasibility, usability and acceptability by health care providers and patients in the Ottawa Hospital’s Thoracic Cancer Assessment Clinic. Methods: The TSQIC has been migrated onto the Microsoft SQL server within the server farm of the TOH. All recording and reporting screens (eCRFs) are currently undergoing evaluation and further refinement with live patient data entry and reports. The scientific evaluation of the TSQIC will be performed after this run-in phase, with a clustered randomized controlled trial, with clustering of evaluations by clinic date. For each clinic date, all patients in the clinic will be randomly selected either to the intervention (TSQIC) or current documentation system (paper-based). Anticipated outcomes include: improved documentation accuracy and completeness (including legibility and comprehensiveness), promotion of communication among clinicians and patients, and enhancement of practice and information management within the TOH’s Thoracic Cancer Assessment Clinic. Results and Conclusions: This poster presentation will describe the process of TSQIC development, including content and software design. We will highlight the current and future objectives related to quality improvement, patient empowerment, and clinical research.

59. Determining the validity of the AMA guidelines: A retrospective analysis of the ADReS and rate of crash in older drivers. Andrew Woolnough1,2 (MSc Student), Danish Salim2 (B.Sc. Student), Malcolm Man-Son-Hing2,3,5 (MD MSC FRCPC), Shawn Marshall2,3,4,5 (MD MSC FRCPC) 1 Institute for Rehabilitation Research and Development 2 The CIHR Team in Driving in Older Persons (Candrive II) Research Program 3 University of Ottawa 4 The Ottawa Hospital Rehabilitation Centre 5 Clinical Epidemiology Program, Ottawa Health Research Institute Background: As a result of the aging population, the number of older persons holding a drivers license has increased [1, 2]. The chronic health conditions associated with aging can lead to changes in driving ability [1, 3]. Candrive is a CIHR-funded 5-year, prospective study aiming to develop an in-office screening tool that will help clinicians identify potentially at risk older drivers. Currently, no tools exist to directly predict crash risk. However, the American Medical Association (AMA) has designed guidelines to screen for older driver fitness. The AMA recommends that physicians use the Assessment of Driving Related Skills (ADReS) as a test battery to measure vision, cognition and motor/somatosenory functions related to driving. This functional assessment consists of visual acuity test (Snellen E chart), visual fields by confrontation test, trail-making test part B, clock drawing test, rapid pace walk, manual test of range of motion and motor strength. Objective: The following study aimed to use the data from the Candrive common cohort of older drivers to evaluate the validity of the ADReS protocol. Methods: Participants (n=932, aged 70 or older) were recruited from 7 Canadian cities and completed a comprehensive clinical assessment at initiation of the study. The assessment included all tests selected as part of the ADReS. A retrospective analysis was performed to investigate the association between the results of the ADReS tests and the motor vehicle crash rate. Retrospective crash data were obtained from their respective jurisdictions for all sites.

Data were collected on crashes that occurred within a 2-year time period preceding the baseline assessments. Data were analyzed in SPSS version 19 using the Pearson Chi-Square test. Results: Data obtained from the ministry of transportation indicated that 60 of the 932 participants (6.4%) were involved in a crash within the 2 year time period preceding the baseline assessment. The ADReS scores for vision, cognition and motor/somatosensory tests did not differ significantly between the crashers (n=60) and non-crashers (n=873) (p>0.05), which was contrary to what was expected from AMA guidelines. The lack of differences between groups could not be explained by other factors since no significant differences were observed between non-crashers and crashers for the driving habits, and physical and social activity status. Conclusions: Although limitations are inherent in a retrospective analysis, the results of the ADReS did not correlate with crash rate. This suggests the need for more sensitive tools to properly assess crash risk of older drivers. Reference: 1. J Am Geriatr Soc 2007, 55(6):878-884. 2. Gerontologist 2004, 44(2):237-244. 3. Traffic Inj Prev 2008, 9(4):291-298.

Neurosciences Program

60. Effect of ketamine on neural progenitor cell differentiation and astrocyte morphology in vitro Ushananthini Shanmugalingam (MSc Student), Matthew Coyle, Xudong Cao, Eve C. Tsai Neuroscience, Ottawa Hospital Research Institute, Ottawa Hospital (Civic Campus) University of Ottawa, Ontario, Canada Backgroud: Post central nervous system injury, astrocytes can form a glial scar that can restrict axon regeneration. Ketamine has been reported to interfere in the development of immature neurons. However, the effects of ketamine on glial development is less well understood. Objective: The aim of this study was to determine the effects of increasing doses of ketamine on brain and spinal cord derived endogenous neural progenitor cell (eNPC) differentiation and morphology. Methods: Adult female Sprague Dawley rat eNPCs were isolated from the subventricular zone (SVZ) of the brain and the sub-ependymal zone (SEZ) of the spinal cord. eNPCs were allowed to proliferate in the presence of epidermal growth factor and fibroblast growth factor for seven days. Following the proliferation period, equal numbers of neurospheres were plated on 96 well plates and exposed to concentrations of ketamine ranging from 0-100 ug/mL. The neurospheres were allowed to differentiate in serum-free media for two and seven days, after which the cells were fixed and stained for glial fibrillary acidic protein (GFAP) for astrocytes, brain lipid binding protein (BLBP) for radial glia, O4 for oligodendrocytes and Hoechst for the nucleus. Glial morphology was assessed following two days of ketamine exposure, and the proportion of differentiated glial cells was assessed following two and seven days of ketamine exposure. Results: We found that in the presence of ketamine, both SVZ and SEZ eNPCs differentiated into astrocytes, radial glia and oligodendrocytes. Following, a seven day long exposure to 100 ug/mL of ketamine, there was a significant increase in the proportion of differentiated astrocytes derived from SVZ eNPCs (ANOVA, p<0.05). However, no significant difference was found in the proportion of differentiated astrocytes derived from SVZ eNPCs following two days of ketamine exposure and SEZ eNPCs following both two and seven days of ketamine exposure (ANOVA, p>0.05). Hence, long term ketamine exposure increases the proportion of differentiated astrocytes derived from SVZ eNPCs but not SEZ eNPCs. Following, a two day long exposure to 100 ug/mL of ketamine, we found a significant decrease in astrocytic process length in both SVZ and SEZ derived eNPCs (ANOVA, p<0.05). Conclusions: Therefore, the administration of high doses of ketamine may be useful in restricting the growth of astrocytic processes, thereby decreasing the density of the glial scar allowing regenerating axons to pass through the injury site.

61. Transcriptional requirements for Parkinson’s-linked parkin gene expression Ao HS (PhD Student)1,2, Noble S3, Tomlinson J1,2, Lim KL4, Ekker M3, Schlossmacher MG1,2* 1Division of Neuroscience, Ottawa Hospital Research Institute; 2Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada; 3Department of Biology, University of Ottawa, Ottawa, ON, Canada; 4Genome Institute of Singapore, Singapore Background: Mutations in the Parkin gene are the most frequent known cause of heritable PD. Studies show that the Parkin protein can prevent cell damage and cell death. A reduction in normal human Parkin gene expression accompanied by lower Parkin protein activity leads to increased risk of PD. By inference, I hypothesize that increasing the level of normal Parkin expression represents a rational way to prevent or slow down the process of PD. However, currently little is known about how the Parkin gene is regulated. Examining the cis- and trans-regulatory elements of human Parkin gene has been problematic because of its large size (~1.4 Mb). Fortunately, the recent identification of fugu parkin has made it possible to begin addressing these outstanding questions. Fugu parkin and human Parkin are highly similar in their genomic organizations, tissue expression pattern, and protein function; and fugu parkin is approximately 4 kb which is 350-fold smaller than human Parkin, making it a useful model to investigate the transcriptional regulation of Parkin. Objective: To identify the cis- and trans- transcriptional regulatory elements and signaling pathways controlling Parkin expression; and to screen for small molecules and FDA approved drugs that can increase Parkin expression. Methods: I use fugu parkin genomic DNA in cellular and mammalian models. I have cloned several fugu and fugu-human hybrid parkin-reporter constructs that allow for monitoring of gene expression of the entire vertebrate parkin gene tagged with a reporter cDNA (eGFP or Luciferase). Results: Using these constructs, I have identified a strong repressor element in the -820 to -311 bp region of the 5’-flanking region of fugu parkin; and I found that the 310-bp core promoter can facilitate the transcription of fugu parkin. With these tools, I am now focusing on screening a library of human transcription factors to identify activators of Parkin expression. My preliminary findings suggest that GATA transcription factors can increase fugu parkin expression in cells. I will further determine the signaling pathways regulating Parkin transcription by studying different mitochondria stressors, UPR-related pathways, and human kinases by using an unbiased kinomics approach. Moreover, I will screen libraries of FDA approved drugs to identify compounds that can drive Parkin expression.

Conclusion: This study will enhance our understanding of the transcriptional mechanisms that regulate Parkin. Ultimately, we hope to be able to increase the expression of human Parkin pharmacologically to slow, stop or reverse the process of PD.

62. A role for integrin-linked kinase in oligodendrocyte mediated myelination of the central nervous system John-Paul Michalski (PhD Student), Ryan O’Meara (PhD student), Rashmi Kothary (Supervisor) Recent studies have shown integrin-linked kinase (ILK) to be a major structural binding partner of β1 integrin, an essential component of oligodendrocyte (OL) myelin sheet formation in the central nervous system (CNS). To study the role of ILK in CNS myelination in vivo and avoid embryonic lethality associated with ILK deletion, we have created a conditional knockout mouse model employing the Cre-loxP system. At P14, ILK deficient mice display marked amyelation of small caliber axons. However, by P28, these same fibers appear fully myelinated. The initial development loss of myelinated axons is not due to turnover or loss of ILK deficient OLs, nor P28 recovery to an influx of WT progenitors. Rather, the myelin deficiency is related to an inability of ILK depleted OLs to elaborate processes and properly form myelin membranes, suggesting an essential role for ILK in the early steps of OL-mediated myelination. Future work is directed towards identifying downstream signalling pathways that are perturbed in the ILK-deficient OLs.

Regenerative Medicine Program

63. To identify, validate and characterize the mutation on 18p which is responsible for causing Myoclonus-Dystonia

Megan Vanstone (MSc Student), Tara Read (MSc), Fabin Han (MD, PhD), David A. Grimes (MD, FRCPC), Dennis Bulman (PhD, Supervisor) Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The onset of symptoms usually occurs within the first two decades of life. People with MD also have an increased risk of developing psychological conditions such as depression and anxiety. The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds with the SGCE gene. We have identified a second MD locus (DYT15) in a large kindred spanning 5 generations, which maps to a 3.18 Mb region on 18p11. The coding regions and intron-exon boundaries of all known REFSEQ genes in the critical region were sequenced and did not yield the causative mutation. In total, 110 kb of the critical region was sequenced and we identified 358 novel SNPs and insertion/deletions, all of which have been detected in control samples. Large deletions, duplications and inversions have been excluded as a cause of MD in our family. Two meiotically distant patients from our family were chosen to undergo next-generation sequencing. First, NimbleGen Sequence Capture technology was used to enrich for DNA from the chr18:4,619,674-7,800,480 critical region plus 50 kb flanking, then Roche 454 sequencing technology was used to sequence the targeted region on 18p11. We obtained >97% coverage of our critical region with an average depth of coverage >50 per patient. Using NextGENeTM software from SOFTGENETICS, we have identified 2,413 shared novel variants within the critical region. I have assigned a priority to this list of variants and am currently attempting to exclude candidates by confirming each one by Sanger sequencing and validating their disease-causing potential by testing for their occurrence in control patients. To date, 2,325 candidate variants have been excluded and 88 remain, one of which will be the causative mutation of MD in our family.

64. Fasudil, a Rho-kinase inhibitor, improves survival of the Smn2B/- intermediate SMA mouse model Melissa Bowerman (PhD Student), Lyndsay M. Murray, Carrie L. Anderson and Rashmi Kothary Spinal muscular atrophy (SMA) is the most common genetic disease resulting in infant mortality in humans. SMA is characterized by a severe loss of alpha-motor neurons and is caused by mutations or deletions of the ubiquitously expressed survival motor neuron 1 (SMN1) gene. We have previously shown that Smn deficient PC12 neuronal-like cells have increased levels of active RhoA (RhoA-GTP), a well-characterized regulator of actin dynamics. We have further confirmed that RhoA-GTP is upregulated in the spinal cord of an intermediate SMA mouse model (Smn2B/-). To address the effect of RhoA modulation on the SMA phenotype, the Smn2B/- mice were treated with Y-27632, an inhibitor of Rho-kinase, which is a direct downstream effector of RhoA. This resulted in a significant increase in survival as well in the size of neuromuscular junctions (NMJs) and tibialis anterior (TA) myofibers. In the present work, we are assessing the effect of fasudil on the survival and phenotype of the Smn2B/- mice. Fasudil is a US FDA-approved rho-kinase inhibitor and vasodilator, commonly used in cardiovascular, neurodegenerative and cancer studies. We find that fasudil significantly increases the lifespan of SMA mice, without improving their weight or performance on the pen-test. Similarly to our Y-27632 experiments, it appears that the beneficial effects of fasudil are through an Smn-independent pathway. Our preliminary results also suggest that fasudil results in an increase in NMJ and myofiber size. We will thus further our analysis by identifying the pathways affected by fasudil in SMA muscle and assessing the effects of fasudil on motor neuron loss. We have also administered fasudil to Smn-/-;SMN2 ;Smn7/Smn7 mice, a more severe SMA mouse model. In these mice, fasudil did not improve lifespan. This is most likely due to the fact that the pathologies in this mouse are too severe and are not impacted by the benefits of fasudil in the Smn2B/- model. Of note, in both the Smn2B/-and Smn-/-;SMN2 ;Smn7/Smn7 models, we have observed a non-negligible toxicity with fasudil. We have thus demonstrated that two independent rho-kinase inhibitors, Y-27632 and the USFDA-approved fasudil significantly increase the life expectancy of the intermediate Smn2B/- SMA mouse model. This strengthens the idea that modulators of actin dynamics may serve as valuable therapeutic strategies for SMA.

65. Brown adipose determination in adult skeletal muscle satellite cells is regulated by microRNA-133 Hang Yin, (Postdoctoral Fellow) Alessandra Pasut, Vahab D. Soleimani, C. Florian Bentzinger, Patrick Seale, Wilfred van IJcken, Frank Grosveld, Mary-Ellen Harper and Michael A. Rudnicki Brown adipose tissue (BAT) dissipates chemical energy in the form of heat to maintain body thermoregulation. In embryonic development, a lineage switch from skeletal muscle to BAT is governed by Prdm16, a zinc finger transcription factor. Prdm16 activates the brown adipogenic program while repressing the myogenic program. However, whether myogenic cells can be a source of brown adipocytes in adults remains unknown. Here, we report that adult satellite cells can differentiate into myocytes and brown adipocytes. Furthermore, a muscle specific microRNA miR-133 controls the switch from myogenic to brown adipogenic by repressing the expression of Prdm16 in satellite cells. Knockdown of miR-133 in satellite cells during muscle regeneration extensively promotes their brown adipogenic commitment and induces functional brown adipocytes in vivo. Reversely, overexpression of miR-133 represses Prdm16 in brown preadipocytes and impedes their adipogenic differentiation. In addition, we find that Pax7 is a bona fide transcriptional activator of miR-133 in myocytes. These data reveal a central function of miR-133 in controlling lineage switching from muscle stem cells to brown adipocytes and suggest a therapeutic approach for the prevention and/or treatment of obesity by inducing BAT within muscle.

66. Comparative expression profiling identifies CD53 as a p38-dependent protein that modulates myoblast fusion Qi-Cai Liu (Postdoctoral fellow), Xiaohui Zha, Herve Faralli, Hang Yin, Caroline Louis-Jeune, Eusebio Perdiguero, Erinija Pranckeviciene, Pura Muñoz-Cànoves, Michael A. Rudnicki, Marjorie Brand, Carol Perez-Iratxeta, and F. Jeffrey Dilworth Myogenic differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38 MAPK (p38) signaling. As p38 signaling can modulate Myog expression, we developed a cell culture system that allows the uncoupling of these interdependent myogenic factors to evaluate their distinct functions in myogenesis. Using comparative expression profiling, we demonstrate that exogenous Myog expression in the absence of p38 signaling is sufficient for myoblasts to exit cell cycle, express multiple myogenic markers and align. However, these myoblasts fail to form multinucleated myotubes revealing a critical role for p38 in fusion. Analysis of p38 up-regulated genes identified the tetraspanin CD53 as a candidate fusogen. The essential role for CD53 in cell fusion was confirmed in primary myoblasts, NIH3T3 fibroblasts undergoing myogenesis, and during myofiber regeneration in mice. Thus, our comparative expression analysis has identified CD53 as a novel fusion protein required for myogenesis.

67. Modeling Hutchinson-Gilford progeria syndrome using induced-pluripotent stem cells Zhaoyi Chen (Graduate Student), Wing Y. Chang, William L. Stanford Background: Hutchinson-Gilford Progeria Syndrome (HGPS) patients exhibit traits of premature aging which are believed to be due, in part, to an accumulation of mutations in numerous cell-types, causing rapid tissue deterioration. This disease is characterized as a laminopathy-based disease, where a heterozygous mutation in the LMNA gene cause the accumulation of mutant prelamin A protein termed Progerin, leading to downstream effects such as alterations in gene expression and heterochromatin formation, including the disruption in the HP1 proteins distribution. The molecular mechanism of altered gene expression is relatively unknown due to lack of experimental models. Aim: HGPS-induced pluripotent stem cells (iPSCs) were used to model the disease to dissect the molecular mechanism underlying this genetic disorder. Previous studies have shown that reprogramming can reset epigenetic factors in disease-specific cells. Therefore it is also possible that chromatin formation and HP1 expression can be reset in HGPS cells upon reprogramming. By characterizing HGPS iPSCs, we aim to examine whether reprogrammed cells have the capacity to differentiate normally, whether reprogramming can reset HP1 binding in HGPS cells, how mutation in LMNA gene in HGPS cells can affect chromatin formation and the localization pattern of HP1 upon differentiation. Method: Cells derived from Progeria patients were reprogrammed into iPSCs through retroviral infection using four transcription factors: Oct4, Klf4, Sox-2, and c-Myc. Cell potency of each reprogrammed cell line is characterized using alkaline phosphatase staining and immunofluorescence staining of cell surface pluripotent markers. iPSC colonies were induced to form embryonic bodies, which are then plated to allow growth and differentiation into the three germ layers. Their differentiation capabilities were characterized through immunofluorescence staining using differentiation marker antibodies. To characterize the expression of HP1 in iPSCs, immunofluorescence staining was performed on the cultured iPSC lines using the antibodies for HP1-α, HP1-β, and HP1-γ. Stained cells were imaged under fluorescence microscope. Results / Conclusions: iPSCs derived from patients diagnosed with HGPS exhibited traits of pluripotency, and could differentiate into cells characteristic of the three germ layers. Reprogramming can reset perturbed gene expression in iPSCs caused by mutation in the LMNA gene, reverse HP1 binding distribution back to the normal state. However, the localization pattern of HP1 changes upon differentiation upon heterochromatin formation. In general, a disease model to investigate the progression of HGPS was developed from the study.

68. Snf2l regulates Foxg1-dependent progenitor cell expansion in the developing brain Chelsea P. Corcoran1, 2 (MSc Student), Darren J. Yip1, 2, Matias Alvarez-Saveedra1,3, Adriana DeMaria1, Stephen Rennick1, 2, Michael A. Rudnicki1, 3, Claude Messier4, and David J. Picketts1, 2 1Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON Canada; Departments of 2Biochemistry, Microbiology, and Immunology; and 3Cellular and Molecular Medicine; and the 4School of Psychology, University of Ottawa, Ottawa, ON, Canada Background: Control of progenitor self-renewal and/or differentiation during neurogenesis is critical for regulating brain

size and structure. The epigenetic regulators involved in these pathways have not been fully elucidated. Snf2l (Smarca1) is an ISWI family chromatin remodeling protein that was previously shown to be upregulated during brain development and whose ectopic expression in vitro induces neuronal differentiation. Results: Inactivation of the Snf2l gene causes hypercellularity of both early and late-born neurons in the developing mouse forebrain. Progenitor cells have enhanced self-renewal and delayed neurogenesis. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Foxg1 is important in the maintenance of intermediate progenitor cells which are concordantly increased in Snf2l-inactivated forebrain. Reducing Foxg1 gene dosage attenuated the phenotype thereby placing Snf2l and Foxg1 in the same genetic pathway. Finally, Snf2l is bound to the Foxg1 gene at mid-neurogenesis. Conclusion: Snf2l chromatin remodeling is an antagonistic effector of Foxg1 and an important determinant of brain size.

69. Shift estimation for ChIP-Seq data analysis using whole genome cross correlation Parameswaran Ramachandran (Postdoctoral Fellow), Christopher Porter, Gareth Palidwor, and Theodore J. Perkins Background: Chromatin immunoprecipitation (ChIP) followed by tag sequencing (ChIP-Seq) using high-throughput sequencing technologies is becoming the preferred approach for mapping the sites of transcription-factor binding and chromatin modification. An important step in the ChIP-Seq analysis pipeline is the reliable estimation of the average fragment size. Typically, peak-calling algorithms either try to estimate this parameter from a small set of randomly-sampled peaks thereby yielding limited accuracy or leave it to the user to specify a suitable value. In order to reliably obtain an accurate estimate of the fragment-size, it is necessary to take into account the information pertaining to the entire genome. This calls for a technique that is computationally very efficient. Objective: Here we propose a computationally efficient technique for reliably estimating the average fragment size using the data corresponding to the entire genome. Methods: A robust cross-correlation measure between the plus and minus strand reads is proposed and is used to obtain the shift estimate. Results: Our preliminary tests on benchmark datasets have proven that the technique is both robust and accurate. Conclusions: Fragment-size estimation or shift estimation is a critical step in analyzing ChIP-Seq data that affects the accuracy of peak-calling. The proposed technique, based on the cross-correlation of the plus and the minus strand reads across the entire genome, achieves this objective in a reliable and an accurate manner

70. The role of integrin linked kinase in oligodendrocyte development Ryan OMeara (PhD student), John-Paul Michalski (PhD student), Rashmi Kothary (principal investigator) Interactions between the extracellular matrix (ECM) and the β1-integrin signaling pathway within oligodendrocytes (OLs) are key for the production of myelin. A major downstream effector of β1-integrin signaling is Integrin-Linked Kinase (ILK), which is involved in stabilizing focal adhesions and transducing ECM signals. The current study will determine the importance of ILK in the development and myelinating capacity of primary murine OLs. Two primary cell culture models have been developed to achieve this goal. The first, an enriched oligodendrocyte precursor (OPC) culture, results in a purified population of OLs which may be differentiated over a 6 day time course to produce MBP+ mature OLs. In the second model, purified OLs are co-cultured with dorsal root ganglion neurons (DRGNs) to produce myelinating co-cultures. To determine the importance of ILK in OL development, OLs were harvested from Ilkfl/fl;mT/mG mice. In these mice, the exons coding for the ILK kinase domain are flanked by LoxP sites. Upon administration of recombinant TAT-Cre recombinase, the ILK kinase domain is excised and the treated cells are depleted in ILK protein. OL morphological differentiation is perturbed upon ablation of ILK. ILK-null OLs display simple morphology at both DIV3 and DIV6, with reduced arborization and membrane sheets as compared to control OLs. When co-cultured with DRGNs, ILK-null OLs have a reduced capacity to enwrap DRGN neurites as compared to controls. Future work will determine possible changes in downstream signaling cascades that may underlie observed morphological defects. The results of this study will allow for a better understanding of key players involved in the maturation and myelination capacity of OLs.

71. Neuronal Dystonin Isoform 2 is a novel mediator of endoplasmic reticulum structure and function Andrew Ferrier (PhD student) 1, 2*, Scott D. Ryan1*, Tadasu Sato1 , Ryan O’Meara, Yves de Repentigny1, Susan X. Jiang3, Sheng T. Hou3 and Rashmi Kothary1 ,2 1Ottawa Hospital Research Institute, Ottawa, Ontario, Canada K1H 8L6 2Department of Cellular and Molecular Medicine and the Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 3Institute for Biological Sciences, National Research Council Canada, 1200 Montreal Road, Bldg M54, Ottawa, Ontario, Canada K1A 0R6 *these authors contributed equally to the work Dystonin/Bpag1 is a cytoskeletal linker protein whose loss of function in dystonia musculorum mice (dt mice) results in a hereditary sensory neuropathy with a progressive loss of limb coordination. While loss of expression of two major neuronal dystonin isoforms (dystonin-a1 and dystonin-a2) is sufficient to cause dt pathogenesis, it remains unclear whether a single isoform or combination of isoforms are responsible for dt pathology, and what mechanisms are activated upon their loss. Here we show dt primary sensory neurons manifest ultra-structural defects at the endoplasmic reticulum resulting in development of neurodegenerative pathologies including: abnormal Ca2+ homeostasis, induction of the unfolded protein response, caspase activation and programmed cell death. Furthermore, isoform specific loss of function analysis in F11 sensory neurons demonstrates that the observed neurodegenerative pathologies are a result of specific loss of the dystonin-a2 isoform. Blockade of the ER-stress signaling cascade through either inhibition of caspase activation or administration of the ER-stress protective agent salubrinal partially rescued cell death in both dystonin-a2 deficient F11 neurons and dt primary sensory neurons. Ultimately, this study provides insight into the etiology of dt neuropathology and proposes a novel role for the dystonin-a2 isoform as a mediator of normal ER structure and function.

72. Investigating the role of Pax7 DNA binding domains. Andrew Jones ( PhD student), Vincent Punch, Yoichi Kawabe and Michael Rudnicki The postnatal development of skeletal muscle is dependent on a population of myogenic stem cells known as satellite cells. The development and maintenance of the satellite cell lineage is dependant on the activity of the paired-box transcription factor Pax7. Alternative splicing events of the highly conserved paired-box domain (PD) of Pax7 generates four functional isoforms due to the inclusion or exclusion of a glutamine (Q+/-) residue and/or a glycine-leucine (GL+/-) dipeptide. While these various isoforms are believed to exhibit distinct tertiary structures and possess differential DNA binding affinities and specificities, a specific function for the Pax7 alternative splicing events has not been described. Our findings suggest that while all isoforms can induce the proliferative myogenic regulatory factor Myf5 in myogenic cells, only two Pax7 isoforms (Q-GL-and Q+GL-) can initiate Myf5 expression in non-myogenic cell types, suggesting a functional importance for this GL+/- splicing event. In addition, mutational analyses of the closely related Pax3 indicate a functional interaction between the two Pax DNA binding domains; the PD and the homeodomain (HD). To date, the functional specificities of the Pax7 PD and HD remain to be elucidated. In an attempt to determine how Pax7’s ability to bind DNA alters target gene expression, we employed Pax7 mutant constructs containing functionally relevant point mutations in the paired domain (G48S) or the homeodomain (N265A). In addition, the HD has been suggested to mediate potential dimer formation and early studies suggest the HD of Pax7 binds cooperatively to specific DNA sequences. Elucidation of the molecular mechanisms regulating Pax7 expression and DNA binding, and further understanding of the isoform-specific target gene regulation, will provide a greater understanding of the molecular regulation present during myogenesis.

73. Muscle growth and regeneration defects in Atrx deficient mice is caused by mitotic catastrophe in proliferating myoblasts Michael S. Huh (Postdoctoral Fellow) Tina Price O’Dea, Dahmane Ouazia, Bruce McKay, Robin J. Parks, Michael A. Rudnicki, David J. Picketts Ottawa Hospital Research Institute ATR-X mental retardation syndrome is caused by inherited mutations in the human ATRX gene. The ATRX gene encodes a SWI/SNF like chromatin remodeling protein that is enriched at pericentromeric and telomeric heterochromatin. Males affected with ATR-X syndrome have severe skeletal muscle weakness and hypotonia at birth. To investigate the role of ATRX in muscle development a Cre-LoxP approach was used to conditionally inactivate the Atrx gene in skeletal muscle. Atrxf/y:Myf5Cre+/- (cKO) mice displayed a striking reduction in muscle and body mass at P21, yet possessed normal numbers of resident muscle satellite cells. We examined the activation, proliferative, and regenerative capacity of cKO satellite cells in vivo by cardiotoxin (CTX) injection and in vitro by single muscle fiber cultures. CTX injury in cKO mice demonstrated a marked regeneration deficit and cultured cKO muscle fibers gave rise to a 10-fold reduction in total myoblasts. Furthermore, by using Ad-Cre infected Atrxf/y (KO) myoblasts we ascertained that Atrx was not essential for myoblast differentiation but important in maintaining genomic stability while transiting through S-phase. We determined that Atrx protein colocalized to late replicating chromatin and KO myoblasts were delayed through late S-phase. Moreover, activated ATM and elevated p53 levels were detected in KO myoblasts, a response typically associated with replication dependent DNA damage. Consistent with a replication defect, the deletion of Atrx also resulted in a higher percentage of H2AX nuclear foci, increased percentage of binucleated cells, and profoundly disorganized mitotic spindle apparatus. Thus removal of Atrx had a detrimental effect on genomic stability and ultimately the viability of proliferating myoblasts. Taken together, we propose that Atrx chromatin remodeling activity is necessary during late S-phase in the replication or re-establishment of heterochromatin. Hence Atrx function is required during periods of rapid muscle growth and regeneration that require hyper-expansion of myoblasts necessitating multiple rounds of proper DNA replication and the re-establishment of chromatin structure.

74. Motor neuron plasticity in a mouse model of the childhood motor neuron disease Spinal Muscular Atrophy. Lyndsay Murray (Postdoctoral Fellow), Rashmi Kothary Spinal muscular atrophy is a devastating childhood motor neurons disease caused by mutations or deletions within the survival motor neuron 1 (SMN1) gene. It is currently unclear why motor neurons, and specifically neuromuscular junctions are primary pathological targets following a reduction in this ubiquitous protein. Defects in axon outgrowth have been observed in cellular models of SMA however do not appear to translate to developmental defects in mouse models. These defects may therefore result in compromised synaptic maintenance at the NMJ. This is consistent with the observation that developmentally more plastic neurons display a marked resistance to degeneration. In this study we have investigated anatomical plasticity at the neuromuscular junction in muscles which are differentially vulnerable to pathology in SMA to investigate whether differential plasticity can either contribute to or cause pathology in SMA. We have employed the intermediate Smn2B/- mouse model of SMA to investigate the capacity of motor neurons to remodel and adapt to a range of physiological scenarios, including developmental pruning, nerve injury and toxin induced paralysis. Results suggest that a reduction in Smn has no impact upon nerve regeneration following traumatic injury, indicating axon growth occurs normally in SMA. Although there was no change in the rate of re-innervation and the establishment of new synaptic connections, we observed a defect in nerve induced clustering of acetyl choline receptors. We further found a decrease the amount of paralysis induced remodeling in response to botulinum toxin A injection in Smn2B/- mice. This work will give import insight in to the physiological impact of reduced levels of Smn on motor neurons.

75. Towards a functional understanding of PHF6, a nucleolar zinc finger protein mutated in Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia. Matthew A.M. Todd (PhD Student) and David Picketts.

Mutations in genes that modify chromatin structure have been recognized as a cause of X-linked intellectual disability (XLID). These genes encode proteins that regulate DNA methylation (MECP2), modify histones (JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Börjeson-Forssman-Lehmann syndrome (BFLS) is an XLID characterized by mild to severe mental deficit, truncal obesity, gynaecomastia, large ears, hypogonadism, and digit abnormalities. Mutations in the PHD finger protein 6 (PHF6) gene (Xq26.3) were identified as the cause of BFLS. These mutations occur throughout the gene and include missense, nonsense, and inactivating mutations. More recently, we and others have identified inactivating PHF6 mutations in patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML), suggesting the involvement of PHF6 not only in developmental disease, but also in acquired disease. Moreover, we have reported on two patients with PHF6 mutations afflicted with BFLS and either T-ALL (R342X) or Hodgkin’s lymphoma (R257G). PHF6 protein contains two atypical PHD zinc fingers that share homology with the MLL family of histone methyltransferases although the function of PHF6 remains largely unknown. To begin to elucidate a role for PHF6, we present results from a multidisciplinary approach. Using tandem affinity purification, we have been able to co-purify several interacting proteins that suggest PHF6 is a component of an ATP-dependent chromatin remodeling complex. Moreover, we have used immunocytochemistry to track the localization dynamics of PHF6 within the nucleus and in nucleoli throughout the cell cycle. Finally, gain- and loss-of function studies have been used to determine its importance to cellular growth. Taken together, the results from these experiments are helping us to better understand the molecular mechanisms that link PHF6 mutations with the BFLS and T-ALL disease states.

76. The ISWI homologue Snf2h is essential for cerebellar development. Matías Alvarez-Saavedra (PhD Student), Pamela S. Lagali, Chelsea Corcoran, Keqin Yan and David J Picketts. Regenerative Medicine, OHRI & Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada. Background: The cerebellum is involved in sensorimotor function and balance control and more recently has been implicated in a wide range of cognitive functions. The developing cerebellum is biochemically, functionally and anatomically compartmentalized by genetic signals arising from three types of neuronal precursors: granule neuronal precursors (GNPs), Purkinje cells (PCs) and neurons of the deep cerebellar nuclei (DPN). The appropriate temporal migration, differentiation and cytoarchitecture of these cell types orchestrate cerebellar histogenesis. The ISWI proteins Snf2h and Snf2l are ATPase-containing subunits of multiple protein complexes with diverse functions that include nucleosome assembly and spacing during DNA replication as well as transcriptional regulation. In the developing brain, Snf2h expression is prevalent in progenitor cells, while Snf2l expression increases during terminal neuronal differentiation. Importantly, the in-vivo role of these chromatin-remodeling proteins in the mammalian brain is not understood. Objective: Using a conditionally targeted Snf2h mouse model, we have eliminated Snf2h expression in cerebellar progenitors. This mouse model will allow us to examine the role of Snf2h in cerebellar development. Results: We show that the mammalian ISWI homologue Snf2h is dynamically expressed in the cerebellum and its deletion in cerebellar progenitors results in decreased cell proliferation. In addition, we observe lamination abnormalities resulting in a disorganized Purkinje cell layer. Concomitantly, these abnormalities result in deregulated cerebellar foliation causing severe ataxia and early death. Interestingly, mutant mice display abnormal expression profiles of a wide variety of signaling molecules, including Sonic Hedgehog (Shh). In addition, we have discovered that the homeobox transcription factors Engrailed-1/2 are also misregulated. Conclusion: This work provides the first evidence that the ISWI homologue Snf2h is dynamically expressed in the cerebellum and is essential for the proper development of the cerebellum. Snf2h loss in cerebellar progenitors results in abnormal cell proliferation, lamination and PC layer formation. In combination, these cellular phenotypes lead to abnormal foliation and cerebellar topography. We propose that Snf2h is a master regulator of cerebellar development acting upstream of the evolutionary conserved Engrailed homeobox transcription factors. This work provides insight into the neurogenetic mechanisms required for the development, establishment and maintenance of neuronal circuits.

77. Determining the mutation underlying a novel 46, XY Disorder of Sexual Development (DSD) using next-generation sequencing strategies Hood, R.L.(PhD student)1,2,3, Douglas, S. 3, Goldsmith, C.3, Bulman, D.E.2, and Boycott, K.M.3 1Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario; 2Ottawa Hospital Research Institute, Ottawa, Ontario; 3Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, Canada. The development of next-generation sequencing technologies has revolutionized modern genetics and enabled researchers to elucidate novel biological applications at unprecedented speeds. Through this technology one can experimentally determine the causative gene(s) or variants underlying a particular disease. We describe a family with a novel 46, XY disorder of sexual development (DSD) inherited in an autosomal dominant manner. Intrafamilial variability was evident with clinical presentations ranging from normal male (obligate carrier) to varying degrees of hypospadias to female external genitalia in the 7 affected male family members. The genetic cause of this disorder is currently unknown as mutations in genes known to be involved in the sexual development pathway, including SRY, NR5A1, DHH, NROB1, SOX9, WT1, and MAP3K1, were excluded via clinical testing and segregation analysis with linked markers. The involvement of WNT4 could not be excluded by linkage, although it is considered an unlikely candidate as duplication, the known mechanism of mutation, was excluded by analysis of array data. Therefore, we plan on utilizing next-generation sequencing to experimentally identify the DSD gene and its causative mutation. It will then be possible to determine the biological significance this gene has in human sexual development. This study will elucidate additional molecular components and mechanisms underlying secondary sex-characteristics, thereby contributing to our overall understanding of sex differentiation and determination.

78. The application of whole-exome resequencing identifies a homozygous mutation in WDR62 in Franco-Ontarian siblings with epilepsy, microcephaly and cognitive impairment. L. M. McDonel1 (M.Sc. Student),3,4, D. Foster 2, F. C. C. FORGE Canada Consortium3, D. E. Bulman1,4, K. M. Boycott3,4 1 Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; 2 Algoma Public Health Unit, Sault Ste Marie, Ontario; 3 Children ’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario; 4 Department of Biochemistry Microbiology and Immunology, University of Ottawa, Ottawa, Ontario. Homozyogsity mapping in consanguineous families coupled with whole-exome resequencing has been successful in identifying the causal variant for more than nine Mendelian disorders including Ochoa syndrome and van Den Ende-Gupta syndrome. Exome resequencing facilitates the rapid and efficient discovery of novel mutations by revealing variants in regions which are identical-by-descent (IBD) amongst affected individuals. This approach enables the study of rare disorders in small families where the region(s) of interest is potentially large and/or gene-rich. Here we report a consanguineous Franco-Ontarian family with four affected siblings; two males and two females. The parents are first cousins. The disorder in this family is characterized by early-onset severe epilepsy that is difficult to control requiring the use of multiple anticonvulsants, microcephaly with occipital-parietal circumference of -3SD and severe cognitive impairment. The siblings are now adults and all live in a supported care setting. Language is limited to short phrases in the two female siblings and is essentially absent in the male siblings. Dysmorphic features, minor anomalies or other neurological features are absent. Fragile X testing, MECP2 and metabolic investigations were unrevealing. The four affected family members were genotyped using SNP 6.0 arrays (Affymetrix). Homozygosity mapping using Homozygosity Mapper lead to the identification of multiple regions of IBD on chromosomes 2, 13 and 19 involving in total 326 genes. Exome resequencing of one affected family member using the Agilent Sureselect 50Mb target enrichment method coupled with the Illumina Hi-Seq platform identified a homozygous frame shift mutation in WDR62, a recently recognized autosomal recessive primary microcephaly gene.

79. Circulating microRNAs as blood-based biomarkers of pulmonary arterial hypertension Kenny Schlosser (Postdocotoral Fellow), James White (Scientist), Duncan J. Stewart (Principal Investigator) Background: Pulmonary arterial hypertension (PAH) is a disease characterized by a progressive increase in lung vascular resistance that leads to right heart failure and premature death. The prognosis for PAH patients is grave, with a survival after first diagnosis of only 3-5 years. Early diagnosis and treatment can decrease morbidity and mortality; however, diagnosis remains a challenge as the early stages of PAH can be asymptomatic, and frequently present with only non-specific symptoms such as laboured breathing and fatigue. The detection of the disease is further limited by the poor accessibility of the pulmonary blood vessels, which necessitates the use of a specialized procedure (i.e., right heart catheterization, RHC) to obtain accurate blood pressure measurements to confirm the diagnosis. Objective: We sought to identify a new practical blood-based biomarker of PAH that could facilitate diagnosis and even monitor the response to therapy. Methods: Human plasma was obtained from 9 treatment-naive patients with confirmed PAH (as determined by RHC), and 4 normal control individuals. Total RNA was extracted from plasma and the levels of 85 different microRNAs (miRNA) were measured using RT-qPCR. MiRNA expression levels were normalized to the geomean of several novel internal miRNA reference genes that were identified specifically for this model system by systematically evaluating the expression stability of each miRNA using two different mathematical algorithms. Results: Forty-eight different microRNAs were detectable at varying levels across patient plasma samples. The mean expression level of one miRNA in particular, miR-30e, was significantly higher in PAH patients compared to normal controls (~3 fold; P<0.05). The area under the receiver-operator characteristic curve of miR-30e was 0.86 (P<0.05), suggesting that this miRNA can distinguish between PAH and normal individuals with significant sensitivity and specificity. Conclusions: Plasma-derived miR-30e represents a novel biomarker candidate for PAH. We anticipate that future genome-wide profiling of plasma miRNA levels in larger patient cohorts will identify additional biomarker candidates that collectively may provide a robust disease-specific signature of PAH.

80. Caspase 3 promotes skeletal muscle regeneration by limiting satellite cell self-renewal Sarah A. Dick (PhD Student)1,2 and Lynn A. Megeney1,2 1 Regenerative Medicine Program, Ottawa Health Research Institute, Sprott Centre for Stem Cell Research, Ottawa, Canada. 2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. Satellite stem cells are indispensable for the growth and regeneration of skeletal muscle. Nevertheless, the mechanisms that determine whether activated satellite cells self renew or differentiate into committed muscle progenitors remains unknown. Caspase proteases are traditionally viewed as conserved cell death proteins, yet recent observations have demonstrated that caspase activity is required for muscle stem cell differentiation. Here, we sought to identify whether caspase 3 influenced cell fate by altering the balance between self renewal and differentiation. Using the isolated single fiber model, we have observed that caspase 3 activity is elevated during early stages of satellite cell derived myogenesis. Specifically, we have noted that caspase activity is restricted to Pax7(+) satellite cells and not the mature myonuclei. Peptide inhibition of caspase 3 activity resulted in increased numbers and clusters of the self-renewing Pax7(+)/MyoD(-) satellite cells with a corresponding decrease in the number of proliferating Pax7(+)/MyoD(+) cells. These results suggest that caspase 3 is a key determinant in cell fate decisions and promotes differentiation in part by limiting satellite cell self renewal.

81. Development of a drug screen platform to study the molecular regulation of muscle stem cells in their niche Yu Xin (Will) Wang (MSc Student), Michael Rudnicki (PI) Muscle regeneration in injury and disease requires the activation, proliferation and differentiation of muscle satellite cells. Recent research has demonstrated that satellite cells are self-renewing and compose of a heterogeneous population of stem cells and progenitors. Our lab has identified the satellite stem cell population (Kuang et al., 2007; Cell) and demonstrated that increasing the tissue content of these cells results in a direct enhancement on regeneration kinetics (Le Grand et al., 2009; Cell stem cell). However, the maintenance and regulation of satellite stem cells is not well understood. To study the cell signalling network controlling the homeostatic levels of satellite stem cells and their response in regeneration, we have developed a novel drug screen platform to examine the proliferation kinetics of satellite stem cells on ex-vivo cultured muscle fibers. Screening against a library of compounds targeting developmental pathways (~380 compounds) will allows us to identify cellular pathways that modulate the proliferation of satellite stem cells. Characterization of these target pathways will unveil new insight into the molecular regulation of these stem cells during regeneration. In addition, compounds with a positive effect have the potential to be directly translated into therapeutic options for degenerative muscle disorders.

82. Pax7 dependent regulation of Notch 1 is required to promote the expansion of muscle stem cells Alessandra Pasut (PhD student), Vahab Soleimani (PhD), Michael Rudnicki (PhD) OHRI Regenerative medicine program and University of Ottawa, Faculty of Medicine, CMM department. Muscle regeneration, the process by which new fibers are generated, heavily relies on the contribution of resident stem cells called satellite cells. After an injury satellite cells activate to give rise to a population of rapidly dividing muscle progenitors (myoblasts) that eventually differentiate into elongated myotubes. Precocious differentiation leads to poor muscle regeneration. Pax7 is a major regulator of satellite cells fate and it is expressed by satellite cells as well as myoblasts but down regulated upon differentiation. Previous studies suggest that Notch signalling is required to delay muscle differentiation and allow stem cell proliferation. Here we performed global gene expression analysis and show that Notch signalling is up-regulated in proliferating myoblasts compared to differentiating myotubes. siRNA mediated knockdown of Pax7 in primary myoblasts cultures leads to a 60-70% down regulation of Notch receptors and target genes and up-regulation of differentiation genes. On the contrary, over-expression of Pax7 or Notch1 leads to increased cell proliferation and delay differentiation. Additionally over expression of Notch 1 partially rescues the precocious differentiation phenotype observed in Pax7 knock out mice. Chromatin immunoprecipitation followed by real time PCR (ChIP-QPCR) shows that Pax7 binds to a -125Kb element upstream of Notch1 starting sites. Altogether these data show that Notch 1 is a direct target gene of Pax7 and that active Notch signalling is required to promote muscle stem cell proliferation.

83. MLL dependent and MLL independent activation of Pax7 target genes

Gregory C. Addicks (PhD Student), OHRI, Regenerative Medicine Program, University of Ottawa, Department of Cellular and Molecular Medicine, Michael A. Rudnicki, OHRI, Regenerative Medicine Program, University of Ottawa, Department of Cellular and Molecular Medicine Background: Following muscle injury or strength training exercise, satellite cells residing in the muscle proliferate and fuse, renewing or increasing the size and strength of the muscle. The number of satellite cells and regenerative capacity of muscle decrease during aging and in diseases such as muscular dystrophy. During development and in the adult, the transcription factor Pax7 regulates genes necessary for maintenance and proliferation of satellite cells and is required for survival of these cells. Objective: This project aims to build a better understanding of the requirement for Pax7 in satellite cells and satellite cell derived myoblasts, and how Pax7 functions to regulate these cells. Methods: Myoblasts were isolated from genetically modified mice in which the Mixed Lineage Leukemia (MLL) H3K4 methyltransferase gene is knocked out by addition of tamoxifen via Cre-recombinase. In normal and MLL knockout (KO) myoblasts, Pax7 and Pax7 target gene expression were determined by immunostaining, western blot, qPCR and microarray analysis. Proliferation was determined by hemocytometer. Results: We have found that to activate genes, Pax7 recruits MLL, a histone methyltransferase, which relaxes the binding of DNA to histones, facilitating access for transcription. Previous studies have found that MLL is required for expression of developmental regulators that are necessary to maintain other types of adult stem cells. In cultured MLL KO myoblasts, expression of Pax7 is weakly reduced, but expression of Pax7 targets is lost, as is proliferation. Over-expression of Pax7 overcomes the MLL KO defect and Pax7 target gene expression and proliferation are recovered. In microarray analysis of MLL KO cells, it is expected that expression of Pax7 targets and possibly other critical Pax7 independent developmental regulators will be decreased. Conclusions: Since MLL KO only weekly effects Pax7 expression but Pax7 target gene expression is completely lost it seems that MLL is necessary for Pax7 to activate its target genes. Over-expression of Pax7 in MLL KO cells is able to rescue this effect; with Pax7 over-expression, Pax7 target genes are expressed in the absence of MLL. Since sufficiently high levels of Pax7 expression can overcome the loss of MLL, high amounts of Pax7 may be able to activate genes without the aid of epigenetic modification, or high amounts of Pax7 can recruit a weekly interacting substitute H3K4 methyltransferase to activate target genes. Alternatively, MLL may be required for expression of an unknown transcription factor that normally co-activates Pax7 target genes but is unnecessary with Pax7 over-expression.

84. Transcriptional networks controlling human embryonic stem cell fate Bingyu Betty Li (Msc Student) Background Human embryonic stem cells (hESCs) not only have enormous therapeutic potential, they can also be used

to elucidate disease pathology related to development. Uncovering the genetic network regulating hESC self-renewal and lineage commitment is essential to developing new restorative treatments. Previously in our lab, an integrative approach combining data from transcription factor binding with temporal expression microarray analysis has identified novel regulators of the mESC. From the 63 predicted nodes for mESC self-renewal and commitment, 3 targeted nodes were selected for further analysis in hESC: Sall4, Dach1, Evx1, and Zbtb8a. Sall4 was identified as essential in mediating mESC self-renewal. Dach1 and Evx1 were up-regulated following self-renewal factor withdrawal. Our lab also has shown that Zbtb8a knockdown is associated with Oct4 mRNA down-regulation. Objective The goal of my work is to build transcriptional networks using the 4 selected transcription factors (TF), and then validate the networks in hESC. Method First, construct a modular, multi-featured, inducible over-expression and knockdown piggybac vector for each gene of interest. Then analyze the impact of inducible shRNA knockdown and cDNA over-expression on hESC fate using RT-PCR on critical pluripotent and lineage markers to validate its function as critical node governing hESC fate. Following that, perform RNA-sequencing on inducible shRNA knockdown and over-expression hESCs upon withdrawal of self-renewal factors. This allows us to analyze target TF function under differentiation conditions. Finally, perform ChIP sequencing assay on undifferentiated hESCs for pluripotent genes, and hESCs following self-renewal factor withdrawal for commitment genes to define binding sites to build transcriptional network. The network will be tested using compound knockdowns. Results We are currently constructing a piggybac vector with selected cDNA/shRNA expression through doxycyclin induction for each cDNA and shRNA. The vector is multi-featured, which includes a Hist-Flag tag for immuno-precipitation, selection markers, and insulators to prevent epigenetic silencing. Our lab has previously obtained high transfection efficiency with piggybac vector containing a constitutive crimson expressor gene, validating the piggybac system. Currently, the piggybac constructs are to near completion for selected cDNA and shRNA. Conclusion Identifying novel transcriptional nodes will allow us to draft transcriptional networks of hESC and further our understanding of pluripotency and commitment regulation. This process can be re-iterated to construct a more comprehensive gene regulatory network. Overall, knowledge in gene regulation will provide us with further understanding of human development and disease.

85. MicroRNA misregulation in multiple sclerosis - impacts on oligodendrocytes and their ability to differentiate, mature and myelinate axons in the central nervous system. Samantha F. Kornfeld (MSc Student)1,2 Rashmi Kothary 1,2 1 Ottawa Hospital Research Institute, Ottawa ON 2Faculty of Medicine, University of Ottawa, Ottawa ON Multiple Sclerosis (MS) is a progressive, demyelinating disease of the central nervous system (CSN). MicroRNAs (miRNAs) have been identified as factors that are consistently misregulated in brain lesions from patients with MS. However, the impacts of these misregulated miRNAs in MS have yet to be deduced. In the course of MS, oligodendrocytes (OLs) - the cells responsible for the myelination of axons in the CNS - appear to have a lessened ability to repair damaged myelin as well as a reduction in maturation from precursor cells. Since miRNAs have been shown to have strong control over the differentiation and myelination processes in OLs, it is likely that misregulation of miRNAs during MS has negative effects on OL maturation and myelination. Three miRNAs misregulated in MS, miRNAs -20a, -223 and -145, are all predicted to target cytoskeletal factors that may be important to OLs’ capacity for membrane extension and myelin deposition around axons. Our preliminary research has revealed that these three miRNAs are expressed in Oli-neu cells, an immortalized mouse OL cell line. To further characterize their role in OL maturation, we have investigated their expression patterns by RT-PCR in Oli-neu cells across three time points. Future work will include manipulation of the expression levels of these miRNAs using lentiviral vectors initially in Oli-neu cells and primary mouse cultures, and finally in a mouse model of MS. Target validation for miRNAs -20a, -223 and -145 will be also be explored by protein microarray. Ultimately, rescue of myelination by OLs through manipulation of miRNA levels along with miRNA target validation may reveal valuable therapeutic targets in the treatment of MS

86. Cellular and molecular mechanisms underlying Atrx-mediated retinal interneuron survival Pamela S. Lagali (Postdoctoral Fellow)1,2,3; Chantal F. Medina 1,3; Keqin Yan 1,3; Alan J. Mears 2,4,5; Valerie A. Wallace 2,4,5 and David J. Picketts 1,3 1Regenerative Medicine and 2Vision Programs, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; 3Department of Medicine, Biochemistry, Microbiology, Immunology and 4Department of Ophthalmology, University of Ottawa, Ontario, Canada; 5University of Ottawa Eye Institute, University of Ottawa, Ottawa, Canada Background: Retinal degenerative diseases involve the progressive loss of photoreceptor cells of the retina and are the leading cause of blindness in the developed world. Therapeutic strategies that aim to replace or bypass the lost photoreceptors require the integrity and proper connectivity of the remaining retinal neurons. The survival and functional circuitry of retinal interneurons downstream of the photoreceptors is essential for visual signal processing and transmission leading to effective visual perception. We have generated a mouse model in which retinal amacrine and horizontal cells, the inhibitory interneurons critical for modulation and integration of synaptic activity in the retina, are selectively lost. We are using this model system to delineate the mechanisms that govern retinal interneuron homeostasis and communication in normal and disease states. Objective: To determine the neuronal circuitry and genetic regulation underlying the loss of retinal amacrine and horizontal cells in mice lacking the chromatin remodeling protein Atrx Methods: We use conditional knockout approaches to selectively remove Atrx from different retinal cell populations in vivo, including the production of transgenic mice that express Cre recombinase and the ocular injection and electroporation of Cre-expressing plasmids targeted to specific cell types. Atrx-deleted tissues are analyzed by immunohistochemistry and fluorescence microscopy. Retinal function is examined by electroretinography. Gene expression changes are assessed with DNA microarrays and quantitative RT-PCR. Results: Amacrine and horizontal cell disorganization and loss occurs when Atrx is deleted in multipotent retinal progenitor cells using a Pax6alpha-Cre transgenic mouse driver line, but not when Atrx is inactivated in post-mitotic amacrine and horizontal precursor cells targeted with a Ptf1a-Cre driver line.

Electroretinograms show functional deficits in interneuron communication within the inner retina of the Pax6alpha-Cre-driven conditional knockout mice, suggesting a role for bipolar neurons in the observed phenotype. Deletion of Atrx postnatally through the injection and retinal expression of Cre-encoding plasmids recapitulates some features of the early knockout phenotype, further suggestive of a causative role played by later born neurons such as bipolar cells. In addition, genetic profiling of the mutant mice reveals misexpression of the bipolar cell marker gene Nk3r, as well as genes that function in retinal synaptic communication such as Tac2 and Chrnb2. Conclusions: The loss of amacrine and horizontal cells from Atrx-deleted retinas appears to occur through a cell non-autonomous mechanism. Electrophysiological analysis, cell type-selective gene inactivation in the retina and gene expression profiling implicate a role for bipolar cells in mediating Atrx-dependent retinal inhibitory interneuron survival and function. Atrx may be involved in the regulation of specific genes that play a role in retinal neuron homeostasis, synaptic activity, and connectivity.

87. Intrinsic properties of aged satellite cells affects myogenic regeneration Feodor Price (Phd Student), Julia von Maltzahn (PhD, Post doc), Gareth Palidwor (Msc, Bioinformatasist), Alexander Grayston (Honors Student), Michael A Rudnicki {Background} Adult skeletal muscle possesses a remarkable regenerative capacity due primarily to the satellite cell, a resident adult stem cell. A progressive loss of muscle mass is observed in aged mice and is due to a combination of factors including denervation, changes in the local satellite cell niche, the systemic environment and a potential age related decline in satellite cells. (Objective) Our experiments set out to identify differences between fetal (E17.5), adolescent (3wks) adult (3months) and aged (1yr) satellite cells in an attempt to better explain an age dependent deficiency in skeletal muscle regeneration. (Methods) Pure populations of satellite cells were isolated from limb skeletal muscle of the Pax7zsgreen reporter mouse. Freshly isolated cells were subsequently analyzed by microarray as well as cultured to determine their capacity to proliferate and differentiate. (Results) We observed differences in the cell surface complement of satellite cells as they age. To determine functional differences in muscle regeneration sorted satellite cells were transplanted into mdx mice. An increased regenerative capacity, as assessed by dystrophin positive fibers and zsgreen positive cells, was observed upon transplantation of fetal and 3 week old satellite cells in comparison to isolated adult satellite cells. (conclusions) Although previous age related experiments dealing with skeletal muscle regeneration indicate that the local and systemic environment are critical in regulating skeletal muscle regeneration, our results support the notion that there exist satellite cell intrinsic properties that affect their ability to contribute to muscle regeneration. Further analysis of the mechanisms that regulate these functional properties is of paramount importance as a therapeutic goal and as a means to better understand the basic biology of satellite cells.

88. The role of endothelial apoptosis in the pathogenesis of pulmonary arterial hypertension Heather A.M. Goldthorpe (MSc Student), Jin-Yi Jiang, Shirley H.J. Mei, and Duncan J. Stewart Ottawa Hospital Research Institute and University of Ottawa, Ottawa, Ontario, Canada Introduction: Pulmonary arterial hypertension (PAH) is a complex, lethal disease. PAH is characterized by progressive pulmonary vascular remodeling and increased pulmonary vascular resistance (PVR), leading to increased pulmonary arterial pressure and ultimately right heart failure. Recent evidence points to endothelial cell (EC) apoptosis at the level of distal pulmonary arterioles as the underlying mechanism of this disease. Objective:To establish a titratable, conditional transgenic system to induce EC apoptosis in order to test the hypothesis that lung EC apoptosis is necessary and sufficient to cause PAH. Methods and Results: In a pilot study, the Fas-Induced Apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promotor in transgenic mice (i.e. EFIA mice). Administration of a small molecule dimerizing agent, AP6283, resulted in lung microvascular apotosis, and modest dose-dependent PAH, which was associated with obliterative and highly proliferative vascular lesions localized to distal lung arterioles in a small proporation of mice. However, due to the low level of transgene expression in our current EFIA mouse lines, we have re-designed the transgenic vector by incorporating a more robust endothelial promoter (superTie2), which consists of a 2.1 kb Tie2 promoter and a 1.7 kb fragment from the first intron of the Tie2 sequence. The FIA construct has been successfully sub-cloned along with a reporter gene into this superTie2 expression vector. The new EFIA vector is currently undergoing validation in comparison to our previous EFIA construct in vitro, and will ultimately be used for the generation of new EFIA mice. Conclusion: Our pilot data suggest that EC apoptosis alone is sufficient to induce a PAH phenotype with the characteristic complex lung vascular lesions. The supertie2 transgenic model will enhance the expression of the EFIA transgene, and allow us to better explore the mechanism that links distal lung EC apoptosis with reactive vascular cell proliferation in the pathogenesis of this devastating disease.

89. Development of gene therapy for spinal muscular atrophy Benoit Goulet1,2 (MSc Student), Rashmi Kothary1, and Robin J. Parks1,2 1 Regenerative Medicine Program, Ottawa Hospital Research Institute 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa. Background : Spinal muscular atrophy (SMA) is a disease characterized by the degeneration of the lower motor neurons and affects approximately 1 in 6000 live births, making it the most common genetic disorder that leads to death in early childhood. In SMA, this degeneration is due to the loss of survival of motor neuron (SMN) protein. SMN, a 38 kDa protein, is ubiquitously expressed and localizes to the nucleus where it forms gems. Gems are closely associated to Cajal bodies, regulators of the biogenesis of small nuclear ribonucleoproteins. In humans, there is a duplication of the chromosomal region containing the SMN gene, with the loss of the telomeric version (SMN1) causing severe disease. Objective : Our research focuses on the introduction of SMN into cells as a therapy. Use of viral vectors to deliver a full-length copy of the SMN gene has shown great promise in animal models of SMA. However, most gene therapy vectors lead to very high

level expression of the therapeutic protein in both affected and non-affected tissues. The effect of overexpression of SMN on normal cell function has not been evaluated. Methods : In the hopes of addressing the concerns of overexpression of SMN, we have used adenoviral vectors to deliver SMN in a variety of tissue culture cell lines, and evaluated its effect on SMN localization and function, and on cell growth. Adenovirus vectors were created that stably expressed full-length FLAG-tagged SMN protein. Several common cell lines were infected with different quantities of virus to produce varying levels of SMN. Localization of SMN was examined by immunofluorescence and subcellular fractionation. We also evaluated the ability of the over-expressed protein to interact with its normal cellular binding partners. Finally, cell growth assays were performed to determine the effects of SMN over-expression on the overall health of the cells. Results : Over-expression of SMN lead to an accumulation of the protein in both the nucleus and cytoplasm. In the cytoplasm, FLAG-SMN formed large aggregates, while in the nucleus it co-localized with Cajal bodies. Through co-immunoprecipitation studies, we show that FLAG-SMN physically interacts with Gemin2, a major scaffolding subunit of Gems. Finally, even at levels greater than ten-fold above the endogenous protein, over-expression of SMN did not adversely affect cell growth or function. Conclusion : We showed that expression of SMN from a viral vector produces protein that localizes to gem-like structures in the nucleus, and that the protein interacts with its normal binding partners. However, significant over-expression of the protein does not appear to affect overall cell function. Thus, viral delivery of SMN is an effective and safe therapy to re-establish SMN expression in cells. Our future studies will examine the effect of SMN overexpression in mouse models of SMA.

90. Caspase-dependent pathways governing cardiac hypertrophy Charis Putinski (MSc Student), Mohammad Abdul-Ghani and Lynn Megeney Department of Cellular and Molecular Medicine, University of Ottawa; Ottawa Hospital Research Institute Background: Cardiac hypertrophy occurs when the heart size increases to maintain cardiac output at times of stress. Since cardiomyocytes are terminally differentiated, heart growth results from an increase in individual cardiomyocyte cell size rather than by cell proliferation. This is initially an adaptive response; however, chronic stress on the heart is a maladaptive process that often leads to end-stage heart failure. Members of the caspase protease family have been well characterized for their role in apoptosis. Interestingly, caspases have also been shown to play non-apoptotic roles in mediating skeletal and cardiac muscle differentiation. Current evidence in the literature also suggests that the inhibition of caspase-3 limits the ability of the heart to undergo pathological cardiac hypertrophy, suggesting a role for caspase-3 in the hypertrophic growth process. Objectives: Using primary neonatal rat cardiomyocytes: 1) Determine if an increase in caspase-3 and/or caspase-8 activity occurs during the early stages of cardiomyocyte hypertrophy. 2) Determine if caspase-3 and/or caspase-8 activation is required for the development of cardiomyocyte hypertrophy. Methods: Primary cardiomyocytes isolated from 1 to 4 day old rat pups were treated with the hypertrophic agonist phenylephrine (100 uM) for 30 minutes, 1 hour and 3 hours followed by cell staining using the cardiac-specific marker (alpha-actinin) and active caspase-3 or caspase-8. Caspase localization and hypertrophic induction were analyzed by confocal microscopy, and caspase-specific fluorogenic substrates were employed to measure caspase activity. Results: Preliminary results indicate there was a prominent increase in the number of cardiomyocytes displaying active caspase-3 after exposure to phenylephrine. Localization to peri-nuclear and nuclear locations was observed with a 114%, 116% and 75% increase in active caspase-3 nuclear foci within 30 minutes, 1 hour and 3 hours of phenylephrine treatment, respectively. Treatment with the hypertrophic agonist induced a 40%, 111% and 135% increase in cell size after 30 minutes, 1 hour and 3 hours, respectively. Conclusions: Our preliminary data suggests that there is an increase in caspase-3 activity during the early stages of cardiomyocyte hypertrophic induction. These observations suggest that caspase activation is required for hypertrophic cardiac growth to occur. Further confirmation of the requirement for caspase-3 signaling during early stages of cardiac hypertrophy will be sought through the use of caspase inhibitors. Also, analysis of upstream and downstream pathway components will provide further insight into how caspases elicit their effects on cardiomyocytes during hypertrophy. Specifically, the MEF2 and NF-KB pathways will be evaluated as caspase-3 directed targets.

91. Improving the efficacy of oncolytic adenovirus through expression of p14 fusion-associated small transmembrane (p14FAST) protein Carmen M. Wong 1,2 [MSc Student], Carin Christou 1, John C. Bell 3 and Robin J. Parks 1,2 1OHRI Regenerative Medicine Program 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa 3OHRI Cancer Therapeutics Program Background: Adenoviruses (Ad) have been used extensively as oncolytic viruses to treat cancer. Oncolytic adenovirus, however, has a limited ability to spread throughout the tumour mass. To help oncolytic adenovirus spread, the reptilian reovirus p14 fusion-associated small transmembrane (p14FAST) protein may be an ideal candidate as it mediates cell membrane fusion. Objective: To determine if p14FAST protein expression can improve oncolytic vector efficacy and enhance virus spread throughout the tumour. Methods: An early region 1(E1)-deleted virus expressing p14FAST protein was generated and used to infect 293 human embryonic kidney and A549 human lung adenocarcinoma cells. Infected cells were observed using fluorescence microscopy post infection. Infected A549 cells were monitored for cell growth using a crystal violet assay and apoptosis using a live/dead assay. Results: The E1-deleted virus expressing p14FAST protein replicated in 293 cells which lead to extensive cell fusion. Cell fusion was also observed with A549 cells infected with the same virus. P14FAST expression in A549 cells inhibited cell growth and induced apoptosis. Conclusion: These results suggest that p14FAST protein may improve oncolytic adenovirus efficacy. Future work involves generating an oncolytic virus that has E1 under the control of the telomerase promoter and p14FAST expression under the control of the adenovirus major late promoter.

92. Periostin induced pancreatic regeneration Johnathan Smid (Postdoctoral fellow) Sharlene Faulkes (Research associate) Fan Xiao Michael Rudnicki Background: Periostin (Postn) is a secreted cell adhesion protein involved in promoting cell mobility by inducing an epithelial to mesenchymal transition. Microarray analysis performed in our laboratory has shown that Postn expression is upregulated following a 70% pancreatectomy. Further analysis revealed that the pancreatic Postn mRNA was a novel isoform lacking exons 17 and 21. In addition, Postn knock-out mice show a 50% reduction in pancreatic weight and a smaller sized pancreas 5 days after a pancreatectomy. Lastly, intrapancreatic injection of Postn induced a mesenchymal stroma, tubular complex formation and an increase in the endocrine progenitor marker, Ngn3, all of which are evidence of regeneration. Objectives: To determine if Periostin can induce pancreatic regeneration to ameliorate Diabetes. Methods: Postn was injected once a week for 6 weeks into the pre-diabetic Non-Obese Diabetic (NOD) mouse model to investigate its use in treatment of diabetes in an autoimmune model. In addition, Postn was also injected directly into the pancreas of streptozotocin (STZ)-induced diabetic mice to further test its feasibility in ameliorating Diabetes. Postn-induced regeneration was evaluated by immunohistochemistry, blood glucose levels and pancreatic insulin content. Results: In the NOD mouse model, Postn delayed the onset of Diabetes from 12 weeks to 16 weeks and significantly lowered the fasting blood glucose levels during this period. However, there was no significant difference in the incidence of diabetes between the control and Postn group. In the STZ-induced Diabetic model relative to BSA controls, Postn significantly decreased blood glucose levels one month after the injection (22.1 ± 3.8 vs 11.3 ± 1.8 mmol/L), increased the number of islets (13.2 ± 2.8 vs 25.1 ± 3.5 islets per field of view), and increased pancreatic insulin content (224.7 ± 42 vs 79.6 ± 20.6; p<.05). Although promising, the pancreatic insulin content is below that needed to ameliorate Diabetes. To gain further insight on the dosage of Postn needed, we are investigating a new technique which involves injection via the common bile duct. Preliminary results showed evidence of increased Ki67+ (proliferating) cells 24 hours after Postn was injected via the common bile duct compared to albumin. In addition, we observed an increase in Ngn3 expression 48 hours after the injection of Periostin into the common bile duct. Conclusions: Postn plays a role in regeneration of the pancreas, however, our current method of delivery by IP or direct intrapancreatic injections are not widespread enough to have a significant therapeutic effect. Injection via the common bile duct represents a novel method to effectively deliver periostin to ameliorate diabetes.

93. Echocardiographic assessment of the Sugen (SU5416) and chronic hypoxia model of pulmonary arterial hypertension Mohamad Taha (M.Sc. Student), Dr. Baohua Jiang, Dr. Yupu Deng, Dr. Duncan Stewart Background: Pulmonary Arterial Hypertension (PAH) is characterized by increased pulmonary artery pressure caused by resistance within the lung microcirculation. This increase in pulmonary artery pressure leads to right ventricle hypertrophy and eventually right heart failure and death. Despite all modern therapies, there is currently still no cure for PAH and its pathogenesis is not fully understood. A promising model of PAH combines SU5416, a vascular endothelial growth factor receptor inhibitor, with chronic hypoxia exposure to induce PAH in rats. Objective: To use echocardiography to assess right ventricle structure and function as well as pulmonary artery flow in the SU5416 plus chronic hypoxia rat model of pulmonary arterial hypertension. Methods: The model was established by combining a single initial subcutaneous injection of SU5416 (20mg/kg) (or vehicle) with three weeks of chronic hypoxia exposure (10% oxygen) (or normoxia). At the three weeks time point, echocardiography (ECHO) assessment was performed using the Visual Sonics Vevo 2100 Imaging system using the MS-250 rat transducer. Right ventricle structure was evaluated by assessment of the right ventricle (RV) anterior wall thickness and RV dimension while right ventricle function was evaluated using the tricuspid annulus plane systolic excursion (TAPSE) parameter. As for the pulmonary artery flow, it was assessed by measuring pulmonary acceleration time (PAT) and pulmonary ejection time (PET). Results: Animals in the group that received the combination of drug (SU5416) and three weeks of hypoxia showed significant increase in the right ventricle wall thickness when compared to control animals, while showing a decrease in the TAPSE parameter indicative of decreased function of the right ventricle. Furthermore, that group showed a decrease in the PAT and PAT/PET parameters, in line with increased pulmonary artery pressure. Conclusions: Our ECHO assessments show that the combination treatment leads to elevated PAH parameters and some distortion to the function and structure of the right ventricle, in line with the previous findings about the model. This leads us to believe that ECHO would make a good non-invasive procedure to evaluate PAH parameters.

94. Genomic and proteomic characterization of hHemogen in erythropoiesis Brinda Somasundaram (MSc Student) and Marjorie Brand Regenerative Medicine Program, OHRI Department of Cellular and Molecular Medicine, University of Ottawa Background: Hematopoiesis is a dynamic biological process in which different types of blood cells are formed from a multipotent cell called Hematopoietic stem cell (HSC). Red blood cells are formed from HSCs through a process called erythropoiesis which is regulated in vivo by several transcriptional regulators and signal pathways, deregulation of which could result in leukemia. Hemgn is a novel transcriptional regulator that positively regulates erythroid and megakaryocytic differentiation Hemgn is also highly expressed in leukemic cell lines. An et al., (2005) showed a correlation between Hemgn expression and occurrence of AML in patients. Hemgn expression levels are linked to AML patients’ positive therapeutic response. Though the importance of Hemgn in Acute Myeloid Leukemia has been established, the molecular mechanism by which it plays a role in erythropoiesis remains poorly understood. Objective: To elucidate the molecular mechanism of Hemgn activity during erythropoiesis Materials and Methods: Murine Erythroblast leukemia cells (MEL) were used as a model system for the study. Doxycycline inducible shRNA expressing MEL cells were generated and the effect of Hemogen on proliferation and differentiation of MEL cells were studied. To further study the molecular mechanism by which hemogen plays a role in erythropoiesis, we used Immunoprecipitation, Mass Spectrometry and

Chromatin Immunoprecipitaion(ChIP) techniques. Results and Conclusion: Hemogen plays an important role in the terminal differentiation of erythroblast by regulating the transcription of various hematopoiesis regulatory proteins

95. Role of miRNAs in endothelial progenitor cell differentiation and activity. Behbahani J (PhD Student), Stewart DJ (PI) Background and Rationale: Endothelial progenitor cell (EPC) therapy is emerging as a novel therapy for the treatment of vascular injury and ischemic cardiac disease. Despite much success with this form of therapy, work is still needed to address two major limitations in this field: 1) improving the lack of selection of an optimal cell population and 2) rectifying host factors (i.e. age, lipids, diabetes, and hypertension) that strongly impact of EPC number and regenerative activity. While there is no agreements on what surface markers define the “true” circulating progenitor cell population, highly angiogenic mononuclear cells (MNCs) called “early outgrowth EPCs” or “circulating angiogenic cells” can be readily derived by defined periods of attached cell culture. Prolonged culture of these cells in endothelial growth media results in emerging clusters of highly differentiated cells with strong endothelial phenotype called “late outgrowth EPCs” or “blood-derived endothelial cells”. Recently, it has been recognized that small, non-coding microRNAs (miRs) act as key post-transcriptional regulators of gene expression (and processes like cell differentiation) largely via incomplete complementary base pair interactions with target mRNA sequences. An estimated 30% of genes in the human genome are regulated by miRs. Numerous studies have shown a correlation between specific miR expression profiles and cardiovascular disorders, even in pathological states associated with EPC dysfunction. Hypothesis: Changes in miR expression profiles play a key role in the differentiation of MNCs to early to late outgrowth EPCs. Moreover, EPC dysfunction induced by a host of cardiac risk factors is at least in part mediated by alterations in angiogenic and endothelial miR levels. Objectives: 1) To establish the role miRs in endothelial differentiation of EPCs in attached culture and 2) define specific patterns of miR expression profiles that contribute to loss of EPC activity and differentiation potential. Research Plan: MNCs are isolated from the blood via Ficoll gradient centrifugation and cultured for 24 hours on fibronectin in defined endothelial growth medium. Expression profiles for 13 angiogenic and endothelial miRs will be determined at 0, 3, 5, 7 and 10 days (for early EPCs) and 21 days (late outgrowth EPCs) using qRT-PCR. EPCs will be characterized for relevant endothelial (CD31, CD144, vWF, KDR) and MNC cell markers (CD14, CD45) using flow cytometry. Assessment of regenerative activity and a variety of other parameters including proliferation (BrDU), survival (activated caspase 3), migration (Boyden chamber) and in vitro angiogenesis (Matrigel) will also be made. Using these techniques we will gain an understanding of the mechanisms involved in EPC dysfunction in CAD patients with respect to the specific role of miRs. We will then attempt to manipulate miR activity in vitro using antagomiRs and synthetic oligonucleotides to direct differentiation and modulate activity. Our goal is to manipulate miR levels to reverse dysfunction in EPCs from CAD patients. Conclusion: We hope to use our findings in this study to establish an understanding of the mechanistic role miRs play in the differentiation of EPCs and their potential in therapeutic applications.

96. H3K36me3 in muscle differentiation: Epigenetic regulation of tissue-specific gene expression by H3K36-specific methyltransferases Tarunpreet Dhaliwal (MSc Student) Tarunpreet Dhaliwal1,2,3, Arif Aziz1,2, F. Jeffrey Dilworth1,2,3 1Sprott Center for Stem Cell Research, 2Regenerative Medicine Program, Ottawa Hospital Research Institute, 3Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5. Background The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. For instance, trimethylation mark at H3K4 is characteristic of active chromatin state, whereas, trimethylation mark at H3K27 is characteristic of repressive chromatin. While the H3K4me3 is present around the transcription start site (5’ end), H3K36me3 is predominantly found on the gene body (3’ end) of active genes. Although it has been mentioned in the literature that H3K36 methylation antagonizes H3K27me3 and that these two marks rarely coexist, no data exists that correlates H3K36 methylation with muscle-specific gene expression. Objectives To determine the role of H3K36me3 in mediating muscle-specific gene expression and elucidate the mechanism by which H3K36 methyltransferases regulate tissue-specific gene expression. Methods and Results Recent work in the lab has demonstrated tissue-specific gene expression to be regulated by epigenetic modification of histones. Currently, it is unknown whether H3K36me3 regulates developmental genes. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at genes upregulated during muscle differentiation. An enrichment for H3K36me3 at the 3’ end of these genes was observed which is consistent with the methylation pattern observed in other systems. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of all known H3K36 methyltransferases (HMTs) by shRNA was performed in muscle myoblasts. Two of these HMTs – Hypb and Ash1l displayed striking but opposite phenotypes when their expression was knocked down during differentiation. Upon Hypb knockdown, differentiation was not inhibited, whereas upon Ash1l knockdown, differentiation was inhibited with a significant drop in myotube formation. Global analyses of histone methylation revealed the HMT Hypb to be responsible for the majority of H3K36 trimethylation. N-ChIP was also performed on differentiated Hypb and Ash1l knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. A drop in H3K36me3 enrichment levels was observed on myogenin and Ckm genes for both cell lines. Conclusion In general, the gene bodies of myogenin and Ckm genes showed reduced enrichment levels, possibly indicating trimethylation by HMTs at these loci. Given the postulated role for H3K36me3 in regulating gene transcription, cross-linked chromatin immunoprecipitation (X-ChIP) would be undertaken to look at HMT and cofactor occupancy on genes involved in muscle differentiation. Also, phenotype rescue experiments would be done to validate the role of H3K36-specific methyltransferases in muscle differentiation. These experiments would help elucidate the mechanism by which HMTs regulate muscle-specific gene expression.

97. Investigating the role of Interferon Regulatory Factor 3 in response to genotoxic stress

Adam R. Davidson (MSc Student) and Robin J. Parks Regenerative Medicine Program, Ottawa Hospital Research Institute Department of Biochemistry, Microbiology and Immunology, University of Ottawa Background – Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is also known to be involved in maintaining cellular functions, such as programmed cell death. Programmed cell death and the response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. Objective – Examine the involvement of IRF3 in response to a variety of genotoxic stresses. Methods – We have previously identified a non-activating phosphorylation of IRF3 in response to Adenovirus. We examined the phosphorylation state of IRF3 in a variety of human cell lines in response to different genotoxic stresses using immunoblotting. Using Adenoviral vectors and transfection of plasmid DNA, we over-expressed wild-type, as well as mutant IRF3 clones and examined their effects on cell-viability, protein stability and protein activity in response to infection and genotoxic stress. Results – Infection by Ad causes a change in the phosphorylation state of IRF3. The specific residue that Ad induces phosphorylation on is Ser173 located in the N-terminus of the protein and is thus not an “activating” event. Genotoxic stress also results in the phosphorylation of S173. Cells expressing an IRF3 S173A mutant demonstrate decreased IRF3 stability, as well as diminished cell viability. Conclusions – We have identified a previously unknown phosphorylation event that occurs in response to adenovirus infection as well as genotoxic stress in the forms of UV irradiation and chemotherapeutic agents. The significance of this phosphorylation event appears to have implications for protein stability, cell viability and potentially interaction with Bax, a pro-apoptotic protein. Our continuing studies will identify novel roles for IRF3 as a mediator of the DNA damage response, as well as the inflammatory response to viruses.

98. The OHRI Proteomics Core Facility Lawrence Puente (Senior Research Associate) The Proteomics Facility provides OHRI with access to advanced mass spectrometry capabilities, as a core service for routine protein identification, and as an enabling platform for advanced research projects. Protein identification is provided as a routine service. Mixtures of up to several hundred proteins can often be identified. With appropriate sample preparation, analysis of post-translational modifications and determination of relative or absolute protein abundance is also possible. The services of the Core Facility are available to all OHRI and University of Ottawa researchers on a fee-for-service basis

99. Development of a protein-based therapy for treatment of spinal muscular atrophy (SMA) Joseph Burns (MSc Student), Dr. Robin Parks (Senior Scientist). Regenerative Medicine Program, Ottawa Hospital Research Institute Department of Biochemistry, Microbiology, and Immunology, University of Ottawa Background: One of the leading genetic causes of death among infants is spinal muscular atrophy (SMA), a neurodegenerative disease in which the motor neurons of the spinal cord are progressively lost, resulting in paralysis and, in severe cases, death. Deficiency in full-length survival motor neuron (SMN) protein is the cause of the disease. The severity of the disease phenotype decreases in cases of lesser deficiency, and studies in which exogenous full-length SMN protein was introduced yielded promising results. In order to be effectively delivered to motor neurons, SMN must be fused to a protein transduction domain (PTD), such as the PTD from the trans-acting activator of transcription (TAT) from HIV. The TAT PTD allows the fusion SMN to be taken across the cell membrane. A gene therapy approach in which an adenoviral vector delivers a transgene encoding a secreted TAT-SMN to the liver would allow sustained production, secretion, and systemic delivery of the therapeutic SMN to motor neurons in an SMA patient. Objectives: To develop an effective therapeutic protein for spinal muscular atrophy by fusing an HIV TAT domain and a secretory peptide (SP) to SMN. We will first evaluate the capabilities of the transduction and secretion domains using green fluorescent protein (GFP) as an indicator before continuing into development of the fusion SMN. Methods: We generated DNA constructs in DH5α Escherichia coli cells. We made large-scale preparations of the DNA from positive clones, then transfected the DNA into 293 cells. We assessed expression of the fusion GFPs using fluorescence microscopy and verified the results with Western blotting. We precipitated the secreted fusion GFPs from media using trichloroacetic acid (TCA) and analyzed the precipitates with Western blotting. Results: The TAT-GFP fusion proteins were successfully expressed following transfection into mammalian cells. We were able to purify fusion GFPs from cell lysates using attached His tags. We managed to precipitate the SP-GFPs from the media following transfection and media changes. We have generated some, but not all, of the DNA constructs necessary to begin testing fusion SMNs. Conclusions: The TAT-GFP proteins are being expressed and the secretion peptide is working, though with varying levels of success among the constructs. Considering the less effective secretion observed with the native TAT PTD we will move forward with SMN constructs for the two TAT PTD variants we are investigating.

100. Structure of the adenovirus DNA-protein complex in the infected cell Andrea N. Giberson (M.Sc. Student)and J. Robin Parks Regenerative Medicine Program, Ottawa Hospital Research Institute and Department of Biochemistry, Microbiology, and Immunology at the University of Ottawa Background: Human Adenovirus (Ad) is a widely studied DNA virus. Although it is a pathogen that can cause mild respiratory infections with flu symptoms, it is better known as a viral vector in gene therapy. However, little is known about the structure of Ad DNA once it enters the cell nucleus in a productive infection, and the impact of the nucleoprotein structure on the transcription of viral genes. Previous work with helper-dependent Ad (hdAd) indicated that the Ad DNA

binding protein, pVII is rapidly removed and replaced with cellular histones to form nucleosomes within hours of infection. Of the two main histone 3 (H3) variants, it was found that histone 3.3, which is deposited independently of replication, was preferably deposited on the hdAd DNA. Objective: To establish whether the wild type Ad DNA undergoes chromatinization and identify cellular proteins which are important in mediating this event. Method: Association of DNA binding proteins (pVII, histones) and wtAd DNA was characterized by chromatin immunoprecipitation and other biochemical techniques. Southern blots following micrococcal nuclease digestions of infected cell were used to investigate the assembly of viral DNA into nucleosomes. Results: Most of pVII is lost within an hour and a half of infection and this loss is independent of transcription, although a small amount of pVII remains associated with the Ad DNA. Cellular histones associate with the viral DNA, with a preferential deposition H3.3. Late in infection, the level of associated histones is much lower, and there is no transition to the deposition of H3.1 after DNA replication. Southern blots of the viral DNA supports the lower histone association levels, as can be seen by the lack of laddering, which is observed at 6hrs. Conclusions: Our work suggests that, like hdAd, wtAd undergoes decondensation (pVII loss) and is associated with cellular histones within the first hours of infection, with a preferential deposition of H3.3. Unlike other DNA viruses, a switch to the replication-dependant H3 variant (H3.1) was not observed after DNA replication. The assembly of Ad DNA into nulceosomes at late times is also greatly diminished and may indicate the need to investigate the association of Ad DNA with other DNA binding proteins.

101. Characterization of Pax7+Myf5- satellite stem cells and Pax7+Myf5+ satellite cells in culture condition Yuichi Tomita (Postdoctoral fellow) Background: Our groups reported that satellite stem cells are Pax7+Myf5- and they can contribute to muscle regeneration in vivo as satellite cells. Though Pax7+Myf5- satellite stem cells can generate Pax7+Myf5+ satellite cells, each cells phenotype is still unknown. Objective: In this study, the characteristics and differentiation potency of Myf5-YFP+ & YFP- cells were investigated. Methods: Myf5-YFP mice were generated by mating Myf5-Cre female mice and Rosa-YFP male mice. Freshly FACS-isolated Pax7+Myf5- and Pax7+Myf5+ cells were cultured in several methods. Expanded Pax7+Myf5- and Pax7+Myf5+ cells were characterized both in undifferentiated and differentiated states. Results: Myf5-YFP- cells could differentiate into myosin heavy chain+ myocytes without Myf5 expression. Myf5-YFP+ cells could generate more slow MyHC+ myocytes than Myf5-YFP- cells. Conclusion: Pax7+Myf5- cells have distinct differentiation potency from Pax7+Myf5+ cells.

Vision Program

102. Investigating the role of sortilin in sonic hedgehog trafficking

Charles Campbell (PhD Student), Shawn Beug, Chantal Mazerolle, Valerie Wallace Background: Sonic hedgehog (Shh) is one of a family of secreted morphogens that patterns the development of many systems, most notably parts of the mammalian central nervous system. In the mammalian retina, Shh is produced from Retinal Ganglion Cells (RGCs), and affects cell fate decisions both short range in the retina, and long range in the optic nerve. Long range Shh signaling in the optic nerve is likely mediated by intraneuronal transport of the protein in RGC axons. Previous work in our lab has established that Shh travels down axons via the regulated secretory pathway, a feature of specialized cells, in Dense Core Granules (DCGs) and Synaptic Vesicles. Consistent with Shh sorting to regulated secretory vesicles, Shh secretion from neuronal cell lines is increased under depolarizing conditions. The mechanism of Shh sorting to the regulated secretory pathway is, however, not known. Sortilin (Sort), a receptor protein known to be involved in sorting to the regulated secretory pathway, was identified in an affinity purification screen as a candidate Shh interacting protein. Sortilin interaction with Shh was verified by reciprocal co-immunoprecipitation (co-IP). Objective: To characterize the Shh-Sort interaction, and investigate the role of Sort in intraneuronal Shh trafficking. Methods: Co-localization of endogenous and transfected Sort and Shh has been performed in primary neurons and COS cells, while the requirement for Sort in Shh trafficking in neurons is being determined by perturbation of Sort function using a dominant negative, truncated Sort (tSort) construct (which lacks regulated secretory pathway targeting), as well as by knocking down Sort expression using short hairpin RNA (shRNA). Results: In COS cells, Sort colocalized with Shh in a reticular pattern, while tSort overexpression perturbed Shh trafficking to peripheral granules. Perturbation was not seen in Shh mutants lacking the cholesterol modification, suggesting its importance in the interaction. In primary neurons, Sort colocalized with Shh in the soma and dendrites, but not in the axons, while tSort overexpression abolished Shh targeting to the axons. Loss of function of Sort in cortical neurons suprisingly resulted in increased Shh signal in the axons and dendrites, both in synaptic vesicles and on the plasma membrane. Conclusions: Sort appears to regulate trafficking of Shh away from the regulated secretory pathway, perhaps to the lysosomes or endosomes, via an interaction that may depend on the Shh cholesterol modification.

103. De-differentiation in mammalian muscle cells following treatment with limb-derived newt regeneration extract S. Kawesa(PhD student)and C. Tsilfidis Background: Dedifferentiation is a process wherein cells, such as muscle cells during amphibian regeneration, lose their differentiated characteristics to become undifferentiated cells. Recent studies show that dedifferentiation occurs in differentiated mammalian muscle cells when treated with the right trigger. An assessment of the reversal of differentiation (dedifferentiation) in mammalian muscle cells is of essential importance in designing treatment strategies for muscle diseases. Purpose: The purpose if this study is to examine the ability of mammalian muscle cells to undergo dedifferentiation following treatment with limb-derived newt regeneration extract. Methods: To assess the dedifferentiation process, C2C12 muscle cells transfected with MCK-cre/ pCMV-EGFP/RFP plasmids were treated with 0.3mg/ml of newt

regeneration extract. Control cells were not treated or treated with heat-inactivated newt extract. Treated differentiated cells were stained for BrdU, p21, and MF20 to assess cell cycle re-entry and loss of muscle characteristics. Dedifferentiation was also examined in primary muscle cells derived from transgenic mice (MCK-cre/lox-rosa-YFP). To examine the proliferation, dedifferentiated cells were treated with newt extract and followed with time lapse microscopy or florescence microscopy. Results: The experimental results suggest that C2C12 differentiated cells treated with newt regeneration extract are capable of re-entering the cell-cycle. This was shown by BrdU incorporation in the nuclei of the myotubes. The newt regeneration extract also reduces the expression of the cyclin-dependent kinase inhibitor, p21, in C2C12 differentiated cells. The present data also show that the newt extract blocks the myogenic differentiation of skeletal myoblasts and does not promote cellular proliferation in vitro. Following a myotube with live imaging microscopy shows that newt regeneration extract causes fragmentation and the fragmented mononucleated cells were seen to undergo cell division when put back into growth medium. Conclusion: Dedifferentiation is not normally seen in mammalian regeneration. In this study, we have been able to show that mammalian muscle cells treated with newt regeneration extract are able to dedifferentiate. These findings could have implications for therapeutic intervention in muscle diseases using genetic engineering and myoblast implantation.

104. The role of Gli in Hedgehog dependent neural progenitor proliferation Randy A. Ringuette (PhD Student), Alan J. Mears, Valerie A. Wallace Background: The Sonic hedgehog (Shh) signaling pathway is a critical regulator of growth and patterning in a variety of tissues and organs. In the developing retina, a tractable model system of central nervous system development, Shh signaling from neurons targets the adjacent neural progenitor cells. Studies of conditional Shh inactivation in vivo and gain of function in vitro have demonstrated that Shh is required to maintain progenitor proliferation and control cell fate. How this important signaling pathway affects these different developmental processes is largely unknown, but is likely to involve the downstream mediators of the Shh signaling pathway, the Gli transcription factors. In the retina, Shh target gene expression and proliferation require Gli2, but not Gli1. We hypothesize that transcriptional and posttranscriptional regulation of Gli2 is essential for neural progenitor self-renewal and differentiation Objective: To determine the levels of Gli2 at the protein and message level over the course of retinal development and investigate how interactions with known Gli2 interacting proteins regulate Gli2 function and stability in neural progenitor cells. Methods: Acutely dissected and cultured neonatal retinal tissues were subjected to western blot or quantitative real time PCR. To over express Gli2, explants were transfected using electroporation. Results: During retinal development Gli2 message is initially expressed in the developing neuroblast layer and becomes restricted to a thin row of cells in the inner nuclear layer, likely the Müller glia, the only cell type in the adult retina that can re-enter the cell cycle. Similarly, Gli2 protein is detectable only at early stages of retinal development and loss of Gli2 protein correlates with loss of retinal progenitor cells, suggesting that down regulating Gli2 transcription may be part of the neuronal differentiation program. Consistent with the inverse correlation between neuronal differentiation and the presence of Gli2 protein, we find that ectopically expressed Gli2:GFP fusion proteins in retinal explants only exert a transient effect on proliferation and are down regulated after several days, which corresponds to the timing of cell cycle exit and neurogenesis. In addition, ectopic Gli2 expression in retinal explants resulted in only minimal Shh target gene induction (compared with activation of the Shh pathway upstream of Gli2 transcription factors), suggesting that additional regulators are required for full Gli2-mediated transactivation of target genes. Conclusion: Based on results thus far, restricting Gli2 activity might be a mechanism utilized by RPCs to activate the differentiation program and forced expression overcomes this process, resulting in proliferation.

105. Chitosan microparticles for the delivery of XIAP to the retina. Sarah Wassmer, (MSc Student) Mehrdad Rafat, Adam Baker, Wai Gin Fong and Catherine Tsilfidis Background: Cell death by apoptosis is the final common pathway for many types of retinal degeneration. The X-linked inhibitor of apoptosis (XIAP) protein directly inhibits the proteins responsible for cell death (caspase 3, 7 and 9). Our research has shown that over-expression of XIAP is effective in preventing the death of retinal cells in many types of retinal disease. Nanoparticles or microparticles allow a controlled and stable release of the encapsulated protein or drug. This system can provide safe and successful delivery of XIAP protein to retinal cells. The main goal of this project was to test the efficacy and toxicity of chitosan microparticles for protein delivery to retinal cells. Three concentrations (0.5 mg/mL, 1.0 mg/mL and 10.0 mg/mL) of microparticles containing TAT-EGFP (a control protein) were tested in vitro for toxicity. Subsequently, two concentrations were tested in vivo (1.0 mg/mL and 10 mg/mL). Methods: Chitosan microparticles containing encapsulated TAT-EGFP were tested for cellular toxicity in 661W (mouse photoreceptor) cells at three concentrations: 0.5 mg/mL, 1.0 mg/mL and 10.0 mg/mL. In addition, chitosan microparticles containing encapsulated TAT-EGFP were injected in the subretinal space under two conditions: 10.0 mg/mL and 1.0 mg/mL. The retinal function of the rats was assessed using full-field ERGs. The retina was imaged using a Fundus Camera (Phoenix Res Lab Inc.). ERGs and fundus imaging were performed at weeks 2, 4, 6 and 8 post-injection. At week 8 the eyes were sampled for histology and immunohistochemistry. Results: In vitro results showed that 10mg/mL of chitosan microparticles significantly reduced 661W cell viability. The lower concentrations showed no toxicity. In vivo, the trends suggested that the higher concentration may be slightly more toxic, but there was no significant difference in ERGs when comparing 1.0 mg/mL and 10.0 mg/mL. Fundus imaging showed that particles clumped together and did not spread from the injection site. Immunohistochemistry of histological sections showed significant cell disruption at sites where clumping of microparticles occurred. Consequently, little retinal misconfiguration was observed in areas with few (only one or two) microparticles or GFP diffusion. Conclusions: Our studies suggest that 1 mg/mL of chitosan microparticles is a safe concentration to use for TAT-XIAP protein delivery to the retina. Multiple injections may be required to ensure more widespread delivery of the therapeutic protein. In addition, particles must be treated to prevent aggregation as it disrupts the retinal layers. In conclusion, a low concentration of microparticles that do not cluster may be an effective vector for

protein therapy.

106. A novel characterization of a function for Norrie disease pseudoglioma (Ndp) signalling in cerebellar development. Nicholas Tokarew (MSc student), Erin Bassett (Postdoctoral fellow), Valerie Wallace (Principal Investigator) Brian McNeill (Postdoctoral fellow) and Chantal Mazerolle (Research associate) Norrie Disease Pseudoglima (Ndp) is an X-linked cysteine-rich secreted protein that is best known for its role in regulating angiogenesis in the developing retina. Norrie disease is caused by various mutations in the Ndp gene. In humans, the resulting phenotypes are congenital blindness, sensorineural deafness and in some cases, a delay in cognitive processes. Recently, we showed that Ndp is a direct target of Gli2, an important transcription factor that functions downstream of Shh signalling in neuronal progenitor cells of the developing eye. During cerebellar development, the neuronal progenitor cells located in the external granule layer (EGL) undergo a proliferative expansion predominantly under the control of the secreted morphogen Sonic Hedgehog (Shh). Our study aims to investigate the possible functional and modulatory roles that Ndp plays in this region of the brain. In order to understand Ndp's biological relevance, we will be examining the spatial and temporal expression profile as well as the role of Ndp in neuronal proliferation and survival. This investigation will be carried out in mice and GNP culture models. Preliminary data shows that Ndp is expressed in the external granule layer at a stage when GNPs are undergoing Hh-mediated proliferation. Our investigation into NDP's role in cerebellar development will provide insight into its non-angiogenic functions in neuronal stem cells and neuronal progenitors during

the development of the cerebellum.

OHRI, Hospital or University Staff

107. Factors influencing a specific histological diagnosis of non-small cell lung cancer. Jeff Sulpher (Medical Oncology Resident), Scott Owen (Medical Oncology Research Fellow), Henrique Hon (Research Associate), Kim Tobros (Research Associate), Francis Shepherd (Medical Oncologist), Elham Sabri (Statistician), Geoffrey Liu (Medical Oncologist), Christina Canil (Medical Oncologist), Paul Wheatley-Price (Medical Oncologist) Background: Non-small cell lung cancer (NSCLC) not otherwise specified (NOS) was historically sufficient information to guide systemic treatment decisions. Increasingly specific classification of NSCLC is required to select optimum therapy. We performed a multi-centre retrospective review of NSCLC cases to identify factors associated with a specific histological diagnosis. Methods: With ethics approval, non-operative and palliative NSCLC cases at the Ottawa Hospital Cancer Centre (2008) and Princess Margaret Hospital (2007-2010), Toronto, were identified. Charts were reviewed for patient demographics, disease stage, treatment intent, and methods of obtaining the diagnosis. The number of cases and factors associated with attaining a specific histological diagnosis are reported. Results: Of 739 patients, 51% were male, the age range was 31-91 years (median 66 ), 85% were treated palliatively and 61% had good performance status (ECOG 0/1). 53% of patients were diagnosed in an academic cancer centre, and 59% were diagnosed by respirologists or thoracic surgeons. Cytology alone was available in 65%; histology was available in 35%. In 68% of cases the tissue source was the primary lung mass. CT-guided biopsy was the most common method (50%) followed by bronchoscopy (24%). CT biopsy yielded cytology alone in 88% of cases. Pleuroscopy yielded histology in 100% of cases, followed by thoracoscopy (83%) and bronchoscopy (57%). The final common diagnoses were adenocarcinoma (38%), squamous cell carcinoma (10%) and NOS (30%). Overall, 54% were considered to have a specific diagnosis. Factors associated with obtaining a specific diagnosis were presence of histology rather than cytology alone (p<0.0001), and diagnosis obtained in a community hospital (p=0.0002) or teaching hospital (p<0.0001) rather than an academic cancer centre. Conclusions: Almost half NSCLC cases do not have a specific subtype diagnosis, which usually requires histology rather than cytology. While CT-guidance is the most common method of diagnosis, it is the least likely method to attain sufficient histology.

108. Design of a wearable, clinically feasible sEMG analysis system. Adam Freed (MASc Student), Dr. Edward D. Lemaire, Dr. Adrian D.C. Chan, Dr. Avi Parush. Background: Surface Electromyography (sEMG) has long been an important part of research into muscle activity and the study of functional motion. However, due to the complex nature, time and costs associated with conventional sEMG analysis systems, their benefits have been mostly unseen in a clinical setting. For a sEMG system to be usable in a clinical setting, such as a physiotherapy clinic, factors such as cost, size, complexity and reusability must be accounted for. Advances in microelectronics have provided the means to develop small, lightweight, unobtrusive wearable technology at a reasonable cost. In addition, materials used in reusable dry electrodes have shown comparable performance to the disposable gelled kind generally employed in conventional sEMG analysis. By combining these components, we have designed a prototype for a novel sEMG analysis system Objective: To design a clinically feasible, wearable sEMG analysis system. Methods: In an effort to reduce the time associated with placement of conventional electrodes, a functional prototype of the wearable sEMG system has been developed employing an electrode array of dry reusable electrodes. The array, a set of 8 evenly spaced electrodes, allows 6 channels of sEMG data to be collected simultaneously. Software is then used to determine the best quality pair based on certain characteristics. The electrode mount, a material sleeve that houses the electrodes is currently designed to be used on the lower leg. SEMG data has been captured from the tibialis anterior as well as gastrocs medialis in both isometric and dynamic contractions, as well as

in walking trials. Frequency and time domain results were compared to ensure intra-system repeatability as well as for comparison with the conventional sEMG system. Results: Pilot tests on one subject have shown comparable performance of the novel sEMG system to those of the conventional system, as well as consistent performance between trials. Conclusion: To gain more data for further analysis, testing will be performed on 10 subjects; however, since pilot tests have proven successful, we are confident that the upcoming trials will only confirm our initial conclusion, which is that we have successfully designed a wearable sEMG data collection system

109. Change-of-state determination to recognize mobility activities using a Blackberry smartphone Wu H.H.1,2 (MSc Student), Lemaire E.D.1,2 (Associate Professor), Baddour N.2 (Associate Professor) 1The Ottawa Hospital Rehabilitation Centre 2Mechanical Engineering, University of Ottawa, Canada A Wearable Mobility Monitoring System (WMMS) can be a useful tool for rehabilitation decision-making. This paper presents preliminary design and evaluation of a WMMS proof-of-concept system. Software was developed for the BlackBerry 9550, using the integrated three axes accelerometer, GPS, video camera, and timer to identify mobility changes-of-state (CoS) between static activities, walking-related activities, taking an elevator, bathroom activities, working in the kitchen, and meal preparation (five able-bodied subjects). This pilot project provides insight into new algorithms and features that recognize CoS and activities in real-time. Following features extraction from the sensor data, two decision trees were used to distinguish the CoS and activities. Real-time CoS identification triggered BlackBerry video recording for improved mobility context analysis during post-processing.

110. The effect of perturbation magnitude on walking stability during visual and platform perturbations 1 Emily H. Sinitski (University of Ottawa MSc Student) 1,2 Kevin Terry 1 Jason M. Wilken 2 Jonathan B. Dingwell 1. Department of Orthopedics and Rehabilitation, Center for the Intrepid, Brooke Army Medical Center, Ft. Sam Houston, TX, USA 2 University of Texas at Austin, Austin, TX, USA Background External perturbations applied to the walking surface or visual field can challenge an individual’s ability to maintain stability during walking. Accurately quantifying and predicting changes in stability during walking will further our understanding of how individuals respond to challenges encountered during daily life and guide the development of assessments and rehabilitation interventions for individuals at increased risk of falling. Objective The objective of this study was to determine how orbital and local dynamic stability metrics, including maximum Floquet multipliers and local divergence exponents, change in response to continuous medial-lateral visual and surface perturbations of different amplitudes. Methods Eleven health individuals walked at normalized speeds in a fully immersive virtual environment. Participants completed two 3-minute trials each of walking with either no perturbation, one of three platform perturbation amplitudes (P1-P3), or one of five visual perturbation amplitudes (V1-V5). All perturbations were applied as continuous pseudo-random oscillations in the medial-lateral direction. A marker on the C7 vertebra was tracked at 60 Hz using a 24-camera Vicon system. Orbital stability was quantified by calculating maximum Floquet multipliers. Local stability was quantified by calculating divergence exponents. These measures were calculated using a 5-dimensional delay-embedded state space of C7 medial-lateral velocity with a time delay of tau = 20 samples. One-way repeated measures ANOVAs were used to determine statistical differences between amplitudes. Results During surface and visual perturbations, individuals were significantly more orbital and locally unstable compared to unperturbed walking (p < 0.001). As platform perturbation amplitude increased, individuals were more orbitally (but not locally) unstable between the lowest amplitude (P1) and higher amplitudes (P2-P3) (p < 0.01). As visual perturbation amplitudes increased, individuals were more locally (but not orbitally) unstable between lower and higher amplitudes (p < 0.01). Conclusions Overall, these dynamic stability metrics were much less sensitive to changes in perturbation amplitudes than to differences between unperturbed and perturbed walking, or to differences between mechanical and visual perturbations. This suggests that the type of perturbation applied has far greater impact than the magnitude of those perturbations in determining the response that will be elicited. Disclaimer: The views expressed herein are those of the authors and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Army, Department of Defense or the U.S. Government.

111. SPIO enabled MR imaging of biodegradable PLGA channels for spinal cord injury Sagedeh Sadat Shahabi (MSc student, U of Ottawa), Karunanithi Rajamanickam(Postdoctoral Fellow, OHRI), Eve Tsai (Neurosurgeon, Civic Hospital)and Xudong Cao (Associated Professor, U of Ottawa) Introduction Spinal cord injury (SCI) causes traumatic paralysis with little or no recovery of function. Bioengineering strategies have been proposed to improve the extent of functional recovery after the SCI. To this end, we have designed degradable PLGA follow fiber channels (HFCs) to create a permissive local environment for axonal regeneration. While the degradation of the PLGA HFCs has been well established in vitro, little is known about the fates of the HFCs once implanted in vivo. In this study, we demonstrate the feasibility of using superparamagnetic iron oxide (SPIO) nanoparticles as a contrasting agent and incorporating SPIO into the degradable HFCs to enable non-invasive long term monitoring of HFCs using MRI. Channel Design and Fabrication HFCs are prepared using degradable PLGA. To expedite the evaluation of these channels, we incorporated SPIO nanoparticles within the channel with different concentration and assessed non-invasively using magnetic resonance imaging with a 1.5 T Siemens Quantum Symphony clinical MRI machine. PLGA channels were constructed through dip coating of PLGA onto a glass rod, and the channels were subsequently foamed with supercritical carbon dioxide process. The SPIO nanoparticles were incorporated directly into the channel during the dip coating. Magnetic Resonance Imaging of the channels MRI imaging of the channels was performed with a spin echo multi contrast sequence for measuring the transverse relaxation rate with a repetition time (TR) of 4000 ms and different echo times (TE) starting from 13.3 to 319.2 milli seconds at 24 intervals. FOV 210 mm

image resolution 256x 256, with 2 times averaging. T2 mapping was computed using in-house algorithm developed in Matlab by pixel by pixel basis. Least square fit was fitted to find the slope of the decay curve by plotting log intensity on the y-axis and echo time on the x-axis. The addition of the SPIO nanoparticles enabled assessment of the location of the PLGA channels noninvasively by serial MRI over time. The SPIO signal decreased over time suggesting that SPIO nanoparticles can be used to assess biodegradation noninvasively. We are currently working on improving the characteristics and contents of the channel to improve regeneration and functional recovery after spinal cord injury. Biodegradable channels hold promise in facilitating the combination therapy required to enable a therapeutic strategy to be applied to humans with spinal cord injury. PLGA + SPIO (n=6) at three different concentrations (0.05% of SPIO, 0.10% of SPIO and 0.20% of SPIO) were fabricated for this study. MRI imaging of the channels demonstrated that while biodegradable PLGA channels could not be seen on MRI, the SPIO nanoparticle labeled biodegradable PLGA channels could be visualized non-invasively by MRI. Results The transverse relaxation time in milli second was tabulated for all the channels. The difference in the transverse relaxation time (T2) was significantly (p=0.001) decreasing with respect to the increase in the concentration of SPIO. Conclusion This preliminary observations confirmed that the biodegradable channels can be fabricated with encapsulation of super paramagnetic iron oxide in different concentrations that might facilitate the magnetic resonance imaging in in-vivo conditions. The biodegradability of the channels are monitored on weekly basis using the T2 measurements: Table-1: Mean and SD of transverse relaxation (T2) of channels in milli seconds SPIO A B C D E F Mean of all channels Mean± Mean± Mean± Mean± Mean± Mean± Mean± SD SD SD SD SD SD SD 0.05% 1142±34 1116±37 1249±17 1170±81 1285±26 1119±93 1180±70 0.10% 672±60 684±71 687±44 690±45 708±21 776±36 702±83 0.20% 562±23 521±12 555±90 535±53 512±71 548±21 538±19

112. Autosomal dominant congenital nonprogressive spinocerebellar ataxia is associated with a missense mutation in the ITPR1 gene Lijia Huang (Postdoctoral Fellow)1, Ruobing Zou2, Melissa Carter3, Stuart Douglas1, Dennis E. Bulman2, Kym M Boycott1 1Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, Canada; 2Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada; 3Hospital for Sick Children, Toronto, Ontario, Canada The spinocerebellar ataxias (SCA) are a group of genetically heterogeneous disorders characterized by slowly progressive incoordination of gait and often associated with poor coordination of hands, speech, and eye movements. In contrast to the common SCAs, congenital nonprogressive spinocerebellar ataxia (CNPCA) is characterized by early gross motor delay, hypotonia, gait ataxia, and mild dysarthria and dysmetria. Cognitive impairment may or may not be present. The clinical presentation improves or remains stable throughout the life of an affected individual. Families with autosomal dominant CNPCA, with and without cerebellar atrophy, have been reported approximately nine times since 1985. It is likely that autosomal dominant CNPCA is a genetically heterogeneous condition. Linkage to 3pter has been demonstrated in an Australian family and the locus designated as SCA29. We present a three-generation family with autosomal dominant CNPCA. The proband is a 3 year old girl with global developmental delay, hypotonia, truncal ataxia, and cerebellar vermis atrophy on MR imaging. Her father had early gross motor delays and did not walk until the age of 6 years. He has an ataxic gait, mild dysarthria, saccadic eye movements and poor balance. His sister and mother are similarly, but less severely, affected. The SCA29 locus overlaps with the SCA15 locus at 3pter. SCA15 is a dominantly inherited slowly progressive cerebellar ataxia with mid-life onset; heterozygous ITPR1 deletions are disease-causing. Given the significant clinical overlap between the SCA29 family and our family, we genotyped the affected and healthy family members with microsatellite makers from 3pter and found the region containing ITPR1 cosegregated with the disease. Sequencing all of the exons and the exon/intron boundaries of the ITPR1 gene in one affected individual identified a missense change (c.1804A>G; p.N602D), which cosegregated with the disease phenotype. The change is located at a highly conserved amino acid position, is predicted to be pathogenic and was not found in over 100 normal Caucasian controls. ITPR1 is an intracellular ligand-gated Ca2+ release channel, playing a critical role in modulating intracellular Ca2+ signaling. Mutations in CA8, an inhibitor of ITPR1, causes a recessive form of CNPCA, supporting our finding that mutations in ITPR1, or genes in the ITPR1-related pathway, can give rise to CNPCA. Analysis of the original Australian SCA29 family is currently underway.

OHRI IMPACT (Identification of Marketable Products, Applications and Commercializable Technologies) Award Posters

113. From reactive to anticipatory medicine in the ICU: development of composite multiorgan variability monitoring. Andrea Bravi, André Longtin, Andrew JE Seely

114. Putrescine supplementation to tTreat premenopausal infertility and to reduce the risk of birth defects in older women. Yong Tao, Supervised by Johné Liu. Ottawa Hospital Research Institute, Ottawa Hospital Civic Campus, University of Ottawa, 1053 Carling Avenue, Ottawa, K1Y 4E9. Canada.

115. LOVV makes the world go round. Chantal Lemay & David Conrad StemCore Laboratories Genomics Facility

OTHER OHRI Core Facilities

116. StemCore Laboratories Genomics Facility Katayoun Sheikheleslamy, Caroline Vergette, Atieh Jalali-Pakdaman, Pearl A. Campbell. Abstract: StemCore Laboratories www.stemcore.ca is OHRI’s high-throughput genomics facility located at the General Campus of the Ottawa Hospital Research Institute. Our mandate is to provide access to research-enabling technologies which are beyond the scope of individual laboratory operations, thereby facilitating biological and medical research through the use of high-end technology and state of the art equipment. StemCore is continuously developing a world-class genomics services infrastructure, and is capable of facilitating large-scale scientific research and biotechnology projects. StemCore provides DNA sequencing services, Affymetrix Genechip microarray solutions, and a recently acquired Next-Generation sequencing platform. StemCore seeks out projects that are challenging, cutting-edge, extend the boundaries of biological knowledge, and will positively impact the state of human health

117. The MoFlo Flow Cytometry and Fluorescence Activated Cell Sorting (FACS) Facility Paul R. Oleynik, Alessandra Pasut, Michael A. Rudnicki, Pearl A. Campbell Abstract:Flow cytometry is the science of examining physical and chemical properties of live cells or other biological particles as they pass in a fluid stream through a measuring apparatus. This technique uses laser light scattering or excitation of fluorophores and signal detection to measure. Some flow cytometers are equipped to separate and collect cells of interest in a technique called Fluorescence Activated Cell Sorting (FACS). Our Flow Cytometry and Cell Sorting Facility is jointly operated by the Ottawa Health Research Institute (OHRI) and StemCore Laboratories. We provide academic and corporate clients with access to flow cytometry analysis as well as high-speed cell sorting services. We also offer comprehensive training and education as well as expert consultation services to enable our users to utilize this technology to enhance the scope and quality of their research. Our Beckman Coulter MoFlo instrument can be used for high speed cell analysis and sorting (up to 20,000 cells/second). This recently upgraded system contains four excitation lasers, two light scatter detectors, and ten fluorescence detectors, allowing for the analysis of up to nine fluorescence colors at once. Specifically, we are equipped with a UV laser, as well as a 405 nm (violet), 488nm (blue), 561nm (green), and 640nm (red) laser. The two light scatter detectors are forward scatter (cell size) and side scatter (cell granularity). Additionally, the ten fluorescence detectors are completely customizable to the experiment at hand.

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