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2010 Undergraduate & International Summer Research Program

RESEARCH DAY

Wednesday, August 04, 2010

Medical Sciences Bldg., University of Toronto

Supported by

Partners

UHN Center for Research

The Life Sciences Undergraduate Student Research Opportunity Program, University of Toronto

Keenan Research Centre at the Li Ka Shing Knowledge Institute

DEPARTMENT OF SURGERY

Sponsors

Supporters

Institute of Medical Science, Faculty of Medicine University of Toronto

2010 Undergraduate and International Summer Research Program RESEARCH DAY

Wednesday, August 4, 2010 9:00 a.m. ­ 3:30 p.m.

2172 Medical Sciences Building, 1 King's College Circle

9:00 am 9:05 am 9:15 am

Welcome ­ Room 2172 · Dr. Neeru Gupta, Director, IMS Summer Research Program Introduction of Keynote Speaker · Dr. Ori Rotstein, Director, Institute of Medical Science Keynote Address "Personalized Medicine ­ The End of the Beginning"

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10:15 am 10:30 am 12:00 am 12:45 pm 3:00 pm

Dr. Katherine Siminovitch, Director, Genomic Medicine Division & Gene Profiling Facility University Health Network, Professor, Departments of Medicine, Immunology, Molecular Genetics, University of Toronto / Mount Sinai Hospital Coffee Break ­ Stone Lobby, Room 2171 Student Oral Presentations ­ Room 2172 Lunch ­ Macleod Auditorium Lobby, Room 2158 Poster Competitions ­ Stone Lobby, Room 2171 Award Presentations ­ Macleod Auditorium, Room 2158

We thank our 2010 Seminar Series Lecturers

Dr. Karen Davis, Associate Director of IMS, Professor of Surgery Dr. Richard Gilbert, Professor of Medicine Dr. Prabhat Jha, Professor, Department of Public Health Sciences Dr. Jay Keystone, Professor of Medicine Dr. Donald E. Low, Professor of Medicine Dr. John Semple, Professor of Pharmacology Dr. Vasundara Venkateswaran, Assistant Professor, Department of Surgery Dr. Nick Wooldridge, Professor and Director, Biomedical Communications

We Thank Our Generous Benefactors

Partners

UHN Center for Research

The Life Sciences Undergraduate Student Research Opportunity Program University of Toronto

Sponsors

Supporters

Keenan Research Centre at the Li Ka Shing Knowledge Institute

DEPARTMENT OF SURGERY

RESEARCH DAY AGENDA

Medical Sciences Building, University of Toronto 8:00 am 9:00 am 9:05 am 9:15 am Poster set-up and registration by students Welcome Dr. Neeru Gupta, Director, IMS Summer Research Program Introduction of Keynote Speaker Dr. Ori Rotstein, Director, Institute of Medical Science Keynote Address ­ "Personalized Medicine ­ The End of the Beginning" Dr. Katherine Siminovitch, Director, Genomic Medicine Division & Gene Profiling Facility, University Health Network, Professor, Departments of Medicine, Immunology, Molecular Genetics, University of Toronto / Mount Sinai Hospital Coffee Break Student Oral Presentations Samir Basmaji - Cardiovascular Patient Age as a Leading Determinant of Function of Autologous Stem Cells for Cardiac Cell Therapy Mohammed Hajiha - Brain Rhythms and Memory: is monitoring brain encephalographic activity a good measure to prevent awareness during anesthesia? Kimberly Shek - Detection of microRNAs in sera of oropharyngeal cancer patients Suzan El-Rass - Transposon Mediated Gene Trapping in Zebrafish Heart Development Liu D. Liu - Effect of levodopa on bidirectional synaptic plasticity in the substantia nigra pars reticulata (SNr) of Parkinson's disease (PD) patients Ali Ibrahim - Sensitivity to change of radiographic scoring instruments in axial psoriatic arthritis Lauren Diamond - MVP Registry: Efficacy and Safety of Different Bridging Regimens of Parenteral Anticoagulation after Mechanical Valve Surgery Jeffrey Gibson - Characterizing Factors Influencing the Inflammatory Infiltrate in a Neural Precursor Cell Transplantation Protocol for Rodent Spinal Cord Injury Lunch Poster Presentations Award Presentations Stone Lobby, 2171 2172 2172 2172

10:15 am 10:30 am

Stone Lobby, 2171 2172

12:00 pm 12:45 pm 3:00 pm

Macleod Auditorium Lobby, 2158 Stone Lobby, 2171 Macleod Auditorium, 2158

POSTER COMPETITION INSTRUCTIONS Poster set up and registration takes place from 7:45 to 8:45 am on Wednesday, August 04, 2010 in the Stone Lobby, 2171 Medical Sciences Building, 1 King's College Circle. Poster boards (3 feet deep by 6 feet wide) and Velcro to mount the posters will be provided. Poster presentation and judging takes place between 12:45 and 2:45 p.m. and takes place where your posters are displayed. Each student is assigned to a presentation group, with three judges. Please be at your assigned location on time to present your poster to the assembled group. Supervisors may attend their students' poster presentation.

Poster Group Poster Judges

A B C D E F G H I J K M N O

Dr. Bharati Bapat, Dr. Nades Palaniyar & Dr. Douglas Lee Dr. Xiao-Yan Wen, Dr. Ren-Ke Li & Dr. Arun Ravindran Dr. James Eubanks, Dr. Amira Klip & Dr. Muhammad Mamdani Dr. Shereen Ezzat, Dr. Qinghua Wang & Dr. Allan Kaplan Dr. Keith Jarvi, Dr. Dimitri Anastakis & Dr. Johann Hitzler Dr. Joe Fisher, Dr. Alan Lazarus & Dr. Ronald Heslegrave Dr. Stephen Meyn, Dr. Warren Lee & Dr. Elizabeth Pang Dr. Jeffrey Medin, Dr. David Clarke & Dr. Ariel Graff Dr. Carol Swallow, Dr. Magdy Hassouna & Dr. Eva Grunfeld Dr. Rebecca Wong, Dr. Kim Connelly & Dr. Ranju Ralhan Dr. Raisa Deber, Dr. Fiona Webster & Dr. Heather Ross Dr. Andrea Kassner, Dr. Andreas Laupacis & Dr. Anthony Levitt Dr. Gary Remington, Dr. David Jenkins & Dr. Warren McIsaac Dr. Tony George, Dr. Gary Rodin & Dr. Roger McIntyre

Each student is allocated a MAXIMUM of 7 minutes to give an ORAL PRESENTATION of his or her work (4-minute talk plus 3-minute question period). Abstracts will appear in the abstract booklet and on the IMS web site. The presentation should consist of a review of your poster and abstract. Poster judges will listen to each oral presentation, ask questions, and rank your abstract, poster, and presentation on · Quality of science (impact on field) · Quality of oral presentation (clarity, organization, hypothesis) · Visual impact of poster (layout, organization) · Response to questions (knowledge in area) · Timing (too long ­ too short) Posters should be removed after 3:30 p.m. Award presentations take place at 3:00 p.m. in the MSB 2158. Prizes will be given for · Best Oral Presentation st 1 prize - $200.00 2nd prize - $150.00 3rd prize - $100.00 Best Poster Presentation 1st prize - $200.00 2nd prize - $150.00 3rd prize - $100.00

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ABSTRACTS

Student Adleman, Alanna Alarifi, Mohammed Alharbi, Reem Aljubali, Ahmad Alobaidi, Ryyan Alrijraji, Eman Alsaaran, Rasha Al Subaihin, Abdulmajeed Alyahya, Reem Bai, Anthony Basmaji, Samir Berall, Laura Supervisor Dr. Chris Hudson Dr. John Parker Dr. Mark Minden Dr. Stephen Lye Dr. Sandy Logan Dr. David Mazer Dr. Gregory Borschel Dr. John Vincent Dr. Magdy Hassouna Dr. Rae Yeung Dr. Terrence Yau Dr. Joseph Beitchman Dr. Dafna Gladman Dr. Vasundara Venkateswaran Dr. Jeff Daskalakis Dr. David Urbach Dr. York Pei Dr. David Mazer Dr. Michael Fehlings Dr. Jeff Daskalakis Title The Effect of Hypercapnia and Hyperoxia on Intraocular Pressure Effect of the Antiplatelet Drug Clopidogrel on Ischemia-Reperfusion Induced Endothelial Dysfunction in the Human Forearm The Role of Colony Stimulating Factor 1 Receptor (CSF1R) in Acute Myeloid Leukemia (AML) Identification of Neutrophils in Mice Decidue and Their Angiogenic Function Assessing heart rate variability as a measure of sympathetic nervous system activity in controlled and refractory hypertensive patients Recruitment variability in a clinical trial: St. Michael's Hospital's experience Survey of Acute Pediatric Fracture Mapping of a new Joubert Syndrome gene in Consanguineous Pakistani Family Intravesical instillation of botulinum toxin A (BTXA) inhibited thoracic and lumbar C-fibre activity in high thoracic spinal cord injury rat model Determinants of morbidity due to systemic steroid therapy in childhood rheumatic disease Cardiovascular Patient Age as a Leading Determinant of Function of Autologous Stem Cells for Cardiac Cell Therapy A Genetic Association Study of the Vasopressin/Oxytocin System and the Oxytocin Receptor in Childhood Aggression The Role of KIR 3DL1 alleles in Psoriatic Arthritis The use of Metformin to Improve the Effectiveness of Androgen Deprivation Therapy in Prostate Cancer Clozapine-Triggered GABAB Receptor Internalization Prevalence of Autoimmune Disease in Patients with Esophageal Achalasia Identifying Novel Therapies for Autosomal Dominant Polycystic Kidney Disease (ADPKD) Transfusion Triggers in Cardiac Surgery Characterizing Surface Markers on IPS derived Neural Precursor Cells The Long-Term Potentiation (LTP)-like Plasticity (PAS-LTP) in the Motor Cortex Group A C H E K F J F G H ORAL N

Berinstein, Jeffrey Besla, Rickvinder Singh Blichowski, Monica Booy, Jason Budman, Joshua Burns, Laura Chan, Timothy Chara, Gurpreet Singh

O I M K A B D N

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Chen, Johnson Yi Hao Chen, Yi An Chen, Yu-Chih Chen, Yuhan Cheung, Yan Yui Benedict Chin, Kyle Choi, David Chun, Yi-Min Darmawikarta, Denise Diamond, Lauren Dossa, Fahima Du, Chao Ehrlich, Rachel El-Rass, Suzan Esen, Emre Cem Fazalullasha, Fatima Fernandes, Kimberly Fingrut, Warren Fung, Alan Gerster, Kate Gibson, Jeffrey

Dr. Charles Tator Schachar, Russell Dr. Tianru Jin Dr. John Semple Dr. Howard Mount Dr. Fang Liu Dr. Yigal Dror Dr. Marc de Perrot Dr. Jill Hamilton Dr. Stephen Fremes Dr. Sandra Black Dr. Liang Zhang Dr. Gary Rodin Dr. Xiao Yan Wen Dr. Bernard Le Foll Dr. Paul Links Dr. Donald Branch Dr. Ori Rotstein Dr. Seema Mital Dr. Fei-Fei Liu Dr. Michael Fehlings

The Efficacy of Neural Stem/Progenitor Cell Transplant on Function Recovery After Endothelin1 Induced Focal Spinal Ischemia in Rats Genetic Epidemiology of ADHD and OCD in a General Population To investigate the potential role of curcumin in the activation of Wnt signalling pathway in a high fat feeding induced insulin resistance mouse model Platelets and their Role in Preventing Severe Transfusion Related Acute Lung Injury (TRALI) Reactions Object memory is impaired in mice expressing a mutant human Presenilin 1 transgene Novel Peptide for the Treatment of Stroke Genetic analysis of the first reported solid tumour in Shwachman-Diamond Syndrome Role of VEGF in pulmonary hypertension development following acute pulmonary emboli Evaluating levels of physical activity in obese boys and girls using accelerometry MVP Registry: Efficacy and Safety of Different Bridging Regimens of Parenteral Anticoagulation after Mechanical Valve Surgery Associations between White Matter Hyperintensities and Hippocampal Atrophy in Alzheimer's Disease Telemetry (Wireless) recordings of physiological parameters in behaving mice Pathways between Pain and Depression in Cancer Transposon Mediated Gene Trapping in Zebrafish Heart Development Medicinal Use of Cannabis and Cannabinoid Derivatives The Relationship between Personality Disorders and Axis I Psychiatric Disorders: Distinct or Not? A Comparison of Methods to Detect Cell-Surface Globotriosylceramide (Gb3) Ceramide Mediates Increased Surface Toll Like Receptor 4 Translocation Increasing Burden of Environmental Risk Factors for Congenital Heart Disease MicroRNA Conservation in Human Cancers Characterizing Factors Influencing the Inflammatory Infiltrate in a Neural Precursor Cell Transplantation Protocol for Rodent Spinal Cord Injury Effect Of Feeding Anti-Cd3 Monoclonal Antibody On In Vivo T Cell Response To T Cell Receptor Activation

C M B E G D I O A ORAL M B N ORAL O K C G E J ORAL

Goethel, Ashleigh

Dr. Kenneth Croitoru

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Gonul, Ozge Gu, Chichang (Sam) Hajiha, Mohammad Halpern, Noah Hariri, Waseem Abdulrahman H. Hong, Jonathan Hu, Tina Huang, Darryl Huang, Guan Ibrahim, Ali Israelian, Nyrie Jain, Ashu Nitin Jayakar, Jai Prashanth Johnston, Calvin Jomah, Mohammed Abdulkader Kawaguchi, Sarah Khosravi, Ramak Kirshenblatt, Shanna Kommaraju, Kamya Koumpan, Yuri Kucukbas, Gokce Naz Kumarappah, Ananthavalli

Dr. Christoph Licht Dr. Andras Kapus Dr. Beverley Orser Dr. Steven Gallinger Dr. Sandro Rizoli Dr. Mark Peterson Dr. Greg Hare Dr. Paul Fortin Dr. Robert Chen Dr. Dafna Gladman Dr. Berge Minassian Dr. Thomas Waddell Dr. Dafna Gladman Dr. David Urbach Dr. Jeffrey Medin Dr. Michael Jewett Dr. Michael Fehlings Dr. Michael Fehlings Dr. Stephen Fremes Dr. Warren Lee Dr. Minna Woo Dr. Carol Westall

Complement Factor H Mutation In Atypical Hemolytic Uremic Syndrome Role Of Glycogen Synthase Kinase-3 In EpithelialMesenchymal Transition Brain Rhythms And Memory: Is Monitoring Brain Encephalographic Activity A Good Measure To Prevent Awareness During Anesthesia? Familial Pancreas Cancer: The Search For A Common Gene Variant Amongst Fpc Patients The Early Traumatic Coagulopathy: A Review Of Pathophysiology Mitroflow Aortic Pericardial Bioprosthesis: Clinical Performance And Functional Class Assessment Maintained Cerebral Perfusion With A Novel -1 Antagonist With Vasodilatory Properties The Prevalence Of Liver Abnormalities In Patients With Systemic Lupus Erythematosus The Effect Of Ap And Pa Directed Currents Induced By Transcranial Magnetic Stimulation On Long Interval Intracortical Inhibition Sensitivity To Change Of Radiographic Scoring Instruments In Axial Psoriatic Arthritis Glycogen Synthase And Its Role In Lafora Disease Design Of A Bioreactor For A Tissue-Engineered Trachea Prevalence Of Metabolic Syndrome In Psoriasis And Psoriatic Arthritis Decision Analysis As An Aid To Determining The Management Of Early Low Rectal Cancer: Does Current Knowledge Change The Balance? Lentiviral Vector Correction Of Fabry Disease Encompassing a Cell Fate Control Safety Element Post-operative outcomes are favourable for patients with cystic renal cell carcinoma Characterization of the blood-spinal cord barrier (BSCB) and vascular changes following spinal cord injury Development of a glutamate-specific biosensor The Graft Imaging to Improve Patency (GRIIP) Substudy Mechanisms of induction of microvascular leak by anthrax lethal toxin Does nfil3 transcription factor affect glucose regulation? Is Tuberous Sclerosis a co-morbidity factor for Vigabatrin-associated retinal toxicity in children with Infantile Spasms

D H ORAL I M G B K C ORAL D E N K F O A H K I A M

3

Lakovic, Katarina Larjani, Soroush Law, Wyanne

Dr. Loch Macdonald Dr. Muhammad Gelareh Zadeh Dr. Lucy Osborne

Lee, Claire Bo Lee, I-Ju Lee, Justin Leon-Carlyle, Marisa Li, Guo Liang, Yun-Tzu Lin, Boken Lin, Dong Lipszyc, Joshua Liu, Annie Liu, Liu (Dave) Mayer, Dana McCurdy, Nina McLean, Katherine Mok, Rebecca

Dr. Howard LeongPoi Dr. Jean-Philippe Pignol Dr. Cindi Morshead Dr. David Urbach Dr. John Semple Dr. Peter Liu Dr. Michael Fehlings Dr. Onil Bhattacharya Dr. Susan Tarlo Dr. Mingyao Liu Dr. William Hutchison Dr. Jane Aubin Dr. Jennifer Jones Dr. Seema Mital Dr. Elise Heon

Mui, Carween

Dr. Cindi Morshead

Effect of Experimental Subarachnoid Hemorrhage on Long-Term Potentiation in Mice Role of bone marrow derived progenitor cells in intracranial tumor neovascularisation Expression levels of genes flanking the targeted region in the Gtf2i- and Gtf2ird1-deficient mouse models of Williams-Beuren syndrome do not appear to be altered Survivin Gene Therapy Prevents the Deterioration of Heart Function in a Rat Model of DoxorubicinInduced Cardiomyopathy AuNP Radiosensitization Assays and Comparison with a Theoretical Model Endogenous stimulation of neural precursor cells leads to neural regeneration and functional recovery following stroke in adult mice. A Comparison of Instruments to Assess Male Sexual Function Following Treatment for Rectal Cancer Platelet MHC Antigens Inhibit Platelet-specific (CD61) Cytotoxic T cell (CTL) Mediated Thrombocytopenia Apelin Deficiency Contributes to Fibrosis in the Heart: Synergies with Angiotensin II Evaluation of the Sodium-Glutamate Blocker Riluzole in a Preclinical Model of Cervical Spinal Cord Injury Clinic managers' insight into organization of healthcare in First Nations in Canada as part of the CIRCLE study Survey of the Outcomes of Work-Related Asthma Examining the effects of peptide-gold nanoparticle hybrids on the activation of myeloid dendritic cells Effect of levodopa on bidirectional synaptic plasticity in the substantia nigra pars reticulata (SNr) of Parkinson's disease (PD) patients Enrichment of Vascular Endothelial Cells in the Highly Purified Osteoprogenitor Cell Fraction of Murine Bone Marrow Stromal Cell Populations Let's Talk: A brief intervention for improving family caregivers' symptom assessments Capturing common genetic variations that influence bioavailability of drugs used in children with heart disease Receptor guanylate cyclases as modifiers of BardetBiedl syndrome phenotypes in Caenorhabditis elegans Cyclosporin A has pro-survival effects on temporally distinct neural stem and progenitor populations in the adult central nervous system

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E J D K F I H O M G ORAL A N B D

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Mundil, Dhanwantee Ngan, Jessica

Dr. Leticia Rao

Oxidative stress and the risk of osteoporosis: The role of dietary polyphenols and nutritional supplements Interferon Regulatory Factor-3 (IRF-3) promotes cardiomyocyte apoptosis following myocardial infarction Evaluation of acute bioengineered delivery of a CD44 function blocking antibody to treat severe spinal cord injury in rats Determinants of hypertension due to systemic steroid therapy in juvenile rheumatic disease LTP is preserved in surviving hippocampus following severe ischemic-hypoxic episodes in mice Is treatment of hypertension and hypercholesterolemia successful in patients with systemic lupus erythematosus? In situ differentiation of transplanted neural stem cells into the injured spinal cord Loss of Regulator of G Protein Signalling 5 Possibly Accelerates Diabetic Kidney Injury Testosterone-Guided Schedule of Androgen Deprivation Therapy as an Alternative to a Fixed Schedule in the Management of Prostate Cancer The role of Snail transcription factor during skin wound healing Clinical outcome after traumatic spinal cord injury: creation of a database and presentation of clinical measures The Influence of the Word Cancer on Patient Treatment Preference TMS-induced Interhemispheric Signal Propagation and Five Factor Model of Personality Detection of microRNAs in sera of oropharyngeal cancer patients Examination of Steroid Dose and Steroid-related Damage in an International Inception Cohort of Systemic Lupus Erythematosus Patients Characterization of the interaction between the Cbl ubiquitin ligase and the lymphoid tyrosine phosphatase (LYP) Polypathology and its impact on supportive care needs: Cancer patient perspectives on existing selfreport questionnaires Cognitive Factors Associated with Belief Formation in the General Population Prevalence of co-existing medical conditions in patients with congenital heart defects

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Dr. Peter Liu

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Oh, Yoonsun Ono, Akane Patel, Nisarg Pek, Elisabeth Peng, Valerie Qin, Zhongming Joel Ragupathi, Loheetha Sattarova, Victoria Schoenhoff, Nicholas Secord, Stephanie Senel, Basak Shek, Kimberly Shi, Hang

Dr. Michael Fehlings Dr. Rae Yeung Dr. Zhang, Liang Dr. Murray Urowitz Dr. Molly Shoichet Dr. Jim Scholey Dr. Ian Tannock Dr. Benjamin Alman Dr. Michael Fehlings Dr. David Urbach Dr. Jeff Daskalakis Dr. Fei-Fei Liu Dr. Murray Urowitz

G I H O B C M D I K N ORAL O

Siu, Adrienne Siu, Maggie

Dr. Katherine Siminovitch Dr. Jennifer Jones

E M

Sokolowski, Helen Moriah Somascanthan, Priya

Dr. Albert Wong Dr. Seema Mital

F A

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Spano, Stefania

Dr. Michael Fehlings

Spiegle, Gillian Sroa, Jyotika Tam, Alex Tang, Michael Tian, Ye Ting, Katrina Tseng, Yun Fan Christine Tulba, Wesam Hussain Vincent, Matthew Vitko, Kyra Wallace, Tessa Wang, Yao Wedeles, Christopher Wheeler, Kathleen Wiebe, Meagan Wong, Lilian Lee Yan Wu, Ke

Dr. David Urbach Dr. Michael Cusimano Dr. Neeru Gupta Dr. Mark Cattral Dr. Dr. Chung-Wai Chow Dr. Cynthia Goh Dr. Michael Fehlings Dr. Mansoor Husain Dr. Carol Swallow Dr. David Malkin Dr. James Kennedy Dr. Edward Cole Dr. Mei Zhen Dr. Rebecca Wong

Dr. David Urbach Dr. James Wright Dr. Paul Arnold

Assessment Of Neural Precursor CellOligodendrocyte Interactions Following Neural Precursor Cell Transplantation In A Rodent Model Of Subacute Spinal Cord Injury Physicians' Awareness Of Cytoreductive Surgery And Intraperitoneal Chemotherapy For Colorectal Carcinomatosis A systematic review assessing alcohol abuse interventions for a post-traumatic brain injury population Quantum Dot in vivo Imaging Shows Lymphatic Drainage from the Mouse Eye A novel in vitro method of generating murine dendritic cells using supernatants containing flt3ligand--supplemented bone marrow cultures PM induces co-localization of Syk and dectin-1 in airway epithelial cells (AEC) Collagen Does delayed AdV-ZFP-VEGF administration following traumatic spinal cord injury reduce neural cell death? Proposed Derivation Of Pure Populations Of Human Smooth Muscle Cells Using Transgenic Human Embryonic Stem Cells Effect of Polo-like kinase 4 (PLK4) on progression of colorectal cancer (CRC) Biological effects of intracellular pH on function of a mutant p53 protein specific to adrenocortical carcinoma Genetic association study of polymorphisms in serotonin receptor 2C, leptin and leptin receptor genes with antipsychotic induced weight gain Within-patient variability of tacrolimus levels as a risk factor for transplant glomerulopathy in kidney transplant patients The RNA binding protein UNC-75 regulates alternative splicing in the C. elegans nervous systems Strategies to Reduce Patient Discomfort and Anxiety during Non-Invasive Radiotherapy and Diagnostic Radiology Procedures ­ A Systematic Review Understanding Barriers To Access In The Management Of Colorectal Cancer Hepatic Metastases: A Qualitative Approach Long-Term Follow-up of Unit-Rod Instrumentation in Spina Bifida Scoliosis Patients Population-based association study of glutamatergic candidate genes in obsessive compulsive disorder (OCD) and response inhibition

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J N I G B B C D D F F H I J

J J O

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Wu, Mei Hsuan

Dr. Lucy Osborne

Wu, Qiu Jing Young, Christine Zaslavsky, Kirill Zayne, Kory Zeng, Seda Zhai, Lihua Zhang, Christine Zhang, Ning

Dr. Sheena Josselyn Dr. Angela Cheung Dr. Paul Frankland Dr. Peter Kim Dr. Suzanne Trudel Dr. Wolfgang Kuebler Dr. Seema Mital Dr. Jeffrey Medin

Expression levels of c-Fos in Gtf2i- and Gtf2ird1deficient mouse models of Williams-Beuren syndrome following exposure to a fearful environment Olfactory impairment in DJ-1 knockout mice as a characteristic of Parkinson's disease Prevalence and Incidence of Vertebral Deformities by VFA in Postmenopausal Women with Osteopenia: Companion Study to the ECKO Trial Structural changes in mossy fiber axons following spatial learning The role of kinesin protein Kif7 in the formation of the diaphragm Pre-clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Multiple Myeloma Tumors Role of platelets in a one-hit model of transfusion-rela lung injury (TRALI) Effect of Cord Blood/Bone Marrow Transplant on Cardiac Function in Pediatric Patients Generation of targeting vectors for construction of mouse models for Farber disease and human malignancies

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A J C E E G H G

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The Effect of Hypercapnia and Hyperoxia on Intraocular Pressure

Alanna Adleman1, Chris Hudson1,2, 1Department of Ophthalmology and Vision Sciences, Toronto Western Hospital and Institute of Medical Science, University of Toronto, 2School of Optometry, University of Waterloo Background: A sequential re-breathing circuit allows the precise targeting and stable control of the endtidal partial pressures of carbon dioxide (PETCO2) and oxygen (PETO2). Previous studies suggest that inhaled hypercapnic and hyperoxic stimuli influences intraocular pressure (IOP) but all work to-date has not maintained tight control of PETCO2 and PETO2 values. Purpose: To determine the magnitude of response, if any, of IOP to standardized and stable inhaled gas provocations using a sequential re-breathing circuit and automated gas blender. Methods: Twelve healthy volunteers (mean age 20-25) were enrolled in the study. Participants were asked to abstain from caffeine, alcohol and red meats for 12 hours, and exercise and water for 2 hours immediately prior to the study. Participants were fitted with a sequential re-breathing circuit and exposed to five different inhaled gas stages. At each stage, IOP was measured in a randomly selected test eye. In the first stage, baseline was established while participants breathed room air. In the second and third stages, PETCO2 was increased by 10% and 20% (order randomized between participants). In the fourth and fifth stages, PETO2 was increased by 150% and 400% (order randomized between participants). Results: Based on previous studies using alternate methods of inhaled gas provocation, IOP is expected to be unchanged during hypercapnia and to decrease as hyperoxia increases. From these results, a doseresponse curve of IOP can be established. Conclusion: The results of this study will provide a standardized measure of the response of IOP to varying levels of inhaled gas provocation.

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Effect of the Antiplatelet Drug Clopidogrel on Ischemia-Reperfusion Induced Endothelial Dysfunction in the Human Forearm

Mohammed Ibrahim AlArifi, Dr. John Parker The current treatment strategy in patients following a myocardial infarction (MI) is to restore perfusion to the infarcted area. Unfortunately, reperfusion itself can contribute to subsequent tissue damage, a phenomenon known as ischemia reperfusion (IR) injury. In the heart, IR injury has been shown to cause arrhythmias, vascular endothelial dysfunction, myocardial stunning (reversible loss of myocardial contractility) and cellular death. studies have demonstrated that exposure to brief periods of ischemia (termed "ischemic preconditioning" (IPC)) can reduce myocardial sensitivity to IR-induced injury. Of clinical value, it has been shown that certain pharmacological agents are able to induce preconditioning protection in the myocardium and other tissues through mechanisms similar to IPC. For example, pretreatment with the angiotensin-converting enzyme (ACE) inhibitor captopril has been able to reduce infarct size and improve endothelial and cardiac function in animal models of IR injury. Most recently, our group provided the first line of evidence that HMGCoA reductase inhibitors (statins) have pharmacologic preconditioning effects in humans by using the forearm study . Given the wide spectrum of patients affected by IR injury, it would be beneficial to investigate other agents that are capable of inducing preconditioning-like protection. Whether or not a clopidogrel can provide protection from IR injury exists in humans has yet to be investigated. The purpose of this study is to assess clopidogrel as a preconditioning agent in the prevention of IRinduced endothelial dysfunction in the human forearm vasculature as assessed by flow mediated dilatation (FMD) in forearm conduit vessels.

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The Role of Colony Stimulating Factor 1 Receptor (CSF1R) in Acute Myeloid Leukemia (AML)

Alharby, Reem, Dr. Mark Minden, Mohamed Fateen Ontario Cancer Institute, Princess Margaret Hospital, University of Toronto Introduction Colony stimulating factor 1 receptor (CSF-1-R/FMS) is a transmembrane tyrosine kinase receptor expressed on early hematopoietic stem cells and macrophages and is responsible for their survival and proliferation. There is a variable association of FMS with stem cell marker CD34 on AML cells and this suggests different structures of the AML hierarchy in different patients. Objective The present study aims to determine if in some AML cases FMS is associated with stem cell compartment of myelomonocytic leukemias (M4 and M5 of FAB classification) and whether the expressed FMS is important for growth and survival of AML stem cells in these forms of the disease. Methods Mouse stromal cells (MS-5) have been transfected with human CSF-1 because mouse CSF-1 is inactive on human FMS. Protein expression of CSF-1 is confirmed by Western blot. Co-culture of AML cells on top of MS-5 or MS-5-CSF1 cells and the cultures are assessed for the number and size of cobble stone areas (a surrogate for AML stem cells). Preliminary Results: AML samples with high FMS (CSF-1-R) expression have shown an increased adhesion to MS-5-CSF1 cells compared to MS-5 cells. In addition, the proliferation of FMS expressing AML cells is enhanced on the CSF1 expressing stroma. Conclusion The finding of improved growth of FMS positive cells on CSF-1 expressing stroma would provide evidence that in some AML cases the stem cell population expresses and is responsive to FMS stimulation. This will provide a novel therapeutic approach for some AML cases with already available FMS inhibitors.

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Identification of Neutrophils in Mice Decidue and Their Angiogenic Function

Ahmed Aljubali, Hagai Amsalem, Caroline Dunk, Dr. Stephen Lye. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto.

Background: Preeclampsia in one of the most common and serious complications of pregnancy that affects both mother and baby. It is caused by failure of the mothers' uterine arteries to increase in size to supply enough blood to the developing fetus.This utero-placental vascular insufficiency is thought to result from defective extravillous trophoblast invasion that leads to failure of uterine spiral arteries remodeling. A large population of decidual immune cells also plays a major role in the angiogenesis and remodeling of these arteries. The newly discovered presence of neutrophils associated with these vessels in the second trimester decidua suggests that they may also possess angiogenic functions. However, in order to confirm that the accumulation of neutrophils in the decidua is not due to an inflammatory process associated with the termination of pregnancy we need to assess neutrophil levels in an animal model of pregnancy across gestation. Hypothesis: We hypothesize that decidual neutrophils will display an accumulation in the murine decidua during the stages of spiral artery angiogenesis between days 8-10 of gestation. Objectives: To establish that neutrophils are associated with active decidual angiogenesis in both humans and mice. Methods: In mice we collected uterine samples including myometrium ,decidua, mesometrial triangle with placenta in different gestational times (day 6,8,10,12,14 and18).samples were processed for immunohistochemistry using monoclonal anti-neutrophil antibody 7/4 to identify neutrophils. Neutrophils associated with the spiral arteries were counted manually using Visiopharm Integrated system with NewCast stereology software. Results: Immunostaining confirmed the presence of neutrophils in the mouse decidual tissue . Neutrophils were observed adhered to endothelium and infiltrating the vascular wall of the uterine spiral arteries. A large accumulation of decidual neutrophils was observed on day 8 of pregnancy that was maintained to d10 and then decreased to non detectable levels by d18 Conclusion: Our results clearly demonstrate the presence of neutrophils in the second trimester decidual tissue in both human and mice confirming their true biological role in this tissue. Although their exact role is still unknown, our data supports their potential role in decidual angiogenesis and vascular remodeling. We hope that this data will lead to advances in the understanding of the developmental pathogenesis of preeclampsia.

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Assessing heart rate variability as a measure of sympathetic nervous system activity in controlled and refractory hypertensive patients

Alobaidi Ryyan, Dr. Alexander (Sandy) Logan, Professor of Medicine, Samuel Lunenfeld Research Institute - Mount Sinai Hospital. Background: Hypertension is a disease that affects approximately 1 billion individuals worldwide. Refractory or resistant hypertension is conventionally defined as a systolic or diastolic blood pressure that remains uncontrolled despite sustained therapy with at least three classes of antihypertensive agents and affects 5-30% of the general population with hypertension. Patients with refractory hypertension have an increased risk of stroke, aortic dissecting aneurysm, myocardial infarction, congestive heart failure and renal failure compared to other hypertensive patients. During the last two decades a significant relationship between autonomic nervous system and cardiovascular mortality was recognized and heart rate variability (HRV) represent one of the best quantitative markers of autonomic activity.

Methods & Results: Overnight polysomnography was performed in 41 patients (26 refractory & 15 controlled). Three 10-minute segments each of wake period, stage 2 and REM stage from each patient were visually identified and ECG signal exported for HRV analysis. The result still pending but we anticipate a high sympathetic activity among the patients with refractory hypertension.

12

Recruitment variability in a clinical trial: St. Michael's Hospital's experience

Eman Alrijraji, MD, Sanjay Yagnik, MD, C. David Mazer, MD, Dept of Anaesthesia, Li Ka Shing Knowledge Institute in the Keenan Research Center, St. Michael's Hospital, University of Toronto Background: 80% of failures of clinical trials are caused by the inability of research sites to find and enroll sufficient patients and meet timelines. Those trials fail not because of the drug, but because the trial's patient recruitment and retention goals were not achieved. Participant recruitment has been called "the most difficult and challenging aspect of clinical trials," * During my summer research elective in Toronto, I've been involved in an ongoing multicenter clinical trial of recombinant Factor XIII in cardiac surgery and had the opportunity to experience and explore the challenges of the recruitment process. Purpose: To determine recruitment rates and variability for the period of Dec 2009 - July 2010 at St. Michael's Hospital, and to examine factors that might have affected the recruitment process. Methods: A retrospective approach was used. Data is gathered for patients' numbers, eligibility, recruitment rates and variability each month. Factors affecting recruitment are tabulated. Results: Preliminary results show that the recruitment was at its peak in the spring and summer months whereas it somehow lagged in the winter months. Percents of total patients recruited per month in order were as follows (3.8%, 0.99%, 4.1%, 4.6%, 9.4%, 5.1%, 6.3%, and 11.4%). April and July had the highest recruitment rates and January was the lowest despite having higher possible number of subjects. Most of the recruited subjects were approached in the pre-admission facility rather than in the ward indicating that the timing and location of first subject contact may play a significant role in the recruitment process. Discussion: Based on the preliminary results, the recruitment process was affected by several factors: the availability of research volunteers, the investigator's enthusiasm for the study, the timing of first subject's contact seem to have a great influence on the recruitment rates. The fact that the site is a teaching hospital with a variety of patients, interests and backgrounds provided a rich recruiting environment. Possible barriers were the lack of time and staff. Holidays might also have an effect on the rates, and language was a notable barrier.

* L.Lovato, K.Hill, S.Hertert, B.Hunningshake, J.Probstfield, "Recruitment for Controlled Clinical Trials: Literature Summary and Annotated Bibliography," Controlled Clinical Trials (1997).

13

Survey of Acute Pediatric Fracture

Rasha Alsaaran, Ronald Zuker, Dr. Gregory Borschel and Emily Ho Division of Plastic Surgery, The Hospital for Sick Children (SickKids) Background: Children with acute fractures are often initially seen by General Practice, Emergency Department, or Walk-in Clinic Physicians. Referrals to Plastic Surgeons may be required for children with acute facial and hand fractures for consultation and potential treatment. A needs assessment of children with acute fractures serviced by the SickKids Division of Plastic Surgery has not been conducted. This type of survey is critical in understanding the characteristics of the patient population being served and how to best provide clinical care. Purpose: Conduct a prospective needs assessment of the children with acute fractures seen by the Division of Plastic Surgery in the summer months. Determining the type of fractures, the spectrum of injury and current medical management will provide essential information in preparation of clinical practice guidelines. Methods: Prospective review of all patients between 0-18 years of age referred for consultation to Plastic Surgery from June 14 to August 13, 2010. Prospective review is required to ensure that all information is collected, as incomplete records are a confounder of retrospective studies. Results: The survey is still ongoing at this time. Preliminary results showed that children aged 10-12 years are the most frequent age group presenting with acute fractures. Sport related injuries are the most common etiology. The study also showed that there was 1to 5 days interval period between the initial assessment by a Plastic Surgery Division physician and the initial assessment by SickKids Emergency Department or General Practice physicians respectively. The average time from the injury to the initial assessment by Plastic Surgery Division physician was 5 days. Conclusion: Upon completion of the survey we will be able to develop clinical guidelines for evaluating children with acute facial and hand fractures at SickKids. Also, surveying the etiology and environmental factors that influence the incidence of these fractures may provide insight to risk prevention strategies to reduce future injuries.

14

Mapping of a new Joubert Syndrome gene in Consanguineous Pakistani Family

AA Alsubaihin, MA Rafiq, JB Vincent Molecular Psychiatry and Development Lab, Centre of Addiction and Mental Health

Background: Joubert syndrome, a rare autosomal recessive disease, affects 1 in every 100,000 individuals in the United States. Clinical features include intellectual disability, developmental delay and cerebellar vermis hypoplasia in brain MRI. Mutations in 8 genes were related to Joubert syndrome, these are INPP5E, AHI1, NPHP1, CEP290, TMEM67, MKS3, RPGI1L, ARL13B and CC2D2A. The products of the aforementioned genes were found to localize in the primary cilium; a multiprotein complex that plays a critical role in vertebrate cellular signaling and development. Many phenotypically diverse genetic disorders share similar genetic defects with Joubert Syndrome, they are collectively known as Ciliopathies. Objective: Mapping of novel genes in Pakistani Families affected with Joubert Syndrome. Methods: A family from Pakistan with consanguineous parents was recruited, DNA was extracted from 4 affected and three unaffected individual. All affected and one unaffected individual were typed by Affymetrix 500k SNP microarray. dChip software was used to localize homozygosity segregating in all affected individuals. Exons and splice junctions of 8 candidate genes in the region of homozygosity have been sequenced. Results: No pathogenic changes were found in candidate genes that were sequenced and analyzed until now. We anticipate performing exome sequencing at Next Generation Sequencing platform to crack any underlying mutated genes in our locus of interest. In conclusion, mapping of a new gene will provide new information about the primary cilium and explain phenotypic diversity of Joubert Syndrome and other Ciliopathies.

15

Intravesical instillation of botulinum toxin A (BTX-A) inhibited thoracic and lumbar C-fibre activity in high thoracic spinal cord injury rat model

Reem Alyahya, Dr. Hassouna, Department of Urology, Toronto Western Hospital Background and hypothesis

Bladder dysfunction after SCI includes detrusor hyperreflexia (DH) and autonomic dysreflexia which involves life-threatening episodes of paroxysmal hypertension and bradycardia. BTX-A is currently thought to affect sensory nerve fibers in addition to its bladder muscle paralysis. It is still however unknown, if BTX-A may affect primary C-afferent fibres sprouting and putative spinal neurons involved in DH and AD following SCI. In this study, we examined the changes in CGRP concentration in T4, L5 and L6 dorsal root ganglia in spinalized rats and in intravesically BTX-A treated spinalized rats.

Objective

The effect of BTX-A on C-afferent fibres sprouting and putative spinal neurons involved in DH and AD following SCI.

Methods

Rats were divided into three groups; normal controls (C); spinally transected at T4 (S); and spinally transected with intravesically BTX-A (B). Under general anesthesia complete spinal cord transection was performed at T4 vertebra level. Three weeks after spinal transection, the bladder of the BTX-A treated group was emptied of urine and slowly filled with BTX-A. CGRP was extracted from the dorsal root ganglia of the T4, L5 and L6 roots of all study groups and quantified by radioimmunoassay. Univariate ANOVA was used to analyse the data and statistical significance was set at P <0.05.

Results

SCT significantly increased T4 CGRP concentration (59.00± 5.29 fmol/ml) than the control group (31.0± 2.64 fmol/ml; P< 0.001); BTX-A significantly reduced CGRP concentration in T4 (21.0 ± 1.0 fmol/ml; P< 0.001). Furthermore, BTX-A significantly reduced CGRP concentration in L5 (24.33 ± 10.78 fmol/ml) than the spinally transacted group (50.0 ± 7.9 fmol/ml; P = 0.03).

Conclusion

Intravesical BTX-A administration seems to inhibit C-fibre activity at the thoracic and lumber levels in rats following spinal cord injury. This provides the first evidence that intravesical BTX-A could affect AD in a preclinical SCI animal model.

16

Determinants of morbidity due to systemic steroid therapy in childhood rheumatic disease

Anthony Bai Supervisor: Dr. Rae S.M. Yeung, Department of Cell Biology, Hospital for Sick Children Background: Corticosteroids are used as anti-inflammatory agents in childhood rheumatic diseases including central nervous system vasculitis(CNS vasculitis), juvenile dermatomyositis(JDM) and juvenile idiopathic arthritis(JIA). Systemic steroid treatments have many adverse side effects including weight gain and hypertension. Factors that contribute to the development of these adverse side effects are not clearly understood. Purpose: To identify disease and patient related risk factors for the development of steroid related morbidity using 3 well characterized cohorts of children with rheumatic disease. Method: JIA inception cohort was obtained from a Canadian JIA study (ReACCh OUT). CNS vasculitis and JDM inception cohorts were obtained from ongoing Sick Kids disease specific registry. Clinical and laboratory data at baseline and 6 months follow-up were collected and validated. Results: Preliminary results included JIA cohort as non-steroid inflammatory control and JDM cohort as steroid inflammatory group (table1). Primary outcome measure of steroid morbidity was change in BMI (kg/m2) between baseline and 6 months follow-up as a surrogate marker of body habitus change. In both cohorts, univariate analysis revealed that patient characteristics including age, sex, baseline BMI and individual disease activity measures had no significant correlations with BMI change. However, steroid dosage per weight had a significant correlation with BMI change in JDM cohort (r=0.33,p=0.009). Conclusion: Based on preliminary results, demographic features, starting body habitus and measures of disease activity were not associated with BMI increase as a marker of systemic steroid therapy associated morbidity. Only steroid dosage had a significant correlation. Another steroid treated group will be analyzed to validate these findings.

Table1 JDM(n=84) Diagnosis Age mean SD 7.7±3.9 Female n(%) 56(67%) European Descent n(%) 46(64%) Baseline BMI mean SD 17.0±3.6* Statistic significant difference: *p<0.05 JIA(n=280) 8.2±4.9 201(72%) 198(72%) 18.8±5.41*

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Cardiovascular Patient Age as a Leading Determinant of Function of Autologous Stem Cells for Cardiac Cell Therapy

Samir Basmaji, Mingxi Li, Yumei Zhang, Keith Brunt, Ren-Ke Li, Richard Weisel, Katherine Tsang, Terrance Yau Department of Cardiovascular Surgery, Toronto General Hospital

Background: Stem cell therapy is an evolving approach to restore heart function after a myocardial infarction (MI). Initial clinical trials were not as beneficial as anticipated; stem cells in elderly patients with diffuse vascular disease (DVD) did not improve heart function compared to younger patients. Purpose: This study evaluated the effects of age and DVD on the functional capacity of bone marrow stem cells used in regenerative therapies. Methods: Bone marrow (50 mL) was isolated from 10 patients undergoing cardiac surgery. Mononuclear cells were separated by Ficoll gradient centrifugation. Stem cell numbers were recorded during culture and their differentiation was measured by counting the number of blast forming colonies.

A

Results: Age is a major predictor of stem cell proliferation and differentiation in cardiovascular patients. We show that human mesenchymal stem cell (MSC) proliferation was significantly greater in younger patients between 50 to 60 years than patients 60 to 70 years (Figure A,B p<0.05). The number of differentiated stem cell blast forming colonies in 3D-methylcellulose culture was significantly decreased with age (Figure C, p<0.05).

Age & Differentiation Correlation Age & Growth Correlation C Conclusions: The proliferation and B 80 80 *Pearson, P<0.036 *Pearson, P<0.026 differentiation capacity of human bone 70 70 marrow stem cells is significantly reduced in older patients. Regenerative 60 60 stem cell therapy may only rescue 50 50 ventricular function in elderly patients if 40 40 the functional stem cell capacity can be 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 0 10 20 30 40 50 60 70 80 90 100 Growth Rate Total-CFU rescued prior to their implantation into the heart. Several new approaches are being tested in the laboratory to rejuvenate stem cells during their preparation, which may permit a rapid return to normal function.

18

A Genetic Association Study of the Vasopressin/Oxytocin System and the Oxytocin Receptor in Childhood Aggression

Laura E. Berall, Joseph H. Beitchman Child and Adolescent Psychiatry, Child, Youth and Family Program, Centre for Addiction and Mental Health Background: Aggressive behaviour in childhood has been associated with negative outcomes seen later in life. Such outcomes include: criminality, substance abuse, and low income. Callous and unemotional traits (showing a lack of guilt or empathy) define a subgroup of aggressive children who display a more extreme and pervasive form of aggressive behaviour. The vasopressin (AVP), oxytocin (OXT) and oxytocin receptor (OXTR) genes have been linked with aggression in preclinical studies. However, no genetic studies exploring the relationship between these genes and aggression in child populations have been published. Purpose: To examine the association between DNA variants within the AVP, OXT and OXTR gene regions with childhood aggression. Methods: 164 clinically referred children displaying extreme and pervasive aggression were included in this study. The callous-unemotional subscale of the Antisocial Process Screening Device (PSD) was used to determine levels of the callous-unemotional trait. Four DNA variants (rs2740204, rs2740210, rs2770378 and rs4813627) in the AVP/OXT gene region and three DNA variants (rs237898, rs237885 and rs2268493) in the OXTR gene region were analyzed. The frequency distribution of these variants was compared between aggressive children and healthy adult controls. Results: The frequency of DNA variants in the AVP/OXT and OXTR genes were not significantly different between aggressive children and healthy subjects. However, rs237885 in the OXTR gene was significantly associated with callous-unemotional scores. Conclusions: : While results from our examination of selected DNA variants in the AVP/OXT and OXTR genes do not support a role of these genes in child aggression, there appears to be an association between the OXTR gene and the callous-unemotional trait.

19

The Role of KIR 3DL1 alleles in Psoriatic Arthritis

Jeffrey Berinstein, Remy Pollock, Fawnda Pellett, Vinod Chandran, Dafna Gladman. Center for Prognosis Studies in Rheumatic Diseases, University Health Network Introduction: Psoriatic Arthritis (PsA) is a chronic immune-mediated inflammatory arthritis associated with psoriasis (Ps), an inflammatory skin disease affecting 3% of the population. 30% of patients with Ps develop PsA. HLA genes within the major histocompatibility complex (MHC) are risk factors for both Ps (HLA-B57, HLA-B13) and PsA (HLA-B27). HLA-B molecules are ligands for the killer-cell immunoglobulin-like receptor (KIR) 3DL1 expressed on NK and NK T cells which are found in psoriatic plaques and synovium of PsA patients. Hypothesis: KIR3DL1 alleles will differ between patients with PsA, Ps and healthy controls. Purpose: To develop an allelic typing system for KIR3DL1 and investigate associations with PsA and Ps compared to healthy controls. Methods: Genomic DNA from 99 unrelated PsA patients satisfying CASPAR criteria, 98 Ps patients (evaluated by a rheumatologist to exclude psoriatic arthritis), and 99 healthy unrelated controls were genotyped by a PCR-SSP method developed in-house and validated using reference DNA with known KIR3DL1 alleles. Chi square analysis was used to compare allele frequencies between sample groups. P-values 0.05 were considered significant. Results: PsA patients (60% females, mean age 44 years, mean psoriasis duration 11.2 years), Ps patients (66%females, mean age 44.6 years, mean psoriasis duration 12.8 years ), and controls (66% females, mean age 44.7 years) were tested for the 13 most frequent alleles. Of these, KIR3DL1*001, *002, *004, and *005 were the most common. KIR3DL1*001 occurred in 13% PsA, 11% Ps, and 13% of controls, *002 occurred in 13% PsA, 9% Ps, and 12% of controls, and *005 occurred in 14% PsA, 17% Ps, and 14% of controls. KIR3DL1*004, which is known to not be expressed on the cell surface, occurred in 19% PsA, 17% Ps, and 23% of controls. There was no significant difference in the frequencies of any alleles between either patient groups and controls. Conclusions: An effective allele typing method for KIR3DL1 alleles was developed. Preliminary results suggest KIR3DL1 alleles are not associated with either Ps or PsA. Sample size needs to be increased to confirm these results.

20

The Use of Metformin to Improve the Effectiveness of Androgen Deprivation Therapy in Prostate Cancer

Rickvinder Besla1*, Alexandra Colquhoun1, Natalie Venier1, Hiroshi Sasaki1, Michael Pollak3, Neil E. Fleshner2, Laurence H. Klotz1, and Vasundara Venkateswaran1. Division of Urology, Sunnybrook Health Sciences Centre, 2Division of Urology, Princess Margaret Hospital and , Toronto, ON, Canada, 3 Division of Urology, McGill University, Montreal, QC. Background: The incidence of prostate cancer (PCa) varies dramatically by geographic location, with developed countries exhibiting significantly higher levels of disease. Some attribute this to the `Westernized lifestyle' of high energy diets coupled with lack of physical activity and consequent obesity. Rising obesity levels have been mirrored by increased diagnoses of non-insulin dependent diabetes (NIDDM). Metformin (an oral biguanide used to treat NIDDM) possesses anti-neoplastic properties in vitro and in vivo. Studies have also shown that metformin treated diabetics experience less cancer diagnoses and/or prostate cancer-specific mortality. Purpose: To assess the potential additive benefit of combining bicalutamide, an androgen deprivation therapy used to treat prostate cancer, with metformin and delineate a potential mechanism of action. Materials and Methods: Androgen receptor positive and negative human PCa cells (LNCaP and PC3 respectively) were treated with bicalutamide (0-10 µM) and/or metformin (0-10 mM). Growth inhibition was assessed using clonogenic assays. Alterations in key signaling molecules following 24 hour treatment with bicalutamide and/or metformin were assessed by Western blot analyses. Results: Exposing cells to bicalutamide (M) or metformin (mM) resulted in significant dose-dependent reduction in cellular proliferation. Combined treatment caused a further significant reduction in cell proliferation (p<0.005). Western blot analyses revealed alterations in PSA, IR, caspase-3, BAX/BCL-XL ratio, and PARP, depending on androgen receptor status. Conclusion: This combination regimen may improve prostate-cancer specific survival via direct antineoplastic properties, possibly by modulation of apoptosis. Acknowledgements: IMS award to RB, CIHR grant to VV, NCIC to MP

1

Fig 1: Dose-dependent reductions in cell proliferation of LNCaP and PC3 cells lines. * denotes significantly reduced colony formation rate compared to either monotherapy alone, p<0.005

21

Clozapine-Triggered GABAB Receptor Internalization

Monica Blichowski, Dr. Daskalakis1, Dr. Snead2

1

Schizophrenia Program; 2Program of Neuroscience and Mental Health1; Centre of Addiction and Mental

Health; 2The Hospital For Sick Children GABAB receptors are G protein-coupled receptors that are considered promising drug targets for the treatment of neurological and mental health disorders. The atypical antipsychotic clozapine has shown to have superior therapeutic efficacy in schizophrenia and neurophysiological evidence suggests that this efficacy may be linked to the GABAB receptor. Previous studies in our lab have also shown that clozapine facilitates the binding of the ligand [3H]-CGP54626A to the GABAB receptor, suggesting that clozapine modulates the GABAB receptor allosterically. In the current study we investigated the ex vivo effects of clozapine on GABAB receptors. Mice were paired and split into two groups: a control group given a vehicle solution and a treatment group administered clozapine (5 mg/kg) through interperitoneal injections. The mice were sacrificed at three different time points: 15 minutes, 1 hour or 4 hours. Crude synaptic membranes were prepared with homogenate of whole brain for binding assays conducted with [3H]-CGP54626A, a radiolabeled GABAB antagonist. Radioactivity was detected by liquid scintillation counting. A significant decrease of [3H]-CGP54626A binding was observed in the clozapine-treated mice versus control mice. These results differed from our previous in vitro observations of an increase in [3H]CGP54626A binding in the presence of clozapine. A possible explanation is a decrease in the number of GABAB receptors on the neuronal surface due to clozapine-triggered GABAB receptor internalization: an important regulation of receptor function that may be affected by allosteric modulators. GABAB receptor internalization may relate to clozapine's allosteric modulating effect and has not been previously shown with this compound.

22

Prevalence of Autoimmune Disease in Patients with Esophageal Achalasia

Student: Supervisor: Co-Authors: Affiliation: Jason D. Booy BSc David R. Urbach MD MSc Julie Takata MA, George Tomlinson PhD Division of General Surgery, Toronto General Hospital

Background Achalasia is a rare disease of the esophagus that has an unknown etiology. Genetic, infectious, and autoimmune mechanisms have each been proposed. Autoimmune diseases often occur in association with one another, either within a single individual or in a family. There have been separate case reports of patients with both achalasia and one or more autoimmune diseases, but no study has yet determined the prevalence of autoimmune diseases in the achalasia population. Hypothesis The prevalence of autoimmune disease is higher in patients with esophageal achalasia than in the general population. Methods We retrospectively reviewed the charts of 193 achalasia patients who received treatment at Toronto's University Health Network between January 2000 and May 2010 to identify other autoimmune diseases and a number of control conditions. We determined the general population prevalence of autoimmune diseases from published epidemiological studies. Results The achalasia sample was, on average, 10-15 years older and had slightly more males than the control populations. Compared to the general population, patients with achalasia were 5.4 times more likely to have type I diabetes mellitus (95% confidence interval [CI] 1.5-19), 8.5 times as likely to have hypothyroidism (95% CI 5.0-14), 37 times as likely to have Sjögren's syndrome (95% CI 1.9-205), 43 times as likely to have systemic lupus erythematosus (95% CI 12-154), and 259 times as likely to have uveitis (95% CI 13-1438). Overall, patients with achalasia were 3.6 times more likely to suffer from any autoimmune condition (95% CI 2.5-5.3). Conclusions Our findings are consistent with the impression that achalasia's etiology has an autoimmune component. Further research is needed to more conclusively define achalasia as an autoimmune disease.

23

Identifying Novel Therapies for Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Joshua Budman Dr. York Pei Nephrology Toronto General Hospital (TGH) Polycystic Kidney Disease (PKD), an autosomal-dominant disorder with no proven therapy, is characterized by extensive and bilateral cyst formation in the kidneys, leading to impaired kidney function. Cyst growth in the kidneys is caused by high levels of cAMP in renal cells. Four factors believed to be associated with high levels of cAMP are the downregulation of the pathway responsible for the release of the "anti-aging" protein KLOTHO and the upregulation of Canonical Wnt (specifically beta-catenin), COX2 and IGF pathways in renal cells. The purpose of this project is to characterize these pathways and to determine whether adjusting their level of function can ultimately lead to diminished cyst growth in patients with PKD. We believe altering the levels of function of these pathways should play a pivotal role in decreasing the volume and quantity of renal cysts in patients with the PKD mutation. By proving this relationship, we hope to identify novel drugs to accomplish these tasks and novel therapies for this disorder. To verify these pathways play a role in renal cyst growth, a cell culture system was created with CD1/GFP mice so that renal cysts will fluoresce. Embryonic kidneys were extracted at three separate time points. Half were treated with 8-Br-cAMP to promote cyst growth, while the other half acted as controls. rtPCR, with a Qiagen mini-kit, was performed using RNA extracted from cystic and non-cystic kidney tissue and the transcriptomic expression of the aforementioned proteins was determined. Once completed, interventional studies to test the specific effect each of these pathways have on the severity of ADPKD were ready to begin.

References Piontek, K et al. A Critical development Switch Defines the Kinetics of Kidney Cyst Formation after loss of Pkd1. Nature Medicine 13. 1490-1495 (2007)

24

Transfusion Triggers in Cardiac Surgery

Laura Burns Dr. C.D. Mazer Dept. Anesthesia St. Michael's Hospital A high proportion of patients who undergo cardiac surgery receive red cell transfusions perioperatively, yet the efficacy of transfusions in this population has never been established. At a time where there are new emerging pathogens and worsening blood shortages, it is essential that patients be transfused appropriately to minimize their risk of poor outcome related to red cell transfusion. Currently, there is no established optimal hemogloblin concentration for triggering a transfusion in patients who undergo cardiac surgery. As morbidity and mortality may be dependent on the severity of anemia and its corresponding requirement for increased transfusions, determination of an optimal haemoglobin level at which physicians should be transfusing patients is necessary to provide a better quality of care for future cardiac patients. However, preliminary studies are required to provide assurance that there will be adherence to any transfusion strategy proposed for any large study aimed at the question of optimal transfusion therapy. At this time there is no clear uniformity of transfusion practice that is used by physicians at St. Michael' Hospital or at most other hospitals across Canada. The objective of this study was to help determine whether physicians will adhere to proposed transfusion strategies and the rationale for non-adherent transfusion events. This study is a single center randomized controlled trial of two transfusion strategies. 50 cardiac surgery patients known to be at high risk of receiving red cell transfusions were enrolled. These patients were randomized to one of two groups. The "restrictive" transfusion strategy which received blood if their haemoglobin was 70g/L intraoperatively during cardiopulmonary bypass (CPB) and 75g/L postoperatively, once separated from bypass. The "liberal" arm received red cell transfusions when the haemoglobin concentration was 95g/L or less during CPB intraoperatively and 100g/L following separation from bypass. The transfusion protocols were to be adhered to until discharge from the hospital. Preliminary statistics suggest that physicians adhered to the proposed transfusion strategies. Reasons for non-adherence include patient safety (high potassium levels) and to provide urgent response (patient was actively bleeding), some patients refused transfusions when feeling well. Complications and SAEs have not been found related to the transfusion strategy.

25

Characterizing Surface Markers on IPS derived Neural Precursor Cells

Timothy Chan, R. Salewski, M. G. Fehlings Genetics and Development, Toronto Western Research Institute

Background: Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state similar to embryonic stem (ES) cells. Since iPS cells are readily available, avoid the use of embryos and, when patient specific, circumvent immune rejection, they are ideal for regenerative therapies such as cell transplantation in spinal cord injuries (SCI). Previously, we have shown that transplanted neural stem cells (NSC) derived from iPS cells differentiated to neural cells in vitro and in vivo. Furthermore, transplanted iPS cells promoted myelination in myelin deficient Shiverer mice. However, since these cells are derived from a pluripotent source, teratoma formation is always a concern. Increased tumorogenic potential may be a result of a heterogeneous transplanted cell population. Hypothesis: Characterizing surface markers on iPS-derived NSC will identify cellular sub-populations that can be used as positive or negative selection criteria for cell sorting to optimize safety and neurogenic potential. Methods: iPS/ES cells are directed to NSCs using clonal, free-floating, serum-free conditions. LIFdependant primitive NSC and FGF-dependant definitive NSC from iPS/ES cell sources will be analyzed using flow cytometry for pluripotency and NSCs surface markers: SSEA-1, CD44, and CD184. Anticipated Results: We expect to show distinct sub-populations within the iPS derived NSCs that reflect the various stages of differentiation. As cells become more differentiated, we expect to see fewer cells markers of pluripotency (SSEA-1). Also, specific markers for NSC, CD184 and CD44, can be used to identify neuronogenic and gliogenic NSCs, respectively. Conclusion: Identifying surface markers on iPS-derived NSC is an important step towards their eventual use in cell-based therapies. Further purification and optimization, to reduce unwanted growth and promote regeneration, is essential for iPS cells to be safely and efficiently used in SCI repair and regeneration.

26

The Long-Term Potentiation (LTP)-like Plasticity (PAS-LTP) in the Motor Cortex

Gurpreet Chara, Dr. Jeff Daskalakis, Dr. Tarek Rajji, Schizophrenia Program, Centre for Addiction and Mental Health (CAMH), 250 College Street, Toronto, Ontario, M5T 1R8

Schizophrenia is a devastating mental disorder that affects 0.5 - 1.0 % of worldwide population. Impairment in neuroplasticity is thought to underlie several aspects of schizophrenia. Paired associative stimulation (PAS0 is a novel transcranial magnetic stimulation (TMS) paradigm that assesses neuroplasticity in humans. It has been shown to result in long-term potentiation (LTP)-like plasticity (PAS-LTP) in the motor cortex. PAS involves pairing of electrical stimulation of the median nerve in adbuctor pollicis brevis muscle with TMS of the contralateral motor cortex. PAS-LTP is expressed through potentiation of the motor evoked potentials (MEP). A more direct way of measuring PAS-LTP in motor cortex through TMS-Electroencephalography (TMS-EEG) has not been explored. Through TMSEEG, PAS-LTP could be measured through potentiation of cortical evoked activity (CEA) in the motor cortex. Our preliminary data show a positive correlation between MEP and CEA ratio in healthy subjects. Our next steps include looking for PAS-LTP in patients with schizophrenia and in the dorsolateral prefrontal cortex which is particularly relevant in schizophrenia.

27

The Efficacy of Neural Stem/Progenitor Cell Transplant on Function Recovery After Endothelin-1 Induced Focal Spinal Ischemia in Rats

Johnson Chen, Andrea Mothe, Charles Tator Division of Genetics and Development, Toronto Western Research Institute, Krembil Neuroscience Centre. Introduction Spinal cord stroke is a rare condition caused by the reduction of blood flow in the vertebral vasculature, leading to ischemia and cell death in the affected regions. Currently, there are no effective therapies for spinal stroke and the resulting motor deficits; however, neural stem/progenitor cell (NSPC) transplantation has shown promise in promoting functional recovery after spinal stroke via cellular replacement and neuroprotection. Purpose In this study, the efficacy of transplantation of rat spinal cord NSPC was examined using a model of transient focal spinal ischemia induced by the vasoconstrictor endothelin-1 (ET-1). Methods Injury was induced by intraspinal injections of ET-1 at the T8 spinal level. Animals were randomized into two groups, and received media or cells at the lesion core one week after injury. Functional assessment of the hind-limbs was conducted weekly using the BBB locomotor open-field test. Results We found that functional recovery was higher in the NSPC group, though not statistically significant. Most of the NSPCs had differentiated one week after transplantation into multiple types of neural cells. Phenotypic analyses using immunohistochemistry with cell type specific antibodies demonstrated that more than 60% of the NSPCs differentiated into oligodendrocytes, while 6% and 12% expressed neuronal and astrocytic markers, respectively. Also, cavitation analyses revealed that NSPCs show limited effects in minimizing the size of the lesions. Conclusions These results show that NSPC transplantation shows limited recovery after ET-1 induced spinal stroke, but further work will be required to enhance efficacy.

28

Genetic Epidemiology of ADHD and OCD in a General Population

Yian Chen (National Chiao Tung University, Taiwan), Jennifer Crosbie, Paul Arnold, Russell Schachar, The Hospital for Sick Children, Toronto. Introduction: Attention deficit hyperactivity disorder (ADHD) and obsessive- compulsive disorder (OCD) are common child psychiatric conditions affecting 5% and 3% of the population respectively. ADHD and OCD often present in the same clinical patient, however, it is not known whether they occur together because of referral bias. Purpose: To examine the co-occurrence of ADHD and OCD in a large community-based sample. Methods: The study sample was 5865 children, age 7 to17, visiting the Ontario Science Center. Following informed consent, participants or their parents provided ratings of ADHD (using ADHDSymptoms and Normal-Behavior Scale, SWAN, range -3 to +3), OCD (Toronto Parent Rating of OCD symptoms, TPOCS, range -3 to +3) and reported past psychiatric history of ADHD or OCD. In addition, participants with scores at or above the 95 %ile were designated as "affected". Result: Increasing number of ADHD symptoms on the SWAN scale was significantly associated with increasing number OCD symptoms on the TPOCS (p<.02) although the relationship was not strong (every increase in SWAN of 1 point was associated with only a .05 point increase in TPOCS). Participants with a history of ADHD did not have elevated TPOCS, but those with self-reported OCD had elevated SWAN scores (p<.01). Of the 221 participants (3.8% of the sample) with SWAN ADHD scores at or above the 95 %ile, 12 (5.4%) were at or above the 95 %ile for OCD; but, of the 36 participants (.6% of the sample) with OCD scores at or above the 5%ile, 12 (33.3%) were above the 5%ile for ADHD. Conclusion: For the first time in a general population of children and adolescents, we show that ADHD and OCD are significantly, although weakly correlated suggesting that clinical observations of comorbidity are real rather than a product of referral bias. The relationship is asymmetrical: Individuals with OCD are very likely to have ADHD, but those with ADHD rarely have OCD. ADHD+OCD could reflect a distinct subtype. Next, we will estimate the heritability of ADHD and OCD and co-heritability of ADHD and OCD.

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To investigate the potential role of curcumin in the activation of Wnt signalling pathway in a high fat feeding induced insulin resistance mouse model

Yu Chih Chen, Weijuan Shao, Zhiwen Yu, Tianru Jin Dept. of Medicine and Physiology, Banting and Best Diabetes Center, Toronto Medical Discovery Tower, MaRS Building. 101 College Street, Toronto, Ontario, M5G 1L7 Obesity, a major risk factor for T2D and other metabolic diseases, has been recognized as a proinflammatory disease. Curcumin is a polyphenol extracted from the rhizomes of Curcumin longa, which is a commonly used dietary spice in Asia. Extensive examinations have shown that curcumin has anti-inflammatory effects and that it blocks the effect of HFD-induced obesity. Evidently, curcumin lowers plasma cholesterol level and represses adipogenesis via inhibiting the expression of PPAR and CCAAT/enhancer binding protein , two key transcription factors in adipogenesis and lipogenesis. Very -catenin nuclear translocation, possibly recently, an in vitro study using the 3T3-L1 cell model suggested that curcumin suppresses adipocyte differentiation via Wnt/ -catenin pathway activation. It induces through the inhibition of the expression of GSK-3 , CK-1 and axin. Our lab has been investigating the

effect of curcumin in preventing obesity and insulin resistance in an HFD C57BL/6J mouse model. We found that curcumin blocked the effect of HFD induced body weight gain, fat mass and insulin resistance, associated with reduced expression of ChREBP, a key transcription factor for lipogenesis. We are now examining whether these effects are at least partially attributed to Wnt activation in adipose tissue and in hepatocytes. We have, at this stage, observed that GSK-3 expression in fat tissue is increased by HFD and reduced by curcumin. We will assess nuclear in diabetes and other metabolic disorders. -catenin level and Wnt target gene expression in our experimental animals. Results obtained will deepen our understanding of the beneficial role of curcumin

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Platelets and their Role in Preventing Severe Transfusion Related Acute Lung Injury (TRALI) Reactions

Yuhan Chen, Dr. John W. Semple, Transfusion Medicine Research of St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, M5B 1W8, Canada Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion. Its definition is based on acute respiratory distress, non-cardiogenic lung edema and hypoxia. The pathogenesis of TRALI is not clearly understood but previous data has suggested that platelet depletion can protect the mice from TRALI. We hypothesized that platelet depletion protect SCID mice from severe TRALI reactions and death. We used a mouse model of TRALI where SCID mice were depleted of platelets by either the iv or ip route prior to TRALI induction. Platelet depletion by an ip injection prior to anti-MHC I antibody challenge caused significant mortality in SCID mice recipients (50% survival) whereas iv injection only cause a 20% mortality. Moreover, the wet/dry ratios of the platelet-depleted animals challenged with MHC I mAb were out of the normal range despite platelet depletion by either the ip or iv route of injection. Similar results were observed with rectal temperatures (a measure of systemic shock). Our results suggest that platelet depletion does not protect the SCID mouse from TRALI but instead, the route of platelet depletion may play a role in the protective TRALI effect.

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Object memory is impaired in mice expressing a mutant human Presenilin 1 transgene

Benedict Y. Y. Cheung1, Keith T. Ho1,2, Beverly M. Francis1,3, Zohar Weinbrand1, Paymon Azizi1 and Howard T. J. Mount1,2,3,4 Centre for Research in Neurodegenerative Diseases, 2Institute of Medical Science, 3Department of Physiology and 4Department of Medicine (Div. Neurology), University of Toronto. Toxic gain of function mutations in the gene encoding presenilin 1 (PS1) have been linked to familial forms of early-onset Alzheimer's disease (AD). PS1 is part of a multimeric -secretase complex that cleaves amyloid precursor protein (APP), generating toxic beta-amyloid peptides. These peptides in turn accumulate as plaques in the AD brain. Murine beta-amyloid does not share this propensity to accumulate. Thus, expression of wild type or mutant human PS1 transgenes (Tgs) does not produce ADlike plaque pathology, or clear spatial memory impairment in the mouse. Nonetheless, a growing list of substrates for PS1-regulated -secretase cleavage suggests that cleavage of species other than APP might contribute to the disease phenotype. Consistent with this notion, our lab has reported marked impairment of LTP, as well reduction of spine density in the brains of 39-45 week old mice expressing L286V mutant human PS1. To determine whether these electrophysiological and morphological changes might be reflected functionally, we have now begun to assess behavioural phenotypes of mice expressing L286V and wild type human PS1 Tgs. We report that L286V PS1-Tg mice exhibit a modest increase in spontaneous open field activity relative to non-transgenic littermates. More strikingly, we have found short-term object recognition memory to be abolished in mutant PS1-Tg mice, but unaffected in wild type human PS1-Tg animals. Our data point to profound impairment of the entorhinal cortex in the mutant PS1-Tg animals. As entorhinal cortex is also severely compromised in the AD brain, this phenotype may provide insight into aspects of AD pathology linked to mutant PS1, but independent of effects on betaamyloid accumulation.

1

32

Novel Peptide for the Treatment of Stroke

Kyle Chin Supervisor: Dr. Fang Liu Molecular Neuroscience, Department of Neuroscience Centre for Addiction and Mental Health Individuals who have a stroke have a 25% chance of dying or becoming severely disabled. Ischemic strokes are able to damage not only the penumbra but also peripheral cells due to excessive levels of glutamate. Glutamate is released from a presynaptic cell to activate receptors such as N-methyl Daspartate (NMDA) or -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. High levels of glutamate cause an imbalance in intracellular ionic concentration leading to cell death. Clinical treatment of strokes utilizing AMPA antagonist molecules have proven ineffective, due to receptor kinetics they suppress the postsynaptic glutamate response needed for normal functioning. We have identified a peptide that could block the neurotoxic effect of AMPA in ischemic strokes. The peptide was administered intravenously to adult Sprague Dawley rats after middle cerebral artery occlusion (MCAO) and before reperfusion. The infarct volumes were significantly smaller in the rats treated with 3 ­ 0.03 nM/kg. Our peptide is capable of decreasing the area of stroke damage, and may be utilized in future treatments.

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Genetic analysis of the first reported solid tumour in Shwachman-Diamond Syndrome

David Choi, Dr. Yigal Dror, Department of Cell Biology, The Hospital for Sick Children. Background. Shwachman-Diamond Syndrome (SDS) is the third most frequent inherited bone marrow failure syndrome and second leading cause of pancreatic insufficiency in children. Hematological cancers are commonly seen in SDS patients; however, no solid tumors have been reported in SDS patients thus far. Review of SDS patients in the Canadian Inherited Marrow Failure Registry revealed one patient with a solid tumor. This patient was diagnosed with pancreatic ductal adenocarcinoma. Purpose. To determine whether differences between the patient tumor and documented pancreatic cancers exist, and to identify molecular events underlying malignant transformation in the pancreas of the patient. Methods. A genome-wide analysis was performed using the Affymetrix single-nucleotide polymorphism (SNP) 6.0 array on patient tumor and germline DNA. Copy number alterations (CNAs) and regions of homozyogsity (ROH) were analyzed using commercially available software. Results. Analysis by the genomic segmentation algorithm in Partek Genomics Suite resulted in 65 CNAs overlapping coding regions in the tumor, but not in the germline. Additionally, we used Affymetrix's Genotyping Console software, which identified 16 CNAs overlapping coding regions in the tumor, but not in the germline. ROH analysis by a Hidden Markov Model identified 130 regions unique to the tumor. These CNVs and ROHs overlap with various cancer-related genes, such as STK11 and MTA1. Conclusion. Genome-wide analysis enables the detection of multiple genetic alterations of potential etiological significance. The identification and ongoing validation of these mutations and their disrupted genes will provide insight into pathways that lead to malignant transformation of the pancreas in the patient.

34

Role of VEGF in pulmonary hypertension development following acute pulmonary emboli

Yi-Min Chun, Marco Mura, Marc de Perrot Latner Thoracic Surgery Laboratories, Division of Thoracic Surgery, University of Toronto

Pulmonary hypertension (PH) is a lung disease characterized by increased blood pressure in the lung vasculature that can progressively lead to heart failure. Vascular endothelial growth factor (VEGF) is a mitogenic and survival factor for endothelial cells; however, there are conflicting reports of both beneficial and detrimental effects on the lung in the literature. In our preliminary study comparing the gene expression signatures of pulmonary fibrosis patients with or without severe PH, we found that VEGF expression was higher in those lacking PH. To determine whether a lack of VEGF favours the development of PH or is simply a result of the PH disease process, we wished to identify the role of VEGF in clot resolution during PH development following acute pulmonary emboli (PE). We hypothesize that VEGF may have a protective role in the development of PH and further investigated this speculation by developing a simple and effective rat model of chronic thromboembolic pulmonary hypertension (CTEPH). In this model, clots are formed ex vivo and then injected into an autologous rat via the jugular vein to simulate acute PE and anticipate a subsequent development of CTEPH. We hope to utilize this model to reveal the impact of VEGF and its inhibitor, SU5416, on the development of PH. Moreover, development of this model will be useful for the future study of both mechanistic aspects and potential therapies for PH.

35

Evaluating levels of physical activity in obese boys and girls using accelerometry

Denise Darmawikarta Dr. Jill Hamilton Endocrinology, Hospital for Sick Children

Background: Accelerometry provides an objective means of measuring physical activity (PA). Studies have shown that obese children perform less moderate to vigorous physical activity (MVPA) than lean children. To capture children's PA, accelerometers are often programmed to use 15-s or 60-s epochs. However, children may participate in shorter bursts of PA, thus a shorter epoch may more effectively quantify their PA. Purpose: 1) To evaluate duration of MVPA/day in obese boys and girls and compare weekday and weekend activity, 2) To determine the relationship between PA and BMI z-scores. Methods: PA was measured in 16 obese children (11M, 5F) ages 8-18y using 3-s epochs. Subjects wore an Actigraph GT3X accelerometer for 7 days and kept a log to indicate wear time. The data were manually edited to employ cut-points of >3200 counts per minute (cpm) and <800 cpm to define MVPA and sedentary activity, respectively. Results: Higher BMI z-scores were associated with less total MVPA (R=-0.64, P=0.02), while sedentary activity increased with age (R=0.74, P=0.004). Additionally, girls spent significantly less time in MVPA than boys (5.9±5.8 min MVPA/day vs. 21.9±13.0 min MVPA/day, p=0.008). There were no significant differences between weekday and weekend physical activity. Conclusions: Preliminary findings indicate that MVPA declines with increasing BMI z-scores. Older children spent more time being sedentary, and obese girls are less active compared to obese boys. Future directions include evaluating a larger number of children across a spectrum of BMI and studying correlations between physical activity and other variables including insulin resistance and cardiorespiratory fitness.

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MVP Registry: Efficacy and Safety of Different Bridging Regimens of Parenteral Anticoagulation after Mechanical Valve Surgery

Lauren Diamond, Supervisor: Dr. Stephen Fremes Division of Cardiac and Vascular Surgery, Sunnybrook Health Sciences Centre Background: The optimal method and intensity of anticoagulation for mechanical heart valve recipients in the immediate postoperative setting is uncertain. The MVP Registry will be the first large-scale study to evaluate the risks of thromboembolism, bleeding, and heparin-induced thrombocytopenia (HIT) associated with different anticoagulation strategies. Purpose: To determine whether (i) postoperative bridging therapy with full-dose heparin anticoagulation is superior to low-dose heparin and (ii) whether bridging therapy with low molecular weight heparin (LMWH) is superior to unfractionated heparin (UFH) in the immediate post-mechanical valve implantation period. Methods: This study is a multi-centre retrospective chart review of all mechanical valve replacements done during a 7 year period between January 1, 2003 and December 31, 2009. Data will be collected at participating centers on case report forms and entered into the database (iDataFax) for analysis. Preliminary Results: There were 263 patients who underwent mechanical heart valve replacement at Sunnybrook from January 1, 2003 ­ December 31, 2009. As of July 2010, a total of 83 patients at Sunnybrook have been reviewed, 10(12%) of which have had a total of 14 complications (4 patients had 2 events): 3(21%) arterial events, 1(7.1%) venous thrombosis, 7(50%) major bleeding events, 1(7.1%) HIT positive and 2(14%) deaths. Conclusions: According to the preliminary results obtained, bridging therapy with LMWH is superior to UFH in the immediate postoperative setting. Additionally, treatment with full-dose antithrombotic therapy has shown to be a safer method of bridging therapy compared to low-dose treatment.

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Associations between White Matter Hyperintensities and Hippocampal Atrophy in Alzheimer's Disease

Fahima Dossa Dr. Sandra Black Medicine (Neurology), Sunnybrook Health Sciences Centre Background and Purpose: Cerebrovascular disease (CVD) is common in patients with Alzheimer's Disease (AD). White matter hyperintensities (WMH), areas of high signal intensity in white matter seen on T2-weighted or FLAIR MRI, are considered a biomarker of small vessel CVD and a risk factor for cognitive impairment and dementia. Hippocampal volume (HV) loss is a reliable biomarker of AD, closely linked to memory and cognition. This study aimed to elucidate possible associations between HV, WMH, and cognitive measures. Methods: Participants included 9 AD, 11 AD+CVD, 27 with minor subcortical stroke/transient ischemic attack (TIA), and 17 normal controls. Mini Mental State Examination (MMSE) and Mattis Dementia Rating Scale (DRS) were used as global cognitive measures. HV was manually measured on 3D T1weighted MR acquired at 3 Tesla. Global WMH volumes were determined with an in-house semiautomated lesion segmentation protocol. Hippocampal shape analysis was performed using the FMRIB's Integrated Registration and Segmentation Tool (FSL-FIRST). Results: In the whole group, HV correlated with age, MMSE, and DRS (r=-0.426, p<0.001; r=0.434, p<0.001; r=0.414, p<0.001); age also correlated with WMH volumes (r=0.285, p<0.05). After covarying age, HV positively correlated with WMH in the AD+CVD group (r=0.788, p<0.01). In the whole group stepwise linear regression, HV was a better predictor than age, education, and WMH volumes combined, of both MMSE (r2 change=0.170, p<0.001) and DRS (r2 change=0.118, p<0.01) scores. In contrast to the left anterolateral hippocampal shape changes in AD and TIA groups, shape analysis in AD+CVD showed more extensive left dorsal anterior and posterior as well as right posterolateral changes. Conclusions: HV was a valid predictor of general cognition. Higher WMH volume correlated with greater HV in AD+CVD. WMH is known to contribute to clinical expression of dementia; hence, patients with more WMH may reach dementia at an earlier pathological stage of AD, as indexed by HV. Hippocampal shape analysis was more sensitive to group differences and may be a valuable additional biomarker.

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Telemetry (Wireless) recordings of physiological parameters in behaving mice

Chao Du1,4, Nisarg Patel1, Chiping Wu1,James Eubank1,3, Liang Zhang1,2 1 Toronto Western Research Institute, University Health Network,2 Department of Medicine (Division of Neurology), University of Toronto,3 Department of Surgery, University of Toronto,4 Shandong University School of Medicine Telemetry technology can provide a good way to measure variety of physiological parameters in behaving animals including EEG, ECG, EMG, body temperature and motor activity. However, the use of such technique in mice is a challenge because of small body size relative to the transmitter currently available. In addition, limited systemic studies are available about intracranial EEG recording in behaving mice. The aim of our study is to explore the technical aspects of telemetry recording in mice. Twelve C57 black mice at initial ages of 4-9 months were used. The telemetry transmitter and the thermal sensor were placed into the peritoneal cavity and the EEG electrode was implanted in the hippocampus CA1 region. Animals were allowed to recover for at least 9 days prior to the recording. All the mice with implantation showed normal behavior and lack of substantial weight loss. The core body temperature was 37.0±0.2 ºC (n=11), and the heart rate was 460±60min-1 (n=6). These observations are in accordance with previous reports (1, 2). Hippocampai EEG recording revealed typical behavioral state dependant activity such as theta rhythm during movement and exploration and the irregular activity/sharp wave during behavior immobility. Such EEG observations are in keeping with our previous wired EEG recording (3). However, it was found that proper positioning of the reference EEG electrode is crucial for reliable EEG assessment.

1. Sanchez-Alavez, M., S. Alboni, et al. (2010). Age (Dordr). 2. Arraj, M. and B. Lemmer (2006). Chronobiol Int. 3. Marco,W and M. Herry(2005). Brain Research Protocols.

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Pathways between Pain and Depression in Cancer

R. Ehrlich1,4, C. Lo1, J. Ellis3, G. Rodin2,3,

Behavioral Sciences and Health Research Division, Toronto General Research Institute, University Health Network, Toronto, ON

2 1

Faculty of Medicine, University of Toronto, Toronto, ON

Department of Psychosocial Oncology and Palliative Care, Princess Margaret Hospital, & Ontario Cancer Institute , Toronto, ON

4

3

Faculty of Arts, McGill University, Montreal, QUE

Background Previous research has suggested that pain may be causally related to depression in cancer populations, although the underlying mechanisms are unclear. In the present study, we investigated whether the effect of pain on depression may be mediated by other intervening physical symptoms. Hypothesis The effect of pain on depression may be mediated by other physical symptoms, including fatigue and changes in appetite. Method Head and neck cancer patients (N=104) completed the PHQ-9, a depression measure, and the ESAS to assess the severity of pain, fatigue, changes in appetite, drowsiness, nausea and shortness of breath. Moderate to severe depression was defined as scoring 15 on the PHQ-9. A multiple mediation analysis was conducted. Results Of the sample, 10% had moderate to severe depression and 36% had moderate to severe pain. Drowsiness, nausea and shortness of breath were dropped from further analysis due to symptom redundancy or lack of relationship with pain and depression. Pain was directly associated with depression, and fatigue and appetite were tested as mediators of that association. Both mediating pathways (pain fatigue depression; pain appetite depression) were statistically significant. Conclusion Pain in patients with head and neck cancer may lead to depression directly or via its effects on energy levels and interest in and capacity to enjoy food. Future research is needed to determine to what extent these or other mechanisms affect the relationship between pain and depression in other cancer sites.

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Transposon Mediated Gene Trapping in Zebrafish Heart Development

Name: Suzan El-Rass Supervisor: Dr. Xiao-Yan Wen Department: Medicine Hospital location of IP: St. Michael's Hospital. Queen Wing 8-019 Introduction: Zebrafish is a superior model organism in revealing the molecular control of cardiovascular development because of its embryonic transparency and rapid development. Gene trapping is a technique used to identify gene function and expression pattern by randomly introducing insertional mutations across the genome. In this research, a transposon-based, highly mutagenic gene trap vector (RP2), carrying fluorescent GFP and RFP markers, is used to randomly trap genes in the zebrafish genome. Strains with fluorescently labeled hearts will be further studied. Hypothesis: We hypothesize that a portion of "trapped" genes in strains with fluorescently labeled hearts play critical roles in heart development. Method: A Tol2 transposon-based RP2 vector and Tol2 transposase mRNA are co-injected into newly fertilized zebrafish eggs by microinjection. The expression of the "trapped" genes can be directly visualized under the microscope during the 2-5 days of embryonic development. Results: After 17 microinjection experiments, a total of ~2,000 embryos were injected. Of these F0 zebrafish, 80 fish expressed GFP in the heart and five of them also expressed RFP in the heart. In about 350 injected embryos, we observed GFP and/or RFP in other tissues such as brain and trunk muscles. We are in progress to generate F1 transgenic fish and identify the "trapped" genes in fish lines with a fluorescent heart. Conclusion: We developed a strategy to rapidly tag and mutate the zebrafish genes in the genome. The created transgenic zebrafish strains with a fluorescent heart will be valuable in dissecting the genetic controls of cardiovascular development.

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Medicinal Use of Cannabis and Cannabinoid Derivatives

Emre Cem Esen 1, Bernard Le Foll 2, 3

1

Hacettepe University School of Medicine, Ankara, Turkey

2

Departments of Family and Community Medicine, Pharmacology and Psychiatry, University of Toronto, Toronto, Canada

Translational Addiction Research Laboratory, Centre for Addiction and Mental Health Toronto, Canada. Background: The medicinal use of cannabis has been a controversial subject for a very long time. Cannabis has been used as a medicine over hundreds of years however individual cannabinoid receptor agonists were first came to market in 1981. There are 3 cannabinoid derivative medicines that are licensed to the date; Nabilone (Cesamet®) (a synthetic analogue of delta-9-THC), dronabinol (Marinol®) (delta-9THC), and Sativex® (contains equal amounts of delta-9-THC and cannabidiol). Nabilone and dronabinol are both indicated for nausea and vomiting of cancer patients who are having chemotherapy and that have failed to respond to other anti-emetics. Dronabinol is also indicated for anorexia with AIDS patients who are having weight loss. Sativex is licensed for relief of central neuropathic pain (CNP) that occurs in patients with multiple sclerosis. There are also other uses for cannabinoid derivatives and other molecules interact with endocannabinoid system, that are currently being researched. Methods: There are numerous articles, reviews, clinical trials on the uses of cannabinoid derivatives, on which this article is based on. Discussion: Cannabinoid derivative medicines show great hope on treatment of central neuropathic pain, nausea, vomiting and in many other areas that are being researched. However it is questioned if the cannabinoid derivatives are safe to use and if it's adverse effects beyond its therapeutic purposes. Also some clinical trials show that these medicines may have abusive potential. With all the controversial articles published, this review points out benefits and drawbacks of medical use of cannabinoid derivatives, from both sides of the debate.

3

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The Relationship between Personality Disorders and Axis I Psychiatric Disorders: Distinct or Not?

Fatima Fazalullasha, Ravi Shah, Dr. Paul S. Links, Arthur Sommer Rotenberg Chair in Suicide Studies, Department of Psychiatry, St. Michael's Hospital Background: The frequent co-occurrence of Axis I Psychiatric Disorders and Personality Disorders (PD) is associated with poor outcomes. The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) has newly proposed five PD types. Purpose: The objective is to systematically review recent literature examining the co-occurrence and relationships between Axis I Psychiatric Disorders and Axis II PDs, specifically the proposed DSM-V constructs, and consider the clinical utility of approaches to classifying comorbidity. Methods: Articles were located using Ovid Healthstar, EMBASE, MEDLINE, CINAHL and PsycINFO. The following keywords were used: Schizotypal Personality Disorder, latent schizophrenia, ObsessiveCompulsive Personality Disorder, Antisocial Personality Disorder, Psychopathic Personality Disorder, Axis I, comorbidity, co-occurrence, and coexistence. The search was limited to English language articles published inclusively from 2001 to July 2010. Articles were included on the basis of providing evidence into the prevalence of co-occurrence, possible etiologic mechanisms, and clinical implications of Axis I and II comorbidities. Studies were excluded if they did not analyze Axis I and II comorbidities, did not focus on or analyze results for the PD of interest, and were reviews without new empirical findings. All discrepancies were discussed and resolved by team members. Results: Our search revealed 174 Schizotypal, 363 Antisocial/Psychopathic, and 308 ObsessiveCompulsive articles. The review is ongoing. Conclusion: This review will address the contemporary debate about whether PDs should be classified with Axis I Psychiatric Disorders. It will evaluate whether current knowledge about comorbidity has clinical utility, and infer how this may affect the revision of DSM-V.

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A Comparison of Methods to Detect Cell-Surface Globotriosylceramide (Gb3)

Kimberly R. Fernandes and Donald R. Branch, Institute of Medical Science (Canadian Blood Services) Cell-surface globotriosylceramide (Gb3) has been implicated in a variety of diseases, including cancer and HIV-1 infection. Over-expression of Gb3 has been shown to decrease HIV-1 infection in vitro; thus, Gb3 has been proposed to protect against HIV-1 infection. The primary target of HIV-1, CD4+ T-cells, are thought normally to express very little Gb3. However, the literature has little data as to the level of cellsurface expression of Gb3 on human lymphocytes and no consensus on how to best detect cell-surface Gb3. To determine the optimal method for detection of cell-surface Gb3 on human cells, fluorescentactivated flow cytometry (FACS) was used to test 3 different, commercially available anti-Gb3 antibodies (BRG23, CD77, 38-13)) plus a natural ligand for Gb3, VT1B. Comparisons with each of the four detection methods used cell lines known to be positive (Burkett's lymphoma-derived and THP-1) or negative (Jurkat and Namalwa) for Gb3, and cell lines having unknown Gb3 status that included various carcinoma cell lines. All four detection methods were shown to work on high expressing Gb3 cell lines; although, differential results were obtained. When using low-expressing Gb3 cells, only 38-13, a rat IgM anti-Gb3-phycoerythrin(PE), or VT1B-Alexa488 were able to detect Gb3. Thus, these two methods of detection of cell-surface Gb3 were deemed the best to use. Subsequent examination of Gb3 on CD4+ Tcells was done with and without activation using PHA and IL-2. Although activation was shown to increase Gb3, the increase was limited to an apparent subset of CD4+ cells. Thus, a Gb3 enrichment method was developed using VT1B-FITC bound to magnetic beads. This allows for the purification and characterization of Gb3 cell-surface expressing cells in the peripheral blood. These methods should be useful for investigators wanting to study the cell-surface expression and function of Gb3 in various cell types

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Ceramide Mediates Increased Surface Toll Like Receptor 4 Translocation

Warren Fingrut, Supervisor: Dr. Ori Rotstein, Department of Surgery, St. Michael's Hospital Introduction: Shock/resuscitation contributes to tissue injury by generating oxidative stress, which primes the immune system for an increased response to subsequent inflammatory stimuli, including lipopolysaccharide (LPS). One mechanism underlying this increased sensitivity to LPS is the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts. Recent studies have shown that the oxidants produced by shock/resuscitation activate the intracellular enzyme acid sphingomyelinase (ASM), thereby inducing generation of the sphingolipid ceramide. These studies have also demonstrated that ceramide is necessary to induce increased cell surface expression of TLR4 receptors. Purpose: To determine if ceramide is itself sufficient to mediate TLR4 translocation. Methods: In the present study, RAW 264.7 cells (a murine macrophage cell line) were treated with C2 synthetic ceramide (dissolved in DMSO) for 15min, 30min, or 60min. Both immunofluorescence microscopy and flow cytometry were used to examine TLR4 translocation in response to ceramide treatments. As a control for C2-ceramide, experiments were conducted in RAW cells using the inactive analogue of C2 ceramide, dihydroceramide. Additional experiments were run to control for the effects of the vehicle (DMSO). Results: Ceramide treatment caused a TLR4 peripheralization in RAW cells by immunofluorescence microscopy and showed a time-dependent increase in TLR4 translocation by flow cytometry, suggesting that it is sufficient to mediate TLR4 translocation. As expected, cells treated with dihydroceramide or DMSO revealed no change in TLR4 surface expression observed by flow cytometry. Conclusion: Ceramide is an upstream mediator of TLR4 translocation. This study elucidates a component of the hierarchy of signalling molecules following oxidative stress which could lead to novel targets for therapy in critical illness and organ injury.

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Increasing Burden of Environmental Risk Factors for Congenital Heart Disease

Alan Fung, Seema Mital MD, Department of Cardiology, 4514 Hill Wing, the Hospital for Sick Children Background: Congenital heart defects (CHD) are prevalent in approximately 1.3m North Americans, occurring in 36,000 babies annually. Genetic and environmental causes are known in <20% cases. Methods: CHD patients <18 years old were prospectively enrolled in a population-based registry. Prenatal exposure to environmental risk factors and medications were captured by questionnaires and compared to National Birth Defects Prevention Study and Baltimore-Washington Infant Study controls. Associations were measured by odds ratios (OR), trends were measured between eras (1990-1999 vs. 2000-2010). Results: 1860 CHD patients were enrolled: conotruncal defects (CTD) 31.2%, left-to-right shunts (LRS) 28.6%, LV outflow tract obstructions (LVOTO) 15.0%, RV outflow tract obstructions (RVOTO) 11.0%, valvular defects (VD) 5.1%, and others 9.1%. There was higher frequency in the current era of advanced maternal age (OR=1.98, p<0.001), advanced paternal age (OR=2.13, p<0.001), insulin-dependent diabetes mellitus (OR=3.69, p=0.008), and urinary tract infections (OR=2.22, p=0.002). Case-control comparisons revealed CHD association with advanced maternal age (OR=1.33, p=0.001) and gestational diabetes (OR=1.49, p=0.012). Smoking was associated with VD (OR=3.44, p<0.001). Family history of CHD in 1st degree relatives was higher in CHD (3.14%, OR=2.66, p<0.001) and specific lesion subtypes i.e. LRS (OR=4.50, p<0.001), LVOTO (OR=2.80, p=0.030) and RVOTO (OR=4.56, p=0.004). Patients with extra-cardiac anomalies had higher maternal exposure rates to alcohol (OR=1.60, p=0.049), recreational drugs (OR=2.97, p=0.045) and other medications (OR=2.66, p<0.001). Conclusion: Higher incidence of maternal risk factors in the modern era indicates increasing CHD environmental risk burden. Understanding mechanisms by which these factors predispose to CHD will be pivotal in minimizing CHD burden.

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MicroRNA Conservation in Human Cancers

Kate Gerster1,3,4, Fei-Fei Liu1,2,3,4,5

1 2 3

Division of Applied Molecular Oncology, Ontario Cancer Institute Department of Radiation Oncology, University Health Network

Institute of Medical Sciences, Departments of 4Medical Biophysics and 5Radiation Oncology, University of Toronto Introduction: MicroRNAs are short, non-coding RNAs which post-transcriptionally regulate gene expression. Aberrant microRNA expression patterns have been implicated in cancer development and progression, and certain microRNAs have been determined to play oncogenic or tumour suppressive roles. Hypothesis: It is often said that cancer recapitulates development; thus microRNAs which are greatly deregulated in cancer are likely better evolutionarily conserved than non-deregulated microRNAs. Objective: This study aims to show that conservation of microRNAs and their targets is an indicator of cancer relevance; this information will be useful to cancer biologists seeking to identify oncogenic and tumour suppressive microRNA-mediated pathways. Methods: Primary tumour samples from patients with cervical, breast, oropharyngeal, or head and neck squamous cell carcinomas were evaluated for expression of 370 human microRNAs, as were corresponding normal tissues. MicroRNA conservation in mice and rats was determined. MicroRNA targets were predicted by four unique algorithms, and target homology was assessed in mice and rats. Targets of conserved, deregulated microRNAs were examined for enrichment in cancer-relevant Gene Ontology annotations. Results: In cervical cancer, a significantly greater proportion of deregulated microRNAs are conserved, in comparison with non-deregulated microRNAs. Similarly, a significantly greater proportion of conserved, deregulated microRNA targets possess homologs in mice and rats, relative to other microRNAs. Conclusion: While enrichment in cancer-relevant Gene Ontology annotations continues to be investigated, these preliminary results suggest that conservation of microRNAs may be a useful criterion for cancer biologists wishing to narrow in on relevant microRNAs and pathways.

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Characterizing Factors Influencing the Inflammatory Infiltrate in a Neural Precursor Cell Transplantation Protocol for Rodent Spinal Cord Injury

Student: Jeffrey Gibson Supervisors: Gregory Hawryluk and Michael Fehlings Author Affiliation: Neurosurgery, Toronto Western Research Institute Background: Neural precursor cell (NPC) transplantation shows great promise as a novel therapy for spinal cord injury (SCI). A better understanding of the host immune response may allow investigators to improve upon the modest functional recovery that has been consistently reported following NPC transplant. This has been subject to little study, however. Hypothesis: NPC transplantation leads to an increase in the number of inflammatory cells following SCI. These cells will be localized to regions of transplanted NPCs. Methods: Axial spinal cord sections from experimental animals 6 weeks post cell or control transplant were stained with a primary antibody for ED-1 and subsequent Alexa-568 secondary antibody. Fluorescent microscopy and StereoInvestigator software was then used to perform unbiased stereology; counts were performed at 7 sites relative to the injury epicenter (epicenter and 500, 1000 and 2000 microns rostral and caudal to the epicenter. Results: Preliminary analysis shows that despite immunosuppression, NPC transplantation leads to an increase in inflammatory cell infiltrate near the epicenter. Although ED-1 positive cells do not cluster in regions of NPCs nor the transplantation sites, inflammatory cells near NPCs appear to wall-off the exogenous cells, however. Analysis of control groups indicate that implantation of an osmotic minipump releasing EGF, FGF and PDGF for 7d increases the inflammatory response with a suggestion that this increase is greatest near the tip of the catheter. Discussion: NPC transplantation is associated with an increased inflammatory response. Given that ED1 positive cells do not localize preferentially to the transplanted cells, and that all groups implanted with a trophin pump exhibited a similar increase it suggests that the infused trophins may be responsible for the increase in ED-1+ cell numbers. This conclusion is supported by the fact that this increase was more pronounced rostral to the epicenter in the region of the tip of the infusion catheter. PDGF stimulates migration of macrophages and monocytes, which may explain this finding. A control group transplanted with NPCs that did not receive growth factor administration showed superior functional recovery to animals receiving both treatments. Exacerbation of a harmful immune response may explain this finding.

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Effect Of Feeding Anti-CD3 Monoclonal Antibody On In Vivo T Cell Response To T Cell Receptor Activation

A. Goethel1, K. Croitoru1,2,3

1 2 3

Institute of Medical Science, Department of Medicine, University of Toronto, ON; Clinical Sciences Division, Department of Medicine, University of Toronto, ON; Division of Gastroenterology, Mount Sinai Hospital, Toronto, ON Crohn's disease (CD) and ulcerative colitis (UC) affect more than 200,000 Canadians, with

approximately 9,000 new cases diagnosed each year. There is currently no known curative therapy to treat Inflammatory Bowel Diseases (IBD). In mouse models, T regulatory cells have been shown to prevent colitis by diminishing the effect of polyclonal T cell activation, thus having a homeostatic effect. Previous work in the lab indicated that oral treatment with anti-CD3 monoclonal antibody (MAb) was effective in preventing T cell induced colitis in the CD4+CD45RBhi transfer mouse model. This effect could be due to T cell anergy or through the induction of T cell suppression. We therefore examined the effect of feeding anti-CD3 MAb on T cell response to T cell receptor (TCR) activation in vivo. BALB/c mice will be fed either anti-CD3 or control hamster immunoglobulin gamma (HIgG) at 5µg per day for 5 days. T cell activation will be measured by challenging these mice on day 8 by intra-peritoneal injection with anti-CD3 or HIgG. After 24 or 48 hours, small and large intestine will be examined by histology and analyzed for villus to crypt ratios as a measure of T cell induced enteropathy. Cells isolated from spleens and mesenteric lymph nodes will be analyzed for T cell markers CD4, Foxp3 and CD45RB by flow cytometry. Cytokine production by these cells will be measured. Results from this study will provide insight into the mechanisms by which oral anti-CD3 affects T cell responses, leading to further exploration into IBD therapeutics.

49

Complement Factor H Mutation in Atypical Hemolytic Uremic Syndrome

Ozge Gonul Dr. Christoph Licht, Cell Biology Program, Hospital for Sick Kids Atypical hemolytic uremic syndrome (aHUS) is a severe kidney disease affecting children. aHUS is associated with defective regulation of the alternative complement activation pathway, where complement factor H (CFH) acts as an inhibitor of constitutive activation. aHUS patients have been found to carry loss of function mutations in CFH that lead to high levels of alternative pathway activity resulting in tissue damage. The objective of this study was to determine the pathological significance of a CFH C-terminal mutation carried by an aHUS patient by assessing CFH expression and alternative pathway activity in the patient and family members, at least one of whom carries the same mutation. Methods and Results Expression of CFH was assessed via immunoblot analysis of plasma, which showed no significant difference in CFH expression among the patient, family members and normal controls. Alternative pathway activity was measured via a haemolytic assay using sheep erythrocytes. Patient plasma showed strong activity, indicating lack of CFH inhibition. Plasma from a sibling carrying the same mutation gave similar results. Both samples are being tested for the effect of adding purified CFH. Conclusion The CFH mutation carried by the aHUS patient appears to be linked to a loss of CFH inhibition of the alternative complement pathway. The different clinical presentations seen in the patient and an unaffected sibling carrying the same mutation indicates that other factors contribute to aHUS pathology.

50

Role of Glycogen Synthase Kinase-3 in Epithelial-Mesenchymal Transition

Chichang Gu (Supervisor: Andras Kapus, Dept of Surgery, St. Michael's Hospital) Introduction: Epithelial-Mesenchymal Transition (EMT) is the crucial process in organ fibrosis. Progressive scarring of organs is a central feature in a variety of currently incurable diseases affecting the kidney, the lung and the liver. The culprit of organ fibrosis is the myofibroblast (MF), a motile and contractile mesenchymal cell that can originate from the epithelium via EMT. The hallmark of MF is expression of -smooth muscle actin (SMA), which is both a marker of and an active contributor to fibrosis. A critical factor in the induction of SMA (the activation of the myogenic program in the epithelium) is myocardin-related transcription factor (MRTF), the driver of the SMA promoter. Intriguingly, MRTF seems to be rapidly degraded in the absence of the cell contact element -catenin. While -catenin appears to be an important regulator of MRTF stability, the underlying mechanisms are unknown. Hypothesis: We hypothesized that the enzyme GSK-3, the main regulator of -catenin phosphorylation and subsequent degradation may also be responsible for MRTF degradation. Thus, elimination of catenin and/or activation of GSK-3 will augment MRTF phosphorylation and subsequent ubiquitinationdependent degradation. Objective: To explore the role of GSK-3 in EMT and to define the underlying molecular mechanisms. Methods: Western blotting, immunfluorescence microscopy transfection and cell culture. Results: Upon the downregulation of -catenin, fibrogenic stimuli such as uncoupling the intracellular contacts by low calcium medium (LCM) and transformig growth factor-1 (TGF-) induce rapid MRTF degradation. Inhibition of GSK-3 prevents MRTF degradation. These stimuli also provoke the transient dephosphorylation (activation) and perinuclear redistribution of GSK-3 . Conclusion: GSK-3 promotes MRTF degradation, and therefore it is an important negative regulator of the myogenic program during EMT and organ fibrosis.

51

Brain Rhythms and Memory: is monitoring brain encephalographic activity a good measure to prevent awareness during anaesthesia?

M. Hajiha1, H. Ju3, A.A. Zurek,2, B.A. Orser1,2,3, Institute of Medical Science 1, Department of Physiology 2 , Department of Anesthesia 3 Sunnybrook Health Science Center, Toronto, Ontario, Canada

INTRODUCTION: An essential component of the anesthetic state is memory blockade, however, a serious adverse event associated with anesthesia is the unexpected recall of intraoperative sensory events, termed "intra-operative awareness." Monitoring the surface electroencephalogram (EEG) activity, from electrodes on the scalp, has been proposed as a means to prevent awareness in patients. HYPOTHESIS: Mice treated with the anesthetic etomidate will have predictable changes in their surface EEG activity that parallel memory formation. OBJECTIVE: To evaluate the relationship between surface EEG activity and memory blockade during anesthesia. METHODS: Surgery was performed on 6 mice (C57Bl6) for surface EEG electrode implantation. A randomized, crossover design was used and the observer was blinded to treatment. On postoperative days 7 and 21, animals were randomized to receive either etomidate (2mg/kg) or a vehicle control (saline). EEG was recorded continuously during training on the memory task (10 min) and during memory testing (5 min). The EEG power was analyzed; specifically (4-8 Hz) rhythm was used to assess EEG correlation to exploratory memory behaviour. The discrimination ratio (novel object time / novel object time +familiar object time) was measured. RESULTS: Etomidate treatment caused a decrease in memory performance indicated by reduced discrimination ratio in etomidate-treated animals (0.44± 0.15) versus control (0.55 ± 0.11). There was no significant difference in EEG power during novel object recognition between the control and etomidatetreated group (P>0.05). CONCLUSIONS: This study demonstrates that surface EEG recordings using current methods are not an accurate measure of memory formation under anesthetic influence.

52

Familial Pancreas Cancer: The Search For A Common Gene Variant Amongst FPC Patients

Noah Halpern Dr. Steven Gallinger, Senior Investigator Samuel Lunenfeld Research Institute Mount Sinai Hospital Joseph & Wolf Lebovic Health Complex Pancreatic cancer can be divided into two categories ­ sporadic and familial. While the majority of pancreatic cancer cases are sporadic in nature, a small percentage of people presenting with pancreatic cancer appear to have a family history of the disease. The main purpose of this study is to identify a common genetic variant amongst all people suffering from familial pancreatic cancer. Our case study, Family C, is a family in which four individuals were diagnosed with pancreatic cancer, and later died from the disease. The affected individuals underwent exome sequencing and common genetic variants were identified in all affected family members, while none appeared in the controls. The genetic variants were identified using 25 selected genes, verified using PCR and gene sequencing analysis. 350 primer sets were then optimized for temperature and MgCl2 concentration conditions to be used for mass sequencing of genomic DNA in 50 unrelated familial pancreatic cancer patients. Following the initial 50 patient screen, 75 new patients (including 5 positive controls from Family C) will be sequenced using DNA retrieved via whole genome amplification.

53

The early Traumatic coagulopathy: A Review of Pathophysiology

Waseem A, Hariri, MD, MBBS. Sandro B, Rizoli, MD, PhD, FRCSC, FACS. Division of General Surgery, Sunnybrook Health Science Center, University of Toronto Worldwide, trauma remains the leading cause of death among the young. Hemorrhage accounts for 40% of all deaths, mostly due to trauma- induced coagulopathy (TIC). The pathophysiology of TIC is poorly known but recent studies are significantly changing both our understanding of TIC and its management. Methods: Literature review. Results: Recent findings are summarized in two theories: Brohi demonstrated that TIC is primarily caused by shock and tissue destruction, resulting in systemic anticoagulation and hyperfibrinolysis. Endothelial injury and hypoperfusion cause the release of Thrombomodulin (TM) that complexes with activated protein C (APC), leading to systemic anticoagulation. TM also associates with thrombin, reducing its availability to cleave fibrinogen into fibrin. Other studies demonstrated that endothelial injury and ischemia also causes excessive release of Tissue Plasminogen Activator and systemic hyperfibrinolysis. Another theory proposes that large amount of tissue factor exposed in injured tissues, activates the extrinsic pathway, leading to a consumptive coagulopathy. Injury and inflammation also suppress anticoagulant pathways, while plasminogen activator inhibitor-1 mediated inhibition of fibrinolysis is followed by disseminated fibrin deposition in the microvasculature. During such disseminated intravascular coagulopathy (DIC), both bleeding and occlusive microvascular thrombosis occur. The latter causes tissue hypoxia, sustained inflammatory response and development of multiple organ failure (MODS). Thus, DIC is the primary issue leading to a consumptive coagulopathy and MODS. Conclusions: Despite many recent advances in understanding the pathophysiology of trauma-induced coagulopathy, much remains unknown. There is an urgent need of further investigations in hope of reducing the enormous burden of traumatic bleeding.

54

Mitroflow aortic pericardial bioprosthesis: clinical performance and functional class assessment

Jonathan Hong Mark Peterson MD, PhD, FRCSC Division of Cardiac Surgery St. Michael's Hospital Background: The current trend in North America and Europe shows an increasing number of implanted tissue valves due to an aging population. The selection of an appropriate aortic valve bioprosthesis is based on durability, hemodynamic performance and aortic annulus size. The Mitroflow pericardial bioprosthesis (Sorin Group Inc, Mitroflow Division, Vancouver, Canada) has excellent hemodynamic performance but long-term durability remains controversial. Purpose: To investigate the long-term clinical performance of the Mitroflow valve and the functional class of patients post-implantation. Methods/Results: A retrospective chart review of prospectively gathered data of all patients implanted with a Mitroflow valve at St. Michael's Hospital since 1999 (n=326). Demographic, hospital and echocardiographic data from early and long-term follow-up was collected. Variables included were gender, age, type of aortic lesion, and concomitant diagnosis and procedures. All adverse events related to the heart valve prosthesis, such as bleeding, endocarditis, paravalvular leak, structural valve degeneration, thrombosis, reoperation and valve related and all cause mortality that occurred during the follow-up period were recorded. Data from follow-up echocardiograms will assess the long-term valve function. The Kaplan-Meier method will be used to assess the time-related outcome of survival and freedom from valve-related complications and composites. Comparison of curves among patient groups will use logrank test at 0.05 level of significance. Conclusion: The study will determine the long-term durability of the Mitroflow pericardial bioprosthesis in the patient population at St. Michael's Hospital.

55

Maintained Cerebral Perfusion with a Novel -1 Antagonist with Vasodilatory Properties

Tina Hu1, Emily Wright1, and Gregory Hare1

1

Department of Anesthesia and Physiology, St. Michael's Hospital, University of Toronto

Perioperative -blockade is associated with an increase in mortality and stroke in hypertensive and surgical patients. Experimental studies have shown that metroprolol, a 1 selective antagonist, impaired cerebral perfusion by two presumed mechanisms: impaired cardiac output and reduced resistance artery dilation. We wished to determine if a novel -blocker with vasodilatory capacity would have cardioprotective potential (reduced heart rate) while maintaining cerebral perfusion. The impact of nebivolol, a 1 antagonist which causes vasodilation by upregulating endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO), on organ perfusion will be assessed. We hypothesize that tissue perfusion will be maintained after administration of nebivolol. Anesthetized rats were treated with nebivolol (1.25 mg/kg iv) or a vehicle solution. Mean arterial pressure, heart rate, and cerebral blood flow (CBF, laser Doppler) were measured. Preliminary results (mean ± SD) demonstrate that nebivolol significantly decreased heart rate compared to baseline (302.4 ± 41.8 bpm vs. 324.8 ± 34.7 bpm, p>0.05, n=3) and maintained mean arterial pressure (107.7 ± 28.4 mmHg vs. 110.4 ± 26.6 mmHg, n=3) and normalized cerebral blood flow (1.04 ± 0.07 vs. 0.99 ± 0.01, n=3). We conclude that nebivolol may provide the positive therapeutic effects (cardiac protection) without the risk of ischemic organ injury (maintained CBF), possibly due to its ability to promote eNOSderived NO. This is in contrast with metroprolol which impairs cerebral perfusion in this model. Future experiments will assess the impact of nebivolol on cardiac output, tissue eNOS and perfusion following acute hemodilution.

56

The Prevalence of Liver Abnormalities in Patients with Systemic Lupus Erythematosus

D. Huang, E. Aghdassi, J. Su, PR. Fortin. University Health Network, Division of Rheumatology. Toronto, Canada

Studies reporting on liver involvement in Systemic Lupus Erythematosus (SLE) are limited, often focused on liver transaminases and without inclusion of imaging procedures and/or liver biopsies. Furthermore, the etiology for the abnormal transaminases has not been investigated. This study determined in SLE patients: 1) the prevalence of abnormal liver function tests (LFTs) and whether further investigations were done; 2) the differences in SLE-related and/or metabolic factors in patients with and without abnormal LFTs. Patients who met at least 4 of the ACR classification criteria for SLE and had 1.5 times the upper limit for AST or ALT on two visits within a 2-year period were selected from the database registry of the University of Toronto Lupus Clinic. Demographic, and laboratory data at the time of the first appearance of LFTs abnormality were extracted from these patients as well as the age, gender and SLE duration matched controls who had normal LFTs. Disease activity and damage were assessed by SLE- Disease Activity Index (SLEDAI) and SLICC Damage Index (SDI). Chart review was performed to determine the contributing factors to these liver abnormalities. Out of 1533 patients in the database, 135 (8.8%) met the inclusion criteria. The subjects had mean (SD): age of 40.1 (13.7) years, BMI of 25.9 (5.8) kg/m2, SLEDAI of 6.4 (5.4), SDI of 1.5 (1.8) and 83% were female. 65% were Caucasian; 23% smoked; 30% consumed alcohol, 61% had hypertension, 7% had cardiovascular involvement and 68% had other autoimmune disorders. Medication profile of the two year period preceding the first abnormal LFT included: prednisone (67% of patients) with a mean (SD) dose of 12.1 (15.3) mg; antimalarial (63%), immunosuppressants (61%), statins (16%), hormone replacement therapy (13%) and anticoagulants (13%). Laboratory findings were normal for CBC, blood glucose and lipid profile except for serum triglycerides that was elevated (2.10 ± 2.04 mg/dl). The mean (SD) levels for AST and ALT were 73 (67) and 88 (79) respectively. Chart review completed in 133 of the patients, showed that only 30 patients were further evaluated by a Hepatologist, 75 had imaging studies (41 were done specifically for liver investigation), and only 13 had a liver biopsy. Results of these investigations showed 30 fatty liver, 34 drug-induced hepatitis, 6 autoimmune hepatitis and 4 related to lupus disease activity. In the nested case control study of 134 matched subjects, cases had higher prevalence of: hypertension (60% VS. 46%, p=0.02); antiphospholipid syndrome (9% VS. 2% p=0.02); and immunosuppressants use (60% VS. 37%, p<0.001), especially azathioprine (39% VS. 22%, p<0.01) and methotrexate (22% VS. 10%, p<0.01) compared to controls. Although BMI, SLEDAI and SDI were similar, blood biochemistry showed a significantly lower IgM (1.2 (0.7)

57

The effect of AP and PA directed currents induced by transcranial magnetic stimulation on long interval intracortical inhibition

Guan Huang, Dr. Robert Chen Division of Brain Imaging & Behaviour Systems - Neuroscience Toronto Western Research Institute (TWRI)

Transcranial magnetic stimulation (TMS) is a safe, non-invasive and painless way to stimulate the human brain in intact, behaving subjects. TMS of the motor cortex (M1) produces a series of descending corticospinal waves termed direct and indirect (I) waves. The cortical circuits activated by TMS depend on the induced current direction; posterior-anterior (PA) current preferentially recruits the early I-waves whereas anterior-posterior (AP) current preferentially recruits the late I-waves. Intracortical inhibition can be studied by paired pulse TMS. Long interval intracortical inhibition (LICI) is observed at interstimulus intervals between 50 ms and 200 ms with suprathreshold conditioning stimuli inhibiting the responses to test stimuli. LICI is abnormal in some neurological diseases such as Parkinson's disease. The effect of current direction on LICI has not been studied before. The aim of the present study is to investigate the effect of AP and PA directed currents on LICI. We planned to recruit 8 healthy volunteers for the study. Motor-evoked potentials (MEP) were recorded from the right first dorsal interosseous muscle. Paired conditioning and test stimuli in the AP-PA and AP-AP directions were applied to the hand area in the left motor cortex. The time course of LICI was studied with interstimulus intervals of 50, 100, 150, and 200 ms. The MEP recruitment curves were also studied. Results are expected to be finalized before the end of August. We hypothesize that AP current direction generates stronger LICI than PA current direction. The results will help further our understanding of I-wave generation mechanisms.

58

Sensitivity to change of radiographic scoring instruments in axial psoriatic arthritis

Ali Ibrahim, Vinod Chandran, Lihi Eder, Arane Thavaneswaran, and Dafna D. Gladman, Centre for Prognosis Studies in the Rheumatic Diseases, Toronto Western Hospital. Introduction: Psoriatic Arthritis (PsA) is an inflammatory arthritis associated with psoriasis, usually seronegative for rheumatoid factor. 25-75% of patients with PsA have axial arthritis (AxPsA). Various instruments are used to quantify radiographic spinal damage in AxPsA (BASRI, mSASSS, RASSS, PASRI). We recently showed that these systems have good reliability in AxPsA. However, sensitivity to change is still not known. In this study we aimed to test the sensitivity to change of available scoring methods in AxPsA. Hypothesis: PASRI is more sensitive to change in AxPsA than mSASSS, BASRI and RASSS. Methods: Spinal radiographs of 105 patients diagnosed with AxPsA (grade 2 or greater sacroiliitis and/or inflammatory back pain and/or restricted spinal mobility) were retrieved for 2 time points separate by at least 2 years, and subsequently anonymized to name and order of examination. All radiographs were scored by three rheumatologists using a standard form, which allows calculation of mSASSS, BASRI, RASSS, PASRI, along with features that may be associated with PsA or complicate scoring (such as osteoarthritis, osteoporosis, degenerative disc disease). The scores were entered into a computerized database for analysis. An independent expert reader will provide a measure of radiographic progression from an overall impression after reading the radiographs with knowledge of chronologic order, and his assessment of progression will be used a the gold standard. The change in radiographic scores will then be compared to the gold standard. Results: The 105 patients (71 males, mean age at the time of first X-ray 51.9 years, mean PsA duration of 16 years) had on average 8 actively inflamed joints, 1.6 swollen joints, 15 clinically damaged joints, and 17% had dactylitis. BASRI-spine, mSASSS and PASRI showed statistically significant difference in mean scores between the two time points. The BASRI and PASRI scores showed the largest proportions of patients with a worsening score (both 32.4%), followed by mSASSS and RASSS (24.8% and 22.9%, respectively). Conclusion: The PASRI shows sensitivity to change longitudinally, but further analysis with the gold standard is necessary. More studies are also needed to determine the minimal clinically significant change of these scores to reflect radiographic progression in AxPsA.

59

Glycogen Synthase and its role in Lafora Disease

Nyrie Israelian Dr. Berge Minassian, Genetics and Genome Biology, Hospital for Sick Children Lafora Disease (LD) is a rare progressive myoclonus epilepsy syndrome. Seizures begin during early adolescence, followed by neurodegeneration and death within ten years of onset. LD is characterized by the presence of polyglucosan Lafora Bodies, consisting of abnormal poorly branched insoluble glycogen-like polymers that accumulate in most tissues. LD is known to be caused by either a deficiency in laforin dual-specificity phosphotase or malin E3 ubiquitin ligase. Currently, there are two dominant hypotheses behind the formation of Lafora bodies. One hypothesis is that hyperphosphorylation of glycogen leads to insolubility, with subsequent conversion to polyglucosan. The other is that overactivity of glycogen synthase (GS), the enzyme that elongates glycogen strands linearly, leads to polyglucosan formation. The laforin-malin complex is thought to recognize polyglucosans and function to decrease the activity of GS through phosphorylation or polyubiquination of the enzyme, and that mutation of either laforin or malin will lead to increased levels and activity of GS. However, this study shows that there is no significant difference in GS level or activity between wild type and laforin-deficient mice. Mice were run until glycogen stores were depleted, at which point GS activity levels would be highest. Mice were sacrificed and muscle and liver tissues were harvested. Contrary to the hypothesis of increased GS activity, Western blot and GS enzyme activity assays showed no change in protein or activity levels between the wild type or laforin-deficient mice in both tissues. Determining the pathophysiological mechanism of Lafora disease is crucial for developing specific treatments and therapies.

60

Design of a Bioreactor for a Tissue-Engineered Trachea

Ashu Jain, Siba Haykal, Thomas Waddell Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute, UHN Background: Malignancy, subglottic stenosis and traumatic injury to the trachea require surgical resection. When short segments are involved, primary re-anastomosis affords excellent results. Longer lesions, on the other hand, have no currently acceptable solution. Recent progress in airway transplantation has provided renewed optimism for potential solutions for these problems. An area which has emerged to eliminate the need for immunosuppression following tracheal allotransplantation is that of organ decellularization. Decellularized scaffolds can then be repopulated with the recipient's own cells. Bioreactors are required to provide the ideal environment to recellularize these tracheal allografts. Objectives: To design a bioreactor for recellularization of decellularized tracheal allografts. Methods: Tracheas were harvested from Yorkshire pigs (n=12) and decellularized using two decellularization protocols (A and B) involving a combination of detergents and enzymes. Structural integrity following decellularization was measured using a tracheal collapsibility device. The bioreactor was designed to control conditions such as pH, temperature, oxygen tension and shear stress. Results: We have designed a bioreactor which allows for dynamic cell seeding within the three dimensional matrices, to overcome the restraints of a static culture environment and to physically stimulate the developing constructs. The inner luminal surface of the tracheal allografts will be ventilated and perfused with tracheal epithelial cells. The external surface will be perfused with mesenchymal stromal cells. Following cell seeding, media alone will be perfused to maintain cell viability. Conclusions: This bioreactor design follows the principles established in the literature. Cell density, rotational parameters, and flow speed will have to be modified following use of this device. Cell viability within the constructs will also have to be determined.

61

Prevalence of metabolic syndrome in psoriasis and psoriatic arthritis

Jayakar JP, Eder L, Pereira D, Thavaneswaran A, Shanmugarajah S, and Gladman DD Centre for Prognostic Studies in the Rheumatic Diseases (CPSRD), Toronto Western Hospital Introduction: Psoriasis (Ps) is an immune-mediated skin disease affecting 2-3% of the population, of whom about 30% develop Psoriatic Arthritis (PsA), an inflammatory arthritis. The metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors that includes abdominal obesity, dyslipidemia, hyperglycemia and hypertension. It is known that Ps and PsA patients have an increased prevalence of cardiovascular disease. However, the prevalence of MetS in PsA and the differential cardiovascular risk between Ps and PsA are not established. Hypothesis: PsA patients have a higher prevalence of MetS and compared to Ps patients because of higher inflammatory burden. Methods: Patients with PsA are followed prospectively in the Toronto Western Hospital PsA clinic. Patients with Ps without arthritis have been included in an observational cohort since 2006. All patients undergo a clinical examination according to a standard protocol which includes assessment of the components of MetS. Descriptive statistics are used to describe the patients. Univariate analysis including t-tests for continuous variables and chi-squared tests for dichotomous variables was performed to compare MetS in Ps and PsA. Multivariate analysis using a stepwise logistic regression model was used to identify associations between potentially related variables and MetS. Results: 167 patients with PsA (42.5% F/ 57.5 % M, mean age 51.1 yrs, mean Ps duration 25.3 yrs, mean PsA duration 15.6 yrs, mean number of actively inflamed joints 8.6, mean PASI (psoriasis skin severity score) 4.1) and 98 Ps patients (43.9% F/56.1% M, mean age 50.3 yrs, mean Ps duration 18.4 yrs, mean PASI 5.8) were included. Prevalence of MetS was 36.5% in PsA, and 25.5% in Ps (p=0.0773).Multivariate analysis revealed that age, use of anti-TNF agents, and psoriasis skin severity scores (PASI) are associated with MetS, with odds ratios of 1.063 (p<0.0001), 2.455 (p=0.055), and 1.049 (p=0.033) respectively. Conclusions: Our interim results provide the first report of the prevalence of MetS in PsA. Our results further show that MetS is not more prevalent among patients with PsA compared to Ps. However, a larger sample size may be necessary to detect a significant difference. It is notable that markers of severe disease activity, such as use of anti-TNF agents and high PASI scores, are associated with increased development of MetS in patients with Ps with or without arthritis.

62

Decision Analysis as an Aid to Determining the Management of Early Low Rectal Cancer: Does Current Knowledge Change the Balance?

Calvin Johnston David Urbach, Clinical Decision Making and Health Care ­ General Surgery Toronto General Hospital Background: There is an ongoing discussion as to whether it is better for the patient to have an abdominoperineal resections (APR) or a transanal local excision (TLE) in cases of early (suspected T1/T2), low (<5cm from anal verge) rectal cancer. Decision analyses aim to present a rational argument for one over the other based on axiomatic principles. Methods: We developed a microsimulation-based decision analysis to evaluate the individual's choice between transanal local excision (TLE) and abdominoperineal resection (APR) for early, low rectal cancer. Critical transition rates and utilities were elicited by systematic literature reviews and requests from additional parameter estimates from study authors. Sensitivity analyses focused on varying utility valuations and the age of the patient. Results: [Pending] APR was associated with [ ] additional life years, but a loss of [ ] life years when qualityadjusted for time tradeoff utilities; thus a TLE is considered rationally preferable within this model. The preferable option changed when a patient was willing to give up less than [ ]% of their remaining lifespan to avoid a permanent colostomy, or when the patient was younger than [ ]. Conclusion: Though radical surgery for early low rectal cancer may give a patient more life years on average, a TLE may still be rationally preferable based on a measurable willingness to trade life years for life quality.

63

Lentiviral Vector Correction Of Fabry Disease Encompassing a Cell Fate Control Safety Element

Student: Mohammed A Jomah Supervisor: Matthew D. Scaife & Dr. Jeffrey A Medin Department of medical biophysics, Division of stem cells and developmental biology Canadian blood services Introduction: Fabry disease is an X-linked lysosomal storage disease caused by a deficiency in the enzyme activity of alpha-galactosidase A (-gal A), resulting in accumulation of globotriaosylceramide in different tissues of the body, leading to a wide range of clinical complications. Consequences of the diseases result in progressive organ failure of the kidney, heart, and premature death. Lentiviral (LV) gene mediated therapy has been implicated as a treatment method for Fabry disease. Our lab has shown in a number of reports the effectiveness of this approach. Unfortunately, a few clinical trials have reported an adverse effect, a leukemic transformation, when using integrating viral vectors. Therefore our goal is to make safer gene therapy lentiviral vectors for treatment of Fabry disease. This involves introducing a safety gene that is incorporated into the LV. This safety gene should allow for clearing these genemodified cells after activation of normal non-toxic prodrug, given an leukemic transformation occurs. The LV vector being constructed will encode the cell fate control safety cassette, truncated human LNGFR fused to mutant Tmpk (F105Y), with the cDNA for -gal A downstream of this safety cassette. Hypothesis: Our novel LV should fulfill three requirements: (1) truncated LNGFR that will be highly expressed in the transduced cells, allowing for enrichment by FACS if required. (2) Mutant Tmpk will provide sufficient killing with prodrug AZT. (3) -gal A will provide the enzymatic correction for Fabry disease. Objective: Long-term goal for this LV is to be implemented in clinic for safe and effective treatment for Fabry disease patient. Method: Using the DNA cloning techniques, we will PCR amplify the a codon optimized -gal A cDNA then subclone it into a lentiviral plasmid that already contains the safety element, LNGFRTmpk. These results will be confirmed by restriction analysis and DNA sequencing. After results are confirmed, functional studies will be performed on transduced cells: flow cytometry for LNGFR expression, doseresponse to AZT, and -gal A enzymatic activity. Conclusion: Preliminary results from Dr. Medin's lab have been highly suggestive that this LV is sufficient for correction of Fabry disease while protecting against insertional mutagenesis. To date, sequencing results have confirmed the correct sequence of the lentiviral plasmid. We are now in process of synthesizing the lentivirus for functional studies.

64

Post-operative outcomes are favourable for patients with cystic renal cell carcinoma

Student: Sarah Kawaguchi Supervisors: Dr. Michael A.S. Jewett and Dr. Antonio Finelli Department of Surgical Oncology, Princess Margaret Hospital INTRODUCTION: Multilocular cystic renal cell carcinoma (MCRCC) is an uncommon subtype of renal cell carcinoma (RCC) consisting of cysts lined by clear cells within an encapsulated tumor. Evidence from small retrospective case series suggests that this pathologic entity possesses low malignant potential.1-9 PURPOSE: To validate and challenge our current understanding of the behaviour of MCRCC through a retrospective population-level study. METHODS: We searched the Ontario Cancer Registry (OCR) for all cases of cystic RCC treated with nephrectomy between 1995 and 2004. Pathology reports were independently reviewed by two individuals, and cases that did not meet the 2004 WHO definition of MCRCC were excluded.10 RESULTS: Of the 69 cases studied, the majority (59%) were men and the majority of tumors (64%) were under 4 cm in maximal axial dimension. Average tumor size was 3.5 cm (range 0.5 to 11 cm). 51% of cases were treated with partial nephrectomy and 49% were radical nephrectomy. Almost all lesions (97%) were Fuhrman grade 1 or 2, and 91% were pathologic stage T1 or T2. There was no association between stage and grade (p=0.91). All cases were N0M0 and none showed lymphatic invasion. Patients were followed up post-operatively for a median of 5.0 years (range 1.0 to 12.1 years). 6 patients died during follow-up, but none died due to kidney cancer. CONCLUSION: The diagnosis of MCRCC predicts favourable histopathologic features and an excellent post-surgical prognosis. Our observed 100% cancer-specific survival is consistent with previous studies and suggests that we may soon revise our approach to managing these cases.

REFERENCES:

1. Bielsa O et al. Cystic renal cell carcinoma: pathologic features, survival and implications for treatment. Brit J Urol, 1998. 82: 16-20. 2. Corica FA et al. Cystic renal cell carcinoma is cured by resection: a study of 24 cases with long-term followup. J Urol, 1999. 161: 408-411. 3. Nassir A et al. Multilocular cystic renal cell carcinoma: a series of 12 cases and review of the literature. Urology, 2002. 60: 421-427. 4. Koga S et al. Outcome of surgery in cystic renal cell carcinoma. Urology, 2000. 56: 67-70. 5. Han K et al. Cystic renal cell carcinoma: biology and clinical behaviour. Urol Onc Sem Orig Inv, 2004. 22: 410-414. 6. Aubert S et al. Cystic renal cell carcinomas in adults.Is preoperative recognition of multilocular cystic renal cell carcinoma possible? J Urol, 2005. 174: 2115-2119. 7. Suzigan S et al. Multilocular cystic renal cell carcinoma. Am J Clin Pathol, 2006. 125: 217-222. 8. Webster WS et al. Surgical resection provides excellent outcomes for patients with cystic clear cell renal cell carcinoma. Urology, 2007. 70: 900-904. 9. Gong K et al. Multilocular cystic renal cell carcinoma: an experience of clinical management for 31 cases. J Cancer Res Clin Oncol, 2008. 134: 433-437. 10. Eble JN. Multilocular cystic renal cell carcinoma. In: Eble JN et al. Pathology and genetics of tumors of the urinary system and male genital organs. Lyon, France: IARC Press, 2004. World Health Organization Classification of Tumors.

65

Characterization of the blood-spinal cord barrier (BSCB) and vascular changes following spinal cord injury

Ramak Khosravi1, Sarah Figley1, and Dr. Michael Fehlings1,2 1 Institute of Medical Sciences, University of Toronto 2 Department of Genetics and Development, Toronto Western Research Institute, University Health Network Introduction: Spinal cord injury (SCI) initiates local vascular disruption ­ including hemorrhage, thrombosis, and disruption of the blood-spinal cord barrier ­ and progressive ischemia, which contribute to the damage and deterioration caused by the secondary injury. Endogenously, vascular repair processes are initiated following injury; however, endogenous re-vascularization is insufficient to restore complex CNS vascular networks. Various therapies have been developed to target and enhance vascular repair mechanisms, but to effectively interpret the results of an individual therapy, it is critical to characterize SCI vascular disruption. Previous studies have only characterized the vascular changes following weightdrop models of SCI, which have dissimilar characteristics of neuronal preservation and functional outcomes compared to clip-compression SCI models. Aim: We aim to define the functional and structural changes that occur within the microvasculature following clip-compression SCI. Methods: All animals were given daily subcutaneous injections of the immunosuppressant cyclosporin A (10 mg/kg) one day prior to injury and for the duration of survival. Animals received a 35g clipcompression injury at T6-T7, resulting in a moderately-severe SCI. At 3, 5, 7 and 10 days post-injury, animals were injected with Evan's Blue to assess vascular permeability or FITC-LEA to observe vascular function. Tissues were analyzed using densitometry and fluorescence microscopy. Results: Preliminary data show that animals subject to SCI have dramatic changes in blood-spinal cord barrier permeability and reduced numbers of functional vessels as early as 3 days post-injury. Continuing studies will characterize the temporal and spatial profiles of these vascular changes and compare them to the microvasculature of animals treated with a VEGF gene therapy.

66

Development of a glutamate-specific biosensor

Shanna Kirshenblatt, Josef Buttigieg, Michael Fehlings

Genetics & Development, Toronto Western Hospital

During spinal cord injury, surviving axons at the subpial rim are often dysmyelinated resulting in poor saltatory conduction. To improve axonal function mechanisms that involve axonal remyelination, e.g. stem cell therapy, are currently being investigated. Preliminary evidence in neuronal development suggests that glutamate is released, not only from the axonal terminal end, but also along the bare axon. The release of this axonal glutamate is thought to induce oligodendrocyte precursor cells (OPC) to migrate and differentiate into mature oligodendrocytes and myelinate these bare axons. To date, there has been only anecdotal evidence supporting this model due to the lack of a sensitive glutamate sensor. We proposed a method to detect glutamate at a cellular level. We created a biosensor that can be utilized to detect glutamate release through using electrophysiology. Using lipofection and the pLEX-JRed vector, we inserted the glutamate kainate receptor 2 gene into Human Embryonic Kidney 293 cells (HEK 293). The resulting cell line is now able to respond to glutamate. Membrane sections can be isolated, and using the outside-out configuration of patch clamp electrophysiology, can detect quantal glutamate release. These findings will aid in our understanding of neurotransmitter release and stem cell development.

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The Graft Imaging to Improve Patency (GRIIP) Substudy

Kamya Kommaraju; Dai Une; Reena Karkhanis; Stephen E. Fremes, MD, MSc Division of Cardiac and Vascular Surgery, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada

Introduction: Graft Imaging to Improve Patency (GRIIP) was a randomized, single-center, singleblinded clinical trial designed to determine whether intraoperative graft assessment with graft revision according to a priori criteria (Imaging group) would result in improved graft patency 1 year after bypass surgery when compared with standard intraoperative management (Control group). Objective: The purpose of this substudy was to reassess the a priori criteria (transit-time flowmetry (TTF) and indocyanine-green (ICG) findings) for graft revision in GRIIP, as the rate of graft revision was low (3.6%) and the 1 year graft patency was similar in the Imaging and Control groups. Methods: This is a case-control study of the Imaging patients from GRIIP. The preoperative demographics, intraoperative ICG angiograms and TTF results, and 1 year postoperative angiographic results from 55 patients (160 grafts) in the GRIIP Imaging group were analyzed. Preliminary results: There were no differences between cases and controls in clinical variables, ICG results, or TTF diastolic fraction or pulsatility index. TTF mean flow (MF) values in saphenous vein (SV) grafts was different between patent and stenotic/occluded grafts at 1 year (43/64 vs. 21/64, p=0.011). A MF less than 20mL/min was a better predictor of 1-year graft failure than 10mL/min as originally used in the GRIIP trial (p=0.046). Conclusions: A mean graft flow less than 20 mL/min is predictive of postoperative saphenous vein graft failure. Further research is recommended to address whether targeted therapy including graft revision based on ICG angiography and TTF MF measurements can prevent postoperative SV graft failure.

68

Mechanisms of induction of microvascular leak by anthrax lethal toxin

Yuri Koumpan, Susan M Armstrong, Jayesh Tigdi, Changsen Wang, Warren L. Lee, Institute of Medical Science and the Keenan Research Centre of the Li Ka Shing Knowledge Institute of St. Michael's Hospital, and the Divisions of Respirology and Critical Care Medicine, University of Toronto. INTRODUCTION: Anthrax poisoning is a result of lethal toxin and edema toxin, produced by Bacillus anthracis. Lethal toxin (LT) disrupts endothelial cell barrier integrity, resulting in microvascular leak and shock-like symptoms. Studies have shown that LT causes disruption of VE-cadherin, the major component of adherens junctions which helps to maintain an intact endothelial barrier in the vasculature. There is also evidence that LT induces endothelial cell apoptosis, which could lead to vascular leak. However, whether one or both of these factors contribute to LT-induced microvascular leak is currently unknown. PURPOSE: To explore mechanisms of LT-induced endothelial leak by determining the effect of LT on VE-cadherin and apoptosis. METHODS: Experiments were performed using human dermal microvascular endothelial cells. Leak was measured using trans-endothelial electrical resistance (TEER). Immunofluorescence was used to observe VE-cadherin distribution in LT-treated cells. An apoptosis assay using annexin V and propidium iodide staining was performed by flow cytometry. RESULTS: Leak increased upon LT exposure in a time-dependent manner and required catalytically active toxin. Immunofluorescence images of LT-treated cells show loss of VE-cadherin from the cell membrane with accumulation around the nucleus. Preliminary flow cytometry results do not show increased apoptosis in LT-treated cells. CONCLUSION: Preliminary results suggest that LT-induced leak in endothelial cells is accompanied by VE-cadherin internalization and is independent of apoptosis. In future experiments, we will determine the time course and mechanism of VE-cadherin redistribution, as well as identify the specific locations of VE-cadherin accumulation.

69

Does nfil3 transcription factor affect glucose regulation?

Gökçe Naz Küçükba Dr. Minna Woo, Room 8-113, Princess Margaret Hospital, Ontario Cancer Institute Introduction: Circadian rhythm may have an effect on the functions of the organs during the day. Some of the factors in the Circadian rhythm are the transcription factors called Nfil3 and DBP. These two factors work contrary to each other and DBP is higher in the morning whereas Nfil3 is higher at night. It's thought that the functions of the organs responsible for blood glucose level(BGL) may also be affected by the Circadian rhythm as it's shown that people who undergo sleeping disruptions have impaired insulin sensitivities. Therefore Nfil3 and DBP seem to be important factors in BGL regulation. Hypothesis: "If Circadian rhythm affects regulation of BGL then Nfil3 knocked out mice will have impaired BGL regulation." Methods: PCR is used for the genotyping. Western Blot is used to show the presence of the transcription factors in the tissues. BGL regulations are compared between the control and the knocked out groups by Glucose Tolerance Test and Insulin Tolerance Test using BGL measuring kit. Results: The research is still on progress and we are expecting to find impaired regulation of BGL in the knocked out group compared to that of control group. Conclusions: If we find out significant difference between the groups, it means that Circadian rhythm may have an effect upon BGL and with balanced hours of sleep and awakeness the risk of diabetes may be reduced. If there is no significant difference between the groups, the impaired BGL because of Circadian rhythm may not be Nfil3 induced.

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Is Tuberous Sclerosis a co-morbidity factor for Vigabatrin-associated retinal toxicity in children with Infantile Spasms

Ananthavalli Kumarappah Supervisor: Carol Westall, Ophthalmology and Vision Sciences, The Hospital for School Children Vigabatrin is an antiepileptic drug that increases the concentration of the neurotransmitter GABA (gamma aminobutyric acid) in the brain and retina. Vigabatrin is used to treat children diagnosed with Infantile Spasms (IS) and it has been found to be more effective in patients with Tuberous Sclerosis (TS) (Chiron 1991, MacKay et al. 2004). Vigabatrin causes visual field defects in some patients (Lawden et al. 1991), which in infants is diagnosed as a repeatable decrease in age-expected 30-Hz flicker amplitude on an electroretinogram (ERG). This retrospective study reviewed a database of all patients treated with Vigabatrin for IS from 1994 (n=345). TS status was identified by review of hospital charts. There was no significant difference in the prevalence of toxicity between patients with TS and patients with other underlying etiologies. Of the 45 (22 M and 23 F) patients diagnosed with TS, 4 were found to show evidence of toxicity (9%). Of the 300 patients with other etiologies, 24 were found to show evidence of toxicity (8%). There was no significant difference in the age at which toxicity was first identified between the two groups (median age: 24 months p=0.64). However, patients with Tuberous Sclerosis were on Vigabatrin for a longer time period (33±15 months versus 13±8 months in non-TS) before the first signs of toxicity (p<0.01). These results suggest that for children with TS a shorter treatment time (under 12 months of Vigabatrin) will result in a reduction in numbers with retinal toxicity in this group.

71

Effect of Experimental Subarachnoid Hemorrhage on Long-Term Potentiation in Mice

Katarina Lakovic Supervisor: R. Loch Macdonald, MD, PhD Neurosurgery, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, M5B 1W8, Canada

Aneurysmal subarachnoid hemorrhage (SAH) occurs in about 10 of every 100 000 people per year in North America. Approximately 50% of these people survive but they are often left with substantial cognitive impairment. Memory, for example, is frequently impaired and tends not to recover. Why patients with SAH develop cognitive and memory impairment is unclear since they often do not have focal ischemic lesions or evidence of brain damage in the hippocampus and temporal lobe. Long-term potentiation (LTP) is an electrophysiological measurement of activity-dependent synaptic plasticity, and an accepted molecular substrate of memory. We hypothesize that the alteration of LTP in the hippocampus might play a role in the memory impairment following SAH. Therefore, the purpose of this study is to determine the effect of SAH in mice on hippocampal LTP, and correlate this with histological changes in this model. Male CD-1 mice were randomly assigned to a naïve control, saline-injection control, or a SAH group. LTP was recorded in the hippocampal CA3-CA1 pathway either 2 or 7 days after surgery on saline-injection and SAH mice. Hippocampal slices were also collected for future histological studies. Contrary to previous findings in the rat SAH model, these preliminary results do not show a diminished response in LTP in the SAH mice both 2 (n=3) and 7 days (n=3) post-surgery, relative to saline controls. This discrepancy may have occurred due to the use of the analgesic buprenorphine in the mouse model which may have acted as a neuroprotectant. Alternatively, the amount of blood injected into the SAH mice may not have been enough to cause significant impairments. Further work in necessary before definitive conclusions can be drawn.

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Role of bone marrow derived progenitor cells in intracranial tumor neovascularisation

Soroush Larjani 1, Gelareh Zadeh 1,2

1

Brain Tumor Research Center, Hospital for Sick Children

2

Department of Neurosurgery, Toronto Western Hospital

Introduction: Glioblastoma multiforme (GBM) are the most common primary malignant brain tumors and the histopathological hallmark of their malignant transformation is neovascularization. Thus, there is increasing interest in targeting modulators of vascular proliferation in hopes to identify novel therapeutics for these malignant tumors. There are two forms of neovascularization: vasculogenesis and angiogenesis. Vasculogenesis is the formation of de novo vascular network and angiogenesis is the formation of new vessels from preexisting ones. The exact mechanism of GBM neovascularization, whether through angiogenesis or vasculogenesis, remains controversial. We here study the spatio-temporal role of bone marrow derived precursor cells (BMDCs) in GBM neovascularization to determine whether vasculogenesis (de novo vessel formation) occurs in GBMs. We further study the effect of ionizing radiation (IR) on recruitment, migration, and differentiation of BMDCs in tumor vascularization. Methodology: Chimeric NOD/SCID mice reconstituted with bone marrow expressing red-fluorescentprotein were used for creating human GBM xenografts in an intracranial window chamber model (ICW). Two glioma cell lines were used: U87 and U251. To study the effect of IR, mice were treated with hemicranial irradiation at 3x2Gy. Using two-photon laser capture microscopy (2PLM), real-time images of tumor cells, vasculature and contribution of BMDCs were obtained and compared in a longitudinal manner over a course of 21 days. Mice were sacrificed and brains collected for immunohistochemical analysis. Results: In U87 models, 2PLM and histological analysis, demonstrated that BMDCs contribute to perivascular support cells. Additionally, BMDC contribute to tumor vessel formation in a temporal manner, with later stages of tumor growth exhibiting angiogenic versus vasculogenic vessel formation.

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Expression levels of genes flanking the targeted region in the Gtf2i- and Gtf2ird1-deficient mouse models of Williams-Beuren syndrome do not appear to be altered

Wyanne Law1, Emily Lam1, Lucy R. Osborne1,2

1

Institute of Medical Science; 2Departments of Medicine and Molecular Genetics, University of Toronto

Introduction: Williams-Beuren Syndrome (WBS) is a neurodevelopmental disorder characterized by cardiovascular abnormalities, distinctive facial features and mild to moderate intellectual disability, affecting approximately 1 in 20,000 to 1 in 7,500 people. WBS is most commonly caused by deletion of 1.55 Mbp on a single copy of human chromosome 7, which results in the loss of 25 genes that may be linked to specific symptoms observed in WBS patients. Previous studies have suggested that general transcription factors GTF2I and GTF2IRD1 within the WBS deletion region may be responsible for neurological features of the disorder and our lab has generated knock-out mouse models of these genes. Purpose: To determine whether other genes within the WBS deletion region may be affected by genetrapping and/or haploinsufficiency of Gtf2i and Gtf2ird1 in our mouse models. Methods: Gene expression analysis was conducted using mouse models singly and doubly deficient in Gtf2i and Gtf2ird1, and results were compared to wild type (WT) littermates at 15 to 20 weeks of age. Total mRNA was extracted from brain and cDNA was synthesized according to previously published methods. Real-time quantitative PCR was performed to check for changes in gene expression of 12 genes, normalized to two housekeeping genes. Results: Preliminary findings show that there are no significant alterations in mRNA expression of genes flanking Gtf2i and Gtf2ird1. Conclusion: This study confirms that the gene trap and deletion techniques used to generate our mouse models do not affect mRNA expression of other genes examined in the WBS deletion region. Gene expression will be verified with analyses of protein levels using Western blots.

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Survivin Gene Therapy Prevents the Deterioration of Heart Function in a Rat Model of Doxorubicin-Induced Cardiomyopathy

Name: Claire Lee Supervisor: Dr. Howard Leong-Poi Division: Cardiology Hospital: St. Michael's Hospital Background. Heart failure is a growing problem as more patients survive cardiac injury, such as myocardial infarction. Clinical studies have shown that programmed cell death, or apoptosis, plays a role in progression to end-stage heart failure. Hypothesis. We hypothesize that gene delivery of survivin, an anti-apoptotic protein, will reduce cardiomyocyte apoptosis in vitro, and prevent progressive left ventricular (LV) systolic dysfunction in vivo in a rodent model of doxorubicin-induced heart failure. Methods. In vitro: Non-transfected, mock-transfected and survivin-transfected rat H9c2 cells (ATCC) will be incubated for 24 hours in Dulbecco's Modified Eagle's Medium (ATCC) with 10-6M of doxorubicin. Apoptosis will be assessed using Annexin-V staining and terminal deoxynucleotidyl transferase mediated nick-end labeling (TUNEL). In vivo: Fisher rats (n=20) received intra-peritoneal injections of 2.5mg/kg b.w. of doxorubicin every two days for a period of two weeks. Ultrasoundmediated gene delivery (500mcg of survivin plasmid DNA+1x109 cationic microbubbles) to the LV was performed in the active treatment group (n=8) at week 3, while control rats (n=12) received no treatment. Echocardiography was performed on all animals at weeks 0 (baseline), 3 (pre-delivery), and 6 (endstudy). Results. In vitro apoptosis study results are pending. For the in vivo studies, in control untreated animals, LV fractional shortening (FS) decreased by week 6 (37.5±6.7% vs 50.9±3.7% at day 0, p<0.01). In contrast, survivin-treated animals showed no significant reduction in LVFS over time (45.3±3.6% vs 46.6±2.9% at day 0, p=NS). Conclusions. Ultrasound-mediated survivin gene delivery reduces progression of LV dysfunction in a model of doxorubicin-induced heart failure.

75

AuNP Radiosensitization Assays and Comparison with a Theoretical Model

Eric IJ Lee1, Eli Lechtman2, and JP Pignol3

1

Institute of Medical Science, University of Toronto; 2Department of Medical Biophysics, University of Toronto; 3 Odette Cancer Centre, SunnyBrook Health Science Centre

INTRODUCTION: The main purpose of the Radiation therapy to cancer is to achieve the loco-regional tumor control. However, the success of radiation therapy is sometime limited by the intrinsic radioresistance and increasing the dose may induce normal tissue toxicity. One approach to increase the efficiency of radiation therapy is using high-Z radiosensitizers such as iodine, gadolinium and gold. The accumulation of a high-Z material in the tumor region effectively increases the local absorption of low-energy photons through the photoelectric effect. Upon irradiation, this increased absorption results in a highly conformal dose enhancement caused by the localized spray of photoelectric products (photoelectrons, Auger electrons, and characteristic x-rays). In recent years gold nanoparticles (AuNPs) have become a promising candidate for targeted radiosensitization due to gold's high photoelectric cross-section and biocompatibility. Furthermore, the nanoparticle is an effective vehicle for radiosensitization due to the large number of atoms per particle, and a surface well suited for conjugating to tumor targeting antibodies or synthetic oligonucleotide aptamers. Therefore, the combination of tumor targeted AuNPs in conjunction with conformal radiation delivery represents a dually specific tumor treatment strategy. This approach has the potential to broaden the therapeutic window for the possibility to control cancers. OBJECTIVE: 1. To validate a theoretical model of the energy deposition around AuNPs at the sub-cellular scale based on Monte Carlo simulation. 2. To predict the radiobiological effect of AuNP radiosensitization, and to define the optimal use of AuNP for radiation therapy. METHODS: We will grow the PC3 cell line to confluence first, and then culture the cells in media containing different concentrations of two sizes (5 and 30nm) of pegylated AuNP (3000, 10000, 30000, 10000, 300000) nanoparticles per cell) for 24 hours. After 24 hours, we irradiate 2 Gy of X-ray in different energy (150kV and 300kV). The cells are counted and the appropriate number plated to obtain 100 colonies per Petri dish after 10 days. RESULTS AND PERSPECTIVE: We expect that the AuNPs will dramatically increase the cells kill and thereby improve x-ray therapy. We will compare theoretical cell kill as predicted by Monte Carlo simulation to the in vitro cell kill measured by clonogenic assays.

76

Endogenous stimulation of neural precursor cells leads to neural regeneration and functional recovery following stroke in adult mice

Justin Lee, Nadia Sachewsky, Cindi Morshead, Department of Surgery, University of Toronto 1006 Donnelly Centre for Cellular Biomolecular Research The adult brain contains a population of neural precursor cells (NPCs) within the periventricular region of the forebrain. Recent studies have shown that the administration of factors such as Epidermal Growth Factor and Erythropoietin or Cyclosporin A (CsA) leads to NPC activation, effectively promoting tissue regeneration and functional recovery following cortical stroke (Kolb et al. 2007) (Erlandsson et al. 2010). Permanent devascularization of the motor cortex via the pial vessel disruption (PVD) of motor cortex was model used to induce stroke in these studies. Although a commonly used model, the clinical relevance of PVD is unclear. Herein, we have developed a more clinically relevant model of stroke and asked whether the application of endogenous repair strategies is applicable across models. Endothelin-1 (ET-1) is a potent vasoconstrictor that causes restriction of blood flow, followed by reperfusion, hence better reflecting the pathology of stroke. Two groups of mice received ET-1 injections into the motor/sensory cortex, which led to measurable and permanent motor deficits. At the time of stroke, mice received either systemic administration of CsA or artificial cerebrospinal fluid (aCSF) (control) and were subsequently tested in the foot fault task weekly to monitor behavioural recovery. Parallel to what was observed following PVD, mice with ET-1 lesions and CsA treatment displayed complete functional recovery while control aCSF treated animals did not recover. We are currently performing tissue analysis including lesion volume, NPC proliferation, migration and differentiation following ET-1 with the goal of developing regenerative strategies to treat stroke.

77

A Comparison of Instruments to Assess Male Sexual Function Following Treatment for Rectal Cancer

Marisa Leon-Carlyle Dr. Erin Kennedy General Surgery Toronto General Hospital Background: Several studies report a high prevalence of sexual dysfunction in male patients following treatment for rectal cancer. Many of these studies were limited by retrospective designs and failure to use validated instruments. Although the EORTC QLQ-CR38 is commonly used to assess sexual function, only 5 of 38 items pertain to sexual function. Therefore, the purpose of this study was to compare sexual function results obtained on the EORTC QLQ-CR38 to the International Index of Erectile Dysfunction (IIEF), a validated instrument specifically designed to measure sexual function in males. Methods: Male patients with newly diagnosed rectal cancer attending clinics at four medical centres were invited to participate in the study. Participants completed the International Index of Erectile Dysfunction (IIEF), EORTC QLQ-C30 and EORTC QLQ-CR38 at the time of diagnosis (Time 1), after pre-operative chemoradiation when applicable (Time 2) and one year after surgery (Time 3). Results: 82% reported being sexually active before surgery and 69% by 12 months after surgery. Overall, the IIEF scores for each domain were low reflecting a high degree of sexual dysfunction at all time points. The erectile function domain and overall IIEF score substantially decreased. Interestingly, the scores for the male sexual problems domain of the EORTC QLQ-CR38 showed a significant increase reflecting an increase in symptoms relating to erection and ejaculation over time (p=0.00). Conclusions: Based on this study, the EORTC QLQ-CR38 seems to be a relatively sensitive and reliable instrument to measure male sexual function following rectal cancer when compared to the IIEF.

78

Platelet MHC Antigens Inhibit Platelet-specific (CD61) Cytotoxic T cell (CTL) Mediated Thrombocytopenia

Guo L, Aslam R, Speck E, Kim M, Ni H, Semple JW Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by peripheral immune platelet destruction and or platelet production defects. To understand how one antigen system on a platelet can affect the immune response against another antigen, we used our novel mouse model of ITP. CD61 KO mice were transfused with the three different platelet populations that were either single antigen positive (CD61 or MHC class I) or double antigen positive (CD61 and MHC class I) and sera IgG antiplatelet production was monitored by flow cytometry. When the mice exhibited antibody production, they were sacrificed and their splenocytes (either non-depleted (ND) or depleted of CD19+ B cells) were transferred into irradiated SCID mice. When splenocytes from CD61 only immune mice were transferred, they induced a significant thrombocytopenia with a bleeding mortality of 20% within 2-3 weeks posttransfer. Depletion of B cells was used to analyze CD8+ T cell mediated thrombocytopenia. These depleted splenocytes also induced thrombocytopenia and bleeding. In contrast, when splenocytes from double immunized (CD61 and MHC) mice were transferred, the CD8+ T cell mediated thrombocytopenia and bleeding was lost. These results suggest that both antibody and CD8+ T cell mediated immune thrombocytopenia exist and that CD8+ T cells' interaction with allogeneic MHC antigens on platelets paralyzed the pathogenic CD8+ T cell-mediated thrombocytopenia. Thus, platelet transfusions may be a therapeutic option for those patients who suffer from cell-mediated ITP.

Principle investigator: John W. Semple (Head, Transfusion Medicine Research, St. Michael's Hospital. 30 Bond St., Toronto, Ontario, Canada. M5B 1W8. 416-864-5534 416-864-3053 (FAX))

Mentor: Rukshana Aslam

Sponsors and funding agencies: Canadian Blood Services, The University of Toronto, Toronto, Canada and Shandong University, Jinan, China

79

Apelin Deficiency Contributes to Fibrosis in the Heart: Synergies with Angiotensin II

Annie Liang, XiaoXia Wang, Peter Liu Toronto General Research Institute and Heart and Stroke/Richard Lewar Centre of Excellence, University Health Network, University of Toronto. Introduction-- Apelin, a 13 amino acid ubiquitous signaling peptide, processed by the novel ACE2 enzyme from a prepropeptide, is a ligand for the G-protein coupled APJ receptor. To date, apelin has been shown to have significant cardiovascular effects, including modulation of systemic vascular resistance through nitric oxide-dependent pathway, and enhancement of cardiac contraction. Apelin determines cardiac pattern formation during embryonic development, and counteracts angiotensin AT1 receptor signaling. Our lab has demonstrated that Apelin knockout mice develop significant cardiac hypertrophy and heart failure following pressure overload, and that recombinant apelin infusion can improve cardiac function. Recent unpublished data also demonstrated that interstitial fibrosis was enhanced in apelin knockout mice after myocardial infarction. Hypothesis-- We hypothesize that apelin is critical in matrix maintenance and prevention of fibrosis. Apelin deficiency in the presence of angiotensin alone without stress, can lead to fibrosis via the TGFbeta pathway. Methods--Apelin-/- knockout mice in C57/B6 background and wild type littermates were infused with angiotensin II (Ang II, 1.0 ug/kg/day) by osmotic mini pumps for 2 weeks. The hearts were harvested, and evaluated for morphology, fibrosis and function. The fibrosis was evaluated by picrosirius Red staining. Quantitative Real-Time PCR was performed to quantify gene expressions. Results-- Heart Weight/Body Weight ratio was significantly higher in Apelin KO mice compared to WT after AngII infusion. PicroRed staining showed enhanced interstitial fibrosis in Apelin mutant mice compared to WT mice with AngII infusion. mRNA levels of fibrosis related genes, such as collagenalpha, collagen III, and TGF- 1&2 were significantly higher in KO-AngII mice relative to wild type. This is compatible with previous microarray data following banding showing up regulation of Col8a, Postn, Ltbp2, Lox and Tgfb2, amongst other fibrosis genes. Conclusion--Apelin contributes to cardiac fibrosis in the presence of angiotensin II, without mechanical stress through TGF- signaling pathway.

80

Clinic managers' insight into organization of healthcare in First Nations in Canada as part of the CIRCLE study

Dong Lin Onil K. Bhattacharyya Li Ka Shing Knowledge Institute, 193 Yonge Street Aboriginal people in Canada have high rates of diabetes and related complications. While prevention is essential to reduce the incidence of diabetes in Aboriginal communities, quality health services are necessary for those currently living with diabetes. The chronic care model is associated with better care and better health outcomes for diabetes. A survey administered as part of the Canadian First Nations Diabetes Clinical Management and Epidemiologic (CIRCLE) study asked clinic managers about organization of care in their communities. The surveys were completed by 29 clinic managers from 19 First Nation communities across Canada. The surveys completed by this group included questions about the clinic staff (number in the community, level of training, etc.) and the availability and functioning of different programs and services. SPSS software will be utilized to descriptively analyze these surveys (mean, mode, proportion of respondents for each response) and to analyze the data by sub-group (isolation level, community). The results of this survey will enable us to document the different models of care, and to examine potential factors driving these different models (i.e. community size, isolation level, etc.). Comparisons with health outcomes data from the CIRCLE study will help us better understand the factors driving the quality of diabetes care in First Nations communities. In addition to filling gaps in knowledge, the results of this work will guide local and federal decision-making and inform program renewal to fill practice gaps.

81

Survey of the Outcomes of Work-Related Asthma

Joshua Lipszyc, Marcos Ribeiro MD PhD, Susan Tarlo MB BS, FRCP(C) Location ­ Toronto Western Hospital, Asthma and Airway Centre Background: There are two forms of work-related asthma: occupational asthma, where respiratory symptoms are brought on by the workplace environment, and work-exacerbated asthma, in which the symptoms of patients already diagnosed with respiratory illness are exacerbated by the workplace environment. Many of these individuals take steps to reduce workplace exposure, up to and including leaving their place of work. It is important to consider the patient's history with work-related asthma, the type of workplace exposure, and the steps taken by the patient to reduce workplace exposure in order to improve the management and outcome of work-related asthma. Purpose: To examine the outcomes of individuals with occupational and work-exacerbated asthma. Method: Data was collected by patient interview using two questionnaires. The first included 46 questions covering respiratory symptoms, medication use, workplace environment, and demographic details. The second included 15 questions about quality of life. Statistical analysis aimed to compare outcomes and determinants between occupational asthma and work-exacerbated asthma. Results: Initial participants comprised 16 patients diagnosed with work-related asthma between 2000 and 2007, of which 9 were diagnosed with occupational asthma and 7 with work-exacerbated asthma. Only 2 of the 16 remained in the same exposures as when their work-related asthma started. Both of these individuals were diagnosed with work-exacerbated asthma, where leaving an occupation is not always suggested. Ten of the participants reported a decrease of total income following their work-related symptoms. A trend was found for a greater number of participants in the work-exacerbated asthma group (P=0.06) to waken with chest tightness in the past month compared to the occupational asthma group. Conclusions: The vast majority of participants left their workplace to reduce their symptoms, and most lost income. Findings suggest worse control of asthma at follow-up among those with work-exacerbated asthma compared with occupational asthma.

82

Examining the effects of peptide-gold nanoparticle hybrids on the activation of myeloid dendritic cells

Annie Liu, Dr. Hong Yang and Dr. Mingyao Liu (Supervisor), Latner Thoracic Surgery Research Laboratories, UHN

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in innate and adaptive immunity. They have become a major target for immune modulation based therapeutic strategies. Gold nanoparticles (GNPs) are biocompatible materials. The surface of GNPs can be easily modified with peptides via thiol-gold linkage to alter their surface chemistry and to modulate their cellular uptake. The purpose of this present study was to investigate the ability of peptide-GNP hybrids to modulate the DC immune response in vitro. To assess the activation effects of peptide-GNP hybrids, DCs were differentiated from bone marrow (BM) cells of black B57BL/6 mice by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At day 9 of culture, BM-derived DCs (BMDCs) were exposed to GNP hybrids with a diameter of approximately 10 nm. The purity of the resulting DCs was determined by the CD11c positive cell population measured by Flow Cytometry. We found that the peptide-GNP hybrids were internalized by immature BMDC via bright field microscope imaging. Furthermore, the addition of GNPs to immature BMDCs was found to influence both phenotype and function of immature BMDCs. Stimulation by peptide-GNP hybrids on the immature BMDCs led to an enhanced expression of co-stimulatory molecules on the cell surface, including CD40, CD80 and CD86, and a secretion of cytokines in the culture medium. This observation suggests that peptide-GNP stimulation can lead to the activation of BMDCs, and implies a potential application for strengthening vaccine delivery devices or cancer immune therapy.

83

Effect of levodopa on bidirectional synaptic plasticity in the substantia nigra pars reticulata (SNr) of Parkinson's disease (PD) patients

*Liu D. Liu1, Ian A. Prescott1, Jonathan O. Dostrovsky1, Mojgan Hodaie2, Andres M. Lozano2, William D. Hutchison1, 2 1 Department of Physiology, University of Toronto, Toronto, ON Canada 2 Division of Neurosurgery, Toronto Western Hospital, Toronto, ON Canada Levodopa effectively treats motor symptoms of PD, but many patients develop debilitating levodopainduced dyskinesia (LID). Studies have shown that levodopa potentiates corticostriatal synaptic plasticity and suggest that LID is linked to a loss of de-potentiation of the plasticity with low frequency stimulation (LFS). In PD patients undergoing deep brain stimulator insertions, we previously reported that levodopa also potentiates the activity-dependent synaptic plasticity in SNr. In a new cohort of patients, we investigated the ability of LFS to reverse high frequency stimulation (HFS)-induced potentiation. Using dual microelectrodes, one microelectrode stimulated while the second microelectrode recorded the evoked field potentials (fEP). Baseline fEP amplitudes were collected and then conditioning HFS (100 Hz) was delivered. Post HFS, fEP amplitudes were measured again every 30 s until a stable plateau was reached. LFS (2 Hz) was then delivered and followed again by fEP recordings. Similar to our previous finding, levodopa administration in 9 patients enhanced the potentiation following HFS (+61.3 % of baseline at 70.1 s following a plateau), compared to 14 patients in the OFF drug state (+30.5 % at 66.3 s). LFS did not reverse this LTP-like effect in the levodopa ON state, since the fEP amplitude decay in the ON group following LFS (+32.3 % at 165.1 s) was similar to the decay without LFS (+37.1 % at 160 s). In conclusion, levodopa significantly increases SNr plasticity, and this effect was not reversed by LFS, suggesting that LIDs in PD patients may also arise from a lack of depotentiation in SNr.

84

Enrichment of Vascular Endothelial Cells in the Highly Purified Osteoprogenitor Cell Fraction of Murine Bone Marrow Stromal Cell Populations

Dana Mayer1, Kristen McKenzie2, Jane E. Aubin1,2,3, 1Institute of Medical Science, 2Graduate Department of Dentistry and 3Department of Molecular Genetics, University of Toronto Introduction: Bone is a highly vascularized tissue and thus its repair and regeneration require both osteogenesis and angiogenesis [Kanczler and Oreffo, 2008]. We previously fractionated murine bone marrow stromal cells (BMSCs) using a magnetic microbead negative selection process to exclude hematopoietic stem cells, resulting in a highly purified osteoprogenitor (HipOP) cell fraction [Itoh and Aubin, 2009]. HipOPs were demonstrated to have a high potential for regeneration of a bone organ in vivo, including sinusoids, suggesting the presence of an angiogenic subpopulation in the HipOP fraction of cells. Purpose: To determine if the murine HipOP population is enriched in vascular endothelial cells. Methods: Expression of specific vascular endothelial cell markers (VE-cadherin, Tie2, Delta4, and Eselectin) was quantified with QPCR in RNA extracted from BMSCs and HipOPs on the day of fractionation and at day 14 and 21 of culture in osteogenic differentiation medium. Results: E-selection mRNA expression was significantly increased in HipOPs versus unfractionated BMSCs at day 0, and there was a trend toward an increase for the other vascular endothelial cell markers. At day 14 and 21, there was also a trend toward an increase in expression for all markers, although none reached statistical significance. Conclusion: The data suggest that vascular endothelial cells are enriched along with osteoprogenitor cells in the HipOP fraction of BMSCs. Future experiments are directed towards confirming and extending these preliminary results.

85

Let's Talk: A brief intervention for improving family caregivers' symptom assessments

Nina McCurdy PI/Supervisor: Dr. Jennifer M. Jones, Psychosocial Oncology and Palliative Care, Princess Margaret Hospital Background: Up to 70% of terminally ill patients become too ill or cognitively impaired to complete selfassessment measures. In these situations, proxy ratings (i.e. a family caregiver's assessment of the patient's condition) may be an effective way to gather critical information. Studies have found, however, that family caregivers (FCGs) often have difficulty providing reliable estimates of a patient's condition, especially when asked to rate his/her subjective experiences (e.g. psychological domains). It has been suggested that promoting open discussions about the experience and meaning of symptoms and psychological issues may improve the congruence of patient and FCG ratings, but to date no one has studied whether encouraging such discussions is an effective intervention. Purpose: This study piloted a brief intervention aimed at increasing empathic communication between FCGs and patients in order to: (1) refine the intervention; (2) ascertain its feasibility and acceptability; and (3) gain a preliminary estimate of the effect of the intervention on the congruence between patient and FCG symptom ratings. Methods: Forty-one patient and FCG dyads were recruited from the Palliative Care and Oncology inpatient units at Princess Margaret Hospital and St. Michael's Hospital. At baseline (T1), patients completed a perception of empathy scale (BLRI-R Empathy) regarding their relationship with the FCG, and both patients and their FCG proxies independently completed a symptom assessment scale (CMSAS). The dyads then sat down with the research assistant to discuss their responses on the CMSAS (intervention). These discussions were structured using a retrospective "think-aloud" cognitive interviewing framework and were audio taped and transcribed. Three days later (T2), dyads were again asked to independently complete the CMSAS, and exit interviews were conducted to elicit participants' experiences in the study. Results: The intervention discussions and exit interviews will be analyzed using latent content analysis, and the T1 and T2 CMSAS data will be summarized with descriptive statistics and analyzed with Pearson's r, t-tests, intraclass correlations, and mixed linear models. Data will be presented. Implications: Enlisting FCGs at the early stages of cancer diagnosis may provide an effective opportunity for encouraging patient-FCG communication and empathetic perspective taking, so that if the patient does become unable to provide their own information, the FCG will be able to provide more accurate estimates of the patient's symptom experiences.

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Capturing common genetic variations that influence bioavailability of drugs used in children with heart disease

Katherine McLean, Seema Mital MD, Heart Centre, Department of Cardiology, 4514 Hill Wing, Hospital for Sick Children Background: Common genetic variations in drug biotransformation enzymes can affect the rate of metabolism of drugs and significantly influence drug levels and toxicity. Purpose: To identify common genetic variations that can alter drug bioavailability of commonly used cardiac medications. Methods: Pediatric patients with heart disease enrolled in the Heart Centre Biobank Repository were retrospectively reviewed to identify commonly used drugs in cardiac patients. Enzymes involved in drug absorption, distribution, metabolism and excretion (ADME) were identified using the Compendium of Pharmaceuticals and Specialties (CPS) and drug inserts. Common single nucleotide polymorphisms (SNPs) in the genes encoding these enzymes were identified via a literature search on PubMed. Results: We identified 49 commonly used drugs in pediatric cardiac patients including anti-arrhythmics, anti-heart failure, anti-thrombotics, sedatives, vasoactive drugs, and immunosuppressants. 31 (63%) are metabolized by polymorphic enzymes. SNPs of the genes encoding all of the enzymes of 58% of the drugs are represented on Illumina's ADME panel; an additional 16% of drugs are partially covered, and 24% have no coverage. Conclusions: We identified the availability of a SNP panel that captures many common genetic variations that influence the biotransformation and bioavailability of commonly used drugs in cardiac patients; however there is still a need to develop more comprehensive SNP panels. We propose to perform genotyping to identify if these SNPs are associated with drug levels and adverse drug reactions in children with heart disease enrolled in the biorepository. This knowledge will be used to develop guidelines for pharmacogenetic testing in pediatric cardiac patients.

87

Receptor guanylate cyclases as modifiers of Bardet-Biedl syndrome phenotypes in Caenorhabditis elegans

Rebecca Mok, Calvin Mok and Dr. Elise Héon Department of Genetics and Genome Biology, The Hospital for Sick Children Bardet-Biedl syndrome (BBS) is a rare autosomal recessive, genetically heterogeneous and pleiotropic disorder. The primary features of BBS in humans include photoreceptor degeneration, digit anomalies, obesity, cognitive delay and renal abnormalities. BBS proteins are necessary in intraflagellar transport and important for cilia biogenesis and function. In Caenorhabditis elegans, ciliated neurons play the role of sensory organelles for various chemical and physical environmental signals. Currently, 27 receptor guanylate cyclases (rGCs) found in cilia membranes produce the secondary messenger cyclic guanosine monophosphate (cGMP) to activate signaling pathways important to sensory behaviours and developmental processes. The soluble guanylate cyclase GCY-35 modifies various bbs mutant phenotypes including the suppression of a small body size (unpublished data). We hypothesized that loss of rGCs would be similar to GCY-35 in their effect on bbs body size of C. elegans. In these experiments, mutant rGC strains are outcrossed with N2 wild-type animals at least 4 times to reduce the emergence of unwanted recessive mutations. Then mutant gcy and bbs strains are crossed to see the effect of rGC alleles in a bbs-7 mutant background. Primers are designed in order to complete genotyping for homozygosity using polymerase chain reaction. If the small size of bbs-7 mutants is suppressed by the loss of rGCs in a manner similar to GCY-35, then animals with mutations in both gcy and bbs-7 would display a wild-type body size. This work may implicate that rGC function and cGMP regulation are modulated by BBS proteins or affected by intraflagellar transport and cilia biogenesis.

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Cyclosporin A has pro-survival effects on temporally distinct neural stem and progenitor populations in the adult central nervous system

Carween Mui, Jessica Hunt, Cindi Morshead Department of Surgery, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto Neural stem and progenitor cells (collectively called neural precursor cells, NPCs) are found in the adult periventricular region along the entire neuraxis of the adult CNS. NPCs can be isolated using in vitro assays whereby individual cells proliferate to form multipotential, self-renewing colonies. We have recently demonstrated that the immunosuppressant, Cyclosporin A (CsA), has direct effects on NPC behaviour, increasing their survival both in vitro and in vivo, without affecting their proliferation kinetics or differentiation profile. Moreover, CsA administration in vivo activates NPCs to contribute to new tissue formation following injury. We are interested in understanding the intracellular mechanism mediating CsA's survival effects. CsA binds to cyclophilins (CyP), and CsA/CyP's have numerous targets within the cell, including binding to calcineurin. It is known that blocking calcineurin leads to a loss of reactive oxygen species, thus inhibiting cell death. Alternatively, CsA/CyP can block mitochondrial pore formation and inhibit apoptosis. We are using small molecules to selectively manipulate these pathways (FK506 and NIM811, respectively) to determine which molecule mimics the pro-survival effects of CsA in vitro. Finally, we will ask if the pro-survival effects of CsA can be seen in NPCs isolated from older aged animals, as this is a more clinically relevant population of cells for developing neural repair strategies. Our preliminary findings suggest that CsA has similar effects on these temporally distinct NPC populations in vitro. Together, these findings will provide a better understanding of CsA's effects and its clinical relevance for promoting neural regeneration.

89

Oxidative stress and the risk of osteoporosis: The role of dietary polyphenols and nutritional supplements

Dhanwantee Mundil and Leticia G. Rao A randomized, double-blind, placebo-controlled trial is currently being undertaken to test whether Greens+ bone builderTM, an investigational product, reduces the risk of osteoporosis in postmenopausal women. The primary end-point was the correlation between oxidative stress and antioxidant status with bone turnover markers, measured in serum. Out of a total enrolment of 60, only 41 participants were recruited and randomized to either Greens+ bone builderTM (N = 22) or placebo (N = 19) for 8 weeks, after 2-weeks washout period. Fasting blood samples, urine and body measurements of weight, height and blood pressure were collected at weeks 1, 2, 6 and 10. Blood was separated into serum, plasma, buffy coat, and red blood cells and samples were stored at -80oC. Mean age of participants was 56.4 years with an average of 6.1 years postmenopausal. Body measurements remained stable from weeks 1 to 2, 6 and 10 for all participants. Assays for oxidative stress, antioxidant status and bone turnover markers are to be carried out when the required number of participant is recruited. Future directions include optimizing participant recruitment through newspaper and poster advertisements. Therefore, the study remains ongoing until a minimum of 60 participants are recruited. Keywords: Osteoporosis, oxidative stress, polyphenols, antioxidants, bone.

90

Interferon Regulatory Factor-3 (IRF-3) promotes cardiomyocyte apoptosis following myocardial infarction

Jessica Ngan, Geoffrey de Couto, Peter Liu, Toronto General Research Institute, University Health Network, Toronto, ON

Cardiomyocyte apoptosis is a critical determinant of infarct size post-myocardial infarction (MI). During this process, a delicate interplay of the host's innate immune system contributes to cardiomyocyte survival. IRF-3 is a ubiquitously expressed transcription factor that regulates type-1 interferon (IFN) and cytokine production that can potentiate apoptosis. We hypothesized that the transcriptional activation of IRF-3 in cardiomyocytes following myocardial infarction through hypoxic stress would promote apoptosis. Therefore, we allocated 8-week old IRF-3 knockout (KO) mice and their wild type (WT) counterparts to left anterior descending (LAD) coronary ligation. Cardiac tissue was harvested at 3-, 7-, and 28-days post-MI. Over the 4-week time course, IRF-3 KO mice demonstrated improved survival and decreased infarct size. At day 7, IRF-3 KO mice revealed attenuated TUNEL staining as well as caspase protein levels. Moreover, these data were complemented with in vitro mouse neonate hypoxia experiments. These findings suggest that IRF-3 promotes, while a deficiency protects, cardiomyocyte apoptosis following ischemic stress.

91

Evaluation of acute bioengineered delivery of a CD44 function blocking antibody to treat severe spinal cord injury in rats

Yoonsun Oh, James W. Austin1,4, Molly Shoichet3,5 and Michael G. Fehlings1,2,4 1) Faculty of Medicine, Institute of Medical Science, 2) Department of Surgery, and 3) Department of Chemical Engineering, University of Toronto 4) Division of Genetics and Development, Toronto Western Hospital 5) Institute of Biomaterials and Biomedical Engineering Background: Spinal cord injury (SCI) involves inflammation as a key mediator of tissue injury and repair. CD44 expression is increased in many CNS disorders including SCI, playing a role in inflammation. We hypothesized that blocking CD44 acutely through localized injection of a CD44 function blocking antibody, will modulate inflammation and result in functional improvement following SCI. Methods: Activation of the CD44 receptor was inhibited with a function blocking antibody (aCD44-Ab) and was tested in lipopolysaccharide (LPS) stimulated primary microglial cultures. Next the aCD44-Ab was tested in a model of severe SCI in female Wistar rats. Local delivery of the aCD44-Ab was achieved with a blend of hyaluronan and methyl cellulose (HAMC), which was injected intrathecally 24 hours following injury. Results: Compared to isotype antibody or PBS controls, the aCD44-Ab significantly increased microglial IL-10 expression and decreased nitric oxide (NO) production in a dose dependent fashion following 6 and 24 hours of LPS stimulation in vitro, respectively. In vivo, the aCD44-Ab decreased CCL2 gene expression and showed a trend towards increased axonal preservation 2 days following injury compared to aCSF or HAMC/isotype antibody controls. Conclusion: While the mechanism is unclear, blocking CD44 receptor function influences inflammatory signaling in LPS sitmulated microglia in vitro and following SCI in vivo. As there are currently no effective neuroprotective drugs to treat SCI available and given inflammation is key to tissue repair and functional recovery, studies aimed at modulating the immune response, such as acutely blocking the CD44 receptor, represent a promising avenue of research towards the development of a cure.

92

Determinants of hypertension due to systemic steroid therapy in juvenile rheumatic disease

Student: Akane Ono Supervisor: Dr. Rae Yeung, Cell Biology, Hospital for Sick Children Introduction: Corticosteroids are used as anti-inflammatory agents in childhood rheumatic diseases including JDM(juvenile dermatomyositis), CNS(central nervous system) vasculitis and JIA(juvenile idiopathic arthritis). Systemic steroid treatments have many adverse side effects including hypertension. Factors that contribute to the development of these adverse side effects are not clearly understood. Purpose: To identify disease and patient related risk factors for the development of steroid related hypertension using 3 cohorts of childhood rheumatic disease. Methods: JIA inception cohort was obtained from a Canadian JIA study(ReACCh OUT). CNS vasculitis and JDM inception cohorts were obtained from ongoing Sick Kids disease specific registry. Clinical and laboratory data at baseline and 6 months follow-up were collected and validated. Results: Preliminary results included 2 groups, JIA cohort as non-steroid treated inflammatory control and JDM cohort as steroid treated inflammatory group. Primary outcome measure was change in systolic BP(blood pressure) between baseline and 6 months follow-up as a marker of hypertension. Univariate statistical analysis revealed significant correlation between baseline systolic BP and change in systolic BP in both steroid treated JDM cohort(r=-0.63, p<0.001) and non-steroid treated JIA cohort(r=-0.45, p<0.001), indicating that this correlation is independent of steroid therapy. In both cohorts, individual disease activity measures and patients characteristics including age, sex, and baseline BMI had no correlation. Interestingly, 6 JDM patients were prescribed antihypertensive drugs between baseline and 6 months follow-up.(Table1) Conclusion: Comparison of JDM patients treated and not treated with antihypertensive(Table1) showed that age and body habitus were associated with risk of hypertension during steroid therapy, whereas sex, baseline systolic BP and disease activity had no association. Based on preliminary results, other demographic features, starting body habitus and measures of disease activity were not associated with increase in systolic BP as a marker of hypertension. Another steroid treated group will be analyzed to validate these findings.

Table1 JDM (n=84) Antihypertensive Rx(n=6) Age at Diagnosis mean SD 4.56±1.58* Female n(%) 4(67%) Baseline BMI mean SD 14.7±0.78** Baseline Systolic BP mean SD 98.6±25.8 CHAQ mean SD 1.81±1.14 Statistic significant difference level: *p<0.05, **p<0.001 No Antihypertensive(n=78) 7.91±3.97* 52(67%) 17.2±3.69** 111.2±12.6 1.20±0.78

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LTP is preserved in surviving hippocampus following severe ischemic-hypoxic episodes in mice

Nisarg Patel1, Youssef Hanna El-Hayek1, Chiping Wu1 , Liang Zhang1,2 Toronto Western Research Institute, University Health Network, 2Department of Medicine (Division of Neurology), University of Toronto Rodent models of ischemia-hypoxia (IH) have been increasingly used to examine the mechanisms of ischemic brain injury. However, relatively less information is available regarding electrophysiological properties of post-IH brain neurons. The aim of our present study is to examine CA1 Long term potentiation (LTP) in mice following severe IH episodes, as LTP is type of synaptic plasticity hypothesized to be involved in learning and memory. C57 black mice at initial ages of 3-4 months were used. The animals received a permanent unilateral occlusion of the common carotid artery and then underwent a global hypoxic episode (8% O2 for 30 min). When examined 5-6 weeks post IH, the animals showed no evident motor-sensory deficits but gross brain injury in the hemisphere ipsilateral to the carotid artery occlusion. Such ipsilateral brain injury included the temporal cerebral cortex, hippocampus and striatum, whereas the general structural integrity of the contralateral hemisphere was largely preserved. We thus prepared hippocampal slices from the contralateral hemisphere for extracellular recordings in vitro. CA1 field EPSPs were evoked by stimulating the Schaffer collateral pathway, and a theta burst protocol was used to induce LTP. We found that the CA1 LTP was retained in the contralateral CA1 neurons, although slightly decreased from the sham control mice. We show for the first time that the synaptic activity is preserved in the contralateral hippocampus following IH episodes. And it raises the question whether the hippocampus can unilaterally act in hippocampus dependent learning and memory.

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94

Is treatment of hypertension and hypercholesterolemia successful in patients with systemic lupus erythematosus?

Pek, EA., Gladman, DD., Ibañez, D. and Urowitz, MB Center for Prognosis Studies in the Rheumatic Diseases, Toronto Western Hospital

Background: Patients with SLE demonstrate accelerated atherosclerosis and are at an increased risk of coronary artery disease. In fact, the incidence of CAD has been shown to be approximately 10% in various SLE cohorts with women between the ages of 35-44 having a 50 times greater risk than the general population. The prevalence of classical CAD risk factors such as hypertension and hyperlipidemia is also higher in these patients. The importance of controlling these risk factors has gained increased recognition. Recent quality improvement studies demonstrated that increasing numbers of eligible patients are being treated with anti-hypertensive and lipid lowering medications. However, neither the short or long term efficacy of these treatments has been evaluated in this population. Objectives: 1) To determine whether treatment of hypertension or hypercholesterolemia can successfully achieve target blood pressure or serum cholesterol in patients with SLE 2) To determine whether successful treatment leads to a decreased incidence of CAD. Methods: Patients have been prospectively followed at the clinic since 1970. Clinical and laboratory information is collected on a standard protocol and stored in a database. Patients presenting within 1 year of SLE diagnosis and who received treatment with anti-hypertensive and/or lipid lowering medications were included. Success was defined as having met target BP (140/90) or serum cholesterol (TC < 5.2 or LDL < 3.2) during at least 90% of follow-up. Results: Preliminary analysis involved 66 patients treated for hypertension (86% female, mean age at treatment of 43.2 ±15.6, mean disease duration at treatment of 3.7±4.5 years) and 45 treated for hypercholesterolemia (78% female, mean age at treatment of 44.2±13.1, mean disease duration at treatment of 3.5±3.7 years). 20 (30.3%) and 18 (40%) of these met the criteria for successful treatment in the two groups respectively. Conclusion: Treatment of hypertension and hypercholesterolemia does not necessarily result in successful control of these risk factors in the majority of SLE patients. Further analysis will be important to discern the reasons for unsuccessful treatment and to compare CAD outcomes in patients who were successfully treated and those who were not.

95

In situ differentiation of transplanted neural stem cells into the injured spinal cord

Valerie Peng, Howard Kim, Charles H Tator, Molly S Shoichet Department of Surgery, TWH and Department of Chemical Engineering, CCBR Background: Spinal cord injury (SCI) results in the loss of the neuronal circuitry responsible for relaying signals between the brain and the rest of the body. The mammalian spinal cord has very limited capacity for endogenous repair, leaving most victims of SCI with permanent motor and sensory dysfunction. We are investigating the use of adult neural stem/progenitor cells (NSPCs) as a cell replacement therapy to induce regeneration. Purpose: We are interested in how differences in NSPC maturity stage affect transplant survival and differentiation. NSPC cell fate was controlled through exposure to dibutyryl cyclicAMP, a small molecule that directs NPSCs towards a neuronal phenotype. Methods: GFP-positive NSPCs were transplanted into adult rats at the time of spinal cord transection. Three treatment groups were investigated; undifferentiated NSPCs, undifferentiated NSPCs with dibutyryl cyclicAMP drug delivery (in situ differentiation) and pre-differentiated NSPCs (with dbcAMP treatment prior to transplantation). All groups were transplanted within a bioengineered implant consisting of a chitosan channel, polylactide-co-glycolide microspheres, and fibrin scaffold. At two and six week timepoints, the injured tissue was explanted and examined by immunohistochemistry for: cell survival (GFP) and markers of differentiation into the neurons (BIII, MAP2, NeuN), oligodendrocytes (CC1) and astrocytes (GFAP). Results: At two and six weeks, connective tissue bridges were formed between the two previously transected spinal cord ends. Cell survival was observed in all three treatment groups, with the highest survival associated with pre-differentiation. We will complete our analysis to provide insight into the most efficacious method for cell transplantation.

96

Loss of Regulator of G Protein Signalling 5 Possibly Accelerates Diabetic Kidney Injury

Zhongming (Joel) Qin, supervised by Dr. James Scholey, Clinical Science Division Diabetic nephropathy is a common cause of end-stage renal failure; however factors responsible for its development are not fully elucidated. Kidney injury is composed of both functional and structural changes. Renal hypertrophy and fibrosis are common structural changes for diabetic nephropathy. Regulator of G protein signalling 5 (RGS5), a potent deactivator of angiotensin II receptor AT-1, has been shown to protect heart from hypertrophy and fibrosis. The effect of RGS5 in kidney however is unclear. The purpose of my experiment was to investigate the role of RGS5 in diabetic nephropathy. RGS5-/- mice were crossed with Akita mice (Ins2WT/C96Y), a model of type 1 diabetes mellitus. The study was done on 3 groups of male mice at age of 8 week: Ins2WT/WTRGS5+/+, Ins2WT/C96YRGS5+/+ and Ins2WT/C96YRGS5+/-. Blood glucose and body weight were measured every week. 24-hour urine samples were collected using metabolic cages at end of the 8th week. Urinary albumin concentration was determined by ELISA. Both Ins2WT/C96YRGS5+/+ and Ins2WT/C96YRGS5+/- mice developed profound hyperglycemia. Preliminary data suggested that 24 hour urinary albumin excretion rate of Ins2WT/C96YRGS5+/- mice (68.38±8.65 mg/24 hour) is higher than Ins2WT/C96YRGS5+/+ mice (50.37±6.65 mg/24 hour). Hence, loss of RGS5 would possibly accelerate the development of diabetic nephropathy.

97

Testosterone-Guided Schedule of Androgen Deprivation Therapy as an Alternative to a Fixed Schedule in the Management of Prostate Cancer

Loheetha Ragupathi, Anna Dodd, Saroj Niraula, Ian Tannock Department of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto ON In Canada, prostate cancer is the most common cancer in men 1 . At diagnosis, 80-90% of prostate cancers are androgen dependent 2 . Androgen deprivation therapy (ADT) has thus been the standard initial management of men with advanced prostate cancer. This can be achieved either by surgical or medical castration; the latter requires treatment with a luteinizing hormone releasing hormone (LHRH) agonist and, at least initially, a peripheral anti-androgen to prevent flare. Typically, LHRH agonist injections are given at fixed intervals of 3 to 4 months, and the cost of a single dose in Canada is approximately $1250 3 . ADT, however, is associated with subtle toxicities, such as osteoporosis and metabolic consequences which can lead to diabetes or cardiovascular events. Here, we propose to evaluate a schedule of ADT that is not given at fixed intervals, but rather, is guided by serum testosterone levels. In this prospective cohort study, we withhold LHRH agonist injections in prostate cancer patients with castrate baseline testosterone levels (defined here as <1.75nmol/L), who have already received >12 months of ADT, and we monitor testosterone levels at 6 week intervals. We resume LHRH agonist therapy when testosterone levels exceed 1.75nmol/L. We will investigate the temporal relationship of testosterone levels, clinical factors influencing increase in testosterone levels, and we will conduct a cost-benefit analysis comparing the expense of our model and standard management of ADT. Participants will also complete a quality of life questionnaire and a simple test of their physical ability. Our hypothesis is that men will require less frequent injections of LHRH agonists to maintain castrate levels of testosterone, with savings in cost and possible improvements in quality of life. The sample size of this study is 100 men, and at present 21 have been enrolled.

Canadian Cancer Society. What is Prostate Cancer? December 10 2009. http://www.cancer.ca/Canadawide/About%20cancer/Types%20of%20cancer/What%20is%20prostate%20cancer.aspx 2 Heinlein CA, Chang C. Androgen Receptor in Prostate Cancer. Endocrine Reviews. 25(2): 276-308. 2004. 3 Bayoumi AM, Brown AD, Garber AM. Cost-Effectiveness of Androgen Suppression Therapies in Advanced Prostate Cancer. Journal of the National Cancer Institute. Vol. 92, No. 21, November 1, 2000.

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98

The role of Snail transcription factor during skin wound healing

Victoria Sattarova Dr. Benjamin Alman Developmental and Stem Cell Biology Sick Kids Hospital Background The Snail family of transcription factors plays an essential role during embryonic development and cancer metastasis. It has been observed to increase cell mobility and cell invasion by inducing epithelialmesenchymal transition-like mobilization. This is characteristic of the cellular events occurring during wound healing. Objective Therefore understanding the role of snail is of interest in studying skin wound healing. Hypothesis We hypothesize that loss of snail will affect cell number in both keratinocytes and fibroblasts resulting in greater wound healing in mice. Methods Wound healing was analyzed in three conditional snail1 knockout mouse lines that were generated using cre-loxp technology. Snail was knocked out either ubiquitously (using an adenovirus expressing cre recombinase), or specifically in mature keratinocytes and fibroblasts using keratin14 and FSP1 promoters respectively. Results Ubiquitous down-regulation of snail1 increases wound size, robustly altering wound healing. Downregulation of snail1 in fibroblasts, results in larger wounds with lower number of cells and a lower number of cells that stained for the proliferation marker KI67 in the dermis. Down-regulation of snail in keratinocytes, results in smaller wounds with a lower number of cells in the dermis and less KI67+ cells. Conclusion Snail is involved in the regulation of mesenchymal and epithelial cells population size in a wound healing model. This supports the hypothesis that Snail1 is an important regulator of cell motility as suggested by Rowe and colleagues.

99

Clinical outcome after traumatic spinal cord injury: creation of a database and presentation of clinical measures

Nick Schoenhoff, Dr. David Cadotte and Dr. Michael Fehlings, Division of Neurosurgery, Toronto Western Hospital Background Traumatic spinal cord injury has proven to be an immense and costly issue presented to modern society (Fehlings et al., 2001). Currently, progress is determined using outcome measures such as the Functional Independence Measure (FIM), the Spinal Cord Independence Measure (SCIM), the Walking Index for Spinal Cord Injury (WISCI) and the American Spinal Injury Association (ASIA) scale. The objective of this study is to evaluate the use of functional magnetic resonance imaging (fMRI) to gain more accurate and early projections of possible outcomes after a traumatic SCI. Purpose/Objective This work represents the first phase of a larger project whereby clinical measures will be correlated to spinal fMRI data. The specific objective of this work is to create a database by which standard clinical assessments can be entered and an automated output generated on an individual and group level. The database will generate output in two ways: raw numerical data and a graphical representation. Methods There are many approaches to creating a database, but for this study, Microsoft Access was determined to be the best tool, as it offers many options for the storage and presentation of data. Access 2010 was used to design the product due to its relatively easy-to-use platform. Results Currently, the database is still in production. 90% of the outcome measures have been accurately re-created in Access and report design has been initiated. Minor coding revisions are needed in order to reduce input error. Conclusions Although the database is not yet in use, the prospects of this design to generate an automated numerical and graphical output will satisfy the requirements to test the correlation of clinical measures with spinal fMRI readout. References Fehlings M.G., Sekhon L.H., (2001). Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine, (24) S2-12.

100

The Influence of the Word Cancer on Patient Treatment Preference

S Secord, J Takata, D Urbach Department of Surgery, Toronto General Hospital and Toronto Western Hospital, University of Toronto, Toronto, Ontario, Canada. Introduction: When the word "cancer" is used in disclosing diagnostic details, preconceived negative connotations may inappropriately influence patient treatment preference in favor of radical and unnecessary therapeutics. It has been well documented that the framing and terminology used by physicians has significant influence on how a patient perceives the threat of the illness. Research indicates that most patients with low-risk cancers will opt for aggressive therapeutics beyond conventional recommendation. Objective: The objective of this study is to examine the influence of the word cancer in patient therapeutic choice for indolent prostate cancer. Methods: Data will be obtained through interviewing forty subjects with two consecutive surveys based on hypothetical diagnoses with support of patient decision aids. The two-part survey compares treatment preference based on an initial hypothetical diagnosis of "prostatopathy" or "prostate cancer" as compared to subsequent hypothetical diagnosis of the alternate "prostate cancer" or "prostatopathy". Results/Conclusions: Results will inform the perception that the word cancer has unwarranted influence on patient decision-making. This information will advise physicians as to the terminology pertinent in diagnostic framing of low risk cancers.

101

TMS-induced Interhemispheric Signal Propagation and Five Factor Model of Personality

Basak Senel, Zafiris J. Daskalakis Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto BACKGROUND: Personality disorders such as psychopathy have been previously characterized by abnormal interhemispheric processing and callosal function. Five Factor Model (FFM) of personality is used to describe human personality and it involves the five broad domains of personality which are extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience. PURPOSE: In this study transcranial magnetic stimulation (TMS) combined with electroencephalograpy (EEG) was used to measure the propogation of TMS induced cortical evoked activity from the site of stimulation to the contralateral hemisphere. The aim of this study was to investigate the association between the NEOFive Factor Inventory (NEO-FFI) and the TMS-induced interhemispheric signal propogation (ISP) in the motor cortex and dorsolateral prefrontal cortex (DLPFC), a cortical region more closely related to the pathophysiology of psychiatric disorders. METHODS: The NEO-FFI was adminstered in ten righthanded healthy subjects (mean age= 37.8±7.8, range= 27-48years; 4 males, 6 females) in whom TMS was applied to the left motor cortex and left DLPFC. Pearson correlation coefficients were used to assess the relationship between TMS-induced ISP and inventory results. RESULTS: There was a significant positive correlation between agreeableness and ISP from the left to the right DLPFC (p=0.007). However, no correlation was found between agreeableness and ISP in the motor cortex (p=0.79). Moreover, no correlation was found between other personality domains and ISP in the DLPFC or motor cortex. CONCLUSION: These findings provide preliminary evidence to suggest that the cross talk between left and right DLPFC, but not motor cortex, may underlie the personality domain agreeableness in righthanded healthy individuals.

102

Detection of microRNAs in sera of oropharyngeal cancer patients

Kimberly Shek, Dr. Angela Hui, Dr. Fei-Fei Liu Division of Applied Molecular Oncology, Ontario Cancer Institute Introduction: MicroRNAs (miRs) are a class of endogenous, non-coding RNA molecules that negatively regulate gene expression through post-transcriptional silencing. Our previous study identified several aberrantly expressed microRNAs in 87 formalin-fixed, paraffin-embedded (FFPE) tumour samples of oropharyngeal cancer (OPC) patients. Hypothesis: Expression levels of microRNAs can be measured in the sera of oropharyngeal cancer patients and reflect expression levels in the corresponding FFPE tumour samples. Methods: A preliminary investigation was conducted using serum samples from 18 patients with oropharyngeal cancer and 10 patients for normal controls. RNA was extracted from the sera and reverse transcribed. Quantitative RT-PCR was performed to determine expression levels of ten microRNAs that were over-expressed in our previous study of OPC microRNA profiling. Results: Preliminary data show that significantly higher expression of three of the ten microRNAs (miR21, miR146, and miR451) was detected in OPC patient sera, compared to that of normal controls. These microRNAs have been previously reported to play a role in cancer development. Conclusions: We were able to extract and detect microRNAs from serum samples and identified three candidate OPC-related microRNAs. This is the first study to compare microRNA levels in tumours with serum samples from the same patient. We are currently investigating the expression of these three miRs in the tissue and sera samples of an additional 40 OPC patients ranging from stages II to IV. These results will be used to compare miR expression levels in sera to expression levels in their corresponding tumor samples, as well as to patient stages and prognosis.

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Examination of Steroid Dose and Steroid-related Damage in an International Inception Cohort of Systemic Lupus Erythematosus Patients

Hang Shi, Nicole Anderson, Dominique Ibañez, Dafna Gladman, Murray Urowitz Toronto Western Hospital, University Health Network, Toronto, ON, Canada

Introduction: Corticosteroids (CS) remain the mainstay in the treatment of Systemic Lupus Erythematosus (SLE). Significant morbidity and irreversible damage to multiple organ systems are known to be associated with its long term use, but the factors that confer susceptibility to permanent damage in this patient population have not been well elucidated. Aim: To determine the effect of dose dependent factors in the accrual of steroid-related damage in SLE patients. Methods: Patients with SLE from the multicentre SLICC-RAS inception cohort were followed prospectively for 5 years. Annual data collection includes clinical and laboratory features of SLE, past and current therapy, and the SLICC/ACR damage index (SDI). The occurrence of permanent damage, as measured by the SDI, was determined and categorized as definitely related, possibly related, or independent of steroids. Steroid courses over this period were captured and used to calculate accumulated dose and average daily dose. Multivariable stepwise logistic regression and Mantel-Haenszel chi-square tests will be performed to identify significant associations between dose dependent variables and steroidrelated permanent damage. Results: Of the 342 patients in our preliminary analysis (295 female, mean(±std) age at enrolment of 35±13 years), the median cumulative steroid dose increased from 0.49g to 13.2g from enrolment to 5 years follow-up. During the same period, the mean damage (SDI) increased from 0.15±0.50 to 0.97±1.33, of which 0.24±0.60 (25%) was definitely related to steroids. Steroid-related damage was associated with cumulative dose and average daily dose as follows:

Cumulative Dose SDI SDI due to CS 0.67 ± 0.99 0.14 ± 0.47 1.08 ± 1.36 0.24 ± 0.57 1.37 ± 1.70 0.42 ± 0.84 1.39 ± 1.43 0.10 ± 0.30 1.77 ± 1.74 1.00 ± 1.15 <0.0001 0.0001 Average Daily Dose SDI SDI due to CS 0.10 ± 0.32 0±0 1.07 ± 1.41 0.31 ± 0.72 1.10 ± 1.39 0.25 ± 0.59 1.35 ± 1.42 0.22 ± 0.48 1.64 ± 1.75 0.54 ± 0.96 <0.0001 0.004

Dose (g) <10 10-20 20-30 30-40 40+ P value

Dose (mg) 1-5 5-10 10-15 15-20 20+ P value

Conclusion: SLE patients receiving corticosteroid therapy accrue damage over time. Steroid-related damage increases with higher cumulative dose and average daily dose. This suggests that both duration if sufficiently long, and dose if sufficiently large are associated with accumulation of more damage.

104

Characterization of the interaction between the Cbl ubiquitin ligase and the lymphoid tyrosine phosphatase (LYP)

Adrienne Siu, Helen Miliotis, Katherine A. Siminovitch Samuel Lunenfeld Research Institute, Mount Sinai Hospital Introduction PTPN22, also known as lymphoid tyrosine phosphatase (LYP), is a protein implicated in T-cell receptor signaling. Notably, a mutation on amino acid residue 620 from arginine (R) to tryptophan (W) on Lyp is associated with several autoimmune diseases, such as type 1 diabetes and rheumatoid arthritis. Among wildtype LYP and LYP RW's interactors is c-Cbl (Cbl), an E3 ubiquitin ligase important in T-cell receptor signaling. It is known that a mutation from tyrosine (Y) to phenylalanine (F) on amino acid residue 371 abolishes Cbl's E3 ligase activity. Thus, Cbl YF can be used alongside the wildtype in determining Cbl's effect on LYP. . Purpose Our objective is to determine how LYP interacts with Cbl, and whether Cbl ubiquitinates LYP. Furthermore, we will investigate how this interaction is affected by the LYP RW and Cbl YF mutations. Methods Plasmid DNA combinations of LYP, Cbl, and their variants were transfected into COS7 cells and harvested. Specifically, a Cbl construct consisting of only an N-terminus was used, in order to determine sufficiency for binding and/or ubiquitination of LYP. The lysates were immunoprecipitated and immunoblotted with indicative antibodies. Results/Conclusion The data suggests that the N-terminus of Cbl may be sufficient for binding with LYP. Moreover, there is ubiquitin associated with LYP, in the presence of Cbl. Further experiments are needed to elucidate this interaction, and how it differs between the wildtype and variant proteins. Understanding the LYP-Cbl complex will be helpful in delineating their roles in cell signaling.

105

Polypathology and its impact on supportive care needs: Cancer patient perspectives on existing self-report questionnaires

Maggie Siu, Dr. Jennifer Jones1, Dr. Pamela Catton2, Dr. Alex Jadad3 1 Psychosocial Oncology and Palliative Care, Princess Margaret Hospital 2 Collaborative Centre for Health, Wellness and Cancer Survivorship, Princess Margaret Hospital 3 Centre for Global eHealth Innovation, University Health Network Background: With the aging population, clinicians will be treating a greater proportion of patients presenting with pre-existing co-morbid conditions (polypathology). To date there is very little information describing both the nature and severity of polypathology in persons with cancer, and even less exploring its burden on outcomes such as supportive care needs. Methods: In Phase I, a literature review was conducted to identify and evaluate existing self-report measures. Based mainly on documented validity and reliability, patient involvement during development, and comprehensiveness, the Supportive Care Needs Survey and the Self-Administered Co-morbidity Questionnaire were selected and modified for use in the study. As part of Phase II, the questionnaire package is being administered to patients in four cancer sites (breast, gynecological, gastrointestinal and prostate) at the Princess Margaret Hospital. Participants are then asked to provide feedback on the questionnaires' completeness and design. Descriptive statistics, group comparisons, and correlations will be conducted. Thematic analysis will also be used to analyze the feedback. Results: To date n=25 have completed the study. Interim quantitative and qualitative results will be presented. Significance: Cancer patients with co-morbidities are part of a growing, but understudied population with complex and varying needs. Results from this study will contribute to the development of a validated measure to assess supportive care needs in cancer patients with polypathology and will provide preliminary data on their unique needs.

106

Cognitive Factors Associated with Belief Formation in the General Population

Helen Moriah Sokolowski, Albert Wong, Schizophrenia, CAMH Delusions are fixed beliefs, usually seen in individuals suffering from psychotic disorders, held by individuals despite concrete proof to the contrary. These patients show cognitive biases, thought to be associated with delusion formation. A number of social factors, personality variables and cognitive factors are hypothesized to be involved in the willingness to hold strong beliefs. In the current study, we were interested in examining the relationship between cognitive and personality variables and the willingness to form and hold socially accepted and adaptive beliefs. Specifically, we compare three groups of psychiatrically healthy individuals - one group with strong religious beliefs, which represent strong beliefs that are culturally accepted; a group who believe in natural energy based alternative medicine, which represent strong beliefs held only by a small proportion of the population, and a group who believed in neither. All subjects participated in the following measures: strength of religious beliefs, energy healing practices, beliefs in various `paranormal' phenomena, a measure of personality, a measure of delusion proneness, and three tasks associated with cognitive biases seen in patients with delusions. We use these measures to examine relationships between personality and cognitive variables and the willingness to hold strong beliefs in various paranormal phenomena, energy based medicine and religious beliefs. We also quantitatively examined the relationship between group membership and the cognitive biases. Understanding factors involved in the formation of strong beliefs in healthy individuals could be an important route to understanding delusion formation, and in helping to differentiate between adaptive and maladaptive beliefs.

107

Prevalence of co-existing medical conditions in patients with congenital heart defects

Priya Somascanthan, Seema Mital MD. Department of Cardiology. 4514 Hill Wing, the Hospital for Sick Children. Background: Clinical outcomes and survival in patients with congenital heart defects (CHD) may be influenced by the presence of co-existing medical conditions. The frequency of co-existing medical conditions in CHD patients is not known. Objective: To assess the frequency of non-cardiac medical conditions in paediatric patients with CHD. Methods: Medical records were reviewed in 1463 patients <18 yrs with CHD enrolled in the Heart Centre Biobank Registry from 2008-2010. Common medical conditions were coded using ICD-10 coding. Results: Mean age of patients was 8.8 years. 57.8% were male. Overall frequency of non-cardiac medical conditions in CHD patients was 21.5%, with highest frequency in patients with conotruncal defects (CTDs) (6%), LV outflow tract obstruction (LVOTO) (4%) and left-right (L-R) shunt lesions (6.1%). The commonest medical conditions included mental and behavioural disorders, 5.9%, diseases of the respiratory system, 5.6%, - asthma, 5.3%, short stature, 1.2%, epilepsy, unspecified, 1.2%, and autism, 0.8%. 29.8% patients had undergone genetic screening with a positive genetic diagnosis in 54.3%. This was higher than patients with isolated CHD. Conclusion: Additional systemic disorders occur in over 20% of patients with CHD. These patients should undergo genetic screening to identify a possible genetic etiology. The impact of co-existing medical conditions on overall survival, clinical outcomes and functional status of CHD patients requires further assessment.

108

Assessment of Neural Precursor Cell-Oligodendrocyte Interactions Following Neural Precursor Cell Transplantation In A Rodent Model Of Subacute Spinal Cord Injury

Spano S 1,2, Hawryluk GWJ 1,2,3, Fehlings MG 1,2,3,4.

1

Division of Genetics and Development, Toronto Western Research Institute, University Health Network. 2 Institute of Medical Science, University of Toronto. 3 Division of Neurosurgery, University of Toronto. 4 Krembil Chair, Neural Repair and Regeneration, Head, Spinal Program, University Health Network.

Introduction: Following spinal cord injury, axons survive and cross the injury site but are dysfunctional due to oligodendrocyte loss and demyelination. Previous data by our laboratory demonstrate that neural precursor cell (NPC) transplantation leads to functional recovery and remyelination in a subacute rodent thoracic SCI model. Yet other studies indicate that functional recovery can occur in absence of exogenous remyelination, suggesting a neuroprotective effect. It is unclear whether oligodendrocyte sparing and/or regeneration across the injury site contribute to such recovery. Oligodendrocytes are more susceptible to injury than neurons, and thus are a particularly sensitive assay of neuroprotection. Purpose: Use stereological techniques to discern the nature of the NPC-oligodendrocyte interaction, thus characterizing neuroprotective and/or cell replacement effects of NPCs on host tissue 6w post-transplant. Hypothesis: NPC transplants preserve endogenous oligodendrocytes following SCI in addition to replacing those which have been lost. Methods: Adult female rats received a T7 clip compression injury. Two weeks later, eYFP-NPCs were transplanted rostral and caudal to the injury epicenter. Rats were sacrificed 6w later and tissue was immunologically stained for oligodendrocytes (anti-APC). Using StereoInvestigator, NPCs and oligodendrocytes were then quantified at the epicenter and at several bilateral "mirror" sites equidistant to the epicenter. Results: Preliminary data indicate evidence of oligodendrocyte sparing 6w following NPC transplantation. NPCs differentiate into oligodendrocytes 1w post-transplant independent of distance from. Furthermore, there is a strong correlation between oligodendrocyte counts and the pattern of white matter seen in H&E/LFB stains. Approximately half of the NPCs in animals lacking trophin infusion pumps differentiated into oligodendrocytes­ nearly double the amount seen with infusions. However, animals lacking infusions contained fewer total oligodendrocytes on average than other experimental groups. Conclusion: Following an assessment of the progress of the different experimental groups, it seems likely that tissue sparing at 6w is a consequence of pharmacotherapy and/or trophin infusion, and not of transplanted NPCs.

109

Physicians' Awareness Of Cytoreductive Surgery And Intraperitoneal Chemotherapy For Colorectal Carcinomatosis

G Spiegle, S Schmocker, H Huang, C Victor, C Law, ED Kennedy, JA McCart Department of Surgery, Toronto General Hospital, Sunnybrook Health Science Centre and Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. Introduction: Recent studies have shown that cytoreductive surgery and heated intraperitoneal chemotherapy (HIPEC) for colorectal cancer (CRC) carcinomatosis leads to 5-year survival rates of over 30%. In 2007, an international consensus statement recommended HIPEC as the treatment of choice for selected patients with CRC carcinomatosis. Objectives: Since these data represent a significant shift from the traditional treatment paradigm, the objectives of this study were to determine: (1) physicians'awareness of HIPEC and (2) physician characteristics predictive of awareness of HIPEC for the treatment of CRC carcinomatosis. Methods: This study was a mailed, cross-sectional survey of general surgeons (GS) and medical oncologists (MO) in Ontario. The survey consisted of 17 items evaluated on a categorical scale. Results: The overall response rate was 40.5% (257/635). The majority of the respondents were male, <50 years and in practice <10 years. There was a fairly even split between respondents working at academic and community hospitals. Overall, 78% of the respondents reported consultations with <5 of these patients/year and overall only 41% of the respondents would consider HIPEC as a treatment option for CRC carcinomatosis. GSs tended to be more likely than MOs to consider HIPEC as a treatment option (43% vs 34%, p=NS). Multivariate analysis did not show any physician factors to be predictive of awareness of HIPEC. Conclusions: Physician awareness of HIPEC for the treatment of CRC carcinomatosis in Ontario is low. This has considerable implications for healthcare delivery in Ontario as many eligible patients may not be referred for HIPEC and has significant consequences with respect to patient outcome. Therefore, strategies to improve physician knowledge about HIPEC are important to ensure appropriate and timely treatment for patients with CRC carcinomatosis in Ontario.

110

A systematic review assessing alcohol abuse interventions for a post-traumatic brain injury population

Jyotika Sroa, Michael Cusimano, Injury Prevention Research Office a Division of Neurosurgery, St. Michael's Hospital Background: Alcohol is involved in about 50% of all head injuries and the occurrence of alcoholism after traumatic brain injury is a well known problem. Interventions have been designed and implemented to address this issue. Objective: The objective of this systematic review is to assess the effectiveness of various interventions for addressing and overcoming alcohol abuse in a population with traumatic brain injury in order to determine which programs are most successful in eradicating post-TBI alcohol abuse and associated adverse effects such as a secondary TBI. Methods: The review included peer-reviewed articles retrieved from Medline, Embase, PsycINFO and Scholar's Portal. Hand searching journals and references of articles was also done. Predetermined exclusion and inclusion criteria and the Downs and Black's Checklist (DBC) were used to assess the quality and provide a construct through which the studies could be compared. Results: Motivational interview as an intervention showed a significant decrease in alcohol abuse among the intervention group participants and the quality of these studies assessed with DBC show that they are both well conducted. The other interventions did not perform as well as they either did not produce significant decrease in alcohol abuse or their studies were poorly conducted as determined by DBC. Conclusions: Overall, more studies are required for each intervention to truly make any judgments, but from the available literature, motivational interviews seem to be the most effective at reducing the occurrence of alcohol in a post-TBI population. This systematic review is still being written, further analysis is pending.

111

Quantum Dot in vivo Imaging Shows Lymphatic Drainage from the Mouse Eye

Alex Tam, Yeni Yücel, Zhexue Zhang, Neeru Gupta. Ophthalmology & Vision Sciences and Laboratory Medicine and Pathobiology, University of Toronto, Keenan Research Center at the Li Ka Shing Knowledge Institute of St. Michael's Hospital Background: Glaucoma is a leading cause of world blindness estimated to affect 70 million people. Impaired fluid drainage from the eye leads to high pressure in the eye, a leading risk factor for glaucoma. We recently reported the discovery of lymphatics in the human eye and predict that this circulation is present in mice and that the "uveolymphatic" outflow pathway is partially responsible for fluid drainage from the eye. Purpose: To determine whether lymphatics play a role in aqueous outflow from the mouse eye by Quantum dots and in vivo imaging. Methods: Quantum Dots 655 were injected into the anterior chamber of the left eye of mice under general anesthesia (n=7). Hyperspectral fluorescence in vivo molecular imaging was performed 30 minutes, 1, 3 and 6 hours after injection to look for fluorescent nanoparticle tracer. Following sacrifice, high signal regions in the neck were dissected and stained for nuclear stain (Syt Green) and immunostained for basement membrane (collagen IV antibody). Results: In all 7 mice, Quantum dot in vivo imaging showed reduced intensity in the left eye, and increased intensity concentrated in the submandibular neck region 6 hours after injection. Examination of neck region sections counterstained for nuclear and basement membrane stains confirmed the presence of Quantum dot tracer in the left submandibular lymph node. Conclusion: This is the first report of a lymphatic pathway in the mouse eye and demonstration of its role in aqueous drainage. Further studies of this pathway following pharmacological manipulation may stimulate novel glaucoma treatments. Acknowledgment: This work was supported by Glaucoma Research Society of Glaucoma.

112

A novel in vitro method of generating murine dendritic cells using supernatants containing flt3-ligand--supplemented bone marrow cultures

Michael Tang, Jun Diao, Mark Cattral Multiorgan Transplant Program, Toronto General Research Institute, University Health Network Dendritic cells (DCs) are professional antigen presenting cells that play a crucial role in the initiation and maintenance of immune responses. Traditionally, the generation of DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) or flt3-ligand (Flt3L) as principle growth factors. The use of GM-CSF in BM precursor culture results in DCs which exhibit an inflammatory state phenotype, while the use of Flt3L produces a diverse population of DCs, which resembles to steady-status DCs. However, a large quantity of recombinant Flt3L is needed to generate a greater population of DCs that are useful in our studies. Here we describe a novel BM culture system using fibroblast culture supernatant containing secreted Flt3L (FCS-Flt3L). S17 cells, a bone marrow-derived fibroblast cell line, were transduced with a retroviral vector encoding extracellular domain of the human Flt3L gene. Levels of human Flt3L in FCS-Flt3L were quantified by enzyme-linked immunosorbent assay (ELISA). Murine BM cells were isolated and cultured in 20% of FCS-Flt3L for 7 days. Using this method, we were able to generate various types of DCs including DC precursors (CD11c+, MHC II-, PDCA-1+; CD11c+, MHC II-, PDCA-1-). A mixed lymphocyte reaction was employed to confirm the antigen presenting ability of these newly generated DCs from BM culture. As compared to the conventional method of DC generation, a greater yield of DC precursors and DCs were generated by this novel culture method. This novel culture system will be useful in facilitating our understanding of DC development in transplantation research and tumour biology.

113

PM induces co-localization of Syk and dectin-1 in airway epithelial cells (AEC)

Ye Tian Chung-Wai Chow Department of Medicine 1 King's College Circle, Room 6270 Background: Syk, an immunoregulatory protein originally described to be downstream of immunoreceptors such as the Fc and Fc receptors, is increasingly recognized to be bind to diverse receptors, including dectin-1, a pattern recognition receptor which recognizes -glucan of fungi. Our laboratory has identified Syk in the AEC and observed its activation by the particulate matter (PM) of environmental air pollution. The upstream receptor is not known. As PM is composed of inorganic and organic material (including fungus), we hypothesize that dectin-1 may be a receptor that leads to Syk recruitment and activation in AEC following PM exposure. Method: BEAS-2B human AEC were cultured on coverslips and incubated with PM (1.0 mg/ml; SRM

1649b) for up to 60 minutes. The cells were fixed and immunostained with appropriate primary and secondary antibodies for immunofluorescence. Images were obtained using a scanning confocal microscope (60x objective; Zeiss LSM510) and presented as tiff files. Results/Conclusions: Under basal conditions, Syk (red) was diffusely distributed in the cell whereas dectin-1 (green) is localized to the cell membrane and perinuclear punctuate structures resembling endosomes. Little co-localization (yellow; merged images) is observed. Following 15 min exposure to PM, Syk is recruited to the membrane. At 30 and 60 min, significant co-localization of dectin and Syk is observation, primarily in the punctuate perinuclear structures (yellow). To date, our data indicate that PM induces recruitment of Syk and its colocalization with dectin-1 in BEAS-2B cells. We are currently characterizing the nature of the interaction and its functional consequences.

114

Collagen

Katrina Ting, M. Cynthia Goh Department of Chemistry, University of Toronto, Toronto, Ontario, Canada Introduction-- Collagen is the most abundant structural protein in animal connective tissues. It exists in a variety of morphological forms. The most well-studied is the native fibril, which is characterized by a banding pattern with a ~67 nm period. Fibrils that are similar to the native fibril can be produced in vitro, and altering the conditions of assembly process can lead to the formation of different products. Such as fibrous long spacing collagen (FLS) and segment long spacing collagen (SLS). Purpose-- FLS collagen is characterized by a periodicity in the banding pattern larger than that in native collagen, typically ~200-300 nm. SLS is an abnormal packing pattern of collagen molecules formed by adding ATP to acidic collagen solutions, becoming a crystal-like structure. By comparing the ultrastructure of different collagen fibrils can provide insight into the different functions they might have. Methods-- Atomic force microscopy (AFM) is used to observe the structures of different kinds of collagens. AFM is a technique by which surface topography can be measured. It can provide insight into the ultrastructure of collagen. Results-- Altering the conditions of assembly substrates can lead to the formation of different products, and AFM topographs showed structural details of different types of collagen. Conclusion-- Different types of collagen have different ultrastructures and assembly process, and they have great potentials to be used in various biotechnological and medical applications.

115

Does delayed AdV-ZFP-VEGF administration following traumatic spinal cord injury reduce neural cell death?

Yun-Fan Tseng, Sarah Figley1 and Dr. Michael Fehlings1,2

1 2

Institute of Medical Sciences, University of Toronto

Department of Genetics and Development, Toronto Western Research Institute, University Health Network Introduction: Spinal cord injury (SCI) is a major cause of human disability and death. Secondary injury such as ischemia, inflammation and apoptosis can exacerbate damage and contribute to overall morbidity and mortality. Vascular endothelial growth factor (VEGF), known to be the primary regulator of angiogenesis and vasculogenesis, exhibits neurotrophic, neuroprotective and neuroproliferative effects. In our previous research, increasing the endogenous VEGF-A mRNA and protein levels using AdV-ZFPVEGF administration immediately after SCI resulted in a significant decrease in apoptosis, increase in angiogenesis and neuroprotection. Aim: The aim of this study is to investigate if the neuroprotective effects of AdV-ZFP-VEGF are retained when administration is delayed 24 hours. Hypothesis: We hypothesize that 24 hours post-SCI administration of AdV-ZFP-VEGF will result in a decrease in apoptosis and preservation of neurons. Methods: Adult female Wistar rats were randomly divided into four treatment groups: 1) Shamlaminectomy, 2) Injury 3) Injury + AdV-eGFP and 4) Injury + AdV-ZEP-VEGF. All animals were given daily subcutaneous injections of 10 mg/ kg of cyclosporine-A starting 24 hours prior to SCI until the animals were sacrificed 5 days post-injury. Animals received a 1 minute, 35g clip-compression injury at T6-T7 and were injected with AdV-eGFP or AdV-ZFP-VEGF at 24 hours post-SCI. Immunohistochemistry was performed to examine apoptotic cell death (caspase-3) and neuronal cell counts (NeuN). Anticipated Results: We propose that 24 hours delayed administration of AdV-ZFP-VEGF will significantly reduce programmed cell death at 5 days after SCI compared to control animals and that neurons will be preserved as a result of AdV-ZFP-VEGF administration.

116

Proposed Derivation Of Pure Populations Of Human Smooth Muscle Cells Using Transgenic Human Embryonic Stem Cells

Wesam Tulba, Omar El-Mounayri, Sarah Steinbach, Mansoor Husain McEwen Center for Regenerative Medicine, 101 College Street, Mars/TMDT, Toronto Ontario Background: Cellular heterogeneity intrinsic to existing differentiation systems limit our ability to derive specific and homogenous population of smooth muscle cells (SMCs) from embryonic stem cells (ESCs). Transgenic human ESCs enabling the selection of specific human cell types would represent a powerful tool for regenerative medicine. Objective: To construct a targeting vector enabling the selection of hSMCs differentiated from transgenic hESCs. Approach: To generate hESCs carrying a fluorescent reporter under the control of a smooth musclespecific promoter targeted to the HPRT locus. Method: We performed gene targeting to introduce a linearized 1.75kb target vector with a LoxPdocking site flanked by a neomycin expression cassette (Sakurai K et al, Nucleic Acid Research 2010) into the HPRT locus of hESC line, hES-H1. To screen for targeted clones, we first designed and generated a control vector to optimize PCR conditions. A 1.2kb fragment of genomic hES-H1 HPRT corresponding to the sequence 3' of the 3' homologous arm was PCR-amplified from genomic DNA using primers with AscI restriction sites. This product was introduced into the cloning vector pGEM TEasy and shuttled to the target vector. Restriction enzyme analysis and sequencing is underway to confirm integration, directionality and exclusion of concatamers. Conclusion: A reporter system regulated by a smooth muscle-specific promoter targeted to the HPRT locus in hESCs has been designed and a strategy for selection of targeted clones is in hand.

117

Effect of Polo-like kinase 4 (PLK4) on progression of colorectal cancer (CRC)

Matthew Vincent, Dr, Carol Swallow

PLK4 is a member of the polo family of cell cycle kinases and is required for centriole duplication and mitotic progression. Up- or down- regulation of PLK4 have been described in several cancers; in particular, PLK4 expression is increased modestly in primary CRC, and markedly in liver metastases. Recently, our lab has found evidence that PLK4 regulates cellular motility (Rosario et al., unpublished). The purpose of the present study is to determine the effect of PLK4 on the ability of colorectal cancer cells to metastasize. First, we demonstrated that DLD-1 CRC cells transiently transfected with PLK4 migrate at a greater rate than cells transfected with Flag alone. To study the effect of PLK4 in vivo, we stably transfected DLD-1 cells with tetracycline-inducible PLK4, and confirmed expression at RNA and protein levels. We will inject stably transfected cells into the tail vein, portal vein or spleen of 6 week old (use official genotype) mice as a model for the metastatic cascade. Mice will be sacrificed 7-10 days following injection, and liver metastases will be identified by H&E staining and immunohistochemistry. PLK4 is involved in actin cytoskeletal rearrangements as well as MMP production, both of which are important in cell motility. We expect that increased PLK4 will enhance formation of liver metastases.

118

Biological effects of intracellular pH on function of a mutant p53 protein specific to adrenocortical carcinoma

Kyra Vitko Supervisor: David Malkin, Jonathan Wasserman Genetics and Genomic Biology, Toronto Medical Discovery Tower (TMDT)

The TP53gene encodes the p53 tumor suppressor which is vital for the induction of cell cycle arrest, apoptosis, senescence, or DNA repair in response to cellular stress. Human p53 is comprised of four domains with the majority of cancer-associated mutations found within the DNA binding domain. A unique germ-line mutation, R337H, observed in children with adrenocortical carcinoma, is located within the tetramerization domain. Tetramerization is required for tumor suppressive activity. In contrast to wildtype TP53, the stability of the tetrameric form of the R337H mutant has been shown to be pH-sensitive in vitro. This study examined the effects a rise in intracellular pH has on the function of R337H compared to wildtype p53. p53-deficient H1299 lung carcinoma cells were transfected with R337H or wild-type p53 constructs. MTT and luciferase gene reporter assays were conducted to determine whether a slight alkalization in intracellular pH promotes changes in cell viability and p53 transcriptional activity, respectively. Intracellular pH was controlled by pre-calibrated 5% and 2% CO2 atmosphere incubators. Wild-type p53 function was noted in cells expressing R337H, when maintained at 5% CO2 (pHi=7.4). By shifting to 2% CO2 , intracellular pH increased which resulted in a decrease in R337H activity while wildtype activity remained unchanged. These data indicates that R337H-p53 is pH-dependent and exhibits dysfunction with alkalization of pH within the physiological range. This may, in part, explain the unique permissive nature of this mutant in the hormonally regulated adrenal cortex.

119

Genetic association study of polymorphisms in serotonin receptor 2C, leptin and leptin receptor genes with antipsychotic induced weight gain

Tessa JM Wallace1, Arun K. Tiwari1, Olga Likhodi1, Ruth Ohlsen2, Kathy J. Aitchison2, James L. Kennedy1, 3, Daniel J. Müller1,3 1. Pharmacogenetics Research Clinic, Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, ON, Canada 2. MRC SGDP Centre, Institute of Psychiatry at King's College London, UK 3. Department of Psychiatry, University of Toronto, Toronto, ON, Canada Introduction: Antipsychotic induced weight gain (AIWG) is a side effect of antipsychotic treatment that leads to increased morbidity and mortality. The serotonin receptor 2C (HTR2C) knock out mouse develops hyperphagia, resulting in obesity. Leptin is a hormone secreted by adipose tissue that suppresses food intake and promotes energy expenditure. The single nucleotide polymorphisms (SNPs) -697C/T and -759A/G of the HTR2C promoter, -2548A/G of the leptin gene and Q223R of the leptin receptor have been associated with AIWG in previous studies. Purpose: To confirm the association of these SNPs with AIWG in a patient sample from London, IOP. Methods: DNA was extracted from blood collected from 93 patients undergoing antipsychotic treatment. Patients were recruited from The South London and Maudsley NHS Foundation Trust and associated clinics in London, England. Genotyping was performed using TaqMan SNP genotyping assays. Patients were classified as cases if they gained 5 pounds since starting antipsychotic treatment and controls if they gained <5 pounds. Results: The leptin receptor polymorphism Q223R was found to be associated with AIWG in the entire sample (p = 0.031) and in the subgroup of African ancestry (p = 0.042). A trend of allelic, but not genotypic, association was observed for the leptin gene SNP -2548A/G in patients of European ancestry on clozapine or olanzapine (p = 0.078). Conclusions: Our results show a marginal association between the leptin receptor Q223R polymorphism and AIWG as well as an interesting trend for the leptin gene. Our results do not support an association between AIWG and the HTR2C gene.

120

Within-patient variability of tacrolimus levels as a risk factor for transplant glomerulopathy in kidney transplant patients

Yao Wang, Dr. Edward Cole Background/rationale: Transplant glomerulopathy (TG) is a form of chronic rejection of a kidney transplant. Although the incidence of TG is fairly low (affecting 4% of patients within 1-year posttransplant, 20% at 5 years post-transplant), it is a particularly significant pathogenic condition for kidney transplant patients, because it is associated with anti-HLA antibodies and very poor long-term graft survival. Currently, the effectiveness of immunosuppressive therapy is highly dependent upon stable calcineurin inhibitors (CNI) exposure within a narrow therapeutic window. High within-patient variability of blood CNI levels could lead to drug levels being frequently above the therapeutic window, causing nephrotoxicity, or below the therapeutic window, increasing the risk for acute/chronic rejection. Objective: This study aims to examine within patient CNI variability as a risk factor for the development of biopsy-proven TG in adult kidney transplant patients. Study Design/ Methodology: This study is an observational, retrospective, nested case-control study using chart reviews of 130 patients who received kidney transplants at the Toronto General Hospital and were using tacrolimus. This included 26 patients with TG, and 114 controls (1:4 control-to-case ratio) matched on follow-up time via risk-set sampling. Variability of tacrolimus trough levels at a given dose will be measured by the number of standard deviations for all recorded measurements for an individual over a six month window. Other variables such as demographic variables, type of donor (deceased vs. living), and length of dialysis prior to transplant will be analyzed to ensure the TG and control groups are comparable in other respects. Expected outcomes: Since the study is still in progress, the results are pending, but we expect the patients with TG to have significantly greater standard deviation compared to the control group. This may provide transplant physicians a marker of heightened risk for TG and an opportunity to intervene (e.g., drug dose adjustment or assessment of adherence) as a means to reduce this risk.

121

The RNA binding protein UNC-75 regulates alternative splicing in the C. elegans nervous systems

Chris Wedeles, John Calarco, Arun Ramani, Mei Zhen Samuel Lunenfeld Research Institute Alternative splicing is a critical gene regulatory process where multiple transcript variants can be generated from a single precursor mRNA transcript. Genome-wide analyses of alternative splicing have suggested that splice variants are often regulated in a tissue-specific manner. Moreover, organ systems with many diverse cell types such as the central nervous system undergo frequent alternative splicing. However, despite the prevalence of alternative splicing in the nervous system, the complex mechanisms that regulate this process remain poorly understood. It is also unclear how networks of regulated splicing events contribute to nervous system development and function. It is therefore of great interest to understand in greater detail how neural RNA binding proteins influence splicing decisions by interacting with their target transcripts. We are currently studying the functional role of UNC-75, a C. elegans nervous system-specific splicing factor. Loss of unc-75 leads to synaptic transmission defects, suggesting that it regulates one or many alternative splicing events in genes important for proper neuronal function. In order to identify target transcripts regulated by this factor, we have analyzed the transcriptomes of mutant worms that do not express UNC-75 by high-throughput sequencing of mRNA (RNA-Seq). We have identified a number of alternative splicing events dependent on functional UNC-75, and validated these experimentally by RT-PCR assays. To complement these data and to investigate which of these mRNA targets are directly bound by UNC-75 in vivo, we are currently performing UV Cross-linking and Immunoprecipitation experiments. Further investigation of the effects of altered splicing of these target transcripts on proper neurotransmission are currently underway. Taken together, these experiments will further characterize how UNC-75 regulates alternative splicing, and continue to identify other key factors involved in nervous system development and function.

122

Strategies to Reduce Patient Discomfort and Anxiety during Non-Invasive Radiotherapy and Diagnostic Radiology Procedures ­ A Systematic Review

Kathleen Wheeler Supervisor: Rebecca K. S. Wong, MB ChB, MSc, Radiation Medicine Program, Princess Margaret Hospital Background: Pain or anxiety during diagnostic and therapeutic radiologic procedures can lead to a reduction in efficiency, accuracy and quality. Purpose: To examine the effect of interventions (pharmacological and complimentary alternative medicine (CAM)) on pain and anxiety during non-invasive diagnostic radiology and radiotherapy procedures. Methods: A systematic review strategy was used to search Medline (1950 to Jun 2010) and EMBASE (1980 to Jun 2010). The search strategy employed MeSH headings and keywords for anxiety, pain, radiotherapy (RT) and non-invasive diagnostic radiology (DR). Randomized trials of interventions designed in part to reduce pain and/or anxiety during non-invasive radiology procedures were included. Data was extracted by one reviewer and verified by a second. Study quality was assessed following Cochrane Handbook guidelines. The primary outcome of interest was change in pain or anxiety following intervention. Results: 1178 search results were identified, of which 33 (23 RT, 10 DR) met inclusion criteria. 31 of these studies were at high risk of bias. Of the RT studies, 3/12 informational interventions, 3/4 yoga programs, and 3/7 other CAM interventions reduced anxiety. Of the DR studies, 3/5 informational interventions, 2/3 CAM interventions and 1/2 pharmacological interventions reduced anxiety. No interventions significantly reduced pain. Conclusion: Insufficient high quality evidence exists to guide the use of interventions against pain and anxiety in non-invasive radiological procedures, though there is some suggestion that yoga interventions are effective. Further research employing study designs with low risk of bias and adequate sample size is warranted.

123

Understanding Barriers To Access In The Management Of Colorectal Cancer Hepatic Metastases: A Qualitative Approach

Meagan Wiebe, Supervisor: Dr. David Urbach, Dr. Alice Wei Department of Surgery, Division of General Surgery, Toronto General Hospital Background: Colorectal cancer (CRC) is the third most common malignancy in Ontario. Individuals who progress to stage IV disease usually develop hepatic metastases. When the liver is the only site of metastases, hepatic resection offers the potential for long-term survival and cure. However, evidence suggests that liver resection for metastatic CRC is underutilized, and many patients who may benefit from surgery are not being referred. The reasons underlying the underutilization of surgery are not well understood. Objective: To identify the barriers to the dissemination of evidence-based knowledge regarding the surgical management of hepatic metastases from CRC in Ontario. Methods: Two focus groups were held, one with general surgeons (n = 3), and one with medical oncologists (n = 5). Participants were asked semi-structured questions, and their responses were recorded and transcribed verbatim. Qualitative analysis of the transcripts was performed using a modified grounded theory approach, which was done through open and axial coding by three independent coders. Results: Three domains of interest are: processes of care for patients with CRC hepatic metastases, barriers to access, and solutions to the barriers. Extracted themes within the domains are: coordination of clinical care, access to resources, and medical expertise and education. Conclusions: This study demonstrates that general surgeons and medical oncologists are faced with barriers to access to care for patients with CRC hepatic metastases. They have suggested various solutions to these barriers, which will lead to the development of strategies to improve the management of these patients upon implementation.

124

Long-Term Follow-up of Unit-Rod Instrumentation in Spina Bifida Scoliosis Patients

Lilian Wong and Dr. James G. Wright, Orthopaedic Surgery, The Hospital for Sick Children. Spina Bifida (SB) cystica, or myelomeningocoele, is an embryological neural tube defect, resulting in a cyst containing meninges and spinal cord protruding from non-fused vertebral arches. SB patients have multiple abnormalities including flaccid paralysis below their lesion, hydrocephalus, Arnold-Chiari malformation, tethered cord syndrome and scoliosis. Scoliosis, occurring in >50% of patients, is often accompanied by clinical deformity, pelvic obliquity and coronal and sagittal imbalance. Progressive scoliosis causes worsening clinical deformity, impaired sitting balance, decreased pulmonary function and reduced physical function. Surgical intervention, usually involving posterior instrumentation with anterior release, can improve spinal deformity and prevent further curve progression. However, in addition to fusing the spine and high reported complication rates, many studies have reported decreased walking ability and no study has demonstrated clear improvement in functional outcomes such as sitting balance, activities of daily living or pain (Obsebold et al, JBJS 1982; Askin et al, Spine 1997; Muller et al, Acta Paed 1992). Furthermore, no long-term follow-up is available. The purpose of this study is to assess these patients' quality of life into adolescence and adulthood following spinal instrumentation and fusion. Patients will be assessed clinically and radiographically including Cobb angles, coronal and sagittal balance. To assess quality of life (QOL), patients will complete the Spina Bifida Scoliosis Questionnaire (SBSQ) and the SF-36 QOL questionnaire. Finally, post-operative complications will be assessed, including nonunions, infections and non-spine-related spina bifida complications such as ventriculo-peritoneal shunt failure. Even this simple clinical review has encountered several important barriers including significant delays in ethical review, completing ethical review at two sites with different expectations, and ethical review board concerns about using a private investigator to identify patients lost to follow-up. Forty-five patients have been identified, and we are in the process of collecting data. In conclusion, the results of this study will be used to evaluate patients' QOL and the relationship with radiological and clinical outcomes.

125

Population-based association study of glutamatergic candidate genes in obsessive compulsive disorder (OCD) and response inhibition

Student: Ke Wu Supervisor: Dr. Paul Arnold Genetics & Genomic Biology, Hospital for Sick Children, Toronto, Ontario Background: Obsessive-compulsive disorder (OCD) is a common neuropsychiatric condition that affects 1 ­ 3 % of the population worldwide. Individuals with OCD and their unaffected relatives exhibit problems with motor response inhibition, which represents a promising endophenotype of the disorder. Multiple lines of evidence have suggested that dysfunction in glutamate neurotransmission may play a role in both OCD and response inhibition. Purpose: The purpose of this study is to explore seven glutamatergic candidate genes, previously reported to be associated with OCD, for association with response inhibition using a population-based design. Methods: Genotype data for DLGAP3, GRIN2B, GRIK2, GRIK3, SLC1A1, SLITRK1 and SLITRK5 were obtained from an ongoing genome-wide association study of response inhibition based on Stop-Signal Reaction Time (SSRT). Subjects were selected from both extremes of the SSRT spectrum (n=177). Basic association tests and haplotype block analyses were performed on a total of 524 SNPs. Results: Preliminary results showed that SNPs in DLGAP3, GRIN2B, GRIK2 and GRIK3 were nominally associated with response inhibition (P < 0.05). One SNP in the intron region of DLGAP3, rs12120523, remained significant after correcting for multiple comparisons (Padjusted = 0.03349). Haplotype block analysis revealed no evidence for haplotype associations after correcting for multiple comparisons. Conclusions: These results suggest that the glutamate candidate genes may be implicated in response inhibition and OCD, however further study is needed. We are currently genotyping the top hit SNP in DLGAP3 in additional subjects. A separate OCD population (n=27) with neuroimaging endophenotypes will also be analyzed for association with the candidate genes.

126

Expression levels of c-Fos in Gtf2i- and Gtf2ird1-deficient mouse models of Williams-Beuren syndrome following exposure to a fearful environment

Michelle Wu1, Emily Lam1, Edwin Young1, Lucy R. Osborne1,2 1 Institute of Medical Science and 2Departments of Medicine and Molecular Genetics, University of Toronto

Introduction: Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder that is associated with cardiovascular abnormalities, distinctive facial features and mild to moderate intellectual disability. Individuals with WBS are over-friendly with a lack of many normal social boundaries, but also have increased levels of anxiety and specific phobias. WBS is caused by the deletion of 25 genes on chromosome 7, but the exact genes that contribute to the cognitive and behavioral symptoms are still unknown. Our lab has generated mouse models for two candidate genes that are thought to encode for transcription factors, Gtf2i and Gtf2ird1. Gtf2ird1 knock-out mice show increased social interaction and reduced innate fear, and fail to increase c-Fos expression in the brain upon exposure to a fearful environment, suggesting impaired activation of the frontal cortex. Hypothesis: I hypothesize that c-Fos activation in the frontal cortex of Gtf2i heterozygous mice and Gtf2i/Gtf2ird1 double heterozygous mice may be altered following exposure to a fearful environment. Objective: To correlate c-Fos gene expression levels with a behavioral test of innate fear being carried out by another student. Methods: I will use quantitative real-time PCR to measure mRNA expression levels from dissected frontal cortex in mutant and wild-type mice, normalized to two housekeeping genes. Results: The experiments will tell us whether both genes are involved in fear response in mice and whether they act additively or have a synergistic effect. Conclusion/Significance: This study will help determine whether Gtf2i and Gtf2ird1 may contribute to the lack of inhibition seen in people with WBS.

127

Olfactory impairment in DJ-1 knockout mice as a characteristic of Parkinson' disease

Student: Qiu Jing Wu Supervisor: Dr.Sheena Josselyn Dept.: Neuroscience and Mental Health department at Hospital of Sick Children Parkinson`s Disease (PD) is a neurodegenerative disease primarily characterized by motor impairment, but is also associated with cognitive dysfunction, and olfactory deficits. Mutations in the DJ-1(PARK7) gene are linked to autosomal recessive PD. DJ-1 has multiple protective functions against PD such as transcriptional regulation and anti-oxidative stress reaction. To investigate DJ-1­associated impairments in olfaction, we conducted an odour discrimination test in DJ-1 knockout mice. Mice were trained to associate a specific scent with a sucrose reward, then to discriminate between the reward-associated odour, and another unrewarded scent. For 4 days, food restricted mice were first given a training trial where a mint or licorice scent was paired with sucrose pellets (+ scent). Mice were then given a second training trial where they were exposed to the opposite odour (licorice or mint) but received no sucrose reward (­ scent). On day 5, mice were presented with both + and ­ odours, and the time spent digging around each odour was recorded as an index for their ability to discriminate between the rewarded and unrewarded odours. Mice lacking the DJ-1 gene were impaired in discriminating between the odours (relative to wild-type controls), supporting olfactory deficits associated with mutations to the DJ-1 gene.

128

Prevalence and Incidence of Vertebral Deformities by VFA in Postmenopausal Women with Osteopenia: Companion Study to the ECKO Trial

Christine Young, Angela Cheung, Osteoporosis Program, Toronto General Hospital, UHN

BACKGROUND: Previous osteoporotic fractures increases the risk for future fractures, yet 2/3rds of vertebral fractures remain silent. Dual energy X-ray absorptiometry (DXA) measures bone mineral density (BMD) and is now used to assess vertebral fractures. Fractures frequently occur in postmenopausal (PM) women with osteoporosis (lower BMD), but also in those with osteopenia (higher BMD). PURPOSE: We wanted to determine the value of assessing PM women with osteopenia for vertebral fractures by investigating the prevalence and incidence of vertebral compression deformities (VCD) using DXA's Vertebral Fracture Assessment (VFA) in the ECKO trial - Evaluation of the Clinical use of vitamin K supplementation in PM women with Osteopenia. METHODS: The ECKO trial was a 2 to 4 year randomized, double-blind osteoporosis prevention trial involving healthy PM women with osteopenia (lumbar spine, total hip or femoral neck T-score between 1.0 and -2.0), comparing vitamin K1 (5mg) supplementation to placebo. VFA scans were performed on a Hologic QDR 4500A densitometer at baseline, 2-year and 3 to 4-year follow-up visits. Genant's semiquantitative method was used to grade VCDs and descriptive statistics were used in our analysis. Because there were no differences between the vitamin K1 and placebo groups in terms of the number of women with VCDs, we combined both groups for the analyses of this study. RESULTS: 440 women participated in our study and 88% were Caucasian with a mean age of 58.6 years. The mean years since menopause was 10.2. At baseline, 1 in 10 PM women with osteopenia without a clinical history of fragility fracture had VCDs of Grade 1 or greater. 93.8% were grade 1 deformities and 6.2% were grade 2 or grade 3 deformities. At 2 years, 1 in 12 women had one or more new or worsened VCD compared to baseline. At 3-4 years, 1 in 14 women had one or more new VCDs compared to 2 years. The majority of VCDs found in our study at all time points were of grade 1 classification. Interestingly, 58% of women with grade 1 VCDs at baseline improved by 2 years. Similar findings were found at 3-4 years. CONCLUSION: Our study showed that moderate to severe (grade 2 and 3) VCDs are uncommon among osteopenic PM women without a clinical history of fragility fracture. However, incident or worsened VCDs occur 4.3 per 100 person-years of follow up suggesting the importance of assessing vertebral fractures in this population. More research needs to be done to ascertain the significance of grade 1 VCDs in an osteopenic PM population and the elasticity of bone to mild deformity.

129

Structural changes in mossy fiber axons following spatial learning

Kirill Zaslavsky, Leonardo Restivo, Paul W Frankland Neurosciences and Mental Health, Hospital for Sick Children, 555 University Avenue, M5G 1X8 Long-term morphological changes in neuronal processes are thought to underlie memory formation in the hippocampus following spatial learning. In particular, the mossy fiber projection from the dentate gyrus to the CA3 subfield is highly plastic, showing robust synaptogenesis. Mossy fibers are unique in the cortex in that they form specialized synapses based on their target: large boutons synapse on excitatory principal CA3 cells, while filopodial extensions and en passant varicosities innervate inhibitory interneurons. In this experiment, I explore morphological changes of boutons following training in the Morris Water Maze, a spatial learning task, with suppression of excitatory N-methyl-D-aspartate (NMDA) signalling, which has been known to prevent long-term learning. Two groups of mice (n=4 each) were injected with either CPPene (5 mg/kg), a competitive NMDA antagonist, or saline (0.9% NaCl) 15 minutes prior to training. Mice were trained for 8 days, with a probe trial every other day. Following training, the animals underwent surgery to infuse an HSV-GFP vector into the hippocampus to label developmentally generated granule cells. Three days after surgery, the animals underwent another probe test and were sacrificed. The morphology of mossy fiber axons was assessed using laser scanning confocal microscopy. A two-way ANOVA revealed a drug treatment effect, suggesting CPPene injections prevent presynaptic morphological changes (P=0.0106). Furthermore, CPPene delayed water maze learning, with mice developing a bias for platform location only on the 7th probe day, compared to the 5th for the saline treated group. These findings suggest that NMDA signaling is required for axonal bouton plasticity.

130

The role of kinesin protein Kif7 in the formation of the diaphragm

Student: Supervisor: Department: Hospital: Kory Zayne Dr. Peter C.W. Kim Developmental and Stem Cell Biology Hospital for Sick Children Elm Wing 5138A

Background: Congenital Diaphragmatic Hernia (CDH) has an incidence of 1 in 2,000-3,000 newborns and accounts for 8% of all known major human congenital anomalies. Children with CDH suffer from high mortality and morbidity due to pulmonary hypoplasia (PH) and persistent pulmonary hypertension, elicited by abdominal contents advancing through the gap in the diaphragm into the thoracic cavity impeding proper lung formation. Pathogenesis for pulmonary hypoplasia associated with CDH is largely unknown and there are a limited number of genetic models to study CDH. The sonic hedgehog (Shh) cascade is critical for the development of respiratory systems in mammals during embryogenesis. The Kinesin Protein Kif7 has an integral role in the regulation of the Shh pathway by controlling cleavage-mediated degradation of the Shh transcription factors Gli2 and Gli3. Hypothesis: Kif7 is essential in preventing CDH and can be a useful genetic model for studying the pathogenesis of CDH. Methods: Kif7+/- mice were crossed to generate Kif7-/- mice which were dissected at embryonic days 15.5-18.5. Embryos were dissected to determine the percentage of Kif7-/- mice with diaphragm hernias. Histological sections of the diaphragm were stained with Hematoxylin and Eosin to examine differences in cellular architecture. Anticipated Results/Conclusion: It is expected that CDH will be observed at a higher percentage in Kif7-/- mice compared to Wild-type mice; this is a pre-requisite for the protrusion of abdominal contents into the thoracic cavity, preventing proper lung formation. Kif7-/- is a promising genetic model for studying human CDH.

131

Pre-clinical Evaluation of a Small Molecule Inhibitor of CDK1 as Potential Therapy for MYC Expressing Multiple Myeloma Tumors

Seda Zeng, Suzanne Trudel M.D., M.Sc., FRCPC Department of Medical Oncology and Hematology, Princess Margaret Hospital Background and Purpose Purvalanol potently inhibits CDK1, an important regulator of the G2-to-M cell cycle progression. This inhibition was reported to preferentially induce apoptosis in MYC over-expressing tumors(1), and thus an approach targeting deregulated cell cycle progression and MYC may prove effective multiple myeloma therapy. Methods Western blotting and real-time PCR were used to assess cellular MYC levels. MTT viability assay and Annexin-V flow cytometry analysis were used to assess induction of apoptosis. PI staining and flow cytometry was used to assess cell cycle status. Results Purvalanol broadly inhibits a panel of 14 standardized, annotated myeloma cell lines, with IC50s ranging between 5uM-7.5uM. MYC levels were found to be variable within the 14 myeloma cell lines. Comparison of Purvalanol's therapeutic activity against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of MYC, respectively revealed that higher levels of MYC expression demonstrated greater induction of G2-M arrest and apoptosis. Conclusions and future directions Purvalanol exhibits potential as a useful therapeutic agent against myelomas over-expressing MYC. Introduction of a MYC over-expressing gene into constitutively low MYC expressing myelomas can further support current results if Purvalanol treatment produces an increase in apoptosis towards transfected cells. Analysis of Purvalanol efficacy against primary patient marrow cultures needs to be done to confirm preferential inhibition of myeloma cells over the non-myeloma fraction. Bone marrow stroma may provide protection against Purvalanol's therapeutic effects, and thus further analysis of the mechanisms of protection may be done, in addition to drug combination experiments. References Goga A, Yang D, Tward AD, Morgan DO, and Bishop JM. Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC. Nature Medicine. 2007 July 27; 13(7):820-827.

132

Role of platelets in a one-hit model of transfusion-related acute lung injury (TRALI)

Lihua Zhai Supervisor: Wolfgang M. Kuebler Division of lung vascular research, Department of Surgery, St. Michael's Hospital Introduction: Transfusion-related acute lung injury (TRALI) is the leading cause of mortality after blood transfusion. Previous data have suggested that TRALI results from a double-hit of immune priming and subsequent neutrophil activation. Platelet depletion protects from double-hit TRALI by preventing the initial priming response. In contrast to this hypothesis our lab has succeeded to develop a one-hit model of TRALI in severe combined immunodeficiency (SCID) mice, which does not require a priming stimulus. Here, we test whether one-hit TRALI is similarly dependent on platelets, and if so, elucidate underlying mechanisms which are distinct from immune priming. Methods: SCID mice were mechanically ventilated. TRALI and platelet depletion were induced by intravenous injection of an anti-murine major histocompatibility complex (MHC) class I antibody and anti serum for integrin

3,

respectively. Arterial oxygen saturation (SaO2) was monitored for 120

minutes. Wet-to-dry lung weight ratio and bronchoalveolar lavage (BAL) fluid were analyzed. Results: While control mice without TRALI maintained good blood oxygenation (SaO2>95%) throughout the experiments, all mice with TRALI showed hypoxemia (SpO2<90%) and died approximately 30 min after TRALI induction. BAL fluid showed a higher proportion of polymorphonuclear leukocytes in TRALI as compared to control mice (0.44±0.3 % vs 0.28±0.16 %; mean±SEM; n=4 each) without reaching significance. Conclusion: In contrast to existing paradigms, one-hit stimulation by an MHC class I antibody suffices to evoke severe TRALI in SCID mice. We will next test whether this response depends on platelets, and subsequently dissect the mechanisms underlying the role of platelets in TRALI.

133

Effect of Cord Blood/Bone Marrow Transplant on Cardiac Function in Paediatric Patients

Christine Zhang, Seema Mital MD. Heart Centre, Department of Cardiology. 4514 Hill Wing, Hospital for Sick Children. Background: Cord blood and bone marrow transplantation (CB/BMT) are performed for malignant disorders and other medical conditions. The effect of circulating bone marrow progenitors on cardiac function post BMT is not known. We evaluated the effect of BMT/CBT on ventricular geometry and function. Purpose: The aim of this study is to evaluate specific changes in cardiac function that result from the use of Cy in pre-CB/BMT conditioning regimens. Methods: Medical records from patients with history of CB/BMT at the Hospital for Sick Children were reviewed. Parameters of left ventricular (LV) systolic and diastolic function and geometry on 2D echocardiography before and after CB/BMT were compared using a two-tailed Student t-test. Results: 309 patients were reviewed, CBT=18, BMT=291. Mean patient age at transplant was 7.99, 62.1% males. Echocardiograms were compared after a mean interval of 408 days after CB/BMT. Mean EF pre-CBT/BMT was 67.99% and after transplant was 68.05% (p=0.95). Echocardiogram measurements preceding and following CB/BMT showed significant differences in septal diameter (SD), LV end diastole diameter (LVEDD), LV posterior wall diameter (LVPWD), mitral valve annular velocity (MVANN-V). Significant findings included an increase in SD (pre=0.59, post=0.64 cm, p=0.016), decrease in LVEDD (pre=3.81, post=3.73 cm, p=0.028), increase in LVPWD (pre=0.58, post=0.62 cms, p=0.048), and decrease in MV-ANN-V (pre=18, post =15 cm/s, p=0.05). Conclusions: CB/BMT was associated with increase in ventricular wall thickness and LV relaxation abnormalities. Further studies are needed to determine if these cardiac effects are related to immune damage secondary to engraftment of BM-derived progenitors in the heart.

134

Generation of targeting vectors for construction of mouse models for Farber disease and human malignancies

Ning Zhang1, Abdulfatah Alayoubi1, Jeffrey A. Medin1, 2, 3, 4

1

Institute of Medical Science, and 2Department of Medical Biophysics, University of Toronto 3Ontario

Cancer Institute, and 4Toronto General Research Institute, University Health Network Farber disease (FD) is an autosomal recessive disease characterized by lysosomal acid ceramidase (AC) deficiency. In order to further investigate the therapeutic approaches for the fatal disease, gene targeting technology was applied to introduce a human patient mutation into the murine AC gene to generate a knoch-in mouse model that manifest the disease. On the other hand, overexpression of AC was found in some malignancies and may subsequently affect the safety of gene therapy of FD. Therefore, we constructed a transgenic vector to generate a mouse model harboring AC overexpression to high levels in all tissues. To do the knock-in mutagenesis, PCR was utilized to amplify the two homologous arms of the targeting vector flanking the neo sequence which is designed for antibiotic selection. Then, the desired single nucleotide mutation was introduced into the targeting vector. Regarding to the overexpression vector, we subcloned the human AC cDNA, which was driven by elongation factor-1 alpha (EF-1) promoter and tailed with SV40-polyA signal. To date, we have successfully constructed the two vectors and confirmed by digestion and sequencing results. To generate the knock-in mouse model, further work involves screening of antibiotic-resistant electroporated colonies with southern blot; and we will utilize the pronuclear injection technology to deliver the overexpression vector into the mouse germ line to produce the transgenic mouse model.

135

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