Project Code: MGY 1S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Dr. Gabrielle Boulianne Department: Molecular Genetics TITLE OF RESEARCH PROJECT: Generation of Transgenic Drosophila Models of Human Neurodegenerative Diseases NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: The project goal is to create models of human neurodegenerative diseases for use in drug discovery. Disease models will include Alzhimer’s, Parkinson’s, Huntington’s diseases, and ALS. The genetic model system will be Drosophila melanogaster (fruit fly). To generate the models, strains with human disease genes will be crossed with stains that carry fluorescent markers of neurons. Survival of strains will be recorded, along with signs of degeneration. In stocks showing potential as disease models for drug discovery, verification of human transgenes will be conducted using polymerase chain reaction (PCR) and automated DNA sequencing. DESCRIPTION OF STUDENT PARTICIPATION: The student will conduct genetic crosses of Drosophila and record crossing schemes in laboratory notes. The student will compare the longevity of disease and wild type control strains, and record the results in a spreadsheet. For validation of strain transgenes, the student will perform DNA purification and PCR preparations, which are basic molecular biology techniques. This project will require 4 hours of laboratory work per day, 5 days per week for the summer session. Laboratory work will occur in the mornings. From this project, the student will learn to record laboratory notes, conduct basic Drosophila husbandry, understand Mendelian genetics, and gain practical experience in molecular biology methods including DNA purification and PCR. MARKING SCHEME (assignments with weight and due date): Progress report 15% (June 30) Final report 20% (August 20) Oral presentation 15% (schedules for June – July) Quality of lab book 20% (on‐going, evaluated Aug 20) Quality of work 30% (on‐going)
Project Code: MGY 2S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Amy A. Caudy, Assistant Professor Department: Molecular Genetics TITLE OF RESEARCH PROJECT: Metabolomic Characterization of Novel Enzyme Pathways. NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: Every cell on earth processes nutrients to release energy and form the chemical compounds needed for cellular maintenance and growth. The study of metabolism, how cells transform nutrients and produce energy, reaches back hundreds of years but is now undergoing a revolution due to the availability of new methods. Recent advances in analytical chemistry, particularly in small molecule mass spectrometry and 2D NMR (nuclear magnetic resonance) have demonstrated a far more complex small‐molecule landscape than can be accounted for by current maps and models. Our group uses mass spectrometry to identify and quantitate the chemicals. Our group is working to identify previously unknown metabolic pathways within cells by using mass spectrometry to measure the changes in intracellular metabolites that result from the deletion of previously uncharacterized enzymes. There are two tremendous gaps in our understanding of metabolism. First, there are many chemical reactions that are known to occur as cells break down nutrients, yet we do not know the genes that enable these reactions. Second, recent technological advances in mass spectrometry have detected hundreds of chemicals in cells that are not predicted by the current knowledge of metabolism. My group has had success pursuing both types of questions (Cell. 2011 Jun 10;145(6):969‐80., Anal Chem. 2010 Apr 15;82(8):3212‐21.). We recently used these full scan mass spectrometric approaches to discover a major route for the synthesis of ribose, a key building block for DNA and RNA. This project combines cutting edge mass spectrometry approaches with the tools of genetics and biochemistry to discover the function of previously uncharacterized enzymes. We are using budding yeast, a genetically tractable, fast growing and commercially important organism for much of our work. The majority of metabolic reactions can be traced to the origins of life billions of years ago, so we then test our observations in yeast in mammalian cells to compare the roles of new pathways. An area of particular interest is the intersection of these uncharacterized metabolic pathways with the cell division cycle. DESCRIPTION OF STUDENT PARTICIPATION:
The students will construct and design genetically engineered yeast and mammalian cell strains with targeted changes in candidate enzymes. This will involve PCR, DNA sequencing, and other molecular biology techniques to create cells with desired characteristics. The students will be involved in the preparation, mass spectrometric measurement, and mathematical analysis of the data. The data will be compared with existing models of metabolism to identify the route of synthesis and degradation of novel compounds. For those students with the interest and aptitude, there are opportunities for design and fabrication of custom lab hardware and software to further extend our capabilities for high throughput metabolomics analysis and cytometry. MARKING SCHEME (assignments with weight and due date): Project Interim report: Due – July 1: 20% Participation in group meeting and presentations including summer MGY poster session ( 2 times over term): 20% Lab work (evaluations of lab notebook, performed biweekly): 30% Final project report: Due – last day of term: 30%
Project Code: MGY 3S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Julie M. Claycomb, Assistant Professor, Canada Research Chair in Small RNA Biology Department: Molecular Genetics TITLE OF RESEARCH PROJECT: NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: Following the discovery of RNA interference (RNAi), there has been an explosion in the study of small noncoding RNA molecules and their functions in gene regulation. Endogenous small RNA silencing pathways related to RNAi can regulate gene expression by various mechanisms such as translational control, transcript degradation, transcriptional control, and altering chromatin. RNAi‐related mechanisms are governed by the small RNAs, which provide sequence‐specific identification of target mRNA, and Argonaute proteins, which bind to small RNAs and “silence” target transcipts. The soil nematode, Caenorhabditis elegans, possesses 24 Argonaute family proteins, in contrast to eight in humans. Although mutant strains for each of the C. elegans Argonautes have been generated, the functions of only a handful of these proteins have been examined. We aim to systematically characterize the functional roles of different Argonautes, which will lead to novel insights into the involvement of small RNA mediated gene silencing pathways in development, differentiation and evolution, and may even contribute to better therapeutics. This project will combine a variety of genetic and molecular biology techniques (including fluorescence microscopy, PCR, culturing worms and bacteria, among others) to examine Argonaute mutant strains in C. elegans. We will employ immunofluorescence microscopy to examine various defects in these strains and to look at the expression patterns of fluorescently‐tagged Argonaute proteins in wild‐type (normal) worms. We will also conduct RNAi assays and genetic crosses to characterize additional defects present in mutant worms. DESCRIPTION OF STUDENT PARTICIPATION: The student will be directly supervised by a senior graduate student and will be involved in all aspects of the project outlined above. The focus of the student’s work will be in examining worms using microscopy. This will involve caring for worms, PCR, dissecting worms and preparing samples, and immunofluorescence microscopy. Additional experiments involve RNAi assays and performing genetic crosses. The student will gain experience in designing and executing experiments and will be exposed to a variety of molecular biology techniques.
MARKING SCHEME (assignments with weight and due date): Lab presentations: 20% Lab meeting presentation at end of term Lab participation: 20% Journal club (literature review) and lab meeting participation weekly 20% Bench work/attendance 20% Lab notebook, checked weekly Lab report: 20% Final lab report
Project Code: MGY 4S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Lori Frappier, Professor Department: Molecular Genetics TITLE OF RESEARCH PROJECT: Mechanisms of Regulation of PML Nuclear Bodies NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: PML nuclear bodies are important for many processes in mammalian cells including apoptosis and DNA repair and their loss is associated with several cancers. In addition PML bodies act to suppress infection by some viruses, including herpesviruses, and as a result herpesviruses encode protein that function to disrupt the PML bodies. Six different isoforms of PML proteins interact to form the structural basis of the nuclear body but the roles of these specific isoforms and how they are regulated by cellular and viral proteins are not well understood. This project seeks to better understand how PML proteins in nuclear bodies are regulated by viral and cellular proteins. Experiments will involve growing human cells, imaging PML nuclear bodies by fluorescence microscopy and examining PML protein levels by western blotting after altering the expression of various proteins. DESCRIPTION OF STUDENT PARTICIPATION: The student will be taught the above techniques and will conduct the experiments under the supervision of an experienced lab member. The student will keep a detailed lab notebook of the experiments and organize the results for presentation. He/she will also be given detailed topics relevant to the project to research in the literature. MARKING SCHEME (assignments with weight and due date): June 27: 30% ‐ lab performance to date and lab notebook evaluation July 25: 30% ‐ presentation of the project and results to the lab Aug 15: 20% ‐ lab performance and lab notebook evaluation 20% ‐ written summary of experiments performed and results (with figures).
Project Code: MGY 5S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Sevan Hopyan, Assistant Professor Department: Molecular Genetics, Surgery TITLE OF RESEARCH PROJECT: Development of the Mammalian Limb Bud NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: Although we know the identity of many genes that are required for embryonic development, how those genes shape the embryo is still largely a mystery. Our goal is to define how genetic pathways regulate physical forces to shape the embryonic limb bud. The mouse is our primary model organism because of its relevance to human congenital anomalies and the availability of numerous genetic tools. We use time lapse microscopy to observe cell rearrangements and also measure physical properties of embryonic tissue in collaboration with engineers on campus. By manipulating genetic and physical parameters, we deduce how regulators such as Wnt and Fgf transduce forces to alter cell behaviours. DESCRIPTION OF STUDENT PARTICIPATION: Specific projects that students might participate in include defining how mechanical tissue properties are influenced by specific pathways to shape the limb bud, mechanisms by which Iroquois homeodomain genes shape the bud and establish skeletal pattern, and exploration of methods to recapitulate early limb development in a dish. MARKING SCHEME (assignments with weight and due date): June ‐ Midterm evaluation of notebook (10%) and laboratory performance (25%). August – Final lab meeting presentation (15%), written report (15%), evaluation of notebook (10%) and lab performance (25%).
Project Code: MGY 6S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Helen McNeill, Professor Department: Molecular Genetics TITLE OF RESEARCH PROJECT: Regulation of the Ft‐Ds Growth and Planar Polarity Pathway: Identification of Novel Pathway Components Using Drosophila RNAi Screens
NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: The large cell adhesion molecules Ft and Ds regulate cell proliferation via the Hippo kinase pathway, and a form of tissue organization known as planar cell polarity. The ability of Ft and Ds to regulate cell proliferation and organization are conserved to man, and mutations in these genes are implicated in cancer, cystic kidney disease and deafness. The biochemical links between the cell adhesion molecules and the control of growth and planar polarity are still unknown. We have identified a number of proteins that bind to Ft and Ds through mass‐spectrometry and Y2H screens. To determine the function of these Ft and Ds‐binding proteins, we will use transgenic RNAi to knockdown expression of these genes in vivo and measure growth and tissue organization in Drosophila models. DESCRIPTION OF STUDENT PARTICIPATION: Students will establish crosses between transgenic flies to express RNAi in the Drosophila wing. This will involve selecting appropriate flies based on visible markers, and examining offspring under a dissecting microscope. Students will be trained by senior graduate students and technicians in the laboratory. Students will measure wing size and polarity in adult Drosophila to ascertain if the novel genes have roles in the Ft‐Ds pathway. Genes that have effects on growth or planar polarity will be tested for genetic interactions with Ft and Ds. MARKING SCHEME (assignments with weight and due date): Mid‐term report: 30% of final grade: Due June 20 Notebook: 40% of final grade: Notebook and progress will be assessed monthly by the professor and student advisor. Final report: 30% of final grade: due at end of classes. This report will summarize all of the genetic crosses used to test the putative Ft‐Ds pathway members, and propose future experiments.
Project Code: MGY 7S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Marc Meneghini, Associate Professor Department: Molecular Genetics TITLE OF RESEARCH PROJECT: Genetic and Epigenetic Control of Yeast Development NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: The labs interests concern ancient biological mechanisms of eukaryotic cellular differentiation and as a model we use the budding yeast Saccharomyces cerevisiae. A major emphasis in the lab is focused on how histone methylation and demethylation controls developmental transitions that occur during the yeast lifecycle. Specifically, we have discovered that JHD2, a highly conserved demethylase enzyme with specificity for the lysine‐4 residue of histone H3, modulates the transcriptional activity of the nuclear genome in response to metabolic cues emanating from the mitochondria. Moreover, our studies indicate that this function is conserved in human cells, in which the JHD2 ortholog JARID1A controls cancer stem cell survival and oncogenesis. In yeast, JHD2 controls the gene expression programs controlling both gamete differentiation and aging. The student will study the molecular mechanisms by which JHD2 controls gene expression in these developmental contexts. Research objectives will utilize a diverse array of genetic, molecular, and cell biological methods. DESCRIPTION OF STUDENT PARTICIPATION: The student will be mentored by myself and will work closely with a PhD student in the lab. S/He will advance an independent project that is closely related to existing projects in the lab. Genetic crosses using tetrad dissections will be employed to construct yeast strains for experimentation. These strains will be interrogated using quantitative reverse‐transcriptase polymerase chain reactions (qRT‐PCR) to measure gene expression, and chromatin immunoprecipitation (ChIP) to evaluate histone modification changes. These molecular studies will be complimented with experiments to determine the replicative lifespans of mutant strains of interest. MARKING SCHEME (assignments with weight and due date): 33% Weekly participation in lab group meetings/journal clubs 33% Presentation of results in group meeting at the end of course period 33% A brief (2‐3 page) write up of the results will be required at the end of the term
Project Code: MGY 8S
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 SUMMER Name and Title: Derek van der Kooy, Professor Department: Molecular Genetics TITLE OF RESEARCH PROJECT: Learning and Memory Genes NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: Our goal is to use the power and specificity of modern molecular genetics to reveal the component processes of learning and memory. In undertaking a mutational screening approach to learning and memory, we have taken advantage of the best‐known multicellular organism, the nematode C.elegans. C.elegans has proven to be an excellent molecular model for mammalian (including human) biochemical functions. We will use the C.elegans learning and memory genes discovered to find their relevant mammalian homologues. Most important, the C.elegans mutants should allow us to ask if we can separate associative from non‐associative learning, short from long‐term memory, and learning and memory in one sensory modality from that in another sensory modality. DESCRIPTION OF STUDENT PARTICIPATION: The students will participate in the initial screens for new learning mutant worms, as well as all of the behavioural testing to determine if the deficits are in learning, memory storage of the recall of memories. MARKING SCHEME (assignments with weight and due date): TBC Attendance at bi‐weekly Breakfast Club Meeting Attendance at bi‐weekly Wormies group journal club Final presentation