Chief, Section on Organelle Biology Cell Biology and Metabolism Branch National Institute of Child Health and Human Development
Emerging fluorescence technology to study the spatial and temporal dynamics of molecules and organelles within cells
Emerging visualization technologies are playing an increasingly important role in the study of cellular organization and function, capturing dynamic processes at the level of whole organisms down to single molecules. Recent developments in probes, techniques, microscopes and quantification are dramatically expanding the areas that can be analyzed through imaging. Genetically-encoded, photoactivatable fluorescent proteins (PA-FPs) have been particular fruitful in this regard. They become bright and visible upon being exposed to a pulse of UV light. This allows selected populations of proteins to be pulse-labeled and tracked over time. Used for in cellulo pulse chase experiments, the PA-FPs have helped clarify mechanisms for biogenesis, targeting, and maintenance of organelles as separate identities within cells. PA-FPs have further permitted the development of point-localization superresolution (SR) imaging, which dramatically improves the spatial resolution of light microscopy by over an order of magnitude (~10-20 nm resolution). Involving the controlled activation and sampling of sparse subsets of photoconvertible fluorescent molecules, point-localization SR imaging techniques offer exciting possibilities for obtaining molecule scale information on biological events occurring at variable time scales. Here, I discuss several examples where we have used these new fluorescent imaging techniques to dissect different membrane trafficking and cytoskeleton-related topics, including the formation of autophagosomes from mitochondria outer membranes, the dynamics of the actin cytoskeleton and focal adhesions during cell crawling, and transport through the secretory pathway.
Assistant Professor Molecular and Cellular Biology University of Guelph
Fun with the RoboGut: mimicking the human gut in vitro
The human gut contains an extremely diverse consortium of bacterial species, which have recently risen in profile as we come to understand their critical role in the maintenance of host health. Whilst ‘omics’ technologies are teaching us much about this gut microbiota, in contrast there has been much less research focused on the biology of these microbes using culture techniques, partly because the majority of the organisms of the human gut are fastidious anaerobes and many depend on as-yet unappreciated aspects of the consortium as a whole for their survival. Recognizing this, we have set out to build an in vitro chemostat model of the human distal gut, nicknamed ‘Robogut’, and have used this system for a variety of purposes: from modeling gut microbial ecosystems and biofilms, to creating defined experimental microbial communities, to culturing the ‘unculturable’. I will present a medley of my lab’s work to demonstrate the importance of culture to the study of the human gut microbial ecosystem.
Infection and Immunity Division The Roslin Institute University of Edinburgh Scotland Evolution, diversity and recombination in Picornaviruses and other RNA viruses
The virus family Picornaviridae are a large, heterogeneous virus family that cause widespread, often pathogenic infections in humans and other mammals. Picornaviruses fall into a number of distinct genera built to a common plan, with structural genes forming a non-enveloped capsid shell and a series of downstream non-structural genes encoding polymerase, protease and helicase enzymatic activities. Recently applied virus discovery methods have substantially increased knowledge about the diversity of picornaviruses and have led to a dramatic increase in the number of genera and species within the family.From a total of 8 species five years ago, the total number that might be currently assigned has risen to over, following discovery of picornaviruses in a much wider range of hosts (eg. fish, reptiles and birds) and several new groups infecting humans and other mammals (eg. cosaviruses, klasseviruses). These new discoveries are of enormous clinical and veterinary importance in the investigation of infectious diseases of unknown aetiology.Extraordinarily rapid recombination occurs within human enteroviruses and many other genera, as part of a process of their population turnover and renewal. Recombinant forms of echovirus 9, 11 and 30 show a 2-11 year life-span among virus populations circulating in Northern/Western Europe over the last decade. Differences in recombination dynamics between these serotypes correlates with larger scale changes in their incidence and disease associations evident from virus surveillance. While there commonality in the dynamics and constraints of recombination in most picornavirus genera, its evolutionary basis remains entirely uncertain. Understanding its occurrence may provide an important key in future studies of RNA virus epidemiology and pathogenesis.
Professor and Head Department of Medicine Queen's University
The Role of Mitochondrial Fission and Fusion in Hyperproliferative Syndromes: Therapeutic Applications in Lung Cancer and Pulmonary Hypertension
Conflicts of Interest: 1. NIH funding 2. A patent for the use of pyruvate dehydrogenase kinase inhibitors to treat cancer 3. A provisional patent for the inhibition of fission to treat cancer
Upon completion of this activity, participants will be able to: 1. Identify the mediators of mitochondrial fission and fusion (particularly DRP1 and mitofusin-2) 2. Understand the contribution of excessive cell proliferation and apoptosis resistence to the pathogenesis of both Group 1 PH (pulmonary arterial hypertension) and cancer 3. Understand the posttranslational regulation of mitochondrial fission and its relationship to mitosis 4. Have an appreciation for the ability of strategies which force mitochondrial fusion to slow proliferation and be used to treat experimental PAH and lung cancer
Empire Scholar and Murphy Family Professor of Children's Oncology Research Department of Pediatrics and Microbiology & Immunology Adjunct Professor of Chemistry, Syracuse University
Role of SHIP1 in Cancer, Stem Cell Biology and Inflammation
Using both genetic and chemical targeting of SHIP1 activity in vivo we have recently demonstrated a critical role for SHIP1 in cancer cell survival, mesenchymal stem cell fate decisions and T cell survival. These findings explain the major pathologies, mucosal inflammation and osteoporesis, that occur in germline SHIP1 deficient mouse. In my presentation I will describe our work that explains how SHIP1 expression prevents osteoporosis and mucosal inflammation. In addition, I will expand on our recently published work that shows many hematologic cancers have co-opted SHIP1 activity to promote survival, rather than inhibiting it. Our recent demonstration that SHIP1 can be inhibited by small molecules has enabled us to probe the biological role of SHIP1 in normal healthy adult hosts, but has also suggested therapeutic avenues that might be explored with these novel agents. Throughout the lecture these translational possibilities will be described.
Professor Emeritus of Biochemistry and Chemistry Gyula and Katica Tauber Professor of Biochemistry & Chemistry Member, US National Academy of Sciences
A New Approach To the Treatment of Amyotrophic Lateral Sclerosis (Lou Gehrig's Disease) Based on Genetic and Biochemical Studies in a Model Organism
FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. We have created a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required for nuclear localization and is required but not sufficient for the toxicity of the protein. The human nuclear localization sequence is not efficient in yeast, so the wild-type protein behaves like the pathogenic mutants in our model, confirming that mislocalization is directly linked to toxicity. A genome-wide genetic screen using a yeast over-expression library has identified five yeast DNA/ RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model and in a model of disease in mouse and human motor neurons, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS-mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway may be a major determinant of toxicity and/or suppression. The yeast model has a number of features that make it ideal for a screen for drugs that suppress the toxicity of mislocalized FUS/TLS; such a screen is in process and will also be described. Finally, our work has led to a completely novel therapeutic strategy, and the initial results of implementing that strategy will be presented.
Director, Canadian Research & Development Centre for Probiotics, Lawson Professor, Microbiology & Immunology and Surgery Schulich School of Medicine & Dentistry at Western
The grand delusion: humans in control of microbes
A mere 650 million years ago, give or take a few, bacteria arguably ruled the Earth. If that was us and we decided to create new entities, you can rest assure we'd want control over, and put our own stamp on, whatever we created. Not that bacteria created humans per se, but we certainly evolved from them and they have always had the ability to wipe out every species that came after them, had they so chosen. You only need to appreciate that it is a thin epithelium that prevents trillions of intestinal microbiota from killing us at any time! So, why would it be surprising to find that bacteria are involved in most of the things we do, and that the majority of these effects can be beneficial? Pick up a newspaper or copy of Nature, Science, PNAS etc most weeks, and another link between human health and microbes is discussed. Effects on anxiety, digestion, energy metabolism, reproduction, bone density, allergy, cellular signaling, organ function, the list goes on. In my talk, I will present examples of how bacteria influence our lives, and how administration of certain strains (probiotics) can help restore and maintain health, and even treat conditions. This is not a story of bugs versus drugs, but certainly our preponderance for the latter is proving to be a short sighted approach. My hope would be that by sharing my thoughts on the origin and the destiny of probiotics and their mechanisms of action as we know them, the audience will find something of interest to apply to the diverse areas of biomedical, clinical and molecular sciences. Equally important, I hope that students see the incredible opportunities and applications that can come from studying human microbiology.
Professor and Canada Research Chair in Bioethics and Philosophy Dalhousie University Faculty of Medicine, Novel Tech Ethics
'Pregnant Women and Health Research: An Ethical Imperative'
“Pregnant women get sick, and sick women get pregnant.” These are two powerful reasons to include pregnant women in research. Indeed, for pregnant women to receive appropriate care for themselves and their fetuses, their treating clinicians need pregnancy-specific data about safety, toxicity, dosage, side effects and contraindications both for pregnant women and their fetuses. The most effective way to get reliable data of this kind is to routinely include pregnant women in clinical trials, except when there is a compelling scientific or ethical reason not to do so.
This presentation will: review basic facts about current practice; comment on the reasons for the routine exclusion of pregnant women from research; and argue for the responsible inclusion of pregnant women in randomized controlled trials. Considerable attention will be given to the barriers to inclusion and the need to develop appropriate incentives for inclusion (including the development of innovative study design).
Professor, Obstetrics & Gynaecology Director, Center for Pregnancy and Newborn Research University of Texas Health Science Center San Antonio
'Mitochondrial dysfunction in the placenta with preeclampsia and obesity – role of miR-210'
An adverse intrauterine environment, seen in pregnancies complicated by preeclampsia, obesity or intrauterine growth restriction (IUGR), programs the offspring for disease in later life. Placental function regulates fetal growth and development, transducing the maternal and uterine environment to the fetus, and mediating fetal programming. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through translational repression or mRNA degradation. We found significantly increased expression of miR-210 in placentas from pregnancies complicated by severe preeclampsia or with increased maternal adiposity. This suggests that miR-210 plays a pivotal role in the placental response to an adverse intrauterine environment. Using novel technology for measuring mitochondrial energetics, we described significant mitochondrial dysfunction in placentas of preeclamptic or obese women and that gain- and loss-of-function of miR-210 in primary trophoblast resulted in loss or preservation of mitochondrial activity respectively.
Dr. Simon Haroutiunian Postdoctoral Research Fellow Danish Pain Research Center Aarhus University Hospital, Denmark
'Peripheral neuropathic pain: unraveling mechanisms and developing individualized treatment approaches'
Neuropathic pain may be caused by nerve injury or a wide range of diseases affecting the somatosensory nervous system. The current treatment approach does not consider the individual patient's phenotype, or the underlying mechanism of pain. In this undertreated and challenging condition, understanding the individual pain mechanisms to develop personalized treatment approach may substantially improve patient outcomes.
Dr. Loren Martin Postdoctoral Fellow Department of Psychology McGill University ‘Two Tales of Translational Pain Research: From Genetics to Social Modulation’
For many the term translational research refers to the “bench-to-bedside” enterprise of harnessing knowledge from basic science to develop new drugs, devices and therapies for patients. The current gap between translating basic science research findings into effective pain therapies in humans is a serious challenge, thus necessitating the concurrent use of both animal models and human cohorts. This talk will focus on using different translational approaches to address two very different aspects of pain research. First, I report that single nucleotide polymorphisms in the genes encoding for epidermal growth factor receptor (EGFR) and epiregulin (EREG) are associated with the development of chronic facial pain in three human cohorts. Then, in a reverse-translational approach, I used a variety of techniques in rodent models to demonstrate: 1) the presence of EGFR in pain-processing neurons, 2) that EGFR inhibition is analgesic in mice and rats, and 3) that activation of EGFR by EREG increases pain sensitivity through the mammalian target of rapamycin (mTOR) and matrix metalloproteinase 9 (MMP-9). What is striking about these genes is that they belong to a family of proteins—the ErbB family of receptor tyrosine kinases —that are among the most well-studied in all of biology, and yet their prominent role in pain processing has never been reported. Secondly, I will present data showing that empathy for pain is modulated solely by familiarity and stress in both mice and humans. In both species empathy is stronger when members of the dyad are familiars and I show here that blockade of the (hypothalamo-pituitary-adrenal; HPA) stress axis elicits the expression of empathy in strangers of both species. In familiar but not stranger pairs, increased pain behaviors or ratings accompanied dyadic testing. Pharmacological blockade of glucocorticoid synthesis or glucocorticoid/mineralocorticoid receptors enables the expression of pain empathy in mouse and human stranger dyads. These results demonstrate that empathy is blocked in an evolutionarily conserved manner by the stress of a social interaction with an unfamiliar conspecific, and can be elicited simply by blocking the endocrine stress response.
Dr. Nader Ghasemlou Postdoctoral Fellow Harvard Medical School Boston Children's Hospital
Metallothionein 2 activation of TRPA1 drives inflammatory pain
Pain is initiated by activation of ion channels present on the peripheral terminals of nociceptor sensory neurons. These transduce intense mechanical, thermal, and chemical stimuli into ion fluxes, which lead to hypersensitivity. Although inflammatory mediators can sensitize ion channels in nociceptors, no endogenous protein is known to activate these channels directly. We have found that metallothionein (MT) 2, which is expressed at the site of injury, directly activates the nonselective cation channel TRPA1. This new signaling pathway between injured cells and neurons represents a novel mechanism that helps drive inflammatory pain.
Dr. Javeria Ali Hashmi Postdoctoral Research Fellow Harvard Medical School
'Predictive Dynamics in Brain Networks of Chronic Pain Patients'
The advent of new imaging methods and data modeling techniques has revolutionized our capacity to understand and utilize brain signals. With these techniques, the dynamic interactions between the complex elements of the brain can be used to effectively decode the neurological substrates of pain perception and modulation. The task to discover how intricately connected brain networks process psychological cues and interpret them into analgesia is an important scientific challenge that can leeway into novel clinical tools and improved pain therapeutics. This talk will present evidence that brain network properties can be used to predict the time course of pain experiences. We have reported that synchronizations in specific brain networks in particular BOLD frequency bands forecast outcomes of placebo treatment in chronic back pain patients that were tested in clinical trial conditions. More recently we have discovered that computationally characterized whole brain network alignments predict subsequently induced placebo analgesia in a clinical population of knee pain patients. These findings indicate a role of network configurations in facilitating adaptive pain modulation. On the other hand, brain properties also provide several clues into maladaptive pain processing associated with chronic pain. We have found that brain activity related to the perception of back pain shifts in location from regions involved in acute pain to engage emotion circuitry as the condition persists, thereby providing a percept linked brain signature for the transition to chronic pain.
Dr. Margaret Petroff Associate Professor Department of Anatomy and Cell Biology University of Kansas Medical Center
Infertility in autoimmune-prone mice: AIRE and immune tolerance to reproductive tissues
The Autoimmune Regulator (AIRE) contributes to central immune tolerance by promoting expression of tissue-restricted self-antigens in specialized epithelial cells of the thymic medulla. In humans, defective AIRE expression results in a multi-organ autoimmune disorder known as Autoimmune Polyglandular Syndrome Type-1. Both humans and mice with a deficiency in this gene develop autoreactive T cells and serum autoantibodies that precipitate the disease. Among the array of organs targeted in AIRE deficiency are the reproductive tissues, and both males and females exhibit high rates of infertility. In female mice with targeted mutations of the Aire gene, the ovaries are primary immunological targets: follicles and oocytes are eventually and completely depleted. Prior to depletion, there is a reduction in oocyte quality that leads to delayed implantation and early embryonic mortality. In male Aire-deficient mice, targets include the testes, epididymis, seminal vesicles and prostate. Males lacking Aire are almost completely infertile, most likely due to combined effects of unsuccessful mating and inability of sperm to fertilize healthy oocytes. Collectively, these studies illustrate that male and female reproductive tissues and processes are highly sensitive to inappropriate immune responses, and that inflammatory events, even when subclinical, could have a dramatic effect on fertility.
Dr. Paul Wood Director, Metabolomics Unit Professor, Physiology and Pharmacology DeBusk College of Osteopathic Medicine Lincoln Memorial University
Lipidomics reveals dysfunctional myelin glycosynapses in schizophrenia
Background: Abnormal structural/functional connectivity has been proposed to underlie the pathophysiology of schizophrenia. However, the biochemical basis of abnormal connectivity remains undefined. Methods: We undertook a shotgun lipidomics analysis of over 700 lipids across 26 lipid subclasses in the frontal cortex of schizophrenia subjects and hippocampus of G72/G30 transgenic mice. Results: We demonstrate that glycosphingolipids and choline plasmalogens, structural lipid pools in myelin, are significantly elevated in the frontal cortex obtained from patients suffering from schizophrenia and the hippocampus of G72/G30 transgenic mice. Conclusions: Our data suggest that structural lipid alterations in oligodendrocyte glycosynapses are responsible for dysconnectivity in schizophrenia and that increased expression of G72 protein may play a role in the development of abnormal glycosynapses.
Dr. Sara Nunes de Vasconcelos Assistant Professor Institute of Biomaterials and Biomedical Engineering University of Toronto
Tissue engineering strategies for cardiovascular regeneration
Cardiovascular disorders such as myocardial infarction, stroke and peripheral vascular disease are the leading cause of hospitalization in Canada. Motivated by the need to generate alternative therapeutic avenues to treat cardiovascular diseases, Dr. Nunes de Vasconcelos’ research program focuses on neovascularization strategies and on stem cell-derived cardiomyocyte biology and translational applications. Her long-term objective is to develop effective interventions for the treatment of cardiovascular diseases to be achieved by utilizing vascularization strategies, such as ready-made microvessels, to support the survival of transplanted cells for cell-based regenerative medicine. This will facilitate cardiovascular tissue regeneration in the context of myocardial infarction.
Monday, December 15th, 2014 4:30 – 5:30 pm School of Medicine, Room 132A
Professor, Departments of Medicine, Biochemistry, and Medical Biophysics Western University Schulich School of Medicine and Dentistry Co-Director of the Vascular Biology Group, Robarts Research Institute London Health Sciences Centre
Exploring the microvasculature in ischemic disease
The microvasculature is where oxygen and nutrients are delivered to tissues. However, in contrast to large and medium-sized blood vessels, microvessels constitute a "blind spot" in clinical medicine. We have employed real-time and 3-dimensional strategies to investigate the structure and function of this otherwise hidden part of the vascular tree. We find that the regenerative capacity of microvessels in infarcted muscle is tremendous. However, the process differs from classic paradigms of angiogenesis and the end product is flawed. Moreover, the vulnerabilities in microvessels in ischemic and regenerated muscle are similar to those in tumors, raising the possibility of unifying approaches to managing ischemic and malignant disease.
Dr. Mark Ormiston Intermediate Fellow and Senior Research Associate Department of Medicine – Respiratory Division University of Cambridge
‘Innate Immunity in the Pathogenesis of Pulmonary Arterial Hypertension: Implications for Regenerative Therapies’ Dr. Ormiston is interested in the cellular mechanisms governing vascular remodeling in health and disease, with a particular focus on the mode of action of regenerative cell-based therapies and the capacity of circulating cells of the immune system to regulate blood vessel integrity, growth and repair. This interest is rooted in his study of pulmonary arterial hypertension (PAH), a disease of pathological vascular remodeling that is linked to immune dysfunction and is strongly associated with loss-of-function mutations in the bone morphogenetic protein (BMP) type II receptor (BMPR-II). More specifically, he is interested in the capacity of innate lymphocytes, such as Natural Killer (NK) cells, to influence pulmonary vascular homeostasis. Through his research into experimental therapies for PAH, he has also developed techniques for the generation and characterization of adult blood-derived progenitor cells, such as blood outgrowth endothelial cells, which he has used both as a surrogate for patient-derived endothelial cells and as a substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming.
Wednesday, December 17th, 2014 4:30 – 5:30 pm School of Medicine 132A
Dr. David Kaplan Senior Scientist, Program in Neurosciences and Mental Health, Hospital for Sick Children Professor, Department of Molecular Genetics, University of Toronto
Genes and drugs regulating stem cell function and repair
Our group studies how genes and signaling proteins regulate stem cell function in the developing and adult brain. Stem cells must be maintained, undergo proliferation and differentiation when they receive the appropriate cues to build the brain during development, and regulate aspects of learning, memory and repair in the adult. Here, I will discuss new mechanisms we recently identified on how stem cells are maintained and mobilized, and how environmental influences such as maternal infection and diabetes perturb these processes.
There is a desperate need for new and effective therapies for neurological and wound healing indications. We have developed and used various drug screening platforms as well as translation of our basic research findings to discover drug candidates for cognitive dysfunction as a result of acute head trauma, wound healing, alopecia, peripheral neuropathy, and Parkinson’s disease. Our findings suggest that academic drug discovery efforts can rapidly and inexpensively identify drug candidates for intractable neurological and wound-related conditions, and new signaling pathways and proteins regulating stem cell and axonal function.