The potential for a pandemic outbreak of highly pathogenic avian influenza A strains, such as H5N1 or H7N3, is a serious and growing public health threat. Currently, a major limitation in pandemic preparedness is the difficulty associated with the timely development and distribution of a vaccine, as it is impossible to precisely predict the nature of a coming virus until a pandemic has already begun. Moreover, current antiviral treatments that target influenza virus components can be toxic, and can be overcome if the virus develops drug resistance. An alternative approach to antiviral drug design is to target host cell components that are required for viral infection, which eliminates the chance of antiviral resistance. A key step during influenza infection is entry of the virus into the host cell via fusion of viral and host cell surfaces. This process relies on the cutting and structural change of a virus surface protein, which in avian influenza strains is accomplished by an enzyme from the host cell. Recently, a novel, naturally-occurring inhibitor of this host enzyme was discovered in fruit flies. Heather Braybrook is investigating whether this inhibitor can prevent H5N1 virus entry and subsequent widespread infection. She will evaluate its effectiveness and toxicity in a cell culture model of influenza infection and study the mechanism of inhibition in further detail. Her studies will shed light on whether this type of inhibition could be used to reduce avian influenza infection in humans. Braybrook’s research may contribute to the development of novel and diverse antiviral therapeutics in the face of a potential influenza pandemic.
Program: Trainee
Identification and characterization of a novel keratinocyte-derived collagen inhibiting factor(s) for dermal fibroblasts
Wound Healing in skin is a dynamic process that involves continuous sequences of signals and responses from cells like fibroblasts and keratinocytes. An imbalance in the signals and responses at the wound site may result in an over-healing process known as hypertrophic scar.
This scar, thick and fibrous, might inhibit movement when it results from serious burns over large areas, especially around a joint. In hypertrophic scars, the fibroblasts produce too much extracellular matrix (ECM) proteins – the “scaffolding” between cells – including collagen. They also produce too little matrix metaloproteinases (MMP), an enzyme involved in normal tissue breakdown and remodeling.
Keratinocytes are epidermal cells that release factors that will either prompt fibroblasts to produce MMP or keep them from producing collagen. Previous research has identified a factor called stratifin, which stimulates MMP production. However, the factors associated with the inhibition of collagen production have not yet been described.
Claudia Chavez-Munoz’s research seeks to identify and characterize the keratinocyte-derived factor(s) that may function as collagen inhibiting factors for dermal fibroblasts. Ultimately, she hopes that a better understanding of the factors involved in wound healing will lead to therapeutic strategies in order to improve or prevent hypertrophic scarring.
Control of innate and anti-tumour immunity by mononuclear phagocytes
The innate immune system is the first line of defense against invading pathogens and tumours. Dendritic cells, monocytes, macrophages, and natural killer cells are key cells of the innate immune system, clearing microbial infections as well as tumours. These cells are activated by signalling via pattern recognition receptors that recognize pathogen associated molecular patterns. Down-stream signalling leads to the initiation of antimicrobial and inflammatory responses. Any deviation in the receptor signalling, development, or interactions of these cells can result in an inappropriate immune response, potentially leading to either immunodeficiencies (the inability to clear infections) or chronic inflammatory diseases. Lyn tyrosine kinase is an important enzyme in establishing signalling thresholds in leukocytes. Previous research in mice has shown that alterations in the activity of this protein affect the magnitude of the immune response, and that autoimmune diseases develop when it is absent. Manreet Chehal is investigating this further, determining whether increases and decreases in Lyn activity alter the development of innate immune cells and the responses of specific immune cells to pathogens and tumours. Her preliminary results indicate that an increase in Lyn activity enhances the innate immune response, including increased dendritic cell activation of natural killer cells. Chehal hopes to show that the immune response to pathogens and tumours depends on Lyn activity. Ultimately, her work could contribute to the development of new therapies that target the Lyn pathway to control inflammatory and autoimmune diseases or increase the body’s own natural defenses.
A novel role for nuclear EGFR in breast cancer progression and therapeutic resistance
Breast cancer is a major public health problem worldwide. In the United States alone, 178,480 new cases of breast cancer and 40,910 breast cancer deaths were expected in 2007. This unequivocally makes breast cancer the most common cancer in Western women, and second only to lung cancer in terms of cancer morbidity. Alarmingly, the incidence of breast cancer continues to rise with enormous physical, psychological, and social effects on the women who are faced with cancer diagnosis and treatment. During the last decade, great strides have been made in reducing breast cancer morbidity through increased mammography screening coupled with the advent of multi-agent chemotherapy and Tamoxifen. However, treatment of basal-like breast cancer (BLBC) remains especially challenging as these tumours lack the estrogen, progesterone, and HER2 receptors targeted by many traditional chemotherapeutic drugs. Moreover, the tumours readily develop resistance to new generation chemotherapeutic agents, such as Iressa. Further studies are desperately needed to uncover novel signalling cascades responsible for cancer progression that could ultimately be manipulated to combat this highly aggressive subset of breast cancer. The surface of cells are coated with receptors that “listen” to cues from the surrounding environment to direct cells to proliferate, synthesize and excrete proteins, or even undergo apoptosis (cell death). Traditionally, it was believed that these signals were sent to the cell nucleus exclusively through complex and elaborate cascades of intracellular messengers. However, it has recently emerged that cell receptors can become internalized and trafficked to the nucleus where they can act as transcription factors – in essence proteins that can turn on and off particular genes. This novel pathway has tremendous consequences for cancer biology as it offers a novel mechanism which cancerous cells could be exploiting to proliferate, metastasize (spread to other sites), to even combat the effects of chemotherapy. I have recently demonstrated that a fragment of the epidermal growth factor receptor (EGFR) translocates from the cell surface to the nucleus in BLBC cells. This is an exciting finding because EGFR is overexpressed in 50% of BLBCs, but more importantly, it could explain why these cells are resistant to Iressa, a chemotherapeutic that inhibits EGFR from sending messages via signal transduction cascades. Specifically, a fragment of the receptor could be cleaved to directly activate pro-survival genes in the nucleus. My research proposal is focused on gaining a more in depth understanding of this novel, cleaved form of EGFR, known as nuclear EGFR. Specifically, I am interested in determining the structure of nuclear EGFR and uncovering if it interacts with other proteins in the nucleus. In addition, I want to discover the specific pro-survival genes that are being induced by nuclear EGFR. This work will determine if targeting nuclear EGFR represents a viable strategy for combating cell proliferation, metastasis, and therapeutic resistance in a subset of cancer where treatment options are currently limited.
Application of Pleiades MiniPromoters for studying the role of NR2E1 in neural stem cells and mania-like behaviour
Severe psychiatric disorders affect three to five per cent of Canadians. One of these diseases is called Bipolar Affective Disorder Type I (BPDI). People with this disorder manifest many unusual manic behaviours. They typically do not sleep much, feel like they are on top of the world, have racing thoughts and are easily distracted. However, most BPDI patients also have depressive episodes and 15 per cent will commit suicide. The recurrence rate for BPDI is 90 per cent, making it a life-long disorder. Unfortunately, it is incurable and difficult to manage with current therapeutics. The brain transcription factor Nr2e1 controls the proliferation and differentiation of neural stem cells, which are required for the formation of neurons (neurogenesis). Some people with BPDI show dysplasia of forebrain and neurogenic regions and treatment that improve symptoms of bipolar disorder are known to stimulate neurogenesis. Mice that have a non-functional version of the Nr2e1 gene (dubbed ’fierce’ mice) display similar severe mania-like behaviour and defects in neurogenesis as observed in people with BPDI. Charles de Leeuw is investigating the use of MiniPromoters – DNA constructed from small conserved regions of a gene that tell it when and where it should turn on – to affect gene expression in specific brain regions of fierce mice. He will use the MiniPromoters to deliver Nr2e1 to the neural stem cells that are defective but also in the neurons that are generated from defective stem cells, both types of cells which are involved in the fierce mice defects. If he is able to prompt the gene to be turned on in the correct area of the brain, de Leeuw anticipates he will be able to ‘cure’ some of the mania-like behaviours in mice. His goal is to determine the potential for treating the genetic cause of BPDI through the use of MiniPromoters and human NR2E1. His experiments will also help shed light on the neurodevelopmental causes of manic behaviour.
Fluorescent tracking of RNA in living cells: in vitro selection of fluorescent-dye-binding RNA aptamers
Within cells, RNA molecules perform a number of critical functions. Many of these functions are related to protein synthesis – the manufacture of various substances, including enzymes, hormones, and antibodies, that are necessary for the proper functioning of an organism. RNA molecules regulate gene expression (activation) to control cell reproduction, parent-specific inheritance and cell differentiation. They also interact with certain viruses during the establishment of viral infection. Despite recent advances in studying the dynamic interactions of proteins in living cells, where and when RNA molecules move through the cell to perform these various functions is still poorly understood. Elena Dogosheina is developing a new method to track RNA molecules in living cells as they move in and out of cell compartments. This movement will be visualized with the use of a fluorescent dye that contains microscopic magnetic beads to which RNA molecules will bind. This RNA tracking method could prove useful as a real time reporter for changes in RNA expression over space and time, and can be applied to study RNA splicing disorders and cancers involving differential expression of small RNAs. This method could also be used to study viral pathogenesis by visualizing intracellular organization and intercellular movement of viral nucleic acids in the course of infection.
Characterization of the mechanical properties of collagen using optical tweezers
The collagens are a family of more than 20 different proteins, all sharing the same basic structure. Collagen is the most abundant protein in mammals, comprising more than a quarter of the total protein in the human body. Its main role is in connective tissues, such as bone, cartilage, tendons and skin, where it is a vital structural element providing support and rigidity. Even small mutations can lead to weakened tissues, and genetic diseases such as brittle bone syndrome and osteoarthritis. Understanding the mechanical properties of collagen at the molecular level is important for understanding its role in these tissues, their formation, and their degeneration. In humans it has been found that the melting temperature of collagen – the temperature at which the molecule unwinds and separates – is very close to body temperature. The melting temperatures of various types of collagen have been found to be closely linked to the body temperature of the species in which they are present. This indicates that the thermal stability of collagen may be of great relevance to the structural role it plays. Benjamin Downing is investigating how temperature affects the collagen molecule’s strength and flexibility. He is using optical tweezers – a device that employs a tightly focused laser beam to manipulate micron-sized objects – to stretch the molecule and measure its stiffness and elasticity over a range of temperatures. This will reveal how closely the mechanical and thermal stabilities of the molecule are correlated. Downing’s research will help shed light on how the structure of a molecule gives it a particular strength and flexibility, knowledge that may be useful in the future design of artificial molecules that have specific properties. This information could be relevant in the development of biomaterials with applications in tissue repair.
Chromatin mishandling in Huntington disease: potential links to pathogenesis and points of therapeutic intervention
Huntington disease (HD) is a hereditary neurodegenerative disorder that affects 3,000 Canadians. Patients with a HD mutation experience adult-onset psychiatric symptoms, cognitive impairment and motor disturbances that progressively worsen over many years and lead to death. Despite a massive world-wide research effort, there is still no means of prevention, no treatment, and no cure for HD. Recent studies confirm that psychiatric and cognitive changes occur in at-risk individuals several years before formal HD diagnosis. The ability to understand and reverse these early cognitive changes may lead to a cure for HD. Chromatin is the genomic DNA-protein complex that specifies how to make proteins and when and where to do so. Recent studies show that the way genetic material is packaged into chromatin in brain cells can be altered by early life experiences (such as learning), and can affect behaviour. Alterations in chromatin regulation have been observed in depression, bipolar disorder and schizophrenia. Many effective psychiatric drugs have been found to influence chromatin regulation. Mendel Grant is examining a possible link between chromatin and the onset and progression of HD. She is testing her hypothesis that environmentally-responsive chromatin changes (such as those induced by learning) do not function normally in a mouse model of HD. This could underlie cognitive dysfunction observed in HD patients and animal models. If chromatin is shown to be misregulated in HD, the use of FDA-approved neuropsychiatric drugs that alter chromatin regulation could be a promising therapy. Information yielded from this study may also be applicable to other neurodegenerative disorders including Alzheimer and Parkinson diseases and psychiatric conditions such as depression.
The demi-globule hypothesis: a thermodynamic model for prion protein template-directed misfolding
Prions are the causative agents for many brain diseases of humans and animals. In animals, the most well-known prion-related disease is bovine spongiform encephalopathy, or mad cow disease. The human prion diseases are Creutzfeldt-Jakob disease, kuru, and fatal familial insomnia. All these diseases cause brain cell death leading to difficulty walking, dementia, muscle spasms, and seizures. They are invariably fatal, and there are currently no treatments available. The incidence of human prion diseases is roughly 1 per million people per year. Unlike most other infectious diseases that are spread by bacteria or viruses, prion diseases appear to be caused by misfolded protein molecules. Protein is made of long chains of amino acids, and how a protein folds determines its function. A misfolded prion protein can interact with a normally-folded prion protein and cause it to also misfold, setting off a chain reaction that results in widespread brain cell death. How the misfolded prion causes the normal prion to misfold remains unclear. However, a new theory called the demiglobule hypothesis proposes that the misfolded prion binds to the normal prion and causes a key part of the normal protein structure, called a beta sheet, to come apart. This ultimately results in a misfolded shape. William Guest is using a variety of theoretical and experimental techniques from physics, chemistry and biology to determine whether the demiglobule hypothesis can account for prion protein misfolding. If the results of this project support the demiglobule hypothesis, researchers will know much more about how prion conversion occurs. This knowledge could ultimately enable the development of methods to block prion conversion, thereby stopping the spread of disease.
Development of natural killer (NK) cells from a novel progenitor
Natural Killer (NK) cells are immune cells with an important role in the first line of defense against cancer formation, metastases (spread) of tumour cells, and infections by viruses and other pathogens. Once known solely for their role in the innate immune system, responding to pathogens in an immediate and non-specific way, research now suggests that certain NK cells might also be involved in regulating the more targeted adaptive immune response. It is believed that a subset of NK cells in the lymph node is largely responsible for this function. With new knowledge about subsets of NK cells that have specialized functions, researchers are now looking at how these NK cells arise. It is possible that in addition to the NK cells that develop in bone marrow, other immature NK cells travel to different parts of the body where they mature in specific microenvironments that affect their function. In the lung this could mean a better NK cell response to cancer metastases or virus infection, whereas lymph node NK cells might be better at interacting with other immune system components. Timotheus Halim’s research seeks to find immature NK cells (NK cell progenitors) in sites other than the bone marrow. NK cell progenitors have already been found in the lymph node and lung. Now, Halim is using different mouse models to evaluate how important these progenitors are in forming mature NK cells. He is also determining if the NK cells that arise from these novel NK cell progenitors have specialized functions. A better understanding of NK cell development and function is critical in understanding the overall management of the immune system. Ultimately, this knowledge could help in the development of immunotherapy and other forms of treatment against cancer and infection.