The role of secreted protein acidic rich in cysteines (SPARC) in neurogenesis and gliogenesis in the embryonic and adult mouse brain: applications to models of Cerebral Ischemia Reperfusion Injury

Stroke is the primary cause of adult disability in Canada. Recovering brain function after stroke is dependent on the brain’s ability to rewire itself and replace tissue that has died during the stroke – something that is difficult to achieve in the adult brain. Rewiring the brain requires that existing neurons sprout new fibres (axons) and connect to other neurons in a way that allows proper functioning of neural circuitry. Recovery also involves the birth of new cells to replace dead cells and to form functioning connections with new and existing neurons. These processes all occur within the extracellular matrix (ECM) – a network of fibrous proteins, gel-like sugars and linking molecules – and are promoted by a large number of growth factors and intercellular signalling molecules. Anthony Berndt’s research focuses on the role of the SPARC protein in the generation of new neurons. SPARC binds to the ECM and regulates the potency of growth factors that normally promote cell division and migration. Berndt is examining the influence of SPARC on the development of the embryonic brain and on the generation of new neurons in the adult brain. His studies will determine if SPARC’s presence or absence affects the rate or manner in which brain tissue regenerates after stroke. He hopes to formulate an approach that will prompt neural stem cells normally found in the adult brain to follow the developmental steps required to form functional tissue after stroke. By understanding the function of SPARC after brain injury, he could also determine at what point of recovery such an intervention would be of greatest use. By understanding the role of SPARC, Berndt’s research could eventually lead to improved therapies for treating major brain injuries by augmenting the body’s natural repair processes.

Inhibition of productive influenza A virus (H5N1) infection by secretory pathway SERine Protease INhibitors (serpins): A basis for the development of novel antiviral therapy

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.

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.

Computational simulation of transcription factor binding for the prediction of regulatory regions in DNA sequences

The regulation of when and where a gene is turned on (gene expression) is a complex process, fundamental to how a cell behaves and interacts with the environment around it. Abnormal changes in gene regulation are associated with many diseases, including cancer, asthma, and obesity. One class of proteins involved in the regulation of genes are transcription factors (TFs). TFs recognize and bind to short sequences of DNA near the genes they regulate and act to increase or decrease the expression of their target gene. The binding interaction between TF proteins and DNA is affected by an array of biophysical factors in the cell nucleus, making this complicated process a good candidate for computational modelling through bioinformatics. Bioinformatics is a relatively new field in which computational approaches are used to study biological problems; a field that unites computer science, statistics and the life sciences. Rebecca Hunt Newbury is developing a software simulator to model the TF binding process in a dynamic, interactive setting, with the intent of predicting the locations in a DNA sequence at which TFs will bind and regulate genes. From there, she will begin to incorporate the spatial relationships and combinatorial interactions between TFs that result in the different expression responses of genes. Hunt Newbury’s research will contribute to clearly defining the regions of DNA that participate in regulating a gene. Her work may ultimately contribute to new approaches for combating diseases caused by abnormal gene regulation.

Integrated microfluidic technologies for optimization of hematopoietic stem cell expansion

Blood contains different types of specialized cells. Red cells are responsible for oxygen transport, white cells ensure body’s defense against infections, and platelets initiate clotting to limit the loss of blood after an injury. These cells are constantly renewed, and are manufactured in the centre of the bones in a sponge-like tissue called bone marrow. Hematopoietic stem cells are a small subset of cells found in bone marrow that have the astounding ability to self-renew and divide, and to differentiate into a variety of mature blood cells. They are often used to treat blood-related diseases or given after cancer treatment. Although stem cells have great potential for regenerative medicine, they are extremely rare and they are difficult to expand in the lab, because they very readily differentiate into other cell types. The multiple factors that influence their self-renewal are poorly understood. Véronique Lecault is exploiting the potential of microfluidic technology, an engineering advance that allows thousands of different experiments to be performed in tandem upon a device the size of a microscope slide. Across rows and rows of miniature cell culture chambers, individual hematopoietic stem cells can each be exposed to different chemical conditions and tracked over time. This makes determining the specific environments that will allow the cells to be expanded much more efficient. This technology could lead to the ability to produce more hematopoietic stem cells for use in disease therapies. It could also help researchers gain a better understanding of stem cell biology, perhaps leading to the discovery of new ways to identify and purify these rare cells.

Investigation of genetic networks involving genes that confer chromosome stability in S. cerevisiae and C. elegans

Chromosomes are found in all organisms that have a cell nucleus, and carry the organism’s hereditary material. During cell division, chromosomes divide and distribute equally to the daughter cells. Errors in the division process can result in daughter cells that contain an incorrect number of chromosomes. Known as aneuploidy, this state is responsible for many genetic diseases and is characterized by a specific type of genome instability known as Chromosome Instability (CIN). Because chromosome stability is a fundamental requirement across all organisms, it can be studied using simpler organisms, such as baker’s yeast. Genome wide screens on yeast have identified approximately 300 genes important for maintaining chromosome stability (CIN genes). A subset of these genes has also been found to be mutated at an elevated rate in some human cancers, which suggests that these genes contribute to tumour progression and development. Mutations in these key genome stability genes may also represent an Achilles’ heel for tumours. Enhancing chromosome instability to a point where tumour cells can no longer function and reproduce could halt their division or lead to cell death. Jessica McLellan is studying the subset of CIN genes mutated in colon cancer. She specifically aims to identify genes that, when mutated in combination with a CIN gene mutation, lead to cell death. By exploiting the mutations seen in many types of cancers, this project could lead to the development of novel cancer therapeutics that are less harmful to non-cancer cells than current treatments. McLellan’s research will increase our understanding of the complex biological processes that ensure genome stability and the mechanisms by which these processes can become deregulated.

The Role of beta-catenin Stabilization in the Synaptic Pathology of Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurological disorder characterized by the loss of cognitive function and an inability to process and store new memories, caused by the progressive death of neurons (brain cells). It is becoming increasingly evident that before neurons die, changes can be observed at their synapses – the junction between neurons across which information is transmitted. Deficits in synaptic function, loss of synapses, and a reduced ability to form new synapses are the major correlates of dementia. It is therefore crucial to understand the basic biology of synapses, and how these processes are affected in AD. The cadherin family of cell adhesion molecules and their intracellular partner, b-catenin, play a critical role in regulating the formation and remodelling of synapses. Both molecules also associate with presenilin-1 (PS1), a protein that normally degrades (breaks down) b-catenin. Mutations in the PS1 gene account for nearly 70 per cent of early-onset familial AD cases. Fergil Mills is investigating the effects on neurons when b-catenin is not normally degraded by PS1. Using isolated neurons and a mouse model, he is characterizing the synaptic consequences of stabilizing (maintaining) b-catenin in neurons, and determining the molecular mechanisms of b-catenin in the development of synaptic structures. These studies will help determine whether b-catenin stabilization leads to the synaptic pathology and cognitive deficits seen in AD. Mills’ studies will further our understanding of synapse pathology and cognitive deficits, and could lead to new treatments for patients with AD or other neurological disorders.

Escape from mitotic arrest; compounds that induce mitotic slippage as tools for chemical biology and potential therapeutic agents

One of the main characteristics of cancer is the uncontrolled division and growth of cells. Because tumour cells divide very frequently, tumour growth can be selectively stopped by inhibiting cell division. Commonly-used cancer drugs act by effectively “freezing” cell activity right in the process of division. This is done by interfering with mitosis – the point in the cell cycle where the nuclear chromosomes have been duplicated and separated to form two daughter cells. Unlike normal cells, some cancer cells eventually bypass this frozen state in a process known as mitotic slippage. However, the resulting cells now contain too many sets of chromosomes. Researchers still don’t know what happens to these drug-treated cancer cells as a result of mitotic slippage – whether they are able to start dividing again, whether they remain in an arrested state indefinitely, or whether they die. Jenna Riffell is investigating the fate of cancer cells after drug treatment. She is identifying chemical compounds that can stimulate drug-treated cancer cells to undergo mitotic slippage, and monitoring what happens to these cells. Her hypothesis is that the rate of mitotic slippage can be increased with chemicals, and will prompt growth arrest or cell death. If proven successful, this approach could be used for the development of future cancer therapies. In addition, Riffell’s work will be useful for studying the underlying biochemical mechanisms of cell division.

RNA interference analysis of human embryonic stem cells in a microfluidic device

Embryonic stem cells (ESCs) are considered the pinnacle stem cell due to their unlimited capacity to self-renew. Since ESCs can differentiate to produce precursors to almost all types of cells (pluripotency), they may hold the key to curing diseases that are caused by the loss of function among specific cell types such as Parkinson’s disease, Alzheimer’s disease, spinal cord injuries, and type 1 diabetes. In order to understand differentiation and self-renewal, researchers compare gene activity between ESCs that have undergone differentiation and those that have not. Scientists can also intentionally turn off (silence) specific genes to see if there is an effect on cell pluripotency. Conventional techniques are time consuming and only allow for the analysis of large numbers of cells at any given time. The cells in these populations are rarely homogeneous as the environments around them are not precisely controlled. To address these issues, researchers have developed a cutting edge technology called microfluidics. Microfluidic technology involves constructing small chips with thousands of fluid-filled chambers that can each contain a single cell. This reduces the number of cells used and the cost of each experiment, and allows thousands of experiments to be performed on a single chip simultaneously. Darek Sikorski is constructing a microfluidic device for the purpose of sustaining and examining the behaviour of ESCs. Once validated, this will allow for large-scale experiments to be performed where specific genes are silenced in each cell, allowing for the analysis of thousands of genes at once. This technology has the potential to greatly accelerate research into how ESCs grow, communicate and differentiate. Ultimately, this could lead to ways to use ESC lines to produce specialized tissues that can cure certain diseases.

Cross-species characterization of proteins involved in genomic stability

Cell division is required for the development, growth, renewal and repair of all organisms, including humans. During cell division, the cell’s genomic information – stored in the chromosomes – is replicated (copied) so that each of the resulting daughter cells receives a complete set of genetic material. Proper segregation of chromosomes to daughter cells during cell division is critical to their continued survival, and chromosome missegregation has been observed in a majority of cancers. However, the connection between the increased rate of chromosome missegregation observed in cancer, and the development of the cancer itself, remains unclear. Because chromosome segregation is a fundamental mechanism in all organisms, baker’s yeast can be used as a model to study this process. Many yeast genes and proteins involved in chromosome segregation appear to be conserved across evolution between yeast and humans. One such protein in yeast is Ctf4, which has a human counterpart called Wdhd1. Ctf4 plays a critical role in ensuring faithful chromosome segregation during cell division, though its biochemical function is not fully understood. The yeast CTF4 gene also appears to interact genetically with a number of yeast genes whose human counterparts are known to be mutated in cancer. Derek van Pel is further characterizing both the human and yeast proteins’ roles in this chromosome segregation. He is using biochemical techniques to isolate any other proteins within the cell with which these proteins interact. Such interactions will then offer clues to the function of these proteins. Derek’s research will shed light on the role of Ctf4/Wdhd1 in ensuring proper chromosome segregation, and possibly the role of this process in cancer development. Understanding these genetic interactions may also lead directly to new therapeutic avenues for cancer.