Research Location: University of British Columbia - Point Grey

MicroRNAs (miRNAs) are small RNA molecules that regulate the expression (activation) of genes. Recent studies of miRNA expression implicate these molecules in early development, brain development, cell proliferation and cell death. They are also implicated in disease states, such as chronic lymphocytic leukemia. Determining how, when, and where miRNAs are produced and function in cells and tissues would have profound impact on medical disciplines ranging from embryology to cancer diagnosis and therapy. The genes expressed in miRNA differ between developing and mature tissues, and comparing normal tissues to tumour tissues also reveals different miRNA expression profiles. Further studies looking at differentially expressed miRNAs could help identify those miRNAs involved in human cancer development. Unfortunately, traditional expression profiling techniques are laborious, costly, slow, or lack the sensitivity to effectively screen populations of cells and quantify miRNA content. A promising approach to overcome these limitations is the use of microfluidics technology. This technology involves constructing small chips with thousands of fluid-filled chambers, which 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. Adam White is developing a microfluidic device capable of inexpensive miRNA expression profiling of many single cells at the same time. Upon successful development of this new microfluidic tool, he will work with other scientists to look for differentially expressed miRNAs in blood related cancers such as acute myeloid leukemia. The development of a microfluidic device for single cell analysis of miRNA would greatly accelerate the identification of those miRNAs involved in cancer development, and ultimately improve methods of cancer diagnosis and treatment.

Pathogenic E. coli bacteria cause severe intestinal infection and diarrhea in humans, leading to millions of cases of infection every year. The virulence of pathogenic E. coli and many other gram-negative bacterial pathogens (a bacteria type characterized by its membrane structure) is determined by the type III secretion systems (TTSS). TTSS are multi-protein macromolecular “machines” that mediate the secretion and translocation of bacterial proteins into the cytoplasm of eukaryotic cells – a key step in causing infection. Most of the 20 unique structural components constituting this secretion system are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system, another protein secretion system that has been extensively studied. However, real hard biochemical analysis of TTSS has not been done. Dr. Hendrikje Oldehinkel is investigating how the TTSS is built and how it works. She is dissecting protein to protein interactions and assembly of the type III secretion apparatus in enteropathogenic E.coli and in a mouse pathogen, Citrobacter rodentium. Her work employs a combination of biochemical techniques: electroforesis, immunoblotting, stable isotope labelling, mass spectrometry and electron microscopy. Oldehinkel’s research will contribute to the understanding of the structure of TTSS and the role the components of the type III secretion system play in the architecture and function of the system. Understanding TTSS is important for finding new therapeutic options against not only gram-negative bacterial pathogens, but also against many other disease-causing pathogens.

Urinary bladder cancer is one of the most commonly diagnosed malignancies in North America. The great majority of cases are superficial carcinomas, where the tumour is confined to the inner layer of the bladder wall. The most common treatment method is known as transurethral resection, which involves the surgical removal of tumour nodules from the bladder wall. However, there is a high rate of tumour recurrence after this surgical procedure. Intravesical chemotherapy, which involves instillation of one or more chemotherapeutic agents into the bladder following resection, has become the treatment of choice for superficial carcinoma. Unfortunately, the major limitation of this treatment is the rapid and almost complete washout of the drugs from the bladder on first void of urine, and low exposure of chemotherapeutic agents to the tumour sites. This can lead to treatment failure. Although drug treatments via bladder instillation following resection have decreased tumour recurrence rates, overall mortality rates for bladder cancer have not changed in Canada over the last several years. New approaches are needed to treat this type of cancer. Clement Mugabe is working to develop formulations of drugs that are not easily flushed out of the bladder. This can be achieved by creating drugs in the form of mucoadhesive nanoparticles –so tiny and sticky. Mucoadhesive nanoparticle formulations have the potential to adhere to the bladder wall, increase drug uptake into bladder tissue and thereby increase the effectiveness of drug treatment. Mugabe’s research will lead to novel formulations, and new information about the factors that influence uptake of drugs into the bladder wall.

Modern day vaccines are effective at preventing infections such as tetanus, influenza, polio and many others. To ensure full protection from illness, some vaccines require more than one immunization. This is commonly known as a booster shot. In developed countries, getting vaccinated usually means nothing more than going to the clinic. In developing countries the process is not so straight forward. Limited access to, and availability of vaccines makes widespread immunization a difficult process. The fact that people may have to return for a booster shot only compounds the problem. For all of the above reasons, there is clearly a need for improved vaccines in developing countries. Our laboratory is studying ways to create effective single-dose neonatal vaccines for developing countries. This means the vaccine would be given shortly after birth, and there is no need for a booster shot to ensure complete protection. Such a vaccine would alleviate the previously described difficulties. Specifically, our lab is developing more effective vaccine adjuvants. An adjuvant is simply any component added to a vaccine that will interact with the immune system to improve protection. We believe that a class of proteins known as host defence peptides (HDPs) will act as effective vaccine adjuvants. HDPs are short proteins, found almost ubiquitously in nature (microorganisms, insects, plants and mammals for example). Historically, the function of HDPs has been primarily to kill invading bacteria and viruses. Recent research conclusively shows that some HDPs are capable of altering the way in which immune system responds to an infection. My research will focus on how HDPs interact with and important type of immune cell known as a dendritic cell. Dendritic cells (DCs) circulate in the body in an “”immature”” form. When they encounter anything foreign (for example, bacteria or viruses), they become “”activated,”” capture the invader, and alert the immune system so it can mount a full response. They are now said to be “”mature.”” For this reason, DCs are a very unique type of cell. They are part of the front line of defence, yet they are also critical in generating the full immune response, which develops shortly after. We believe that HDPs will influence DCs in such a way that they will promote an efficient immune response in the context of vaccination. I hypothesize that HDPs impact DC function, activation, and maturation by altering specific genes and proteins important to DCs. This hypothesis has lead me to develop five goals to guide my research. I will provide an overview of these goals: 1) Bioinformatics. My preliminary experiments have tracked how HDPs influence the expression of 16,000 genes in mouse DCs. Such a large amount of data needs to be handled by a computer. Using specially designed programs, I am able to sort through the vast amounts of data and determine the broad trends occurring in response to HDPs. Furthermore, I am able to look at how small groups of genes behave in the context of their larger gene families; 2) IRAK-4. Results show that one peptide altered the behaviour of an important protein called IRAK-4. IRAK-4 is known to be important for specific immune responses. I will further analyze how this protein functions in the presence and absence of HDPs and other immune stimuli in DCs. I will also determine how proteins related to, and dependent on IRAK-4 will behave in response to HDPs; 3) Lyn Kinase. Another interesting finding was the altered production of Lyn, another protein important for proper DC function. I will continue analyzing the behaviour of Lyn in DCs in response to HDPs. I will also study the consequences of Lyn deficiency and determine its effects on HDP function. 4) DC Type. There are different types of DCs depending on where in the body you look, each performing similar, yet distinct functions. Currently it is not known how different types of DCs respond to HDPs. A lot of DC research is done with mouse DCs because they are relatively easy to generate compared to their human counterparts. The comparative responses of human and mouse DCs to HDPs are not well understood. For these reasons, I will be experimenting in multiple DC types, and in both human and mouse DCs. 5) In vivo peptide effects. Using the previously described experiments as a guide, I will examine how HDPs affect whole mice. We have access to mice deficient in all of the genes listed above, and this will be useful in determining the role of specific genes on the scale of a whole animal. At the completion of this project, I will have gained a comprehensive understanding of how HDPs influence DCs, with the goal of using this information to provide better vaccine adjuvant candidates aimed at developing countries.

The ability of an organism to perceive its environment and to respond accordingly is a key survival factor for any species. An important example of environmental sensing is the evolution of antibiotic resistance among bacteria, which is a significant challenge for fighting and containing infections in hospital and community settings. These adaptations by disease-causing bacteria allow them to sense the presence of drugs and respond by producing agents to resist the antibiotic. Multidrug resistant bacterial strains have emerged and are increasing in frequency, making treatment more costly and such infections more lethal. Dr. Gerd Prehna is studying the structures and pathways within bacteria that enable this to happen. He is studying in salmonella a novel antivirulence pathway that regulates bacterial populations within the host. Disruption of this process would lead to an unorganized effort by a bacterial infection to maintain itself within a host, reducing its ability to cause illness. He is also studying methicillin resistant staphylococcus aureus, or MRSA, which has evolved a complex sensor molecule that binds to antibiotics and then relays a signal for the bacteria to express resistance factors. By solving the complete three-dimensional structure of this antibiotic sensor, he hopes to determine the mechanism by which this signal is relayed. By learning more about how disease-causing bacteria detect antibiotics, communicate with each other, and collectively mount a defense against these drugs, Prehna hopes this knowledge might be exploited to block sensory and communication pathways, making the bacteria once more susceptible to antibiotics.

Cardiovascular disease remains the number one killer in British Columbia. These diseases include cardiac arrhythmias, which cause the heart to beat too slowly, too quickly, or in an uncoordinated fashion. Arrhythmias arise from dysfunction of the heart’s natural pacemaker: the sinoatrial node. The sinoatrial node consists of a group of cells responsible for generating the electrical impulse that controls normal rhythmic contraction and relaxation of the heart. In order to generate these electrical impulses, these cells possess a group of proteins known as ion channels. These proteins allow ions to selectively cross the cell membrane barrier, generating an electrical impulse that spreads to neighbouring cells. One particularly important family of ion channels are the HCN or ‘pacemaker’ channels which are responsible for generating the spontaneous activity of the sinoatrial node. The assembly and trafficking of these channels to the cell membrane is vital for ensuring our hearts beat in a regular fashion. How the cell accomplishes this task remains an unanswered question. Hamed Nazzarisedeh’s research attempts to uncover the underlying mechanisms that help regulate or contribute to the trafficking of HCN channels in the heart. Specifically, he is examining the role in which N-linked glycosylation of these proteins may factor in this regulation. His research will contribute to further our knowledge about how various forms of cardiovascular disease associated with HCN channel disruption arise in the heart. Ultimately, this work could aid in the discovery of novel treatment strategies.