Microfluidic instrumentation for microRNA expression profiling in hematopoietic stem cells

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.

Assembly of the type III secretion system in enteropathogenic E. coli and C. rodentium

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.

Intravesical mucoadhesive nanoparticulate drug delivery systems for application in bladder cancer

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.

Causal Attributions and Self-Conscious Emotions in Coping with Alcoholism

Although there are large individual differences in recovery rates from alcoholism, little is known about the emotional factors that underlie these differences. Studies suggest that shame and guilt, two negative self-conscious emotions (emotions that require self-evaluations), may have divergent effects on a range of health outcomes. Specifically, shame promotes a range of negative outcomes, such as depression, whereas guilt has more positive effects, including empathy and high self-esteem. In addition, two distinct kinds of pride — “authentic” and “hubristic” — may also have divergent effects. Dr. Jessica Tracy is researching the influence of these four emotions on recovery from alcohol addiction. She is testing whether newly-sober members of Alcoholics Anonymous (AA) who experience guilt and authentic pride, rather than shame and hubristic pride, enjoy greater health and recovery benefits over time. Tracy is also testing whether the thought processes that promote these emotions contribute to health outcomes, and if so, whether specific self-conscious emotions account for these effects. This research is unique in its emphasis on self-conscious emotions, which may play an important role in addiction. The findings could lead to new treatment methods for clinicians, such as targeting these important emotions.

Combined analyses of the environmental risk factors for Lung Cancer in British Columbia and the resulting burden from particulate air pollution, arsenic and radon exposures

Every year, approximately 2,200 British Columbians die of lung cancer and an additional 2,700 are diagnosed with the disease. While tobacco smoke is the primary cause of lung cancer in BC, approximately 25 per cent of lung cancer cases are not attributable to smoking. In fact, lung cancer in non-smokers accounts for the seventh leading cause of cancer death. Studies have found that environmental factors increase the risk of lung cancer in both smokers and non-smokers, including exposure to particulate air pollution, residential radon (a radioactive gas that leeches from soil and building materials), and arsenic in drinking water. These three exposures are widespread throughout BC and affect a large percentage of the population. However, no study has been conducted that determines how many, where and to what levels the BC population is exposed to these environmental carcinogens, or the risks posed to lung cancer development and the resulting burden on lung cancer in BC. Perry Hystad is creating current and historical provincial exposure models for particulate air pollution, radon and arsenic. These results are linked with provincial lung cancer data collected by the National Enhanced Cancer Surveillance System. His research will include exposure assessment, epidemiological analysis and burden of disease calculations. Hystad’s findings will shed light on the risks posed by these environmental carcinogens across BC. Ultimately, this work could help identify potentially susceptible populations and contribute to the development of prevention measures.

The immunomodulatory effects of host defence peptides on dendritic cells

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.

Environmental sensing and signal transduction in pathogenic bacteria

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.

Examining the role of N-linked glycosylation on the maturation and cell surface trafficking of hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels

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.

Viral host interactions of the Dicistroviridae family

All successful viruses have evolved strategies to infect host cells and disrupt normal cell functions. However, the host can counteract these strategies by using its natural antiviral responses to detect and defend against viruses. Revealing the molecular mechanisms between the battle of the virus and host is vital in the fight against many of today’s viruses. Some viruses use an internal ribosome entry site (IRES) to infect cells. Molecular machines in cells called ribosomes translate genes into proteins, but viruses with an IRES can hijack the ribosome to replicate their viral proteins instead. IRESs are found in a number of human viruses, including polio, hepatitis C, herpes and HIV, but there is limited understanding of how these mechanisms work. Understanding the ways in which a virus hijacks the ribosome function is the focus of Dr. Eric Jan’s laboratory. He uses a unique IRES found in an insect virus called the cricket paralysis virus (CrPV). Jan’s previous work was critical in delineating important CrPV IRES functions. Building on this work, he plans to map the specific IRES elements that interact with the ribosome. He will also determine how CrPV disrupts cellular function that leads to IRES activity in Drosophila (fruit fly) cells, and elucidate the host antiviral response in these cells. The study of Drosophila antiviral responses will contribute to knowledge about fundamental virus-host interactions in humans. The research could lead to new drug targets for inhibiting viral IRESs and therapies that can augment antiviral responses. An exciting future goal will be to exploit viral IRESs to prompt the destruction of virus-infected cells – taking advantage of a viral mechanism against itself.

Evaluating changes in the social, sexual, and drug-using networks of men who have sex with men (MSM) following testing and an enhanced prevention intervention for acute HIV infection: Applying social …

The primary purpose of this research is to provide insight into how to reduce the rising rate of HIV infection among men who have sex with men (MSM) in Canada. The number of HIV infections in Canada continues to rise annually, with MSM accounting for a disproportionate number of these new infections. Evidence now suggests that individuals are highly infectious during the early stage of HIV infection, and these individuals are responsible for generating 11 to 40% of new infections annually. Indentifying individuals as soon as possible after acquiring HIV infection and providing a risk reduction intervention is necessary to curtail this HIV epidemic among MSM. Traditional HIV antibody testing is unable to detect HIV during this early infective stage; a new technology (nucleic acid amplification test-NAAT) that can identify early HIV infection has recently become available and will be used in this study. Traditional epidemiologic methods have focused on individual-level risk factors (e.g. number of sexual partners, condom use) but these have failed to fully explain transmission dynamics or control epidemic growth. Social network analysis (SNA) is a methodology that considers relationships, and interactions among those with varying risk characteristics in the social context. SNA is increasingly being applied to infectious disease epidemiology and research suggests this is a useful approach to the study of HIV transmission dynamics. MSM who acquire HIV are likely to belong to social, sexual, and drug-using networks whose members have similar risk behaviours. We hypothesize that early HIV detection and risk reduction interventions targeting these individuals, and their networks, will reduce the transmission of HIV. This research will employ SNA to examine and describe the social, sexual, and drug-using networks of MSM diagnosed with early HIV infection using NAAT. Network information will be used to identify networks containing high levels of risk behaviour and networks with a high incidence of early HIV infection. An enhanced prevention intervention will be developed with MSM community participation and implemented among individuals within the network who are engaging in high risk behaviour, and who are prominent in the network. In addition, SNA will be used to evaluate how the networks change over time (post-intervention) considering network structure, sexual mixing patterns, rate of partner change, risk behaviour, and to determine forward HIV transmission events.