Research Location: University of British Columbia

Asthma is a chronic lung disease affecting more than 2.8 million Canadians. It is estimated that numbers may rise to 400 million globally by 2025, substantially increasing both human and financial costs.

One possible explanation is that environmental exposures, including diesel exhaust (DE) air pollution (which usually increases as countries develop), may synergize with inhaled allergens in both the development and worsening of asthma, often leading to “lung attacks.” Exposure to air pollution may affect healthy gene expression in the lungs through “epigenetic modifications,” which change how cells “read” DNA. In preliminary studies, we confirmed that DE exposure caused numerous epigenetic changes, but we still need to understand how this causes the worsening of asthma symptoms. Moreover, we do not understand which components of DE (gases or particles) are driving these changes and which are more harmful. Therefore, I will leverage a state-of-the-art human exposure chamber and an ongoing clinical study to determine whether exposure to DE (with or without particles) and specific allergens affects epigenetics and gene expression.

Healthy and mild asthmatic volunteers will be recruited; over the course of four randomly-ordered visits (each separated by a month), they will be exposed for two hours to filtered air, DE, or particle-depleted DE, followed by inhalation of volunteer specific allergen or salt water. After 48 hours, cells lining the lungs will be collected and genetic material will be analysed.

Parallel to this clinical study, I will perform basic research experiments exposing lung cells to DE, and investigate the mechanisms through which these changes may occur. In addition, these experiments will examine how DE alters responses to asthma therapies and thereby the risk of “lung attacks.”

These studies may contribute biological plausibility and deepen our mechanistic understanding of emerging epidemiology, suggesting a role for air pollution in “lung attacks,” asthma development, and clinical outcomes.

Alzheimer’s disease (AD) is the most common cause of dementia. Unfortunately, there are no effective treatments for this devastating disease. The Alzheimer’s Society estimates that without new treatments, 1.4 million Canadians will be living with dementia by 2031.

Patients with AD often experience disrupted circadian rhythms, manifested as disrupted sleep. Although largely attributed to the underlying disease process, recent findings suggest that sleep directly impacts the pathophysiology of AD. A promising, emerging hypothesis for identifying novel treatments is correcting for changes in the body’s internal time-keeping mechanism, the circadian system.

It is largely assumed that disrupted rhythms are caused by the dampening of central suprachiasmatic nucleus (SCN)-driven rhythms; however, bright light and melatonin treatments, which have putative action on central SCN-driven rhythms, have only had limited success improving cognitive and non-cognitive symptoms. Alternatively, AD pathology may be disrupting synchrony between central and peripheral rhythms, which would cause similar symptoms but require different interventions.

Peripheral rhythms control the timing of cellular and metabolic processes in organs (e.g. liver) and brain regions (e.g. hippocampus). Synchrony ensures that physiological processes throughout the body occur at optimal times. In contrast, desynchrony is extremely detrimental to health and affects the clearance and repair mechanisms necessary to combat the misfolded proteins driving pathogenesis.

The goal of my research is to identify the cause of circadian dysfunction and potential targets for interventions. First, I will characterize the circadian phenotype in a mouse model by measuring behavioural rhythms and sleep. Second, I will measure bioluminescence linked to circadian gene expression, as a real-time reporter of oscillators throughout the body and brain. This has never been done in an AD model and allows us to directly evaluate synchrony between oscillators. Third, I will evaluate whether the “hunger hormone” ghrelin, which directly affects circadian rhythms, neuroplasticity, and memory processes, can improve synchrony between oscillators. Finally, in AD patients I will characterize circadian dysfunction and sleep, and evaluate whether ghrelin can aid in restoring circadian synchrony. My project is the first to explore whether the peripheral circadian system can be modulated as a therapeutic intervention in AD.

Brain swelling is a major cause of death following insults such as stroke and traumatic brain injury. This condition is often caused by an underlying swelling of neurons in the brain, leading to cell death. We currently have limited capacity to replace these neurons, and therefore must find ways to reduce swelling-induced cell death. Recent evidence suggests that an ion channel protein, called Panx1, is involved in this process. Ion channels essentially act as conduits between cells and the external environment. These proteins pass important signaling molecules to co-ordinate cellular responses, such as cell growth, movement, or death.

In this project, I will test whether Panx1 conduits promote cell death following neuronal swelling. I will also examine the mechanisms through which Panx1 channels are activated during neuronal swelling. Early experiments indicate that harmful molecules known as reactive oxygen species, which are created within swollen cells, might play a role in this Panx1 activation and neuron death. Reactive oxygen species cause damage to all cellular components, including proteins. Therefore, I will also examine whether these molecules activate Panx1 conduits by modifying parts of the protein structure.

This work contributes to unraveling the complex and still largely unknown mechanisms underlying neuronal swelling and death, and will guide future studies on therapeutic interventions for neuronal death following brain injury.

Our heart beat is a complex biochemical event. It relies on electrical signals, which can sometimes be disturbed, resulting in potentially fatal cardiac arrhythmias. One of the key parts involved in the contraction of heart muscle is a small ion known as calcium. Just prior to the contraction, calcium rushes into heart cells and triggers the contraction. Having the right amount of calcium at the right time is key for regular heart rhythms; too much or too little entry of calcium can be potentially fatal. The different compartments within the heart muscle cell are separated by membranes, which form barriers for many molecules. The calcium ions required for contraction of the heart muscle cells must pass through special gates. The sites where it all happens are formed by highly specialized protein channels that can open and close, thus determining the amount and timing of calcium release from one compartment to another. The most important of these so-called “calcium channels” is a large protein called ryanodine receptor. The gene that encodes this protein is one of the largest known genes, with literally hundreds of mutations documented to be the cause of the arrhythmia in patients.

Our laboratory collaborates with several cardiologists specializing in arrhythmias; we aim to determine how exactly the various mutations in this gene lead to the arrhythmias as a step to developing therapeutics. To do this, it is necessary to understand the overall three-dimensional structure of the calcium channel. Because they are too small to see with regular light microscopes, we will use a highly specialized technique called “X-ray crystallography”. By shooting X-rays at crystals of the channel, we can analyze the way these rays scatter off the atoms in the crystal and determine, through complex calculations, what the 3D structure looks like. By comparing 3D structures of the calcium channels of normal and diseased individuals, we can directly observe the mechanisms of the disease-causing mutations and come up with potential therapeutic strategies.

Inflammation is recognized as multi-cell network dysregulation with an immunological component. Among the many cell types involved in acute inflammation are macrophages, specialized phagocytes involved in many immune responses. These cells exist in different activation states dependent on their biological stimulus and are unknown to play either a target or anti-target role in the context of inflammation.

Understanding the role that macrophages play in inflammation is critical for the development of novel therapeutics and effective treatment strategies to alleviate the burden that this disease imposes on the Canadian public.  Our lab reported in 2014 a striking result in Nature Medicine (Marchant et al 2014) that the extracellular protease matrix metalloproteinase 12 (MMP12) secreted from macrophages traffics to the nucleus of virus-infected cells, binds specific DNA sequences and induces life-saving responses. MMP12 also cleaved intracellular substrates that were regulated at the mRNA level, providing dual regulation.

I hypothesize that MMP12 has autocrine roles in macrophages and a distinct roles according to activation state. The Overall lab has developed effective positional proteomic technologies to identify protease cleavage sites in vivo. Using our mass spectrometric method, Terminal Amine Isotopic Labeling of Substrates (TAILS) to identify MMP12 substrates (at their N terminus) during nuclear translocation in an in vivo murine macrophage model of differentiation (peritoneal macrophages) I will characterize the proteins being cleaved.

Upon stimulation with interferon gamma, macrophages differentiate into inflammatory M1-type, while stimulation with interleukin 4 induces differentiation into M2-like wound healing phenotype. Transcriptional effects of MMP12 will be examined using RNA-seq and Chromatin Immunoprecipitation sequencing (ChIP-seq) with Ilumina sequencing.

Combined with whole proteome characterization by LC-MS/MS and large scale substrate identification, this project will elucidate important molecular mediators of the immunological role of MMP12 in inflammation. These findings will be published in peer-reviewed journals, presented at conference meetings and applied for the development of therapeutics to effectively manage immunological disorders with macrophage-specific components.

Staphylococcus aureus infections are a leading cause of healthcare and community associated infections worldwide. Some strains of the pathogen have developed the ability to resist most of the classic antibiotics including penicillins and cephalosporins. There is an urgent need to develop new drugs that work against these resistant strains including methicillin-resistant Staphylococcus aureus, or MRSA.

A promising new set of antibiotic targets has recently been proposed involving the wall teichoic acid biosynthetic pathway of Gram positive pathogens. These long, acidic polymers are synthesized in the cell, transported out and ultimately attached to the growing outer cell-wall protective layer, a process essential to virulence and survival of MRSA in the infected host and in the environment. Small molecule inhibitor screens have identified compounds that block the action of one of these components, TarG/H, an intimately associated pair of membrane localized proteins that transport teichoic acids from the cytosol to the outer cell-wall layer. Learning more about the structure and function of the TarG/H transporter and its partner proteins in the pathway could allow scientists to design drugs that work much more effectively and specifically against Gram positive pathogens such as MRSA.

The goals of my research are therefore to solve the three-dimensional structure of the purified TarG/H transporter in native, mutant and inhibited forms at atomic resolution using X-ray crystallography and secondly, to characterize the molecular details, using single particle cryo-electron microscopy, of how TarG/H binds with other proteins involved in making and transporting the teichoic acid chains to the outer regions of the cell. The ultimate goal of this work is to enable the structure-guided design of potent new antibiotics that block TarG/H action and MRSA virulence.

Streptomyces bacteria are the source of nearly half of our clinically used natural antibiotics. Production of bioactive compounds is usually linked to complex networks of signal-sensing proteins that regulate genes. How do these complex systems come together? Recent advances in molecular biology provide the tools to uncover the detailed mechanisms that underlie the evolution of vast regulatory networks that create complex biological systems.

This project will examine the molecular mechanisms that allow two regulatory proteins that arose from a single duplicated gene in Streptomyces coelicolor to regulate distinct and critical components of sporulation, quorum-sensing, and antibiotic synthesis.

By using a combination of ancestral sequence reconstruction and experimental protein evolution, this project will explore how these proteins took up new regulatory roles, resulting in the current system. Furthermore, we will recreate the intermediate steps along this evolutionary trajectory, in order to discover how the functions of these essential genes were rapidly separated by evolution. Finally, using a system of experimental laboratory evolution, we will alter these proteins to more effectively regulate their targets in an attempt to accelerate antibiotic production.

Ultimately, we will map out some of the ways in which new regulatory proteins can evolve through duplication and modification of genes for existing regulatory proteins. We will also aim to provide new mechanisms that can accelerate the production of antibiotics for clinical use.