Flicking the switch: cross-species translation of rapid context-based switching between tasks

Successful interaction with a constantly changing world requires behavioral adaptation. Unraveling the mechanisms underlying flexible control is essential to stimulate advances in the treatments of disorders where deficits in these functions are a core symptom, such as schizophrenia and Parkinson’s disease. For humans, this type of behavior is commonly assessed using the task-switching paradigm, which uses cues to instruct on a trial-by-trial basis which of two tasks to perform. Comparing behavior when the task is repeated to when it is switched allows measuring rapid behavioral adaptations. Existing tests of behavioral flexibility in rodents (e.g. set shifting tests) often assess the ability to learn that a rule changed, yet real-life situations often entail contextual cues explicitly indicating that changes in behavior are required. In addition, current shifting paradigms do not allow assessment of trial-by-trial switching between tasks, as human assays do. An important step in preclinical animal research is to develop tests of behavioral flexibility that directly translate between species.

Previous research I have conducted used a combination of brain imaging, stimulation, and pharmacology to assess the neural basis of adaptive flexible behavior in humans. My work revealed important roles for the striatum, prefrontal cortex, and the neurotransmitter dopamine in task switching. However, these approaches lack the spatial and pharmacological specificity required to answer questions about the causal and specific role of these regions and transmitter systems. Thus, to complement my work with human subjects, I used a novel translational version of a human task-switching paradigm that is suitable for testing in rodents.

In my post-doctoral work, I aim to fully explore the contribution of specific brain circuits to these processes (focusing on the striatum and prefrontal cortex). I will also investigate how the transmission of the neurotransmitters dopamine and gamma-Aminobutyric acid (GABA) mediate successful task-switching. This is important because dysfunction in these transmitter systems underlie numerous psychiatric disorders associated with impairments in these functions, such as schizophrenia and Parkinson’s disease. These studies will be complemented by those using temporally-discrete optogenetic silencing. This will allow the trial-by-trial manipulation of brain circuits and clarify the precise moments when activity in these circuits are necessary for facilitating flexible behavior.

High-resolution structures of the cardiac Ryanodine Receptor: a target for arrhythmia-causing mutations

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.

Determining the virulence determinants of Fusobacterium nucleatum to define diagnostic and therapeutic targets for colorectal cancer

A substantial portion of the cancer burden worldwide is attributable to infectious agents (viruses or bacteria). Some of these can directly cause cancers, others can facilitate cancer development, and the rest may have no causative role but their existence can indicate the presence of a cancer or risk of developing one.

Recently, Fusobacterium nucleatum, a bacterium present on mucosal surfaces, has been found to be highly elevated in a subset of colorectal cancers. F. nucleatum is an invasive bacterium that can cause acute oral and gastrointestinal infections and can act as a pro-inflammatory agent, thus it is a reasonable candidate for having a facilitating role in tumorigenesis. However, F. nucleatum is also well recognized as a benign resident of mucosal surfaces in the absence of pathology. The reason why F. nucleatum may in some cases be pathogenic and at other times an apparently benign, commensal organism is not yet completely understood.

The overall goal of this study is to identify gene(s) associated with F. nucleatum virulence, and to determine how expression levels of these genes are modulated during infection using RNA-Seq. The Canadian Cancer Society estimates that currently 12 percent of all cancer deaths in Canada are attributed to colorectal cancers; a tendency toward late diagnosis indicates a dire need for simple strategies to help detect colorectal cancers early. The finding that F. nucleatum is strongly associated with a significant number of colorectal cancers cases raises the possibility of developing a simple diagnostic pre-screen for the disease, enhancing early detection rates. The proposed work will identify the F. nucleatum genes that are associated with the disease, creating a signature that will markedly increase specificity of new screening tests. Moreover, this study will indicate how pathogenic F. nucleatum strains cause disease, dramatically increasing our knowledge of this enigmatic bacterium and its interactions with host cells that lead to oncogenesis.

Armed with this new knowledge, it will be possible to develop novel diagnostics, and create new tools such as vaccines to combat, and even prevent, infection. Knowledge translation activities for this study will include presenting results at conferences, writing papers and building on the network between the BC Cancer Agency and our anaerobic bacteriology collaborators at the University of Guelph.

Exploring the autocrine transcriptional role of the macrophage-specific matrix metalloproteinase (MMP12) in phenotypically distinct macrophages in the context of acute inflammation

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.

Toward personalized immunotherapy: defining mechanisms of immune suppression across the molecular subtypes of ovarian cancer

Ovarian cancer affects approximately 1,700 women per year in Canada. Current treatment involves surgery and chemotherapy, which is initially effective in most cases. However, most patients relapse with chemotherapy-resistant tumors within a few years of treatment; this highlights the urgency for new, effective treatment strategies. Encouragingly, the immune system has a strong influence on survival in ovarian cancer. Tumors that are densely infiltrated by T cells (a type of immune cell) are linked to improved prognosis. However, a large proportion of patients lack dense T cell infiltrates. Instead, T cells are trapped in the surrounding stromal regions of the tumor and fail to make direct contact with tumor cells.

I hypothesize that the infiltration of T cells is inhibited by suppressive mechanisms in these stromal regions and with better understanding, these mechanisms can be reversed by immunotherapy. One objective of this project is to determine whether T cells that are trapped in stromal regions are capable of recognizing tumor cells. If so, then these T cells have the potential to recognize and eradicate tumors. Another objective is to identify and then block the signals by which stromal cells carry out suppressive functions. I will assess the effects on T cell infiltration and tumor regression following this blockade. This project will facilitate the development of new treatments that release T cells from the suppressive effects of stroma to launch more powerful attacks against ovarian cancer and related malignancies. The possibilities of using off-patent fibrosis drugs for cancer treatment will be investigated; this might result in an inexpensive, effective new form of immunotherapy, thus reducing costs and increasing the number of patients benefitting from these approaches. Since the BC Cancer Agency’s Deeley Research Centre (BCCA-DRC) is able to perform clinical trials, the work can be directly implicated into clinical research.

This research will be presented at both national and international conferences and published in international peer-reviewed journals. The BCCA-DRC’s clinical trials program will also provide me with ongoing opportunities to speak to patient support groups, clinicians, and lay audiences at forums focused on education, awareness and philanthropy.

Structural characterization of the transporter protein TarG/H in wall teichoic acid biosynthesis of Gram positive bacterial pathogens

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.

Development of a hierarchical algorithm to investigate the role of long non coding RNA regions in the etiology of asthma

Asthma is a complex disease caused by a combination of genetic, epigenetic and environmental factors.

Although several studies have attempted to identify the specific genes associated with asthma, the underlying genetic mechanisms are still unclear. Genomic imprinting, an epigenetic phenomenon that occurs early in life whereby only one gene copy is active and the other  parental copy is fully methylated and hence inactive (“parent-of-origin effects”), may be involved.

I performed the analysis of the first large-scale genome-wide association study (GWAS) of parent-of-origin effects in asthma on data collected from three Canadian family-based studies/cohorts. Preliminary results strongly suggest the involvement of long non-coding (lnc) RNA.

lncRNAs are known to be involved in genomic imprinting. I hypothesize that lncRNAs identified from the parent-of-origin effects in asthma are involved in imprinting.

Due to their length and low information density, lncRNA regions are very time- and cost intensive to confirm and study. I will develop a hierarchical algorithm that will automate the selection of lncRNA regions and specific sites to investigate for DNA methylation.

The ability to select important lncRNA regions in an efficient and automated manner will result in increased efficiency for researchers, and will save time, materials and personnel costs. The selection algorithm will be added to our collection of web-based tools on the Genapha website and will be widely used by researchers interested in genomic regulation.

Characterizing and optimizing mechanisms of antibiotic synthesis in Streptomyces coelicolor

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.

Implementing Mental Health Recovery Guidelines into Services: A Pan-Canadian Study

The overall objective of this project is to implement and evaluate Canada’s new Mental Health Recovery Guidelines in British Columbia and four other Canadian provinces. The Mental Health Commission of Canada (MHCC) developed the Canadian Guidelines for Recovery-Oriented Practice to move beyond policy to implementation of recovery oriented practices in health care organizations.

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