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The major research focus of my lab revolves around acute myeloid leukemia, and preleukemias referred to as myelodysplastic syndromes. Within this context we are interested in various posttranscriptional mechanisms that drive these cancers, how aging increases the risk of developing leukemias, and how to potentially target genomic and epigenomic vulnerabilities.
The ideal applicant is passionate about discovery, is creative and able to independently drive a research project. They are keen to innovate by incorporating novel methodologies to answer key questions in the field. Excellent verbal and written communication skills are necessary, and they should be keen to foster collaborations within the lab and institution, and wider afield.
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
No abstract available.
MicroRNAs are known to be upregulated or downregulated in various types of cancer, leading to changes in the expression of genes involved in cellular proliferation, anti-apoptosis, migration, and invasion. To study the effects of microRNA loss or gain in different neoplasms, numerous models have been described to decrease or increase expression of microRNAs, but the off-target effects of different methods have not been well investigated. I investigated the possibility of off-target effects in a model of miR-143 knockdown in myeloid leukemia cell lines that implemented a microRNA sponge, or decoy, as a method to reduce microRNA expression. The high expression of a sponge with repetitive sequence elements and low expression of the intended microRNA for knockdown, miR-143, created conditions with increased potential for non-specific microRNAs to bind to the sponge. Therefore, I investigated the potential binding sites present in the sponge and whether any novel microRNAs could bind to these sites. I found a number of potential candidates and eliminated them based on their likelihood of regulating protein targets and their resemblance to a microRNA in structure, leaving one potential candidate. I found genomic evidence of the existence of this novel microRNA, evolutionary conservation of function, and performed assays that confirmed the biological activity. Next, the original sponge was redesigned to inhibit the binding of the potential non-specific microRNA; miR-X, or the miR-143 binding sites were mutated to inhibit the binding of miR-143 and capture miR-X instead. This demonstrated that binding of non-specific microRNA could be abrogated and differential protein abundance specific to the knockdown of each microRNA separately was verified. I conclude that non-specific binding to the sponge is a distinct possibility in experiments using this method of microRNA knockdown, which needs to be taken into account when designing sponges in the future. This work also demonstrates that there remain novel microRNAs awaiting discovery.
Myelodysplastic syndromes (MDS) are a collection of hematopoietic malignancies in which genomic abnormalities within the hematopoietic stem cell (HSC) compartment lead to pancytopenia and eventual bone marrow failure or progression to leukemia. The most common karyotypic abnormality in MDS is the interstitial deletion of chromosome 5q where a commonly deleted region (CDR) has been identified. Two microRNAs (miRNA) located within the CDR have been shown to be expressed at lower levels in del(5q) MDS compared to diploid MDS. Here I describe investigations of the functional consequence of loss of these two miRNAs, miR-143 and miR-145, on hematopoietic stem and progenitor cells. Loss of miR-143/145 in mice resulted in a decrease in hematopoietic stem cells and myeloid progenitors. I also identified a direct link between the loss of miR-143/145 and subsequent activation of both the canonical and non-canonical transforming growth factor beta (TGFβ) signaling pathways, mediated in part via the adaptor protein, Disabled-2 (DAB2). Analysis of del(5q) MDS patient bone marrow revealed an enriched TGFβ-signature compared to healthy controls and we show that TGFβ signaling is activated upon loss of miR-145 or enforced expression of its target, DAB2. Subsequent studies focused on the function of DAB2 within the hematopoietic system. Overexpression of DAB2 in mouse bone marrow resulted in a significant decrease in hematopoietic stem cell frequency, self-renewal, and colony forming activity. In competitive transplants, vector-transduced bone marrow cells were able to outcompete DAB2-overexpressing marrow in both primary transplants as well as in secondary limiting dilution assays. However, a subset of mice with enforced DAB2 expression alone developed a transplantable acute myeloid leukemia. In conclusion, our data suggest a role for miR-145 haploinsufficiency in the inappropriate activation of TGFβ signaling through derepression of DAB2 which leads to abnormal HSC function in del(5q) MDS.
Hematopoiesis is a process essential for the maintenance of cells that mediate many vital functions such as the production of red blood cells necessary for transportation of oxygen and the removal of carbon dioxide from the body. It is also required for the production of platelets which are necessary for clotting, and the various types of white blood cells that make up the innate and adaptive immune systems that protect against viral, microbial, and parasitic infections. The cell responsible for the generation of all the downstream effector cells, the hematopoietic stem cell (HSC), is generated during embryogenesis. Through a series of symmetric and asymmetric cell divisions, the HSC is capable of maintaining all the cells of the hematopoietic hierarchy throughout the lifespan of an organism. A variety of genetic and epigenetic cues are necessary to maintain homeostasis, and perturbations in these cues lead to the development of hematopoietic malignancies and myelodysplastic syndromes. In recent years the innate immune pathway has emerged as an important player in hematopoietic homeostasis. This dissertation examines the role of dysregulation of innate immune signaling in the development of the myelodysplastic syndromes, one of the most common hematological malignancies. Using a murine bone marrow transplantation assay, I show that overexpression of the innate immune signaling adaptor, TIRAP, results in perturbations in normal hematopoiesis. Overexpression of TIRAP in the hematopoietic compartment results in an inability to produce mature hematopoietic cells, leading to pancytopenia and bone marrow failure (BMF). TIRAP-induced BMF is a result of both autonomous and non-autonomous effects mediated by the cytokine IFNγ. Interestingly, in an environment depleted of IFNγ, TIRAP-transplanted mice develop a myeloproliferative neoplasm suggesting that TIRAP activates both myelosuppressive pathways (through IFNγ) as well as myeloproliferative pathways. IFNγ acts in a paracrine manner inhibiting osteoclast proliferation and maturation in the bone marrow microenvironment, thus disrupting the HSC niche. In summary, this thesis shows the importance of immune regulation in hematopoietic homeostasis. Furthermore, it shows how defects in the hematopoietic stem/progenitor compartment can translate into a defect in the stem cell niche, contributing further to marrow failure and disease progression.
Notch signaling is evolutionarily conserved and regulates various developmental and pathological processes. We are interested in the role of Notch signaling in cardiac valve formation and postnatal vascular remodeling. The heart is the first organ to form in a developing embryo and a common site of congenital defects. Valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn with a prevalence of 1-2% and are often a result of deregulated atrioventricular canal (AVC) formation. The process of endothelial-to-mesenchymal-transition (EndMT) is required for the growth and maturation of the AVC and Notch signaling has been shown to induce this process. In chapter 3, we identify a novel link between Notch and Nitric Oxide (NO) signaling during EndMT. We show that Notch-activation directly induces GUCY1A3 and GUCY1B3, the soluble guanylyl cyclase that forms the heterodimeric NO receptor, during EndMT. We also show that Notch-induced expression and secretion of the TGFβ family member, Activin A, results in increased NO production via a PI3-kinase/Akt signaling mechanism. Paracrine activation of NO signaling by Activin A contributes to early onset of EndMT in the developing AVC. Functional arteries are essential for restoring blood flow and tissue regeneration in response to hypoxia, ischemia, or wound healing. Arterial obstruction can cause distal tissue ischemia and requires rapid reperfusion to limit tissue necrosis. Ischemia recovery requires two processes: angiogenesis (capillary sprouting) and arteriogenesis (expansion of existing vessels secondary to mechanical stress or chemical stimuli). Arteriogenesis involves two phases: a rapid vasodilatory phase followed by vascular expansion and remodeling. In chapter 4, we examine the relationship of endothelial Notch signaling and arteriogenesis using a hindlimb ischemia model. As the link between Notch and NO signaling unfolded in the heart study, we turned our attention to the NO pathway in the initial phase of arteriogenesis – vasodilation. The data presented in this dissertation defines a novel link between Notch signaling and NO signaling in the cardiovascular system and may help to explain Notch-induced EndMT in other pathologies.
The Notch signaling pathway, which converges on RBPJ, is deregulated in a number of malignancies. Following pathway activation, RBPJ, the DNA-binding component of the pathway, associates with Notch to activate transcription of target genes. In the absence of Notch activity, RBPJ acts as a transcriptional repressor by recruiting a co-repressor complex that must be displaced to reinitiate the cycle of activation. As RBPJ is a key regulator of Notch signaling andis constitutively expressed in normal cells, we set out to evaluate the effect of RBPJ loss in the context of human cancer. Frequent RBPJ loss was detected in human breast and lung tumors.Moreover, depletion of RBPJ in a human breast cancer cell line accelerated xenograft tumorgrowth, whereas over-expression of a mutated version of RBPJ (which allows retained function as a transcriptional repressor but prevents activation via Notch) reduced tumor growth in amouse model. These findings were confirmed in a lymphoma knock-out cell line, where a complete loss of RBPJ strikingly increased tumor growth in mice. RBPJ-deficient tumor xenografts showed up-regulated expression of HEY family genes, which represent direct canonical RBPJ targets. Blockade of Notch activation had no effect on the magnitude of HEYgene derepression in the absence of RBPJ, indicating that Notch does not participate inderegulated signal activation resulting from loss of transcriptional repression. To identify other aberrantly induced genes that contribute to the oncogenic phenotype with RBPJ loss, we performed a global analysis. RBPJ removal led to enrichment of acetylated histone H4 at induced gene promoters. We therefore used this epigenetic mark as an indirect measure ofpromoter activity to identify processes that were differentially active in the RBPJ-depleted breastcancer cells compared to RBPJ-containing controls. RBPJ loss enriched for a Notch-like signal and increased acetyl marks at genes associated with cell survival. Indeed, resistance to cell death was observed in RBPJ-deficient breast cancer and lymphoma tumors both in vitro and in vivo. This work defines a new role for RBPJ as a tumor suppressor, the loss of which represents an alternate mechanism for deregulating Notch signaling in cancer.
The endothelium plays a critical role in coordinating the innate immune response through the regulation of vascular tone, leukocyte recruitment and transmigration, and hemostasis. These functions are mediated, in part, by the signaling cascades initiated upon recognition of bacterial and viral products by a family of transmembrane receptors known as Toll-like receptors (TLRs). In endothelial cells, exposure to lipopolysaccharide (LPS), a major cell wall constituent of Gram negative bacteria, results in endothelial activation through TLR4. Recruitment of the adapter protein, MyD88, to the receptor facilitates association of serine threonine kinases of the IL-1 receptor associated kinase (IRAK) family. The IRAKs initiate a phosphorylation cascade through TNFR-associated factor 6 (TRAF6) culminating in activation of proinflammatory signaling pathways including NF-κB and c-Jun NH2-terminal kinase (JNK) pathways. This thesis investigates signaling molecules and pathways downstream of TLR4 in endothelial cells. Specifically, contained herein is a description of the role of heterotrimeric G proteins in endothelial TLR signaling. This thesis identifies for the first time the function of these proteins in multiple TLR signaling pathways. In addition, the work presented here describes the identification and characterization of a novel TLR4 signaling molecule, SAM and SH3 domain containing protein 1 (SASH1). SASH1 promotes LPS-induced NF-κB and JNK, by functioning as a scaffold molecule to bind TRAF6, transforming growth factor-β-activated kinase (TAK1) and IκB-kinase (IKK), thereby increasing proinflammatory cytokine production. The distinct functions of the endothelium in innate immunity highlight the need for an understanding of the signaling cascades initiated by LPS in endothelial cells and will be crucial to our understanding of the pathophysiology of sepsis in the clinic.
The vasculature is essential for the delivery of oxygen and nutrients and the removal of metabolic wastes from tissues of the body. The embryonic vasculature is developed through the processes of vasculogenesis, angiogenesis, and arteriogenesis. Once the vasculature is fully developed and stabilized, the adult vasculature shows very little proliferation or cell death. Nevertheless, the endothelium, which lines the lumen of the blood vessels, is actively involved in the control of vascular tone, permeability, blood flow, coagulation, inflammation and tissue repair. An injury to the endothelium is important for progression of diseases such as atherosclerosis and the sepsis syndrome. The Notch signaling pathway has emerged in the recent decade as an important player in multiple vascular processes and endothelial behaviors. This thesis examines the role of the Notch signaling pathway in embryonic arteriogenesis and endothelial survival signaling. The first part of this thesis investigates the developmental source of vascular smooth muscle cells. This study presents the first in situ observation of an immediate smooth muscle precursor cell present in all embryonic arteries. This Tie1⁺/CD31⁺/VE-cadherin⁻ precursor requires Notch signaling to differentiate into vascular smooth muscle cells and to ensure vascular stability of newly formed arteries. However, Notch activation is not required in the precursor cells to maintain the medial layer of the arteries once the vessel is invested with vascular smooth muscle cells. In the second part of this thesis, the mechanism of Notch-induced endothelial survival signaling is examined. In endothelial cells, Notch signaling activates phosphotidylinositol-3 kinase (PI3K) through up-regulation of a secreted factor. Activity of PI3K is required to offset the parallel apoptotic signaling induced by Notch activation and to maintain endothelial survival through the up-regulation of Slug, a direct Notch target with anti-apoptotic activity. Upon treatment with apoptotic stimuli, Notch activation shows context-dependent effects on endothelial survival. Inhibition of PI3K activity and Slug expression by a stimulus abolishes Notch-induced endothelial survival and increases apoptotic death.The work presented in this thesis shows that the Notch signaling pathway is essential for the stability of the vasculature through regulation of vascular smooth muscle cell differentiation and endothelial cell survival.
Master's Student Supervision (2010 - 2018)
Germline alterations can have clinical implications for both cancer patients and their families. Because the tumour genome may contain both germline and somatic variants, the increasingly common practice of clinical tumour sequencing presents an opportunity to also pre-screen for germline variants. This framework is time- and cost-effective because only patients with potential germline variants are referred to downstream confirmatory testing. However, a key challenge is that tumour specimens are commonly formalin-fixed and paraffin-embedded (FFPE), which induces DNA damage that may interfere with molecular testing. Another challenge is distinguishing between germline and somatic variants in the tumour in order to accurately select candidates for follow-up screening. In order to leverage tumour sequencing for identifying germline variants, these challenges must be addressed.To this end, we retrospectively analyzed clinical amplicon sequencing data from 213 patients with a range of tumours, for whom matched-normal samples were available. We assessed formalin-induced DNA damage by comparing amplicon enrichment and sequencing results of FFPE DNA to the matched-normal DNA isolated from peripheral blood mononuclear cells, a gold standard for germline testing. Although formalin-induced DNA fragmentation and cytosine deamination were detectable, we determined that the discrepancies were minor and could be mitigated by using shorter amplicons and enriching for longer DNA templates. We also found that 98.0% of germline alterations identified in the blood were retained in the tumours, suggesting that FFPE tumour DNA can be a reliable source for germline variant calling. Finally, we applied variant allele frequency (VAF) thresholds to delineate germline and somatic variants in tumour-only analyses. We reported that a VAF cut-off of 15% would correctly identify 99% of germline alterations in FFPE tumours, but erroneously submit 14% of somatic mutations (false positives) for follow-up germline testing. This underscores the high sensitivity and positive predictive value of using VAF to discriminate between germline and somatic variants. Collectively, our results demonstrate that clinical tumour amplicon sequencing could also be used to provide cost-efficient first-line germline testing.
Hematopoietic stem cells (HSCs) arise from a specialized population of endothelial cells, termed hemogenic endothelium (HE). HE was first identified in the murine dorsal aorta (DA) at embryonic day (E) 10.5. This process is known as endothelial to hematopoietic transition (EHT). Our aim was to identify genes crucial for the development of embryonic HSCs from the endothelium through the process of EHT. We accomplished this through the use of a transgenic mouse model which expresses GFP under the control of an intronic enhancer of the HE gene Runx1. The expression of this enhancer was combined with endothelial and hematopoietic markers to sort specialized endothelial cell populations from the DA of E10.5 embryos. RNA-seq data was generated from these sorted cell populations and 9 possible upstream transcription factors were identified. These candidate transcription factors include both known and novel regulators of EHT, including a novel regulator Meis1. Additionally, endothelial cell populations isolated from E9.5 embryos were sorted and cultured to develop an ex vivo co-culture assay that supports differentiation of pre-HE cells to HSCs. The effect of oxygen tension on endothelial and hematopoietic cell growth was investigated as a means to better support endothelial and hematopoietic cells. Oxygen transition during culture was found to significantly increase the proportion of wells which produced hematopoietic cells, and may aid in HE cell maintenance in culture. To examine the possibility of using this model to study regulators of EHT, the effect of blocking Notch signalling with a ɣ-secretase inhibitor added to our ex vivo culture was examined. Inhibition of Notch signalling did not significantly affect the generation of hematopoietic cells in our assay. Additionally, we evaluated the hematopoietic activity of tissue isolated from E9.5 Meis1fl/fl VeCre null embryos in this assay. No differences in hematopoietic cell generation were observed between wild-type and Meis1fl/fl VeCre null tissues in culture. Characterization of these embryos at E14.5 suggests that there exists a potential defect in Meis1fl/fl VeCre null embryos in later stages of embryonic hematopoiesis. This project contributes to the further understanding of genes important in EHT, while potentially defining transcriptional networks involved in HSC development.
Proper activation and regulation of innate immune signaling pathways and inflammatory responses are essential to efficiently fight infection. Dysregulation of these responses, however, can result in the development of a severe hyper-inflammatory state, ultimately leading to sepsis. The endothelium has been shown to play a critical role in innate immune responses and the pathogenesis of sepsis. In the presence of Gram-negative infection, circulating LPS is recognized by TLR4 expressed on the surface of endothelial cells, mediating downstream activation of pro-inflammatory signal cascades and immune responses. SASH1 has been recently characterized as a novel scaffold protein in the regulation of endothelial TLR4 signaling. The work presented in this thesis further characterizes the role of SASH1 in the regulation of TRAF6 activation within the TLR4 pathway. LPS-induced auto-ubiquitination of TRAF6 is an essential regulatory step in the downstream activation of TLR4-mediated inflammatory signaling. Co-immunoprecipitation analyses confirmed the interaction of endogenous SASH1 with TRAF6 in an LPS-dependent manner in endothelial cells. Furthermore, SASH1 was required for LPS-induced auto-ubiquitination of TRAF6. To further investigate SASH1 function, a yeast two-hybrid approach was employed to identify novel SASH1-interacting proteins. β-arrestin 1 was identified as the top potential interactor of SASH1. Both β-arrestin 1 and β-arrestin 2 have been shown to act as negative regulators of the TLR4 pathway by impairing the oligomerization and auto-ubiquitination of TRAF6. It was speculated that SASH1 may coordinate the interaction of TRAF6 with the β-arrestins to mediate regulation of the TLR4 pathway. Reciprocal co-immunoprecipitation experiments confirmed the interaction of SASH1 with both β-arrestin 1 and β-arrestin 2. SASH1 was also found to interact with β-arrestin 1 in a complex with TRAF6. Further studies aimed to characterize the role of SASH1 in coordinating the interaction of TRAF6 with the β-arrestins. Although knockdown of SASH1 did not modulate TRAF6-β-arrestin binding, the enforced expression of SASH1 was found to specifically impair TRAF6 binding with β-arrestin 1, but not β-arrestin 2. The significance of this isoform-specific regulation remains to be determined. Overall, these studies further investigate the role of SASH1 as a critical scaffold protein in the regulation of the TLR4 pathway.
- DNA methylation analysis improves the prognostication of acute myeloid leukemia (2021)
- MET exon 14 skipping mutation positive non-small cell lung cancer: Response to systemic therapy (2021)
Lung Cancer, 154, 142--145
- Genomic testing in myeloid malignancy (2019)
International Journal of Laboratory Hematology, 41 (S1), 117--125