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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
No abstract available.
Gap junctions are unique intercellular channels assembled from the canonical gap junction family, connexins (Cxs). These channels connect the cytosols of adjacent cells, allowing direct passages of small ions and molecules for intercellular communication and homeostasis within tissues. A novel family of gap junction proteins, pannexins (Panxs), with low sequence similarity to the invertebrate gap junctions, innexins, was recently discovered in chordates. Similar to Cxs, Panxs are also capable of forming functional hemichannels as well as intercellular channels. Aberrations in gap junctions have been associated with abnormal CNS development and diseases including gliomas. The main purpose of this thesis was to determine if Panxs play a functional role under pathological and normal CNS conditions, each of which is represented by gliomas and neuronal differentiation, respectively. A loss of Panx expression was found in the C6 glioma cell line when compared to its normal counterparts, primary astrocytes. Restoring Panx1 and Panx2 expression in C6 glioma cells by stable transfection induced a dramatically flattened morphology, which is similar to the flat and polygonal shape of cultured astrocytes. Both Panx1 and Panx2 also significantly suppressed the neoplastic phenotype of C6 glioma cells, including in vitro monolayer growth, anchorage-independent growth, and in vivo tumorigenesis in immunodeficient mice. Interestingly, while Panx1 reduced cell motility in C6 glioma cells, Panx2 did not elicit a similar effect. Panx1 and Panx2 exhibited a distinct subcellular localization. Panx1 was detected at the plasma membrane and perinuclear regions, whereas Panx2 was only found in membrane-bound compartments within the cytosol, hence suggesting mechanistically different tumor-suppressive pathways employed by the two Panxs. Furthermore, it was determined that Panx1 and Panx3, but not Panx2, increased neurite numbers and further enhanced neurite outgrowth in PC12 cells during nerve growth factor-induced neuronal differentiation. In conclusion, findings from this thesis suggest a functional role of Panxs in normal and pathological conditions of the CNS, and merit critical future investigations to explore their underlying mechanisms and therapeutic implication in diseases.
No abstract available.
Master's Student Supervision (2010 - 2018)
No abstract available.
Objectives: Numerous genes have been shown to impact craniofacial development. One such gene, Hypermethylated in cancer 1 (Hic1), is a tumour suppressor gene that encodes for the transcription factor HIC1. Hic1 is broadly expressed within embryonic mesenchyme. Hic1 knockout mice demonstrate multiple craniofacial anomalies, including facial clefting, abnormal closure/truncation of the secondary palate, low-set/underdeveloped ears, abnormal eye development, shortened snouts, acrania, and exencephaly, suggesting a role for Hic1 in craniofacial development. Molecular mechanisms underlying the role of Hic1 in craniofacial development remain largely unknown. Our objective was to elucidate the cellular fate of Hic1⁺ cells during craniofacial development. Methods: The fate of Hic1-expressing cells in craniofacial development was studied using lineage tracing analysis. A Hic1CreERT² mouse, in combination with a Cre-dependent reporter mouse, was used to label and follow Hic1-expressing cells. Reporter gene expression was induced at the onset of Hic1 expression (embryonic age (E) of 10 days). Embryos were collected at E11.5, E13.5, E14.5, E16.5 and E18.5. The distribution and fate of reporter-expressing cells was determined using epi-fluorescence imaging. Micromass cultures of cells derived from the mandibular, frontonasal and maxillary prominences at E11.5 were used to study Hic1-expressing cells in cartilage formation. Samples (n=3) were fixed and stained with Alcian blue or COL2A1 to study the relationship of Hic1⁺ cells to cartilaginous nodules. Single-cell RNA sequencing was performed on cells positive and negative for Hic1-expression in the head. Results: Hic1+ cells contribute to the mesenchyme within facial/masticatory muscles, tendon, tongue, meninges, nasal cartilage/ connective tissue, eyes, salivary gland stroma, teeth (dental papilla/pulp), and to populations of perineural and perivascular cells, including those within the brain. Hic1 is not seen within the palatal shelves, or Meckel’s cartilage. In micromass cultures of facial mesenchyme, most Hic1-expressing cells contribute to the area surrounding cartilaginous nodules, while few were incorporated within the nodules, mimicking the pattern of distribution of Hic1-expressing cells in the nasal structures. Single-cell RNA sequencing suggests Hic1-expressing cells are neural crest-like, and represent mesenchymal progenitors. Conclusions: Hic1-expressing cells contribute to various mesenchymal tissues during craniofacial development, and likely represent a population of cranial neural crest-derived cells or similar.
Much of the vertebrate skeleton is formed through endochondral ossification. In this process, a chondrogenic template is laid down, which is subsequently replaced by bone. The first step involves condensation of mesenchymal cells and their differentiation into chondroblasts that initiate elaboration of the chondrogenic template. At later stages, chondrocytes undergo hypertrophy, and produce a matrix for bone formation. To enhance our understanding of molecular programs regulating this process a chemical genetics approach was employed. Our strategies involved the development of screens using primary cultures of murine limb bud-derived mesenchymal (PLM) cells. Chondroblast differentiation is associated with increased SOX5, 6 and 9 activity; while hypertrophic differentiation is associated with reduced SOX5, 6 and 9 activity. Therefore, a SOX5/6/9-responsive reporter gene was used to follow expression of the chondroblast phenotype. Compound libraries representing more than 1400 compounds were screened; 28 compounds were found to increase reporter gene activity greater than 2.5 fold. In secondary screens, 7 of 28 positive compounds stimulated cartilage formation, as assessed by alcian blue staining. Two compounds identified, Butamben (butyl 4-aminobenzoate; BAB) and Phenazopyridine hydrochloride (PHCl), exhibited strong pro-chondrogenic activity and morphologically similar alcian blue staining. BAB is a member of the benzocaine family of analgesics and functions by inhibiting sodium channel activity. However, BAB has also been shown to have potassium channel-blocking activity. Specifically, BAB inhibits the activity of Kcnd2; which through transcriptional profiling was also found to be down-regulated by bone morphogenetic protein-4 (BMP4). We speculated BAB and PHCl may be able to modulate chondrogenesis by acting on potassium channels. To confirm this idea we examined molecular activities of PLM cultures treated with BAB and PHCl at two stages of chondrogenesis: 1. pre-chondrocyte to chondroblast and 2. chondrocyte to hypertrophic chondrocyte. Results confirm, BAB and PHCl increase expression of chondrogenic markers and reduce expression of hypertrophic markers. In addition patch clamp analysis revealed both BAB and PHCl are able to block, at least partially, KCND2 channel activity. We confirmed the dynamic expression pattern of Kcnd2 by qPCR and radioactive section in situ hybridization. Together, these results reveal an unanticipated and novel role for Kcnd2 in chondrogenesis.
Prospective Student Info Sessions
Faculty of Medicine Information SessionDate: Tuesday, 08 December 2020
Time: 11:00 to 12:00
UBC’s Faculty of Medicine is a global leader in both the science and the practice of medicine, and is home to more than 1,700 graduate students across over 20 graduate programs. In this session hosted by Dr Michael Hunt, Associate Dean, Graduate and Postdoctoral Education, we’ll provide an overview of the diverse array of graduate programs available, including cutting-edge research experiences in the biosciences, globally recognized population health education, quality health professional training, as well as certificate and online training options. Dr Hunt will also be joined by program advisors from across the faculty to take an inside look at the application process and provide some application tips to help make your application as strong as possible.