Department of Biomedical and Molecular Sciences Graduate Theses

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    Metabolism of Glycosphingolipids and targeting GM2 synthesis pathway to develop substrate reduction approach in Tay-Sachs and Sandhoff disorders.
    Abidi, Iram; Biomedical and Molecular Sciences; Brockhausen , Inka; Walia, Jagdeep
    GM2 gangliosidosis is a rare genetic lysosomal storage disorder (LSD) in children, with no effective therapies available presently. Tay-Sachs (TSD) and Sandhoff disorders (SD) are caused by a disruption of the catabolic pathway of gangliosides in lysosomes, accumulating GM2 and lyso-GM2, which damage cells and tissues. This leads to symptoms that often include neurological deterioration, such as cognitive decline, motor dysfunction, and seizures. For the purpose to develop substrate reduction therapy (SRT) for GM2 gangliosidosis in TSD and SD, we studied the enzyme beta1,4-N-acetylgalactosaminyltransferase 1, B4GALNT1, responsible for the synthesis of GM2. We attempted to predict its structural features by modelling the enzyme using Bioinformatics tools and characterized B4GALNT1 activity to establish its properties, stability, and inhibition. Additionally, we established an enzyme assay to produce Lyso-GM2 from GM2 using an in-vitro method, to utilise Lyso-GM2 as a possible biomarker for detection of TSD and SD. We used eukaryotic transient expression systems (HEK293 and Expi293cell lines) to express B4GALNT1 in vitro. Western blots demonstrated production of soluble protein in Expi293, which was successfully purified using Ni-NTA chromatography. The predicted 3D structure of B4GALNT1 established highly conserved residues, showing a potential catalytic DXD motif at position 356-358, close to residues Y501 and H483 which are likely to be important for donor binding. R505 is another significant amino acid reported to be mutated in a small number of patients with GM2 gangliosidosis. B4GALNT1 was strongly inhibited by bis-imidazolium salts that are selective inhibitors of glycosyltransferases. These inhibitors could decrease the synthesis of GM2 and other related glycosphingolipids in the biosynthetic pathway, and further avoid accumulation of GM2 and lyso-GM2 in patients with patients with lysosomal dysfunctionalities and neurodegeneration like TSD and SD. This work can lead to potential therapies for these disorders.
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    Activation of the WNK1 Pathway in Macrophages and Across Tissues
    Koner, Sakura; Biomedical and Molecular Sciences; Ghasemlou, Nader
    Lysine-deficient protein Kinase 1 (or WNK1) belongs to a family of unique kinases which lack the conserved lysine residue in the subdomain II of their structure. There are four known members of the WNK family (WNK1-4) that phosphorylate downstream targets SPAK (STE20 (sterile 20)-related Ser/Thr protein kinases or the SPS1 (sporulation-specific protein 1)-related proline/alanine-rich kinase) and OSR1 (Oxidative stress-responsive kinase 1), which phosphorylate sodium-potassium-chloride cotransporters and potassium-chloride cotransporters. WNK1’s role in the central nervous system was first elucidated when mutations in the gene was found to result in the development of Hereditary Sensory and Autonomic Neuropathy Type 2. Since then, it has been shown that WNK1 is activated and upregulated in various models of neuropathic pain. More recently, an evolving role for WNK1 has been found in the immune system. WNK1 acts as a chloride sensor under homeostatic conditions and is activated in response to low intracellular chloride concentration and prevents NLRP3 activation in macrophages. Our study hypothesized that WNK1 and pathway proteins have differential expression across tissues and WNK1 can be activated by modulating environmental salt concentrations in macrophages. We studied baseline expression of total and phosphorylated WNK1 and pathway proteins across various tissues in male and female C57BL/6J mice. Sex-dependent differences in expression patterns were observed for OSR1 in lungs and spleen while other tissues displayed similar expression. WNK1 and other pathway proteins did not show sex-differences but displayed tissue-specific differences. Experiments using the RAW 264.7 macrophage cell line found that challenge with increasing concentrations of salt solutions (sodium and potassium chloride) resulted in increased WNK1 expression at both mRNA and protein levels. This work suggests that WNK1 and most pathway proteins’ expression varies in an organ-specific manner but remains uniform across sexes except OSR1. It also suggests that WNK1 expression can be regulated by increasing salt concentrations in a macrophage cell line. Given that increased WNK1 and salt concentrations are known to result in pain, our work is indicating that targeting this pathway in peripheral immune cells may provide a potential therapeutic target for alleviating neuropathic pain in people living with spinal cord injury.
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    Finding Synergistic Lipid Kinase and Mitotic Kinase Inhibitor Combination Treatments for Metastatic Breast Cancer
    Al Ali, Nadia; Biomedical and Molecular Sciences; Craig, Andrew
    Triple-negative breast cancer (TNBC) and Inflammatory breast cancer (IBC) are aggressive subtypes that lack targeted therapies options in most cases. The frequently activated phosphatidylinositol 3 kinase (PI3K) pathway in TNBC is a candidate but resistance to PI3K inhibitors has been observed. However, a recent functional genomics screen using PI3K inhibitor buparlisib in TNBC cells revealed a synthetic lethal interaction with Aurora kinase A (AURKA) gene. Here, I investigated if PI3K and AURKA inhibitors will act synergistically to eliminate TNBC cell growth and motility in culture models and halt progression in mouse tumor models. Testing dose responses of buparlisib and/or alisertib treatments in TNBC and IBC cell lines revealed synergistic effects that increased cytotoxicity in a dose dependent fashion by up to 10-fold change. The combination of buparlisib and alisertib were also better than monotherapies at reducing TNBC or IBC colony growth and cell migration rates. Combination treatments of IBC tumor-bearing mice with buparlisib and alisertib also reduced tumor growth and spontaneous lung metastases in vivo. Together, these results provide rationale for advancing targeted therapies comprised of PI3K and AURKA inhibitors to improve treatment options for TNBC and IBC.
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    Effects of TGF-β Inhibitor on Chemoresistance in Ovarian Cancer Models
    Bishop, Rachel N.; Biomedical and Molecular Sciences; Craig, Andrew
    High grade serous ovarian carcinoma (HGSC) is the most fatal gynecological cancer, due to the aggressive, heterogeneous nature of the tumours. Despite an initially favourable treatment response, patients with HGSC often develop resistance to the first-line platinum-based chemotherapies. In addition, the few currently available second line treatments, such as doxorubicin (Dox), often fail to elicit a significant response in platinum resistant HGSC. High levels of Transforming Growth Factor-β (TGF-β) was linked to poor prognosis, chemoresistance and metastasis risk in HGSC. Mechanisms include roles of TGF-β promoting epithelial to mesenchymal transition (EMT) and immune evasion via suppression of Granzyme B (Gzmb) and Perforin (Prf1) expression in cytotoxic T cells. We hypothesized that treatments of HGSC models with TGFβR1 inhibitor galunisertib (LY) would increase sensitivity to chemotherapy drugs such as doxorubicin (Dox). Using several human and mouse HGSC cell models treated with LY, we observed reversal of EMT and increased Dox sensitivity. We extended these studies to a syngeneic mouse HGSC model comparing treatments with vehicle, Dox, LY, or the Dox/LY combination. Compared to vehicle control, all treatments significantly decreased the tumour burden with the combination having the smallest tumours. Unfortunately, the Dox dose was not optimal to test for synergy with LY treatments. However, in HGSC tumours from LY-treated mice we detected reduced expression of mesenchymal genes SNAIL and CDH2, while increasing expression cytotoxicity-related genes Gzmb and Prf1 compared to vehicle control. These differences in gene expression were highly significant in mice classified as LY responders versus non-responders. Together, these results provide rationale for further testing of TGF-β inhibitors in combination with chemotherapy and/or immunotherapy to limit HGSC recurrence and progression.
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    Ca2+-dependent inactivation of a neuronal Ca2+ channel is regulated by intracellular Ca2+ handling
    MacNeil, Eammon K.; Biomedical and Molecular Sciences; Magoski, Neil
    Intracellular Ca2+ is a critical regulator of gene expression, neuronal excitability, and neurosecretion. Free cytosolic Ca2+ is controlled by numerous channels, pumps, and exchangers in the plasma membrane, endoplasmic reticulum, or mitochondria. In the marine mollusc, Aplysia californica, ovulation is initiated from the central nervous system by bag cell neurons. Upon brief synaptic input, these neuroendocrine cells fire a synchronous afterdischarge, starting with an ~5-Hz, ~1-min fast-phase, then an ~1-Hz, ~30-min slow-phase, that culminates in the Ca2+-dependent neurohaemal secretion of egg-laying hormone. During the fast-phase, Ca2+ rises due to entry through voltage-gated Ca2+ channels. Repetitive opening results in Ca2+-dependent inactivation (CDI) of the channel, aka use-dependent rundown, which serves as a form of negative feedback. Our laboratory previously showed that mitochondria sequester voltage-gated Ca2+ influx, although how this influences Ca2+ channel function is unknown. Here, I test the hypothesis that intracellular Ca2+ handling, by both membrane transport and organelles, controls CDI. Rundown of isolated Ca2+ current was recorded in single cultured bag cell neurons under whole-cell voltage-clamp using recurring step depolarizations (~70% current remaining at the end of a 5-Hz, 5-sec train-stimulus). Rundown was significantly decreased by swapping Ba2+ for Ca2+ in the external solution, or adding the Ca2+ chelator, EGTA, to the intracellular/pipette solution (both ~90% current remaining). However, blocking either the mitochondrial Ca2+ uniporter or the plasma membrane Ca2+-ATPase significantly increased rundown (both ~60% current remaining). To further understand mitochondrial Ca2+ handling, neurons were loaded with the Ca2+ sensitive dye, fura-PE3, and Ca2+ liberated from the mitochondria by collapsing the organelle membrane potential with the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Compared to control, a prior train-stimulus to elicit Ca2+ entry significantly increased the FCCP-induced percent-change in Ca2+ (from ~175% to ~450%), and this was prevented by inhibiting the mitochondrial Ca2+ uniporter. These findings suggest that both mitochondrial uptake and plasma membrane extrusion play a key role in CDI by buffering Ca2+ during prolonged influx. Overall, Ca2+ dynamics serve as a key regulator of Ca2+ channel function, with the potential to govern Ca2+-dependent processes in general.