Department of Biomedical and Molecular Sciences Graduate Theses

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    The Persistence of Trophoblast Stem Cells in Later Mouse Gestation: Insights into the Signalling Involved in Their Maintenance
    (2024-05-13) McGinnis, Avery Jane; Biomedical and Molecular Sciences; Natale, David
    Our group previously identified trophoblast populations expressing the characteristic mouse trophoblast stem (mTS) cell marker, Eomes, or the established stem and progenitor cell marker, Sca-1, that persist until E16.5 and proliferate in response to placental pathology. These findings support the maintenance of tissue-resident stem cells that help the placenta adapt to pathology throughout gestation. If such a population is identified, its characterization could the discovery of an analogous human TS cell population with clinical potential in the context of human placental pathology. During development, Eomes is restricted to the extraembryonic ectoderm and, by E7.5, to the chorion, after which its expression declines. The placental junctional zone and labyrinth layers are thought to develop exclusively from the ectoplacental cone and chorion, respectively. While it is well established that mTS cells express Eomes and that in vitro, they differentiate to all trophoblast subtypes, it is unknown if Eomes-positive (EomesPOS) trophoblast that reside in the chorion beyond E6.5 are restricted in their developmental potential. However, by utilizing a lineage tracing technique with complementary immunofluorescence staining, we found that EomesPOS trophoblast maintain the capacity to contribute to both placental layers in vivo and do so after E7.5. Approximately 85-90% of mTS cells express Sca-1, though its function is unknown. Transforming growth factor beta 1 (TGFB1), which plays a role in placental development and the proliferation/differentiation of trophoblast cells, is required for prolonged mTS cell maintenance in vitro. Previous investigations have demonstrated that Sca-1 can attenuate TGFB1 signalling, and TGFB1 signalling can attenuate Sca-1 expression. The Sca-1/TGFB1 relationship in mTS cells has not been explored. Our study revealed that Sca-1 expression did not affect TGFB1 signalling, nor did TGFB1 treatment downregulate Sca-1 expression. However, we identified that positive regulation of Sca-1 expression in mTS cells is mediated by activation of the TGFB superfamily receptor, ALK5, particularly by TGFB1. Collectively, these findings support a role for ALK5 signalling in sustaining Sca-1 within mTS cells. My MSc work provided further support that mTS cells may persist in the placenta into late gestation and provided insights into the microenvironmental conditions required for late gestation mTS cell maintenance.
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    Patterns in Cytokine Alterations in Inflammatory Bowel Disease Patients are Suggestive of Underlying Disease Activity
    (2024-04-23) Potdar, Chinmay; Biomedical and Molecular Sciences; Mulder, Daniel
    Background: Personalized treatment of inflammatory bowel disease (IBD) is a difficult clinical challenge. Large variation in clinical presentation between IBD patients is a major reason for this problem. It is important to investigate various cytokines and immune cell populations as they are known to be the driving force behind the inflammatory process in IBD and may be responsible for the large variation in clinical presentation. The identification of multiple cytokine concentrations as biomarkers for IBD could enable more precise classification, shifting from reliance on phenotypic deductions or limited biomarkers towards empirical evidence. Hypothesis: Patterns in cytokine alterations in IBD patients are suggestive of underlying disease activity. Methods: Both IBD and control patients were enrolled in this study (HSREB 6033229). Patient systemic cytokine profiles were investigated using a 17-plex bead-based immunoassay (n = 23). Clinical data was collected by chart review. Localized tissue expression of a target cytokine identified from analysis of the 17-plex cytokine bead-based immunoassay was investigated using immunohistochemistry (IHC) (n=7). Results: Cytokine immunoassay performed on serum from control (n=8), active IBD (n=5), and IBD in remission (n=10) patients, demonstrated that control patients had a higher mean serum levels of macrophage inflammatory protein 1-alpha (MIP-1α) compared to both IBD active and remission patients. A uniform manifold approximation and projection model based on cytokine concentrations across patients showed differences in clustering between the groups. An extreme gradient boosting model identified MIP-1α as a negative predictor of IBD. A receiver-operator characteristic (ROC) curve generated for MIP-1α as a negative predictor of IBD had an area under the curve (AUC) value of 0.808. Analysis of MIP-1α IHC staining showed distinct differences in the percent of MIP-1α positive cells in the lamina propria of the patients across IBD subtypes as well as across active and remission categories. Summary: This study found for the first time that MIP-1α may be a novel biomarker of inflammation in IBD by combining clinical data with immunophenotyping and analysis utilizing machine learning models.
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    How Kinesin-8 Alternates Between Motility and Microtubule Depolymerization to Control Mitotic Spindle Dynamics
    (2024-04-23) Hunter, Byron; Biomedical and Molecular Sciences; Allingham, John
    During mitosis, microtubules undergo dynamic reorganization to form the mitotic spindle that aligns chromosomes at the cell’s equator and then equally distributes this genetic information to daughter cells. Kinesin-8 motor proteins regulate the lengths of microtubules during these events by walking processively to microtubule ends where they influence the addition or removal of tubulin subunits. This form of bimodal operation has not been observed in any other kinesin family, and thus the molecular mechanisms of kinesin-8s have drawn significant attention. Using X-ray crystallography, cryo-electron microscopy, and biochemical analyses of recombinant kinesin-8 motors from Candida albicans (CaKip3), we obtained evidence that this ability to switch between walking and microtubule-shortening activities stems from an extended and flexible loop-2 region, which is unique to the kinesin-8 motor domain. We show that kinesin-8s use loop-2 to form interactions with tubulin that sense the shape of tubulin protofilaments in the microtubule, and then activate either their motile or microtubule shortening activities accordingly. On straight tubulin protofilaments in the middle of the microtubule, loop-2-tubulin contacts restrict conformational changes of the motor domain in a way that couples ATP hydrolysis energy to walking. On curved tubulin protofilaments, which are abundant at microtubule plus ends, the loop-2 region morphs to accommodate the different shape of tubulin. This causes the kinesin to couple ATP binding to conformational changes in the motor domain that further bend and destabilize tubulin-tubulin contacts. When these kinesin-8 activities are eliminated from C. albicans cells by knockout of both copies of the KIP3 gene, the astral microtubules and mitotic spindles of these cells become abnormally long and less dynamic. This loss of microtubule length regulation subsequently prevents the fungus from forming invasive hyphal filaments. Based on these observations, and the unique structural properties of CaKip3, we probed the effects of CaKip3 inhibition with the kinesin-8 inhibitor sovilnesib to assess its potential application for the treatment of C. albicans infections. Together, our structure-function work on CaKip3 and our live-cell analysis of C. albicans provide valuable insight into the mechanism of the bifunctional kinesin-8 family and how these motors enable the segregation of genetic material.
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    Metabolism of Glycosphingolipids and targeting GM2 synthesis pathway to develop substrate reduction approach in Tay-Sachs and Sandhoff disorders.
    (2024-02-15) 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
    (2024-01-23) 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.