Department of Biomedical and Molecular Sciences Graduate Theses

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    Investigating the Role of B Lymphocytes in Urinary Bladder Tumor Development
    Biomedical and Molecular Sciences; Koti, Madhuri
    Urinary bladder cancer (BC) is a highly prevalent disease with 25% of the incident cases presenting muscle invasive BC (MIBC). Immune cell populations in the tumor immune microenvironment (TIME) influence BC progression and response to therapy. While immune cells such as tumorassociated macrophages (TAMs) and T cells have been widely studied in BC, the role of B cells remains unclear. This study aimed to examine the role of B cells, particularly, atypical B cells (ABCs) in the progression of BC. As such, we investigated the impact of B cell depletion on BC progression in a carcinogen-induced murine model. Sex differences in the TIME and disease progression were observed following B cell depletion in carcinogen exposed mice. Female mice exhibited increased number of ABCs in their spleen, while males showed near complete ABC depletion. Spatial profiling of bladders post depletion depicted clear sex differences with female mice having higher total B cell and ABC frequencies. B cell depletion also increased the splenic total and cytotoxic T cell populations in both female and male mice, indicating enhanced antitumor immunity. B cell depletion reduced inflammation and delayed cancer progression, specifically, in female mice. However, discontinuation of depletion during carcinogen exposure resulted in accelerated tumor invasion and decreased survival. Treatment of B cells with TLR7 agonist augmented ABC differentiation and cell survival. Spatial profiling of bladders with muscle invasion revealed higher density of ABCs and lower density of T cells in B cell depleted female mice compared to non-depleted controls. Overall, B cell/ABC depletion appears to temporarily induce a favorable immune microenvironment by eliminating the exhausted populations and reactivating lymphopoiesis. However, in the continued presence of carcinogen, the rejuvenated B cell pool differentiates towards an immunosuppressive ABC phenotype after discontinuing depletion, enabling cancer progression. The more pronounced impact seen in females may be due to higher initial baseline ABC levels and increased response to cell death-induced TLR7 activation. These findings provide evidence that ABCs can be contributing factors to an immunosuppressive TIME that enables BC progression. This study elucidates B cell and ABC involvement in the progression of BC, laying the groundwork for combination immunotherapies targeting ABCs to improve treatment outcomes in a sex-specific manner.
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    RET-receptor mediated invasion in pancreatic ductal adenocarcinoma
    Biomedical and Molecular Sciences; Mulligan, Lois
    Pancreatic ductal adenocarcinoma (PDAC) is frequently associated with invasion along nerves, which generates severe pain. KRAS and TP53 are the most common driving mutations for PDAC, however, these tumours also overexpress the RET receptor which regulates cell proliferation and migration. Since nerves are known to secrete RET ligands, PDAC invasion towards nerves is potentially enhanced through RET signalling. We evaluated the dependency of PDAC invasion on RET using a collagen invasion assay and PDAC spheroids. We found that RET-selective and multikinase inhibitors, such as Selpercatinib and Vandetinib respectively, decreased or prevented invasion. We further determined RET pathways that are important to this process by treating with inhibitors or using shRNA knockdown cell lines for various proteins, required in the downstream signalling of RET, in our invasion assay. We found that proteins associated with RET migration, proliferation and recycling pathways, such as PI3Kinase, MEK, mTOR, GRB2 and ARF6, decrease invasion. However, proteins associated with RET-mediated proliferation and recycling, such as STAT3 and GGA3, showed no significant effects. Our data suggest that RET is required for PDAC invasion and therefore inhibiting RET, or certain downstream signals could aid in reducing PDAC invasion along the nerves. Preliminary experiments were also performed to help build an experimental plan for determining the relevance of these proteins for chemotactic invasion of nerves. MiaPaCa2-EGFP cells and primary mouse neurons were cultured together to observe any interactions between the two cell types. Neurons were observed with axons growing in the direction of groups of MiaPaCa2 cells and two potential phenotypes of neurons were observed (axonal growth vs. no axonal growth). Neuron body area and axonal length measurements were taken to characterize morphological differences between the two. A correlation between axon length and neuronal body area was observed with smaller neurons having longer axons, and a significant difference between areas of neurons with axonal growth and those without. This indicates that there may be multiple subtypes of neurons present and that not all of them may react to the presence of PDAC cells, however further research is required.
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    Engineered Agarase Systems to Enhance Agarose Degradation
    Turner-Wood, Keegan; Biomedical and Molecular Sciences; Smith, Steven
    Environmental polysaccharides represent a vast and renewable energy source. A subset of terrestrial bacteria have evolved multi-enzyme complexes called cellulosomes that degrade cellulosic biomass. The cellulosome comprises a modular scaffold containing a carbohydrate-binding module and repeating cohesin modules onto which complementary carbohydrate-active enzymes (CAZymes) assemble via their resident dockerin modules. The proximity and targeting effects associated with these structural features contribute to the efficient digestion of cellulose. The high-affinity cohesin-dockerin interaction displays species-specific properties that have been used to produce purpose-built multi-CAZyme complexes, termed designer cellulosomes, that take advantage of the proximity and targeting effects to degrade terrestrial polysaccharides. Marine bacteria also produce CAZymes to digest marine polysaccharides, but a cellulosome-like complex has yet to be identified. The designer cellulosome system, and its associated proximity and targeting effects, affords an opportunity to engineer highly efficient agarose-degrading enzyme complexes. The purpose of this thesis was to: 1) assemble cellulosome-like multi-enzyme complexes using endolytic and exolytic agarases from Bacteroides uniformis and assess the impact of the proximity effect on agarose degradation; and 2) quantify the increase in agarose degradation resulting from the targeting effect by incorporation of a B. uniformis agarose-binding protein into chimeric scaffolds. Using molecular biology and biochemical approaches we produced B. uniformis agarase (BuGH2C, BuGH16B, BuGH86, and BuGH117B)-dockerin fusion constructs and complementary cohesin-based chimeric scaffolds and showed that while a mixture of the BuGH86 and BuGH117B constructs function synergistically to degrade agarose, complexation did not lead to further enhancement in activity via the proximity effect. We generated an AlphaFold-based structural model of the B. uniformis SusE-like agarose-binding protein, which revealed four distinct modules, the fourth of which was structurally similar to a characterized carbohydrate-binding module. Lastly, incorporating the agarose-binding protein into chimeric scaffolds onto which the endolytic BuGH86 and exolytic BuGH117B agarase-dockerin fusion proteins were attached allowed us to show that the hydrolytic properties of both enzymes were augmented due the targeting effect, and that this effect was further enhanced when these constructs were applied as a mixture. This study provides novel insights into agarose degradation by engineered multi-enzyme complexes and acts as a foundation for future biotechnological applications.
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    Differential Effects of Remdesivir and Lumacaftor on Homomeric and Heteromeric hERG Channels
    Campagna, Noah; Biomedical and Molecular Sciences; Zhang, Shetuan
    The human ether-a-go-go-related gene (hERG) (KCNH2) encodes the pore-forming a-subunit of the channel (Kv11.1) that conducts the rapidly activating delayed potassium current (IKr) in the heart. The hERG channel is important for the repolarization phase of ventricular action potentials, and mutations causing reduction of its expression in the plasma membrane can result in long QT syndrome type 2 (LQT2). As such, promoting hERG membrane expression is a strategy to rescue mutant channel function. In this study, the rescue effects of two drugs, remdesivir and lumacaftor, on trafficking defective mutant hERG channels were studied by applying patch clamp, Western blot, immunocytochemistry, and Quantitative Reverse Transcription PCR techniques. As our lab has recently reported that the antiviral drug remdesivir, used to treat COVID-19, increases wild-type (WT) hERG current and surface expression, we studied the effects of remdesivir on trafficking defective LQT2-causing hERG mutants G601S and R582 in HEK293 cells. The effects of lumacaftor, a drug used to treat cystic fibrosis that promotes CFTR protein trafficking and has been shown to rescue membrane expression of some hERG mutations, were also investigated. These results show that neither remdesivir nor lumacaftor rescued the current or mature protein expression of homomeric mutant G601S. However, remdesivir decreased while lumacaftor increased the current and mature protein expression of heteromeric channels formed by WT hERG and mutant G601S or R582C. These results demonstrate that drugs can differentially affect homomeric WT and heteromeric mutant-containing hERG channels, extend our understanding of drug-channel interaction, and may have clinical implications for patients with hERG mutations. Remdesivir may exacerbate LQT2, while lumacaftor has potential to mitigate LQT2.
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    Characterizing the Impact of Vitamin D and High Dietary Phosphate on Parathyroid Gland Structure and Function in Chronic Kidney Disease
    Khan, Sono; Biomedical and Molecular Sciences; Adams, Michael; Holden, Rachel
    Chronic kidney disease (CKD) affects around 40 million North Americans and significantly contributes to global cardiovascular disease and mortality. CKD-induced systemic dysregulation of phosphate metabolism promotes secondary hyperparathyroidism (SHPT), in which pathological parathyroid gland (PTG) calcium-sensing receptor (CaSR)-based signalling plays a significant role. Clinical interventions such as vitamin D-based treatments aim to control hypersecreting parathyroid hormone (PTH), producing significant maladaptive effects. However, PTG phenotypes of vitamin D resistance and mineral insensitivity arise in SHPT, lessening the efficacy of these interventions. This study aimed to investigate how calcitriol (active vitamin D) and calcifediol, the precursor to calcitriol, affect the structure and cellularity of the PTG. In addition, to elucidate the CKD time course and role of high dietary phosphate on PTG pathogenesis. In two experiments, a 9-week dietary adenine-induced CKD rat model was employed to determine: (i) the effect of calcitriol and calcifediol treatment on CKD rats; and (ii) the time course of changes in CKD animals at 4 and 7 weeks of low dietary phosphate (0.5%) and at week 9 of CKD induction, but after two weeks of high dietary phosphate (1%). In addition to standard biochemical assessments, CaSR immunohistochemistry was performed on PTGs to visualize changes in the glandular phenotypes. A CellProfiler-based image analysis protocol was developed to quantify parathyroid cellularity and structure. Novel CKD-induced parathyroid tissue phenotypes were identified, including cell hypertrophy, reductions in connective tissue, and the presence of skewed basolateral CaSR expression exclusively adjacent to connective tissue. Cell size quantification indicated a dramatic CKD-induced hypertrophic effect on parathyroid cell size (1.72-fold, p<0.0001), exacerbated by calcitriol (1.84-fold, p<0.0001) and protected against by calcifediol (1.54-fold, p<0.0001). CKD induced a significant increase in parathyroid cell number (1.59-fold, p<0.0001), with proliferation preceding hypertrophy from four weeks into CKD pathogenesis. PTG cellularity measurements correlated strongly with PTH, fibroblast growth factor-23 (FGF-23), phosphate, and creatinine. Overall, our findings demonstrate significant CKD-induced pathological alterations to parathyroid cellularity and morphology. The calcitriol-induced stimulation of hypertrophy suggests it is not an ideal therapy, whereas the lesser calcifediol impact, compared to calcitriol, was promising. Lastly, the emerging relevance of the endocytic receptor megalin is reviewed.