Department of Biomedical and Molecular Sciences Graduate Theses

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    The chromosome-19 microRNA cluster promotes a dedifferentiated, metastatic phenotype in malignant melanoma
    (2024-07-03) Dent, Jayne; Biomedical and Molecular Sciences; Craig, Andrew
    Cutaneous melanoma is an aggressive form of skin cancer that often progresses to metastatic and fatal disease. Despite recent advances in targeted therapies and immunotherapies, few patients with metastases survive. This highlights the need for therapies that block metastasis by better defining the molecular mechanisms. Recently, we profiled microRNA (miRNA) expression in the skin cutaneous melanoma (SKCM) cohort of the cancer genome atlas and discovered amplification of the Chromosome-19 miRNA cluster (C19MC) in approximately 10% of SKCM tumors. C19MC expression is normally restricted to a few tissues, and it regulates placental trophoblast migration and differentiation. Recently, C19MC was identified as an oncogenic driver in some embryonal brain cancers. However, the role of C19MC expression in melanoma has not been reported and we hypothesized that C19MC activation may promote a dedifferentiated, metastatic melanoma phenotype. To test this hypothesis, we induced C19MC expression in A375 melanoma cells using a CRISPR/dCas9-based activation system and validated that several C19MC miRNAs were upregulated compared to isogenic controls. C19MC activation in A375 cells caused reduced cell growth, migration, and invasion in vitro. In human A375 melanoma tumor xenograft assays, we found that C19MC activation reduced primary tumor growth with increased spontaneous liver metastases. C19MC expression also increased liver metastases in experimental metastasis assays. Profiling gene and protein expression changes revealed C19MC+ samples had increased stem cell features and decreased CDK2 expression, which may explain altered growth and metastasis phenotypes. While future experiments are required to corroborate underlying mechanisms, the present study contributes new knowledge that will ultimately help to optimize treatments for this subset of melanoma.
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    Exploring The Functional Selectivity Of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists In Pancreatic and Colorectal Cancer
    (2024-07-02) Bunsick, David; Biomedical and Molecular Sciences; Szewczuk, Myron
    Understanding the role of biased G protein-coupled receptor (GPCR) agonism in receptor signaling may provide novel insights into the opposing effects mediated by cannabinoids, particularly in cancer and cancer metastasis. GPCRs can have multiple active states, a phenomenon called ‘biased agonism,’ ‘functional selectivity,’ or ‘ligand-directed signaling.’ However, there are increasing arrays of cannabinoid allosteric ligands with different degrees of modulation, called ‘biased modulation,’ that can vary dramatically in a probe- and pathway-specific manner, not from simple differences in orthosteric ligand efficacy or stimulus-response coupling. Here, emerging evidence proposes the involvement of CB1 (Cannabinoid receptor 1) GPCRs in a novel biased GPCR signaling paradigm involving the crosstalk between Neuromedin-B (NMBR) neuraminidase-1 (Neu-1) and matrix-metalloproteinase-9 (MMP-9) in the activation of the glycosylated receptors through the modification of the receptor glycosylation state. The study findings highlighted the role of CB1 agonists AM-404, Arvanil, and Olvanil in significantly inducing Neu-1 sialidase activity in a dose-dependent fashion in RAW-Blue (murine macrophages), PANC-1 (pancreatic), and SW-620 (colorectal) cells. This approach was further substantiated by findings that the neuromedin B receptor inhibitor, BIM-23127, MMP-9 inhibitor, MMP-9i, and Neu-1 inhibitor, oseltamivir phosphate, could specifically block CB1 agonist-induced Neu-1 sialidase activity. Additionally, we found that CB1 receptors exist in a multimeric receptor complex with Neu-1 in naïve, unstimulated RAW-Blue, PANC-1, and SW-620 cells. This complex implies a molecular link regulating the interaction and signaling mechanism among these molecules on the cell surface. We found that treating these cells with the synthetic cannabinoids also increased cell viability, migration, and cellular projections in PANC-1 cells. However, migration was not increased in SW-620 cells, except for Olvanil. Moreover, the study results demonstrate that CB1 agonists induce NF-κB-dependent secretory alkaline phosphatase (SEAP) activity in influencing the expression of epithelial-mesenchymal markers, E-cadherin, and N-Cadherin in PANC-1 cells and vimentin in SW-620 cells. In essence, this innovative research begins to elucidate an entirely new molecular mechanism involving a GPCR signaling paradigm in which cannabinoids, as epigenetic stimuli, may traverse to influence gene expression and contribute to cancer and cancer metastasis.
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    Thymic stromal lymphopoietin contributes to endometriotic lesion proliferation and disease-associated inflammation
    (2024-06-25) Aleksieva, Stanimira; Biomedical and Molecular Sciences; Tayade, Chandrakant
    Endometriosis is a chronic disorder in which endometrial-like tissue establishes outside the uterus. Patients with endometriosis have been shown to exhibit aberrant immune responses within the lesion microenvironment and in circulation which contribute to the development of endometriosis. This includes the predominance of Th2 immune responses that prevent lesion clearance and enhance lesion proliferation and angiogenesis. Thymic stromal lymphopoietin (TSLP) is an alarmin involved in cell proliferation and the induction of Th2 inflammation. This contributes to pathogenesis of various diseases, such as asthma, atopic dermatitis, pancreatic and breast cancer. Recent studies have detected TSLP within endometriotic lesions and shown that its concentrations are elevated in the peritoneal fluid and serum of patients compared to controls. However, its role in disease pathophysiology remains unclear. Here, we compared TSLP expression in endometriotic lesions to matched patient endometrium and control endometrium samples by performing immunohistochemistry on a tissue microarray. We also assessed its effect on the proliferation and apoptosis of human endometriosis-representative cell lines through calorimetric assays, as well as on lesion development and inflammation in a C57BL/6 mouse model of the disease. We demonstrated that TSLP expression was elevated in the stroma of patient endometriotic lesions compared to control endometrial samples. In cell lines, TSLP treatment reduced the apoptosis of endometrial stromal cells and promoted the proliferation of THP-I cells. In mice induced with endometriosis, TSLP treatment induced a Th2 immune response within the lesion microenvironment, and led to TSLP receptor modulation in macrophages, dendritic cells, and CD4+ T cells. Furthermore, treatment increased murine endometriotic lesion proliferation, but had no detectable effect on angiogenesis. Overall, these results suggest that TSLP modulates the endometriotic lesion microenvironment and promotes a Th2 immune response that could support lesion development.
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    IL-27 utilizes IRF1 for IFN-I-independent ISG expression during IAV infection of human macrophages
    (2024-06-19) Kalin, Emma; Biomedical and Molecular Sciences; Gee, Katrina
    Influenza virus (IAV) is a respiratory virus spread through aerosols or respiratory fomites causing seasonal epidemics resulting in up to 650,000 deaths annually and with the potential to cause more serious life-threatening pandemics. Recognition of IAV by host innate immune cells, such as macrophages (M), signals to activate the immune response, including key cytokines, like type I interferons (IFN-I) and the antiviral cytokine, interleukin-27 (IL-27). IFN-I signalling induces the expression of interferon-stimulated genes (ISGs), which collectively function to limit virus infection. Relatively new research has indicated a role for IL-27 in the induction of ISG expression either in an IFN- I-dependent or -independent manner. Furthermore, IL-27 has previously been shown to regulate the function of monocytes and M, including skewing M polarization towards a pro-inflammatory, M1 phenotype. However, the effects of IL-27 on polarized M during IAV infection are unknown. Therefore, we set out to characterize the relationship between IFN-I and IL-27 in the induction of ISGs in response to pandemic IAV infection (A/NY/18/2009) and to determine if IL-27 plays a role in M polarization and how this changes IAV infection. Here we show that IL-27-mediated IFN-I-independent induction of ISGs requires the presence of the transcription factor IRF1, and in the absence of IRF1, IL-27 is dependent on IFN-I to induce ISGs. As well, we show that post-infection IL-27 treatment does not confer the ability to reduce IAV infection. These results show potential correlation between IAV replication, M subtypes, and IL-27-modulation of the IFN response, allowing for a better understanding of the antiviral function of IL-27.
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    Effect of Inflammation in Pregnancy on Maternal and Offspring Cardiovascular Systems
    (2024-06-19) Toews, Alexa; Biomedical and Molecular Sciences; Graham, Charles H.
    Complications of pregnancy are often characterised by abnormal inflammation during gestation and are associated with an increased risk of future maternal and offspring cardiovascular disease (CVD). Murine studies have revealed that inflammatory pregnancies are associated with the acquisition of risk factors for CVD, but not overt disease. Therefore, a second inflammatory stimulus, such as one induced by a high fat diet, may be necessary to produce overt dysfunction. This study utilized rodent models of pregnancy loss and fetal growth restriction to assess the effects of aberrant inflammation during pregnancy and subsequent high fat diet consumption on cardiac function, morphology, and biomarkers of CVD in mothers and offspring. In the maternal study, pregnant C57BL/6 mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 50 µg/kg) to induce complete fetal loss on gestational day (GD) 10.5. Fourteen days later, mothers were transitioned to a high fat diet (60% kcal from fat) for 10 weeks. In the offspring study, pregnant Wistar rats were injected i.p. with low dose LPS (10 µg/kg) on GD 13.5 and higher dose LPS (40 µg/kg) on GDs 14.5-16.5 to induce growth restriction. Offspring were weaned at postnatal day 21 and transitioned to the high fat diet for 13 weeks. Echocardiography was used to assess cardiac function. At euthanasia, hearts were collected for histological assessment. Maternal cardiac blood was collected to assess CVD biomarkers. In the maternal model, we saw slight alterations in some parameters of cardiac function, morphology, and biomarkers of CVD. In the offspring model, males that were not growth restricted but were fed a high fat diet post-weaning had significantly altered systolic and diastolic function. Males on the high fat diet, regardless of in utero exposure to inflammation, also had significantly smaller hearts. Results from the maternal study may reflect early development of cardiovascular disease. The offspring model has raised questions regarding the influence of in utero inflammation and high fat diet consumption on cardiac development and subsequent function. Future studies with a longer timeline may further elucidate how these factors influence cardiac function and disease progression in mothers and offspring following a complicated pregnancy.