FGS - Electronic Theses and Practica

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This collection contains University of Manitoba electronic theses and practica.

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Now showing 1 - 5 of 26702
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    Open Access
    Transnational circuits of precarity: a qualitative examination of temporary worker wellbeing in Manitoba
    (2025-02-11) Huynh, Anthony; Chase, Robert (Community Health Sciences); Varley, Emma (Community Health Sciences); Sethi, Bharati (Trent University); Marcelino, Diwa (Migrante Manitoba); Lorway, Robert; Hatala, Andrew
    ABSTRACT Background In western Canada, Manitoba is a critical hub for a large population of migrant workers. Usually with limited English or French language ability and possessing limited rights and protections under the current TFWP, Temporary foreign workers (TFWs) are often tied to a single employer, leaving them vulnerable to employer abuse and the under-reporting of workplace injuries and illnesses due to the threat of deportation. Within the context of the COVID-19 pandemic, when my dissertation research began, the many cases seen among TFWs in Manitoba raises additional important public health questions on the health and wellbeing of migrant workers in Manitoba that I discuss in this dissertation. Methodology In close collaboration with Migrante Manitoba (MB), I conducted a qualitative study to explore the precarious lives of migrant workers during the COVID-19 pandemic. I virtually interviewed 20 migrant workers who entered Canada through the TFWP, employed either as seasonal agricultural workers (n=7) or TFWs (n=13). Thirteen TFWs came from Philippines and seven farmworkers from Mexico (n=6) and Jamaica (n=1). Theoretical contribution I developed the notion of transnational circuits of precarity to understand the multiple temporal-spatial layers of precarity that migrant workers encounter along their journeys to Manitoba. This multivalent concept is comprised of the following interconnected pieces: 1) a broader political economic “force-field” that compels the movement of human labour resources from the global South to the global North; 2) the rigid and regulated pathway put in place to ensure workers arrival at their work destinations; 3) the process of making “model minorities” through training programs that ensure the “smooth” transition of workers in their host country; and 4) the affective economy that is fueled by workers’ hopes, dreams, and desires. All together, these seemingly disparate processes articulate to produce complex temporal and spatial realities that shape the precarious trajectories of migrant workers. Such a paradigm shift away from the narrow temporal and spatial limits of a focus on “occupational health hazards” will be critical if workers are to realize any meaningful and substantive changes to their overall physical and mental well-being.
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    Open Access
    Optimization of in vitro RNA interference assays to reduce variability in target gene knockdown in the phytopathogen Sclerotinia sclerotiorum
    (2025-01-16) Fatema, Reemana; Gerstein, Aleeza (Microbiology); Marcus, Jeffrey (Biological Sciences); Whyard, Steve
    Sclerotinia sclerotiorum is a broad host range fungal phytopathogen that is the causal agent of Sclerotinia stem rot in many crop plants, causing devastating yield losses worldwide. Sclerotinia is partially controlled using broad-spectrum chemical fungicides, but new, more species-specific fungicides, such as double-stranded RNAs, are being considered as safer alternatives. This study evaluated the effectiveness of long double-stranded RNAs (dsRNAs) and paperclip RNAs (pcRNAs) to induce gene silencing in two different fungal inoculants of S. sclerotiorum. The three different genes that were targeted in this study, SS1G_01703, SS1G_00005, SS1G_14116, are all involved in different aspects of S. sclerotiorum’s infection process i.e. pathogenicity, encoding 60S ribosomal unit and mycelial growth respectively. Initial dsRNA experiments using small fungal plugs collected from solid media generally resulted in highly variable levels of transcript knockdown, potentially arising from unequal hyphal amounts in treatment tubes. This led to the development of fungal slurries as the inoculum to provide a more uniform distribution of hyphae across all the samples. The slurry method resulted in significant transcript knockdowns using all three long dsRNAs at different doses, although some variability persisted. The variability using the slurry method was linked to potential differences in hyphal developmental stages, age of hyphae and differences in expression levels of each target gene. In contrast, structurally different and shorter in length pcRNAs failed to induce consistent transcript knockdown in both plug and slurry assays, despite earlier reports of their efficacy in reducing lesion sizes on canola leaves. This inefficacy may stem from pcRNAs' reliance on a single siRNA for gene targeting, potentially insufficient to overcome the structural complexity of target mRNAs. These findings highlighted the potential of long dsRNAs for gene knockdown in S. sclerotiorum using optimized slurry-based assays, while suggesting that pcRNAs require further modifications. Future work can explore alternative siRNA designs, increased pcRNA doses, and identifying accessible mRNA regions to improve knockdown efficiency. Additionally, optimizing the methods to assess hyphal developmental stages and minimizing variability during sample preparation are crucial for reproducibility and accuracy in RNAi-based studies.
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    Embargo
    Multiplex immunofluorescence studies to characterize early cellular dynamics in laser-ablated non-tumor mouse brain
    (2025-02-13) Shanmugam Anandhan, Santhosh; Logue, Susan (Human Anatomy and Cell Science); Kong, Jiming (Human Anatomy and Cell Science); Zeiler, Frederick (Surgery); Klonisch, Thomas; Klonisch, Sabine-Hombach
    Introduction: Laser Interstitial Thermal Therapy (LITT) is a minimally invasive, hyperthermia-based laser cytoreductive method used in the treatment of several severe conditions, including inoperable brain tumors, treatment-resistant epilepsy, and radiation necrosis. Laser-induced heat causes damage to the tissue and alters the brain-tissue microenvironment (TME) at the local treatment site and beyond. The regional effects include activation of resident brain cells, release of mediators of inflammation, and immune cell infiltration, which may activate immune responses. Immunomodulatory responses to LITT in the brain microenvironment are largely unknown. One hurdle is the lack of affordable and efficient imaging protocols that enable the simultaneous spatial visualization of multiple immunoreactive cellular targets in the brain. Here, we describe a flexible and affordable multiplex immunofluorescence (mIF) staining method in our established LITT mouse model. In the post-LITT mouse brain, mIF revealed laser ablation-induced complex spatial responses by the resident brain and innate immune cells. Methods: We developed a customizable and affordable mIF detection protocol that uses standard histological and microscopy equipment to assess TME changes on single sections of formalin-fixed paraffin-embedded (FFPE) mouse brain tissues at day 6 post-LITT. This mIF imaging procedure provided a comprehensive spatial analysis of multiple cellular and molecular immunotargets in resident brain cell responses and immune cell profiles in individual tissue sections, as exemplified with a mouse brain microenvironment on day 6 post-LITT. Results: We optimized mIF imaging methodology on LITT-treated mouse brain FFPE serial sections to visualize the spatial distribution of resident brain cell cellular activation states and the emergence and distribution of immune cell populations at the LITT site. Our mIF imaging protocol of mouse brain regions surrounding the LITT site revealed the activation of resident astrocytes and microglial cells and the presence of a diverse innate immune cell population in this region. Conclusion: The combined use of LITT ablation treatment with multiplexed versus regular immunofluorescence offers a significant advancement and aids in our understanding of post-LITT repair and immune responses in the brain. We characterized the thermal ablation-induced modulation of resident brain cells and innate immune cell infiltrations.
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    Embargo
    The safety and long-term outcomes of opioid exposures in pregnancy
    (2025-01-28) Abulannaz, Omaymah; Ruth, Chelsea (Pediatrics and Child Health); Hatch, Grant (Pharmacology and Therapeutics); Hirani, Shela (University of Regina); Kelly, Lauren; 't Jong, Geert
    Background: The use of opioids during pregnancy has increased in North America. Opioids can cross the placenta and produce signs of withdrawal known as Neonatal Abstinence Syndrome (NAS). In this project, I described the trends of opioid prescriptions among pregnant people and the incidence of NAS in Manitoba, examined factors associated with the development and severity of NAS and investigated the neurodevelopmental outcomes following exposure to prescription opioids during pregnancy. Methods: A population-based cohort study was conducted using the population data repository of the Manitoba Center for Health Policy. Poisson regression models were used to describe the time trends of opioid prescriptions utilization during pregnancy and describe the incidence of NAS (Chapter 2). Multivariable logistic regression models were used to examine factors associated with the development and severity of NAS (Chapter 3) and to investigate neurodevelopmental outcomes following exposure to prescription opioids during pregnancy (Chapter 4). Early Development Instrument (EDI) was used to assess developmental vulnerability in kindergarteners. Results: The proportion of live births exposed to opioid prescriptions during pregnancy increased from 3.7% in 1995 to 7.4% in 2017; however, there has been a reduction in recent years. The incidence of NAS in Manitoba more than tripled between 1995 and 2021 (2.0-7.6 per 1,000 live births). Female sex, normal birth weight, and breastfeeding were protective factors against the diagnosis of NAS. Breastfeeding initiation was the only protective factor against the need for pharmacological treatment. Of the 103,905 eligible children, 51% of the exposed group (n=973) were developmentally vulnerable in 1 or more domains, compared with 32% of the matched control group (n=3,814) (Adjusted Odds Ratio [AOR] 1.46; 95% CI 1.23-1.71). Children in the exposed group had a significantly higher likelihood of being vulnerable in all domains: physical health and well-being (AOR 1.31; 95%CI 1.08-1.59), communication skills and general knowledge (AOR 1.53;95%CI 1.25-1.88), language and cognitive development (AOR 1.53;95%CI 1.25-1.86), social competence (AOR 1.60;95%CI 1.32-1.95); emotional maturity domain was the least affected (AOR 1.25;95%CI 1.01-1.55). There was a higher risk of developmental vulnerability in children exposed to opioids in early pregnancy, longer durations, and higher opioid doses. Conclusions: Prenatal exposure to opioids is increasing in Manitoba and was associated with an increased risk of developmental vulnerability in children. It is of utmost importance to provide children with POE and their families with early and long-term developmental screening and family centered supports to help achieve optimal educational and family health outcomes.
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    Embargo
    Evaluating the genotypic and phenotypic stability of CRISPRi essential gene mutants in Burkholderia cenocepacia K56-2
    (2025-01-31) Batun Hernandez, Zayra Yael; Gerstein, Aleeza (Microbiology); Budisa, Nediljko (Chemistry); Cardona, Silvia
    Essential genes encode the processes required for cell growth and survival within specific environments. Understanding the products of essential genes is important for antibiotic discovery, as many essential genes encode the targets of antibiotics. Also, controlling the products of essential genes is a potential approach to reversibly inhibit growth while exploiting beneficial traits. A genetic tool to investigate essential gene function by controlling gene expression is CRISPR interference (CRISPRi). This tool comprises a nuclease-inactive Cas9 (dCas9) and a synthetic guide RNA (sgRNA), forming a ribonucleoprotein complex that binds to the target gene and sterically blocks transcription. Previously, we constructed a CRISPRi system in strains of the Burkholderia cepacia complex, opportunistic bacterial pathogens with biotechnological potential. Our CRISPRi system uses a chromosomally encoded, rhamnose-inducible dCas9 and a sgRNA constitutively expressed from a plasmid. We and others have observed that CRISPRi mutants spontaneously lose their conditional growth phenotype (CGP), hampering their potential use in controlling bacterial growth. To understand this phenotypic reversion, we performed experimental evolution experiments in selected 15 CRISPRi mutants under dCas9-inducing and non-inducing conditions. At time points, we performed targeted gene repression by RT-qPCR and whole genome sequencing (WGS) in parental and evolved mutants. While analysis across generations revealed diverse CGPs, all mutants reverted to wild-type growth phenotypes. K-medoids clustering identified 3 clusters with different relationships between essential gene functions and the CGP. Under consistent inducing conditions, Cluster 1 exhibited severe gene repression of 78-fold, dropping to 3-fold. Cluster 2 decreased from 46-fold to 0.5-fold, whereas Cluster 3 demonstrated wide diversity in gene repression. There was no evidence of single nucleotide polymorphisms (SNPs) in the dCas9, rhamnose system regions, or the encoded sgRNAs maintained on a plasmid. However, WGS identified SNPs in a porin and a LysR-type global regulator. Immunoblot analysis confirmed the absence of dCas9, indicating a lack of expression possibly due to porin malfunction affecting rhamnose uptake or protein degradation caused by unregulated proteases. Together, while the CRISPRi machinery is genetically stable, compensatory mutations compromised its function at longer times. Our work highlights the strengths and limitations of CRISPRi for extended control of bacterial growth.