Saudi Cultural Missions Theses & Dissertations
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Item Restricted Deletion of the Parathyroid Hormone Receptor in Marrow Adipose Lineage Precursors (MALPs) Prevents Their Negative Regulation of Skeletal Homeostasis(Harvard University, 2021-04-30) Alabdulaaly, Lama; Baron, Roland; Gori, FrancescaBackground and objectives: Parathyroid hormone (PTH) is essential for skeletal homeostasis and PTH[1-34] (teriparatide) is used to treat severe osteoporosis, medication-related osteonecrosis of the jaws, and enhance implant osseointegration. PTH exerts its anabolic actions by acting on osteoblasts, bone lining cells, and osteocytes. Recently, marrow adipose lineage precursors (MALPs) have been reported to suppress osteogenesis and enhance osteoclastogenesis. We hypothesized that, since MALPs express the PTH receptor 1 (Pth1r), they may contribute to the skeletal response to PTH. Methods: We deleted Pth1r specifically in MALPs and their lineage (adipocytes) using Adiponectin Cre (AdipoqCre) and tdTomato was used as a reporter. AdipoqCre;Pth1rfl/fl,tdTomatofl/fl mice (Pth1rMALPs) were used as experimental mice and age- and sex-matched AdipoqCre;Pth1rfl/fl,tdTomatofl/fl and Pth1rfl/fl,tdTomatofl/fl were used as controls. The skeletal phenotype was characterized by microscopic computed tomography (μCT) and dynamic histomorphometry at 4, 7, and 12 weeks of age. Bone marrow adipose tissue (BMAT) was assessed by osmium tetroxide staining and μCT analysis. The proximal one-third of the tibiae was designated as regulated BMAT (rBMAT) and the distal one-third was designated as constitutive BMAT (cBMAT). Colony forming unit (CFU) assays, flow cytometry, and fluorescence-activated cell sorting (FACS) were performed on flushed bone marrow stromal cells (BMSC). Twoway ANOVA and Student’s t-test were used for statistical analysis (a = 0.05). Results: Pth1rMALPs female mice exhibited a 54.2%, 15.8%, and 42.7% increase in trabecular bone volume at 4, 7, and 12 weeks of age, respectively (p = 0.001). The increase in bone volume was associated with a significant increase in labeled surfaces (MS/BS and dLS/BS, p = 0.0066 and p = 0.0429, respectively by two-way ANOVA) but an increase in bone formation rate (BFR/BS) was significant only at 4 weeks of age (p = 0.03). Flow cytometry analysis revealed that osteochondrogenic progenitor cells were decreased by half in Pth1rMALPs mice (p = 0.01). Since mineralizing surfaces were increased, these results suggest a shift of the progenitor pool towards differentiated and functional osteoblasts. Additionally, the number of CFU-F colonies was significantly decreased in Pth1rMALPs cells, further confirming the decrease in the progenitors. In contrast, the CFU-Ob remained similar between the two groups, suggesting an increase in the osteogenic potential of cells from Pth1rMALPs mice despite their decreased number. Only 6.4% ± 4.6% of CFU-Ob cells represented a distinct population of tdTomato+ cells, and 5.7% ± 2.7% had a dim tdTomato+ signal (herein, tdTomato+dim). Interestingly, cells from Pth1rMALPs mice exhibited an 8-fold increase in tdTomato+ dim cells, and unfractionated CFU-Ob cells exhibited a 90.3% decrease in Pth1r mRNA gene expression (p = 0.001). Additionally, Pth1rMALPs mice showed a significantly higher rBMAT density, confirming that PTH suppresses adipogenesis. Importantly, sorted MALPs expressed Rankl, and its expression is increased in MALPs lacking Pth1r. The endosteal osteoclast number and surface was increased in Pth1rMALPs mice, indicating that PTH signaling in MALPs suppress osteoclastogenesis. Conclusions: Deletion of the Pth1r in MALPs partially prevents their suppression of osteogenesis through driving the progenitor pool to a differentiated state. Moreover, PTH signaling in MALPs suppresses adipogenesis and osteoclastogenesis.26 0Item Restricted Contribution of Chronic Myeloid Leukaemia Niche to Metastasis and Treatment Resistance(University of Glasgow, 2024) Albilasi, Hakem; Machesky, Laura; Kirschner, Kristina; Jorgensen, HeatherChronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder hallmarked by the Philadelphia chromosome, leading to the formation of the BCR::ABL1 fusion protein and subsequent uncontrolled cell proliferation. Although the advent of tyrosine kinase inhibitors (TKIs) such as Imatinib (IM) has considerably improved patient outcomes, drug resistance and relapse remain a significant challenge. Leukaemia-related deaths and mortalities are often linked to these challenges which are a major cause of human financial and social costs of the disease. The bone marrow microenvironment (BMM) plays a crucial role in normal haematopoiesis and is also the main protection of leukaemic stem cells (LSCs). In CML, the BMM enhances leukemogenesis through an interaction with LSCs, and in turn, LSCs modify the BMM based on their requirements. Mesenchymal stem cells (MSCs) within the BMM are particularly significant, as they can support haematopoietic cells distinctly. The interactions between CML cells and the BMM, including MSCs and the extracellular matrix (ECM), have been shown to influence CML cell proliferation and responsiveness to IM. Moreover, cytoskeletal dynamics also play a crucial role in CML progression and drug resistance. In CML, alterations in cytoskeletal dynamics have been linked to drug resistance and disease progression. Therefore, this thesis will focus on the contribution BMM to these issues, with a particular interest in the role of MSCs and ECM on CML cells. In this thesis, direct contact with MSCs, treatment with MSC-conditioned media, and ECM component exposure were employed to investigate the role of the BMM and MSCs in the proliferation of CML cells and their interaction with the ECM and cell adhesion molecules. Furthermore, the effect of inhibiting specific cytoskeletal components on CML cell behaviour will be examined. The results of this thesis shed light on the complex interplay between CML cells and the BMM, highlighting the critical role of MSCs in modulating CML cell behaviour. While our results did not find a definitive impact of the ECM components on CML cell proliferation or IM sensitivity, the observations suggest that MSCs have the potential to influence the behaviour of CML cells but not definitively affecting their sensitivity to the TKI, IM. Furthermore, inhibiting cytoskeletal components such as the Arp2/3 complex and FAK did not significantly alter the CML cells' contact with MSCs. However, further investigation is necessary to investigate the molecular mechanisms regulating the interaction between CML cells and their microenvironment. Finally, the relationship between CML cells and MSCs, as shown by our transcriptomic analysis, reveals not just alterations in gene expression but also the promise of identifying novel therapeutic targets. The dysregulated pathways in co-cultured K562 cells and MSCs highlight the potential for therapeutic intervention strategies that could disrupt the supportive role of the BMM in CML persistence. The modulation of integrin alpha 9 (ITGA9) expression in K562 cells under co-culture conditions highlights the complexity of CML-MSC interaction and suggests an adaptive mechanism that may contribute to the survival and drug resistance of CML cells within the BMM. Therefore, targeting integrins could potentially enhance the efficacy of existing CML treatments, which in turn, might lead to more effective management of the disease and an increased rate of successful patient outcomes.16 0