Saudi Cultural Missions Theses & Dissertations
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Item Restricted The Effect of Low-Intensity Pulsed Ultrasound on Extracellular Matrix and its cross-linking and organisation.(University of Sheffield, 2025-06) Almukhlifi, Yazeed; Bass, MarkChronic wounds are characterised by their inability to heal within the expected time frame, significantly impacting patient welfare and even life expectancy, while also imposing considerable financial burdens on the healthcare system. The extracellular matrix (ECM) plays an essential role by interacting with growth factors and providing the physical substrate over which cells migrate during healing. However, in chronic wounds, the ECM components often appear to be dysfunctional. Low-intensity ultrasound, as a form of mechanical stimulation, has shown promising results at the cellular level. This project aims to investigate the effect of Low-Intensity Pulsed Ultrasound (LIPUS) on gene expression, matrix deposition, cross-linking, and the organisation of specific ECM molecules. In this study, two cell lines were used, human foreskin fibroblast (HFF) and telomerase-immortalized fibroblasts (TIF). Then, I performed western blot, Quantitative PCR, Immunofluorescence, migration assay and traction force microscopy to investigate the protein and gene expression changes, matrix alignment and contractility and cell migration after the LIPUS treatment. 1.5-MHz frequency, a 20% duty cycle pulse to generate a 30 mW/cm2 spatial average and temporal average (SATA) were applied to the treatment group. The treatment was performed for 20 minutes per day for five days. The results indicate that matrix composition was altered by LIPUS, with specific effects on ECM molecules involved in healing. Transforming growth factor-beta (TGF-β) and collagen type I showed a reduction in protein expression in the conditioned medium, whereas lysyl oxidase (LOX), collagen type III, and fibronectin exhibited increased expression. These findings reveal that LIPUS treatment changes the composition of secreted matrix by altering the expression profiles of several components and regulatory factors in a manner that is far more nuanced than simply promoting expression. LIPUS treatment also altered fibronectin fibre curvature, leading to more random alignment. This is significant as it suggests that a 20-minute treatment can have long-lasting healing effects by modifying ECM architecture. Traction Force Microscopy revealed reduced cellular contractility after LIPUS treatment, likely due to changes in TGF-β expression, which explains the altered ECM alignment. Overall, these findings show that LIPUS influences both ECM structure and cellular mechanical properties, potentially improving tissue repair and healing outcomes. Together, these findings indicate LIPUS's potential as a therapeutic tool to enhance wound healing and reduce the scar effect, particularly in cases of chronic wounds where ECM dysfunction is common. Also, this project suggests that LIPUS can affect ECM dynamics, showing changes in protein expression and ECM organisation.12 0Item Restricted How cells, when exposed to changing biomechanical and biochemical conditions, modify their extracellular matrix environment(The University of Manchester, 2024) Alghamdi, Hussam Mohammed; Ballestrem, ChristophAbstract Background: Fibrosis, a condition characterized by excessive accumulation of extracellular matrix (ECM) components, is a major contributor to organ failure in chronic diseases such as kidney and liver disease. ECM stiffness is known to play a critical role in modulating cellular behavior, particularly through mechanotransduction pathways mediated by integrins. Understanding how cells, specifically fibroblasts, respond to changes in matrix stiffness is crucial for elucidating the mechanisms underlying fibrosis and developing potential therapeutic strategies. Objectives: This study aimed to investigate the impact of matrix stiffness on fibroblast morphology, adhesion characteristics, and ECM deposition. The specific objectives were to determine how matrix stiffness influences cell shape, focal adhesion dynamics, and the production of ECM components. Methods: Telomerase immortalized foreskin fibroblasts (Tiffs) were cultured on polyacrylamide gels with defined stiffnesses of 1.5 kPa (soft) and 28 kPa (stiff), coated with fibronectin. Cell morphology was assessed through measurements of cell area, aspect ratio, and roundness. Focal adhesion characteristics were quantified by counting the number of adhesions per cell. ECM deposition was evaluated by measuring the thickness of the ECM and fluorescence intensity as a proxy for ECM protein content. High-resolution 11 confocal microscopy and ImageJ Fiji software were used for imaging and analysis. Statistical analyses were performed using unpaired t-tests by GraphPad Prism. Results: Fibroblasts cultured on stiffer matrices exhibited a more elongated morphology, with a significantly higher aspect ratio and lower roundness compared to those on softer matrices. The number of focal adhesions was significantly reduced on stiffer matrices, indicating that stiffer environments promote fewer but potentially more stable adhesions. ECM thickness was significantly greater on stiffer matrices, suggesting enhanced ECM deposition in response to increased stiffness. However, no statistically significant difference in fluorescence intensity was observed between the two stiffness conditions, though a trend toward higher intensity on stiffer matrices was noted. Conclusions: The findings demonstrate that matrix stiffness significantly influences fibroblast morphology, adhesion dynamics, and ECM deposition, underscoring the importance of mechanical signals in regulating cellular behavior. These results have important implications for understanding the pathogenesis of fibrosis and suggest that targeting matrix stiffness could be a viable therapeutic approach. The study's limitations, including the use of a single cell type, 2D culture system, and limited ECM components, highlight the need for future research to explore these phenomena in more complex and physiologically relevant models.12 0Item Restricted An Investigation into How Anti-Fibrotic Agents Rescue Cardiac Insulin Resistance and Improve Cardiac Function in a High Fat Diet-Induced Obesity Murine Model(University of Dundee, 2024-10) Banah, Ayman Kamal; Kang, Li; Lang, ChimIn recent decades, there has been significant advancement in our comprehension of the extracellular matrix (ECM) in terms of its composition, structure, and physiological functions. Moreover, mounting evidence suggests that increased ECM deposition contributes to the pathogenesis of various diseases. Current research endeavours targeting the ECM hold promise for the development of novel therapeutic strategies to address challenging medical conditions. Diabetes and heart failure often coexist, exerting reciprocal influences on each other and thereby affecting disease progression and outcomes. Notably, insulin resistance emerges as a pivotal mediator in this bidirectional relationship between diabetes and heart failure, posing a significant risk factor for heart failure development through compromised cardiac insulin signalling pathways. However, the underlying mechanisms of insulin resistance in heart failure remain incompletely elucidated. A growing body of evidence implicates cardiac ECM remodelling in the pathogenesis of insulin resistance, necessitating the identification of novel therapeutic targets to mitigate or reverse cardiac IR and its associated dysfunction. In this thesis, I specifically tested the hypothesis that reducing heart ECM constituents using clinical and pre-clinical anti-fibrotic agents may alleviate cardiac insulin resistance and improve cardiac function. Results from my PhD study have revealed that pharmacological inhibition of ECM receptor integrin α5β1 enhances insulin signalling in H9C2 cells. Additionally, my studies with the mineralocorticoid receptor antagonist Eplerenone have demonstrated its effectiveness in regulating body weight gain and enhancing cardiac function in obese mice. My findings also establish a novel association between increased ECM deposition, cardiac insulin resistance, and cardiac dysfunction in obesity. Notably, pharmacological reduction of hyaluronan, a key ECM component, using pegylated human recombinant hyaluronidase PH20 (PEGPH20) has demonstrated its potential to ameliorate cardiac insulin resistance and associated functional impairments in obese mice. Further evidence was attained through genetic and pharmacological inhibition of the hyaluronan receptor RHAMM. Specifically, my results indicate that mice deficient of RHAMM (KO) exhibit improved glucose tolerance and lower aortic pressures compared to littermate wildtype (WT) controls, particularly in males fed a high fat diet (HFD). Cardiac ECM remodelling and functional alterations induced by HFD were attenuated in KO males. Conversely, such protective effects were not evident in female mice. The present findings underscore the therapeutic promise of early interventions aimed at cardiac ECM remodelling to alleviate cardiac insulin resistance and dysfunction associated with obesity. It is proposed that strategies intended to prevent pathological ECM expansion may confer protection against the progression to severe cardiovascular complications. As such, the implications of these findings are of considerable importance to the development of effective interventions targeting cardiac ECM remodelling in the context of obesity-associated cardiovascular disease.39 0