Saudi Cultural Missions Theses & Dissertations
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Item Restricted Investigation of PDI Involvement in Leukocyte Adhesion to Endothelial Cell in Inflammatory State Using Gene Editing (CRISPR/Cas9) Technology(Arabin Gulf university, 2025) almashhur, wedad awadh ali; Fathallah, Mohamed DahmaniLeucocyte adhesion to endothelial cells is a critical step in the inflammatory process, influenced by various molecular mediators, including Protein Disulfide Isomerase 1 (PDI1). This study aimed to investigate the broader roles of additional PDI family members in regulating endothelial cell activation and leucocyte adhesion under inflammatory conditions. We isolated endothelial cells with a cobblestone morphology from human peripheral blood and umbilical cords, achieving yields of 1.2 × 10⁵ and 3.5 × 10⁵ cells/mL, respectively. Neutrophils were isolated from blood samples using magnetic bead technology, and their phenotype was confirmed via flow cytometry. To induce inflammation, endothelial cells were stimulated with TNF-α (100 ng/mL). Quantitative real-time PCR was utilized to assess the expression of PDI genes and adhesion molecules, specifically ICAM-1, VCAM-1, and E-selectin, which serve as established markers of endothelial activation. CRISPR/Cas9 technology facilitated the knockout of PDIA2, PDIA3, PDIA4, and PDIA6 in endothelial cells, with validation achieved through sequencing and Western blot analysis. Efficient gene editing was confirmed by the significant reduction in expression levels of the targeted PDIs. The results demonstrated a 62.5% decrease in the expression of ICAM-1 and VCAM-1, while E-selectin expression was reduced by an average of 55% across all knockouts. Functional assays revealed significant reductions in neutrophil adhesion (p < 0.01) to knockout endothelial cells, with specific decreases of 25% in PDIA2, 20% in PDIA3, 35% in PDIA4, and 2% in PDIA6 knockout cells. These findings underscore the essential roles of various PDIs in the activation of endothelial cells and leucocyte adhesion during inflammation. The study illustrates the effectiveness of CRISPR/Cas9 technology in elucidating gene functions and highlights PDIs as potential therapeutic targets for inflammatory vascular diseases. This research provides valuable insights into the molecular mechanisms underlying inflammation and opens avenues for novel therapeutic strategies.2 0Item Restricted THE EFFECT OF FRUCTOSE METABOLISM ON HEPATIC DE NOVO LIPOGENESIS AND ADIPOSE TISSUE INSULIN RESISTANCE(University of Kentucky, 2025-05) Fadhul, Taghreed; Softic, SamirThere is a worldwide epidemic of obesity and metabolic dysfunction. The root cause of obesity is driven by a positive energy balance, which is a consequence of increased caloric intake and decreased energy expenditure. Energy intake is the more dominant force, as increasing energy expenditure is a time-consuming and labor-intensive process. The caloric surplus can be mainly accounted for by increased consumption of high-fat and high- sugar diets. While the risks associated with fat consumption are well-documented, the impact of sugar intake is less understood. Fructose is a simple sugar widely consumed in modern diets. Its intake has been associated with various metabolic disorders such as obesity, insulin resistance, chronic kidney disease, hypertension, metabolic dysfunction associated with steatotic liver disease (MASLD) and others. The most detrimental effects of fructose are observed when co- ingested with a high-fat diet (HFD). Fructose is mainly metabolized in the liver, kidney, and intestines. Its negative health effects have been linked to its strong propensity to induce de novo lipogenesis (DNL). This dissertation investigated whether fructose intake in the absence of HFD is sufficient to induce metabolic dysfunction and if its metabolism by other organs, such as adipose tissue (AT), plays a role in the development of metabolic dysfunction. To investigate whether fructose intake is sufficient to induce metabolic dysfunction, male and female mice were studied on three different normal chow diets: Boston Chow Diet (BCD), Lexington Chow Diet (LXD), and Low-Fat Diet (LFD). The diets had different fat-to-carbohydrate ratios, with BCD > LXD > LFD having the most fat. Additionally, the mice were provided regular water or 30% fructose solution in water. In males, fructose supplementation on BCD led to weight gain, glucose intolerance, and hepatic steatosis, whereas on LXD, fructose did not induce these adverse effects. Moreover, male mice on LFD did not gain weight, but once switched to BCD they did gain weight and developed metabolic dysfunction. Interestingly, female mice did not gain weight and remained insulin-sensitive even on BCD when supplemented with fructose. However, they did develop hepatic steatosis. These findings indicate that metabolic dysfunction associated with fructose intake is not a universal finding but, rather, is influenced by the dietary fat-to-carbohydrate ratio of the diet, duration of dietary exposure, and sex of the mice. AT is an important organ in orchestrating metabolic homeostasis. Fructose intake increases adiposity, but whether fructose can be directly metabolized in AT is not known. Male mice were fed a Chow diet or 60% high-fat diet (HFD) and supplemented with either regular water, 30% fructose-, or 30% glucose-solution for ten weeks. Fructose and glucose had similar effects on fat mass and adipocyte size, but only fructose and not glucose intake reduced beneficial adipokines, such as adiponectin and resistin, in HFD-fed mice. This is associated with increased crown-like structures, elevated inflammatory markers, and impaired insulin signaling (p-AKT) in visceral adipose tissue (VAT) of fructose-fed mice on a HFD. Moreover, when fructose metabolism in the liver was prevented via KHK siRNA, more fructose was available to be metabolized in AT, which worsened AT inflammation and metabolic dysfunction. In terms of its metabolism, fructose supplementation elevated HK1 and HK3 expression in VAT. The specific fructose transporter, Glut5, was absent in AT. However, Glut1 expression was increased with fructose supplementation in AT. In vitro, fructose was as sufficient as glucose to promote the differentiation of 3T3-L1 and stromal vascular fraction (SVF) into fully differentiated adipocytes. In human subjects, fructose supplementation was associated with elevated inflammatory markers and increased HK3 expression. HK3 expression was also increased in obese compared to the lean individuals. These findings indicate that adipose tissue may serve as an inducible site for fructose metabolism under conditions of high fructose intake or when fructose metabolism is prevented in the liver. In summary, fructose intake on a normal chow diet is sufficient to induce metabolic dysfunction only when consumed on diets with high fat-to-carbohydrate ratios. The effects of fructose are also sex-specific, as female mice are better at compensating for the detrimental effects of fructose largely due to estrogen, which enhances insulin sensitivity and promotes fat oxidation. While the liver, kidney, and intestine are the primary sites of fructose metabolism, AT may also participate in fructose metabolism under specific situations which raise serum fructose levels. It remains to be determined which specific cell types in AT mediate fructose metabolism.20 0Item Restricted Insights into Human Oviduct Physiology and Inflammatory Stimuli Using In Vitro Organoids(University of Manchester, 2025) Alkasih, Mohannad; Stevens, AdamThe human oviduct is a crucial organ of the female reproductive tract that plays a key role in the early reproductive events. It includes four distinct segments and each segment contributes uniquely in the function of the oviduct. The epithelial lining of the oviduct is of key importance for human reproduction as it regulates the tubal environment, including epithelial-sperm interaction. In addition, the luminal fluid of the oviduct provides an optimum microenvironment for embryo development. It contains molecules that contribute to support gamete viability and preimplantation development. Chlamydia trachomatis is a bacterium that causes chlamydial infection and one of the most common causes of sexually transmitted disease worldwide. This asymptomatic bacterial infection may remain undiagnosed or untreated, which in turn ascend to the upper female reproductive tract and infect the oviduct. Infection of the oviduct can result in scarring and damaging inflammation within the microenvironment of the oviduct. The release of pro-inflammatory cytokines during Chlamydial infection may lead to functional damage of the oviduct. Despite the importance of the oviduct, detail on how oviduct responds to the release of pro-inflammatory cytokines during infections is not well known. This project has used in vitro oviduct organoids to assess whether organoids could be engineered to allow the investigation of sperm-oviduct interaction and investigates the impact of inflammatory mediators on the epithelial cells and luminal fluid of the oviduct. The project has explored the genomic and physiological responses of oviduct organoids. The data showed that the in vitro organoid model clearly recapitulates the in vivo oviduct tissue by expressing the key cellular structures. A complete inversion of the oviduct organoids is achievable for future research on gamete-oviduct interaction. IFNγ is an effective inflammatory mediator and has a significant impact on the epithelial cells of the oviduct. The downregulation of ANXA5 might play a key role in infertility in patients with Chlamydial infection. Oviduct organoids provide a promising platform and a powerful tool for future investigations and to address questions regarding reproductive system.12 0Item Restricted Exposure to Allergens and Proinflammatory Mediators Modulate Airway Epithelial Cell Innate Responses, Metabolism, and Physiology(University of Alberta, 2024) Alzahrani, Khadija Rashed; Vliagoftis, HarissiosLungs and airways health and protection depend on the integrity of airway epithelium. Insulin is a growth hormone that through activation of several signaling pathways play a central role to maintain cellular growth and activate metabolism. Airway epithelium is in constant exposure to inhaled agents like pathogens, allergens, pollutants, and particular matters. Airborne allergens like cockroach or house dust mite (HDM) contain proteases that can interact with airway epithelium and initiate immune responses through activation of airway epithelial cells and innate immune cells. Airway epithelium cytokines and innate immune cells promote activation of adaptive immune cells like Th2 lymphocytes that produce proinflammatory mediators like interleukin-4 (IL-4), IL-13, and IL-5. T helper 2 (Th2) cytokines amplify the immune responses and stimulate airway epithelial cells to release CCL chemotactic factors/eotaxins that attract eosinophils to the site of inflammation. Airway epithelium exposure to inhaled irritants and excessive inflammatory responses is known to disrupt epithelium integrity and exacerbate inflammatory responses. Th2 inflammation in asthma is associated with epithelial injury, epithelial remodeling, and metabolic alterations. Increased expression of inflammatory mediators tumor necrosis factor (TNF) and IL-6 in metabolically active tissues have been associated with cellular damage and insulin resistance. Inducible AECs local inflammation was shown to trigger systemic inflammation, insulin resistance, and impaired glucose metabolism, and the severity of inflammation correlated with impaired glucose metabolism. Several studies showed that insulin resistance increased the risk of aeroallergen sensitization and developing asthma-like symptoms. In this project we hypothesized that exposure of airway epithelial cells (AECs) to allergens and proinflammatory mediators may modulate Th2 immune cell responses, induce insulin resistance and metabolic shifts. First, we examined the effect of cockroach and house dust mite on IL-13 and IL-4-induced inflammatory responses. AECs were cultured and stimulated with cockroach or house dust mite, IL-13, IL-4, or a combination of an allergen and IL-13 or IL-4. IL-13 and IL-4 induced effects were measured. Then, we tested the effect of cockroach, house dust mite, TNF or IL-6-induced inflammatory responses on physiological epithelial changes and insulin-induced effects. Epithelial proliferation, resistance, energetic phenotype changes, and insulin induced activation of PI3K/Akt were examined. We showed that serine proteases of cockroach extract prevented IL-13-induced expression of eosinophil chemokine CCL26 from AECs. Depletion of CCL26 was previously shown to delay resolution of airway allergic inflammation which may result in prolonged eosinophilia. Prolonged inflammation was linked to altered metabolism and insulin action in several studies. We showed for the first time that house duct mite and TNF modulated insulin effects in AECs. TNF and HDM changed insulin-induced ATP production in AECs. Additionally, TNF reduced insulin-induced Akt phosphorylation, reduced epithelial barrier function and recovery after injury. Our data suggest that cockroach serine proteases and TNF may interfere with the Th2-mediated proinflammatory effects, regulate AECs energetic phenotype, and induce insulin resistance. These inflammatory and metabolic changes exacerbate the pathogenesis of asthma which may alter immune cell responses.5 0Item Restricted Role of C-reactive protein in airway smooth muscle dysfunction relevant to obstructive lung disease.(University of Leicester, 2024-07-31) Alanazi, Amani; Saunders, RuthC-reactive protein (CRP), is an inflammation marker, often elevated in airways conditions such as asthma and COPD. This research investigated the impact of CRP on airway smooth muscle (ASM) cells, which are crucial in the airway remodelling and hyperresponsiveness which is observed in these conditions. By using primary human ASM cells, this study has shown that purified CRP reduced cell number, increased cell size and intracellular complexity, and maintained cell viability. However, these findings were not replicated with recombinant CRP, which lacks endotoxin, suggesting that endotoxin contamination in the purified CRP may have played a role. Lipopolysaccharide (LPS) treatment, an endotoxin component, resulted in increased intracellular complexity but did not completely replicate the other effects of purified CRP on ASM cells. Moreover, a CRP-neutralizing antibody did not reverse the changes induced by purified CRP, indicating the potential involvement of contaminants. The differences between purified and recombinant CRP highlight the challenges in isolating the true effects of CRP from those of other inflammatory agents. Future research will give priority to endotoxin removal or neutralization, using higher concentrations of both CRP and the neutralizing antibody. Additionally, the study will focus on exploring potential synergistic effects between CRP and LPS on ASM cells. Further investigations are needed to fully understand the role of CRP in the ASM dysfunction and the underlying mechanisms, including apoptosis, hypertrophy, and mediator secretion. In conclusion, this study suggests that CRP has the potential to contribute to ASM dysfunction but underscores the importance of strict experimental controls to distinguish its effects from potential contaminants like endotoxin. The research emphasizes the need for further exploration of the complex interplay between CRP, endotoxin, and ASM cells to elucidate their individual or combined contribution to ASM dysfunction in lung diseases.85 0Item Restricted Elucidation of the Role of Methylarginine Metabolism in Regulation of Nitric Oxide Production and Inflammation(University of Glasgow, 2024) Alshuwayer, Noha Ali S; Leiper, James; Mercer, JohnAtherosclerosis is a major global health issue, and inflammation is important in its pathogenesis. Many atherosclerosis risk factors lead to reduced nitric oxide (NO) bioavailability. Asymmetric dimethylarginine (ADMA), an independent cardiovascular disease risk factor and NO synthase inhibitor, is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). DDAH2 is the isoform present in the immune system. A deeper understanding of ADMA metabolism could help reveal new therapies for atherosclerosis. However, it is debated if DDAH2 hydrolyses ADMA. There is evidence that DDAH2 has NO-independent cellular functions, and research in our group showed that DDAH2 regulates macrophage functions. This thesis initially aimed to investigate the role of DDAH2 in regulating inflammation in atherosclerosis models. However, this was derailed by limitations imposed by the Covid-19 pandemic. Therefore, models of inflammation were used. Genes and mechanisms associated with inflammation and atherosclerosis were investigated. RAW 264.7 murine macrophage cell line and bone marrow-derived macrophages (BMDM) were validated for suitability to study the DDAH-ADMA-NOS pathway. To better understand the functions of DDAH2, a macrophage-specific Ddah2 null mouse model was re-derived and validated. RNA sequencing data previously generated by our group from peritoneal macrophages of the same model was re-analysed and revealed almost 5,000 genes to be DDAH-dependent and required for normal immune response. More than 200 Reactome pathways appeared enriched, with apoptosis being the most enriched. The in silico data was validated in vitro in DDAH2-knockout peritoneal macrophages from the macrophage-specific Ddah2 null mouse model. Inferred hypotheses were investigated in DDAH2-Knockout BMDMs from the macrophage-specific Ddah2 null mouse model with confirmatory studies on C57BL/6J BMDMs using ADMA. The in vitro analysis in the BMDMs showed no conclusive evidence supporting the in silico data that DDAH2 regulates the investigated genes (except Il17a), nor did ADMA alter the gene response to LPS. Il17a was shown by the in silico analysis to be regulated by DDAH2 and was validated in vitro in peritoneal macrophages by both RT-qPCR and ELISA. Given the significant role of IL17A in inflammation and its existing use in treating systemic inflammatory conditions such as psoriasis, this thesis proposes DDAH2 as a potential therapeutic target for inflammatory diseases in general and atherosclerosis in particular.15 0Item Restricted The role of ASC inflammasom in Pancreatic Cancer(Saudi Digital Library, 2022-09-17) Kashgari, Bassam; Jenkines, BrendanPancreatic ductal adenocarcinoma (PDAC) is an aggressive and highly lethal malignancy. The vast majority of PDAC patients are diagnosed at an advanced and incurable stage of the disease. Even with the best current treatments, the prognosis for advanced PDAC is less than one year. While molecular targeted treatment is widely used in many malignancies with impressive results, no such “precision medicine” approach is currently used for PDAC. The dysregulation of the immune system plays a role in the progression of the disease; in fact, chronic pancreatic inflammation is a risk factor for PDAC. Inflammasomes are key regulators of innate immunity in chronic inflammatory disorders and autoimmune diseases, but their role in PDAC remains ill-defined. The genetically engineered KPC mouse model mimics the molecular and cellular immunological and oncogenic processes that drive human PDAC. The genetic abolition of the Pycard gene, encoding ASC, dramatically reduced tumor growth in different age groups of KPC mice. This phenotypic reduction was associated with reduced levels of inflammasome activity and maturation for the downstream target of inflammasomes Interleukin (IL)-18 (but increased IL-1B). Subsequently, the elevated levels of IL-18, inflammation, proliferation, and apoptosis were reversed upon the genetic disruption of ASC expression in the KPC mouse model in different development stages of PDAC. Furthermore, our clinical data supports that ASC inflammasomes are over-activated and associated with poor patient survival and responsiveness to chemotherapy in PDAC. In conclusion, the findings in this work show that elevated ASC levels are a key feature of PDAC and supports the role of ASC as a tumor promoter in PDAC (e.g. tumor incidence and PDAC characteristics) through mediating IL-18 rather than IL-1B. Understanding the currently ill-defined role of ASC inflammasomes in PDAC may provide novel biomarkers and druggable candidates for targeted therapy for PDAC patients.a16 0Item Restricted Macrophage Function in Processing of Excess Dietary Fat(Saudi Digital Library, 2023-06) Bajonaid, Amal; Van Dyke, ThomasBackground: Chronic low-grade metabolic inflammation is one of the hallmarks of metabolic diseases. The risk of periodontitis is approximately threefold higher in diabetic individuals. To examine this relationship, we used a strain of transgenic mice overexpressing the receptor for Resolvin E1 (ERV1tg) that have a reduced inflammatory phenotype depicted by higher resolving (M2) to inflammatory macrophage (M1) ratio and are resistant to periodontitis and compared them to wild type (WT). Objective: investigate the role of resolving macrophages in processing of excess dietary fat in ERV1 tg mice and determine the difference that leads to sexual dimorphism in energy utilization by metabolic organs. Materials and Methods: 6-week-old ERV1tg and WT male and female mice were fed HFD for 16 weeks, 5 mice per group. Differences in expression of uncoupling protein 1 (UCP-1), peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1⍺), nuclear respiratory factor 1 (NRF-1), sirtuin1 (SIRT1) and estrogen-related receptor alpha (ERRα) in visceral adipose tissue (VAT), liver and brown adipose tissue (BAT) were assessed using real-time quantitative PCR and western blot. mRNA expression was normalized to peptidylprolyl isomerase A (PPIA) as a reference gene. Protein expression was normalized to Actin-B. to investigate the impact of RvE1 on cholesterol uptake by macrophages and efflux to HDL, we used RAW264.7 murine macrophage cell line and performed in vitro cholesterol uptake and efflux assay and investigated the impact of varying concentrations of RvE1 on cholesterol uptake and efflux to HDL. Statistical analysis: One-way ANOVA with ⍺=0.05. Results: Expression of PGC1-α mRNA in VAT was significantly higher in ERV1 tg female mice (4.4-fold ± 0.66) compared to ERV1 tg male mice (0.65-fold ± 0.12, p <0.0001). Relative NRF- 1 mRNA expression in VAT was significantly higher in ERV1 tg female mice (1.6-fold ± 0.12) compared to ERV1 tg male mice (0.65 ± 0.13, p < 0.0001). ERV1 tg female mice showed significantly higher expression of Sirt-1 mRNA in VAT (3.1-fold ± 0.26) compared to ERV1 tg male mice (1.7-fold ± 0.29, p < 0.0001). NRF-1 mRNA expression in the liver was significantly higher in ERV1 tg female mice (2.5-fold ± 0.21) compared to ERV1 tg male mice (2.0-fold ± 0.59, p = 0.0002). Relative Sirt-1 mRNA expression in BAT was significantly higher in ERV1 tg female mice (2.2-fold ± 0.12) compared to WT female (0.5-fold ± 0.08, p < 0.0001), and ERV1 tg male mice (1.3-fold ± 0.29) compared to WT male mice (0.47-fold ± 0.09, p < 0.0001). Conclusions: This study provides direct evidence for molecular targets of RvE1-mediated regulation of obesity that display sexual dimorphism suggesting that female ERV1 tg mice have higher metabolic flexibility through increased mitochondrial function to adapt to excess energy.22 0