Saudi Cultural Missions Theses & Dissertations
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Item 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 Nucleoside Salvage and Metabolism in Trichomonas vaginalis as a target for new therapeutic approaches(University of Glasgow, 2023-10-28) Alsiari, Tahani; Koning, Harry DeTrichomonas vaginalis is a highly prevalent human urogenital protozoan parasite and the causative agent of the most common non-viral sexually transmitted disease (STD), trichomoniasis. Despite the prevalence of trichomoniasis exceeding that of chlamydia, gonorrhoea and syphilis, T. vaginalis has not received the attention it deserves from the public health community. Numerous studies have highlighted the serious health risks associated with T. vaginalis infection, which occurs in the female lower reproductive tract and the male urethra and can result in serious health complications. These include an increased risk of secondary infections of Human Immunodeficiency Virus (HIV) and other STDs. For almost 50 years, metronidazole has been used as the primary treatment for this disease. As a result of side effects and increased resistance to the drug, the development of a substitute compound has become increasingly urgent. As in all protozoan parasites, T. vaginalis lacks de novo synthesis of purines and pyrimidine nucleotides. Instead, it realises on purine and pyrimidine salvage pathways as a source of these nutrients from the host. Indeed, in order to survive and reproduce, protozoan parasites depend completely on salvage pathways and nucleoside transporters. The salvage of nucleosides and nucleobases is believed to be dependent on the Equilibrative Nucleoside Transporter family (ENT). To date, a total of nine genes belonging to the ENT have been identified in the T. vaginalis genome. The aim of this thesis was to identify and functionally characterise these transporters in T. vaginalis. Due to cross-resistance, and the side effects associated with metronidazole (the current treatment for trichomoniasis), new drug targeting strategies and chemotherapy are urgently needed. The study of the ENTs’ ability to uptake vital nutrients could provide new therapeutic approaches as targets for inhibitors, or conduits for nucleoside antimetabolites or other drugs. To achieve this, a strategy to clone T. vaginalis ENT genes into heterologous expression systems was adopted, so they can be studied individually. To achieve this, a new expression system needed to be developed, with at most a very low level of nucleoside uptake. The strategy selected was to delete all three nucleoside transporters from Leishmania mexicana (LmexNT1.1., LmexNT1.2 and LmexNT2) promastigotes using CRISPR/cas9. The author meticulously implemented this strategy by directly knocking out NT1.1 and NT1.2, while a dedicated member from the HDK group took charge of the NT2 component. This collaborative effort ensured a comprehensive and proficient execution of the genetic modifications required for the removal of targeted nucleoside transporters within the Leishmania mexicana promastigotes. The resulting cell line displayed almost no uptake of purine or pyrimidine nucleosides and survived by salvaging purine nucleobases through its LmexNT3 transporter instead. This new cell line, named L. mexicana ‘super knock-out’ (SUPKO), constitutes an easily manipulated system for studying and understanding nucleoside transport. TvagENT genes 1 to 9 gene have previously been sub-cloned into Trypanosoma brucei brucei expression vectors and a nucleoside transport assay was performed on TvagENT3 and TvagENT6 transfected into the T. b. brucei strain TbAT1-KO. However, these cells still have a high background of uptake of almost all purine nucleosides and nucleobases, making the assessment of the unknown heterologous difficult. In this thesis, the construction of SUPKO is described, followed by the construction of nine SUPKO-based cell lines, each expressing one of the nine T. vaginalis ENT genes for separate characterisation. In addition, some of the genes were introduced in an L. mexicana NT3-KO cell line for assessment of nucleobase transporters. Among notable results from these studies were that TvagENT3, expressed in SUPKO, was found to be a high affinity transporter for adenosine and inosine and TvagENT6 was able to transport uridine, albeit with moderate affinity. High affinity uridine uptake was found to be mediated by TvagENT8, and this was inhibited effectively by cytidine and adenosine. Trichomonas foetus and Trichomonas vaginalis are two closely related parasites that cause distinct diseases in different hosts. T. foetus causes trichomoniasis in cattle, leading to infertility, abortion, and significant economic losses, while T. vaginalis is responsible for trichomoniasis in humans, potentially causing various health complications. Both parasites have a shared characteristic of being unable to produce purine and pyrimidine, making them dependent on host epithelial cells for nutrients. In this thesis, introduces the identification and characterization of TfENT transporters in T. foetus, comparing them to TvagENTs in T. vaginalis. While the genetic sequences reveal close relationships, the distinct host environments may lead to functional differences. Although progress was limited due to unforeseen challenges, this work is foundational base of TfENT further investigation and characterization. Overall, the chapter highlights the significance of these ENT genes and their functions in parasites and proposes potential therapeutic targets. The resistance mechanism of T. vaginalis against TH1012, an adenosine analogue, has been analyzed through genomics, transcriptomics, and metabolomics data. The results show that the parasite has adapted to the drug in a multifaceted and complex way over a long period. The study reveals that resistant clones have distinct and common responses to TH1012. Additionally, genomic analysis identified a specific mutation in TvagENT8, Gln264Lys, in resistant clones. However, this mutation is not the sole cause of resistance, as multiple transporters are likely involved in adenosine transport. The resistance mechanism involves a complex interplay of genetic mutations, gene expression changes, and adaptations in cellular metabolism. Moreover, metabolomic analysis points out differences in energy and purine nucleotide metabolism between WT and resistant cells. The study's findings provide comprehensive insights into the multifaceted mechanisms of drug resistance in T. vaginalis, highlighting the need for further research into its unique metabolism and biochemical pathways. Applying the knowledge gained from this these may lead to development of new anti-trichomonal compounds will open the door to developing improved anti-trichomonal control strategies.38 0