Saudi Cultural Missions Theses & Dissertations
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Item Restricted In vitro and in vivo models to study the effect of biofilms and antimicrobial wound dressings on wound healing(The University of Manchester, 2024-06-25) Baqader, Sajwa; Thomason, Helen; Humphrey, Gavin; McBain, AndrewBackground: Biofilms have been strongly implicated in delayed wound healing. Reduced oxygen and low microbial growth rates within wound biofilms are a recognized driver of tolerance towards antimicrobial agents, and poor treatment outcomes. There is however uncertainty about the contribution of biofilm to delayed healing and the mechanisms involved. This emphasises the need to examine the differences in the wound healing process between the planktonic and biofilm-infected wounds through preclinical models. Improved knowledge in this area may lead to the development of improved treatment strategies. Antimicrobial chemicals, especially silver-based ones, are frequently employed in wound dressings, but little is known about their efficacy and there are few reliably predictive preclinical wound models. This doctoral thesis focuses on visualising the variations in metabolic activity in bacterial cells during biofilm development and studying the differences in the wound healing process between planktonic and biofilm-infected wounds. This thesis also evaluates the antibacterial efficacy of innovative wound dressings that incorporate silver oxynitrate, which produces highly reactive Ag2+ and Ag3+ ions, using in vitro and in vivo models. Methods: Initially, a 96 well plate-based assay was used to determine the bactericidal activity of dressings containing silver oxynitrate and untreated controls against planktonic S. aureus in both log- and stationary phases. A colony biofilm model was then used to assess the efficacy of dressings containing silver oxynitrate in eradicating biofilms over 1, 3, and 7 days. Next, an in vivo excisional wound model either uninfected or infected with 104 S. aureus in planktonic form, or as preformed biofilms was established, and used to assess effects of dressings containing silver oxynitrate compared to non-antimicrobial controls and uninfected wound controls. Bromodeoxyuridine (BrdU), a nucleotide analogue that incorporates into nascent DNA in actively dividing cells was used to visualise proliferating. Biofilms were exposed to BrdU for 4 h before harvesting at 1, 3 and 7 days. Wound healing was characterised by quantification of wound width, area, and re-epithelialisation from histological sections. Inflammation was determined based on neutrophil/macrophage marker immunohistochemistry. Wound biofilms were characterised by anti- BrdU immunofluorescence, fluorescence in-situ hybridisation specific prob, viability mapping, tissue Gram stain, quantitative bacterial culture, and scanning electron microscopy. Results: Strong BrdU staining was observed corresponding to active growth of 1 day S. aureus biofilm and slow metabolic rates occurred following 3 days old biofilms. No BrdU signal was detected in the 7 days old biofilms of S. aureus. Over 7 days, wound width and areas increased significantly in wounds infected with preformed biofilm and in planktonic infected wounds compared to uninfected wounds. A significant reduction in the reepithelialisation percentage was observed in wounds infected with preformed biofilms and infected planktonic wounds. In contrast, uninfected wounds exhibited a small wound width, area, and comparatively high reepithelialisation percentage. The number of neutrophils and macrophages was significantly higher in wounds infected with preformed biofilm than wounds that infected with planktonic bacteria. S. aureus abundance on surface of both planktonic and biofilm infected wounds was markedly increased after seven days. Dressings containing silver oxynitrate significantly reduced planktonic bacterial cell counts more during log phase than stationary phase. Furthermore, this dressing effectively decreased number of bacterial cells in colony biofilm following a 7-day exposure. The dressings containing silver oxynitrate also significantly reduced S. aureus viable counts, wound width, area, inflammation, and increased reepithelialisation more in planktonic infected wounds than biofilm infected wounds over 7 days. SEM imaging revealed that infected wounds treated with silver oxynitrate-incorporating dressings contained fewer bacteria and biofilm extracellular polymeric substance was disrupted. Conclusion: The in vitro colony biofilm method of labelling metabolically active S. aureus with BrdU over 7 days demonstrated the bacterial cells dormancy. The murine wound model provides evidence of the impact of S. aureus in planktonic and biofilm form in delaying healing process. The use of dressings containing silver oxynitrate demonstrated a greater impact on the treatment of infected murine wounds in both states, with greater efficacy against planktonic infected wounds. The in vitro and in vivo models mentioned above can be used to evaluate effectiveness of other wound treatments obtain better wound management and patient outcomes.19 0Item Restricted Anti-atherogenic actions of hydroxytyrosol(Saudi Digital Library, 2023-11-02) Alotibi, Reem Mansour; Ramji, DipakIntroduction: Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of lipids in the arterial wall, and is considered to be a major contributor to cardiovascular disease (CVD). Worldwide, CVD is responsible for a third of all deaths. Current pharmacological therapeutic agents for CVD, such as statin therapy, are not fully effective and have considerable residual risk for the disease. Alternative agents for the prevention and treatment of the disease are therefore required. Hydroxytyrosol (HT) is a polyphenol compound found mainly in olive oil. HT has been reported to prevent CVD predominantly due to its antioxidant effects. HT is the only polyphenol recognised by the European Food Safety Authority as a protector against low-density lipoprotein-mediated oxidative damage. Previous investigations carried out in the host laboratory showed that HT altered multiple atherosclerosis-associated risk factors in wild-type mice fed a high-fat diet (HFD) for 3 weeks, as well as various anti-inflammatory and anti-atherogenic actions on human monocytes/macrophages in vitro. Unfortunately, the actions of HT in atherosclerosis in vivo are poorly understood. The aim of this study therefore was to elucidate its effects on atherosclerosis progression and regression in a mouse model system. Methods: In order to study the development of atherosclerosis in vivo, 8-week-old male or female low-density lipoprotein receptor-deficient (ldlr-/-) mice were given HFD alone or in combination with 10 mg/kg/day of HT for 12 weeks. For regression studies, the mice were fed a HFD for 12 weeks to promote the formation of established lesions, and then switched to normal chow diet (NCD) alone or in combination with HT. The two procedures were then followed up by an in-depth investigations of atherosclerosis- associated risk factors and the plaques that formed in the aortic root. RNA-sequencing and bioinformatic analyses was used to assess changes in gene expression and associated pathways in the thoracic aorta. The liver was also analysed in relation to non-alcoholic fatty liver disease (NAFLD) that is often associated with atherosclerosis and extended via the use of an in vitro hepatoma HepG2 cell culture model system. Results: As part of the progression study, female ldlr-/- mice that had received HT supplemented HFD for 12 weeks had attenuated weight gain, plaque size in the aortic root and neutrophil content in the peripheral blood, while male ldlr-/- mice had attenuated occlusion and T cells in the peripheral blood. In both cases, there was reduced plaque inflammation and improved plasma lipid profile and plaque stability. As part of the regression studies on male ldlr-/- mice, intervention with HT combined with NCD reversed hepatic injury and enhanced plaque stability (to a greater extent than NCD intervention alone). Conclusions: These findings provide support for the anti-atherogenic actions of HT as well as its possible use as an alternative nutraceutical agent for preventing the development of atherosclerosis. This is potentially possible due to the lack of adverse effects as well as the relatively low cost in comparison to that of typical pharmacological treatments. Additional research is necessary in order to determine the mechanisms that are responsible for these favourable anti-atherogenic and other beneficial changes.16 0Item Restricted Optimization Of Enantiopure Tetrahydro-Β-Carbolines as Potent Antimalarials & Exploration of Salicylic Acid Analogs for Combating Multidrug-Resistant Neisseria Gonorrhoeae(2023-05-13) Almolhim, Hanan; Carlier, PaulThe emergence of drug resistance towards existing drugs is a constant challenge in the fight against many diseases including Malaria and gonorrhoeae. To evade resistance, new targets must be engaged, and to do that, new structural classes of anti-infective must be prepared and evaluated. During the course of my PhD journey, I had the opportunity to investigate and optimize the antimalarial candidate (Å})-2-3b, and salicylic acid (4-1a) as an anti-gonorrhea treatment. Malaria is a life-threatening mosquito-borne disease. In 2021, there were 247 million cases of malaria and the estimated number of malaria deaths stood at 619,000. Because of the rapid development of resistance to all current antimalarials, discovery of antimalarials with unexploited mechanisms of action is critical to reduce malaria mortality. In the Carlier group, our initial approach focused on discovery of inhibitors of the methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis, since this pathway is essential for Plasmodium falciparum and absent in human. Application of the isopentenyl pyrophosphate (IPP) chemical rescue screen to the compounds of the Malaria Box, a collection of 400 antimalaria candidates with unknown mechanisms of action, identified tetrahydro-β-carboline 2-1 (MMV008138) as an inhibitor of the MEP pathway. Chapter 2 of this work discusses similarity searching of the Novartis portion of the hit set (5K compounds), from the original 20K compound hit set of the Malaria Box, and identifying tetrahydro-β-carboline GNF-Pf-5009, designated as (Å})-2-3b. Preparation of pure enantiomers, by resolution, demonstrated the pharmacological superiority of (R)-2-3b over (S)-2-3b, which was ound to have good asexual blood stage (ABS) inhibition potency against malarial parasites P. falciparum, and low general cytotoxicity. However, (R)-2-3b was found not orally efficacious in a P. berghei mouse model of malaria. We concluded that the lack of oral efficacy of (R)-2-3b was due to its poor drug-like qualities, in particular its high molecular weight and low solubility. Chapter 3 of this work explores modifications of (R)-2-3b ((R)-3-5Aa) that were expected to improve its properties. We show that the new compounds (R)-3-5Gm and (R)-3-5Gk not only are more potent in vitro than (R)-2-3b ((R)-3-5Aa), but also have molecular weights < 500 g/mol. Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. Due to the increased rates of infection as well as the prevalence of multidrug-resistant N. gonorrhoeae strains worldwide, the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) list N. gonorrhoeae at the highest possible threat level to public health. Dual therapy of azithromycin (AZM) and ceftriaxone has been the standard-of-care for treatment of gonococcal infections. However, due to increasing resistance to azithromycin (>33% in some regions) the CDC removed AZM from the treatment regimen for gonorrhea in 2020. Therefore, ceftriaxone remains the only recommended antibiotic for treatment of gonococcal infections. However, increasing resistance to this treatment option has been reported, consequently there is an urgent need to identify novel therapeutics against N. gonorrhoeae. Drug repurposing is a popular strategy that explores new therapeutic opportunities for approved drugs with available information on their pharmacokinetic data, dosages, and toxicity. Salicylic acid is a highly privileged chemical scaffold. Also, the use of salicylic acid to treat sexually transmitted diseases (including gonorrhea) was reported as early as the 19th century. Recently, Dr. Mohamed N. Seleem reported that salicylic acid (4-1a) exhibited modest activity against N. gonorrhoeae strains including the AZM-resistant strain (CDC-181). Chapter 4 of this work illustrates how the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution. However, we found that substituted naphthyl analogs (4-3b,o,p) have superior activity to salicylic acid itself. In addition, the three analogs showed high selectivity, compared to AZM, against N. gonorrhoeae over the vaginal microbiota.15 0