Saudi Cultural Missions Theses & Dissertations
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Item Restricted Measuring Nitric Oxide/Peroxynitrite in Function/Dysfunction of Neuronal Nitric Oxide Synthase Using Metalloporphyrin Nanosensors – Implication for Neurodegenerative Diseases(ohio university, 2025) Alsiraey, Nouf; Malinski, Tadeusz; Dewald, Howard DeanNitric oxide (NO), an essential inorganic signaling molecule, plays a role in numerous physiological but also therapeutic applications. Its oxidation product, peroxynitrite ion (ONOO¯), is cytotoxic. Due to their short lifetimes, NO and ONOO¯ concentrations in biological environments are usually transient, at the sub–millimolar to millimolar level, and discriminately return to baseline levels. This work investigates the role of NO and ONOO¯ in neurological situations, with a focus on neurodegenerative diseases such as Alzheimer's disease (AD). To monitor NO and ONOO¯ concentrations in real time, electrochemical metalloporphyrin nanosensors (200–300 nm diameter) were applied and precisely positioned 4–5 ± 1 μm from human neural progenitor cell (hNPC) membranes. Healthy hNPCs produced an average of 107 ± 1 nmol/L of NO and 451 ± 7 nmol/L of ONOO¯, according to the data, resulting in a [NO]/[ONOO¯] ratio of 0.25 ± 0.005. While dysfunctional hNPCs treated to amyloid beta 42 (Aβ42) showed a significant imbalance, with ONOO¯ increasing to 843 ± 0.8 nmol/L and NO levels decreasing to 14 ± 0.1 nmol/L, the ratio was substantially reduced by 94% to 0.016 ± 0.0001. Accordingly, the hypothesis of the study validates the role of the [NO]/[ONOO¯] ratio as a potential biomarker for the neuronal nitric oxide synthase (nNOS) efficiency and the dysfunction of hNPCs, which may have relevance for other neurodegenerative disorders, such as AD. It also investigates pharmaceutical interventions targeting the NO/NOS pathway to restore the [NO]/[ONOO¯] balance in pathological conditions. The study proves the suitability of metalloporphyrin nanosensors for the detection of real-time molecular alterations in hNPCs and monitoring of nitroxidative stress that indicates their applicability for early diagnosis and therapeutic approaches of neurodegenerative diseases.12 0Item Restricted Regulation of inducible Nitric Oxide synthase in adipocytes and Perivascular adipose tissue by the Sphingolipid system(University of Glasgow, 2024) Aljaezi, Ibrahim; Kennedy, Simon; Watterson, KennethObesity is a major risk factor for cardiovascular disease, often characterized by chronic inflammation that can disrupt normal metabolic processes. One key factor in this inflammatory response is the upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. The induction of iNOS in adipocytes and adipose tissue leads to increased production of NO which, in turn, contributes to the regulation of adipose tissue function and also influences lipid metabolism, insulin sensitivity, and the modulation of vascular function. Perivascular adipose tissue (PVAT) surrounds blood vessels and has an anti- contractile effect, helping to maintain vascular homeostasis via release of a variety of bioactive molecules. In obesity-induced inflammation, some studies have demonstrated that inflammation within the PVAT can compensate or have an adaptive role to preserve endothelial dysfunction function; via upregulating iNOS protein and increasing NO production. Sphingolipid system, encompassing a complex network of bioactive lipids like ceramides, sphingosine, and sphingosine- 1-phosphate (S1P), regulates various cellular processes in adipose tissue. Although research has revealed that the sphingolipid system promotes inflammation in adipose tissue, it remains unclear how this system regulates iNOS expression and function. In response, this thesis has characterised the role of the sphingolipid system in iNOS-derived nitric oxide production in adipose tissue and addressed how this influences PVAT-mediated anticontractile activity under inflammatory conditions. The aims were achieved by using a 3T3-L1 adipocyte cell line as well as thoracic aortic PVAT from mouse and rat to study vascular function. Interleukin- 1β (IL-1β) or macrophage conditioned media were used in this study to induce iNOS expression and NO production. In this thesis, Chapters 3 and 4 focus on the potential role of the sphingolipid system on IL-1β-induced iNOS expression and NO production in 3T3-L1 adipocytes. Initially, it was found that stimulation of 3T3-L1 adipocyte with IL-1β increases sphingosine Kinase 1 (SphK1) expression and that S1P-produced by SphKs enzymes activates various proinflammatory pathways including the phosphoinositide 3- kinase/ protein kinase B pathway (PI3K/Akt), mitogen-activated protein kinase MAPK kinase (MEK)/extracellular signal-regulated kinase pathway (MEK-ERK), Jun N-terminal kinase pathway (JNK), but apparently had no effect on the nuclear factor kappa B pathway (NF-KB). In addition, exogenous S1P augmented-IL-1β stimulated iNOS expression and NO production in 3T3-L1 adipocytes which was suggested to be through the Akt and MAPKs pathways (MEK-ERK pathway and JNK pathway). Chapter 4 investigated more fully how distinct SphKs isoforms and S1PRs modulate iNOS regulation in adipocytes stimulated with IL-1β. The iNOS expression and NO production stimulated by IL-1 β was inhibited by pre-treatment with a SphK1 inhibitor (PF543) but not a SphK2 inhibitor (ROME). Moreover, it was demonstrated that S1PR2 inhibition with JTE 013 strongly suppressed IL-1β- induced iNOS expression and NO production in 3T3-L1 adipocytes. This effect was accompanied by inhibition of MAPK and PI3K/Akt signalling, suppressing phosphorylation of ERK1/2, P38, JNK and Akt. Inhibition of ABCA1 with glybenclamide suppressed the additive effect of S1P on iNOS expression and NO production in stimulated adipocytes. Collectively, by targeting SphKs/S1P/S1PRs axis pathway, Chapter 3 and 4 provided evidence that SphK1/S1P/S1PR2/ MAPKs and PI3K/Akt axis is required for IL-1β induced iNOS expression and NO production in adipocytes. In addition, chapter 4 has shown that conditioned medium from activated RAW 264.7 macrophages stimulated iNOS expression and NO production in 3T3-L1 adipocyte; however, prior inhibition of SphK1/S1PR2 axis attenuated this. Similarly, inhibiting S1PR2 in adipocytes prior to stimulation with conditioned medium diminishes iNOS expression and NO production. In chapter five, it was found that S1P or S1P agonists did not modulate vascular tone in mouse thoracic aorta. Also, IL-1β did not affect the anticontractile effect of PVAT in mouse experiments and pre-treatment with S1P exerted no effect. However, in rat aorta, it was confirmed that S1P induced a vasorelaxant effect. IL-1β was found to enhance the relaxant effect of PVAT in rat aorta, which was most likely mediated by iNOS upregulation, increased NO production, and potassium channel activation in the vascular smooth muscle cells. Moreover, IL- 1β upregulated SphK1 in rat aortic PVAT; however, inhibiting SphK1/S1PR2 axis did not reverse the hyporeactivty induced by IL-1β. Collectively, IL-1β induces a hyporeactive effect in rat aortic vessels independent of the sphingolipid axis pathway. In summary, the results of this study imply that IL-1β induces an increase in SphK1 expression in 3T3-L1 adipocytes. The increased SphK1 induced by IL-1β appears to increase S1P production from adipocytes which could contribute to IL-1β- mediated iNOS regulation through activating S1PR2. However, in the functional studies on rat aortic vessels with intact PVAT, it is likely that IL-1β-mediated hyporeactivity via iNOS upregulation was independent of the SphK1/S1P/S1PR2 axis.26 0Item Restricted Investigation of the role of nicotinic acetylcholine receptors in the control of vascular tone(Saudi Digital Library, 2023) Alkhammash, Abdullah; Lever, RebeccaThe endothelium is a thin monolayer of cells that lines the interior surface of all blood vessels and plays several important physiological roles, including in vascular tone modulation via endothelium-dependent relaxation mechanisms. Cholinergic agonists mediate endothelium-dependent relaxation by activating muscarinic acetylcholine receptors (mAChRs) on endothelial cells (ECs). Activating mAChRs releases nitric oxide (NO) and prostacyclin (PGI2 ) and facilitates endothelium-dependent hyperpolarisation (EDH); subsequently, relaxation of adjacent smooth muscle cells (SMCs) occurs. ECs express mAChRs and nicotinic acetylcholine receptors (nAChRs). While it has been established that M 3 mAChRs mediate endothelium-dependent relaxation, the functional roles and activities of nAChRs in regulating vascular tone are still unclear. Alpha 7 nAChRs (α7 nAChRs), which are expressed more widely in non-neuronal cells, have been shown to play an essential role in several physiological and pathophysiological conditions. The present work investigated the role that α7 nAChRs play in resistance arteries, particularly in endothelium-dependent relaxation. Wire (tension) myography was used to study pharmacological responses in rat mesenteric arteries to investigate the relative effects of cholinergic receptors in mediating functional responses to acetylcholine (ACh). Phenylephrine (PE) or U46619 was applied to precontracted arterial segments, which were relaxed using cholinergic agonists in the presence or absence of α7 nAChR antagonists. Nitrite (NO2 - ) and PGI2 were also added to conditioned media to investigate the response of cultured ECs to cholinergic receptor agonists and antagonists. The results show that selective α7 nAChR antagonists enhance ACh-induced endothelium-dependent relaxation in rat mesenteric arteries. In summary, α7 nAChRs act in opposition to mAChRs in response to agonists such as ACh, and these receptors may therefore play a functional role in controlling vascular tone.123 0