Saudi Cultural Missions Theses & Dissertations
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Item Restricted Effect of dietary nitrate supplementation on high-intensity exercise performance and skeletal muscle calcium handling(Loughborough University, 2024-03) Alsharif, Nehal; Bailey, StephenSupplementation with inorganic nitrate (NO3−) has emerged as a precursor of nitric oxide (NO) and a promising nutritional strategy to enhance aspects of health and exercise performance. Initial studies reported enhanced performance during continuous submaximal endurance exercise after NO3− supplementation, but its effects on single and repeated bouts of short-duration high-intensity exercise are equivocal. Moreover, despite its purported ergogenic effects, the underlying mechanisms for enhanced exercise performance after NO3− supplementation are not fully understood. Accordingly, this thesis explored the potential for NO3− supplementation to improve performance during single and repeated bouts of short-duration high-intensity exercise, high-intensity intermittent exercise (HIIE) and severe-intensity continuous cycling and some of the possible intramuscular mechanisms that could underpin ergogenic effects of NO3− supplementation. In Chapter 2, a systematic review and meta-analysis was conducted to assess the effects of dietary NO3− supplementation on single and repeated bouts of short-duration high-intensity exercise. NO3− supplementation had small positive effects on some, but not all, performance aspects during single and repeated bouts of high-intensity exercise. Specifically, time to reach peak power (SMD: 0.75, P < 0.05), mean power output (SMD: 0.20, P < 0.05) and total distance covered in the Yo-Yo intermittent recovery level 1 test (SMD: 0.17, P < 0.05) were improved following NO3− supplementation while peak power output (SMD: 0.01, P > 0.05) and total work done (SMD: 0.06, P > 0.05) remained unchanged. Chapter 3 investigated the effects of short-term NO3– supplementation on muscle sarcoplasmic reticulum (SR) vesicle calcium (Ca2+) handling and the number of repetitions completed during HIIE. Compared to NO3--depleted beetroot juice (PL) supplementation, NO3--rich beetroot juice (BR) did not alter SR Ca2+ release rate pre-HIIE or post-HIIE (P > 0.05). While BR supplementation did not alter SR Ca2+ reuptake tau pre-HIIE (P > 0.05), SR Ca2+ reuptake tau was lower (SR Ca2+ reuptake rate was faster) post-HIIE in BR compared to PL (30.4 ± 6.9 vs.35.3 ± 6.5 s, P < 0.05). No changes in the expressions of Ca2+ handling proteins (SERCA1 & 2, PLN, P-PLN, CSQ 1 & 2) were observed (P > 0.05). The total number of repetitions completed in the HIIE test continued to exhaustion were greater in BR compared to PL (13 ± 5 vs 12 ± 4, P < 0.05). Chapter 4 assessed the effect of short-term dietary NO3– supplementation on skeletal muscle mitochondrial function in permeabilised muscle fibres and SR vesicle Ca2+ release and reuptake rates at rest and following severe-intensity cycling. Mitochondrial respiration analysis revealed no significant alterations in complex I leak, ADP-stimulated respiration through complexes I-II, or maximal electron transfer system activity through complexes I-IV following BR supplementation at rest or after severe-intensity cycling (all P > 0.05). Similarly, SR vesicle Ca2+ release and reuptake rates were not different between BR and PL pre- or post-exercise. There was no difference in severe-intensity exercise time to exhaustion following BR compared to PL supplementation (453 ± 156 s vs. 414 ± 147 s, P > 0.05). The principal novel findings from this thesis are that NO3– supplementation: 1) improves some performance aspects of single and repeated bouts of short-duration high-intensity exercise; 2) attenuates the HIIE-induced slowing of SR Ca2+ reuptake without changing muscle Ca2+ handling proteins; 3) does not alter skeletal muscle mitochondrial respiration or Ca2+ release and reuptake rates at rest or following severe-intensity cycling. Collectively, these original findings enhance understanding of the exercise settings and potential mechanisms by which NO3– supplementation can improve exercise performance in healthy humans.10 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 Therapeutic Interventions to Target Fetoplacental Vascular Dysregulation in Fetal Growth Restriction(The University of Manchester, 2024-06-21) Almohammadi, Lujain; Brownbill, PaulFetal growth restriction (FGR) affects 3-7% of pregnancies and is a condition where the fetus fails to reach its genetic growth potential, often due to placental dysfunction. This can lead to stillbirth and in survivors increased risk of morbidity and ill health in later life. FGR is associated with reduced fetoplacental blood flow and elevated placental oxidative stress. Nitric oxide (NO) plays a crucial role in increasing blood flow, but in FGR, NO bioavailability and endothelial nitric oxide synthase (eNOS)-dependent vasodilation are impaired. Shear stress, which is the frictional force between the flowing blood and the endothelium, is a powerful stimulator of NO synthesis and vasodilation in several vascular beds. Flow mediated vasodilation (FMVD) is dysregulated in FGR. NOs is mainly produced by eNOS, using L-arginine as a substrate and various cofactors including tetrahydrobiopterin (BH4). Arginase-2 (ARG-2) competes with eNOS for L-arginine, thereby inhibiting NOS-dependent relaxation. BH4 is an essential cofactor for eNOS enzyme activity; without BH4, eNOS creates superoxide instead of NO. In this thesis, the focus was on investigating the effectiveness of (S-(2-boronoethyl)-L-cysteine) (BEC) as a potent and specific arginase inhibitor, along with BH4 both alone and in combination. Here two hypotheses were tested (a) Arginase inhibition by BEC, alone and with BH4, would augment NO production by the fetoplacental endothelium. (b) Arginase inhibition by BEC will reduce vascular tone and improve FMVD in normal pregnancy (NP) and FGR. The aim was to identify potential therapeutic strategies that could increase NO bioavailability, lower vascular resistance and improve FMVD in FGR. Two placental preparations were used to test the hypotheses (a) primary cultures of human placental arterial endothelial cells (HPAECs) (b) dual perfusion of the human placental cotyledon ex vivo. Experiments were performed on placentas from normal pregnancy (defined as the delivery of a singleton infant at term with an individualised birthweight ratio (IBR) between the 20th and 80th centiles) and FGR (defined as an infant with an IBR below the 3rd centile). Using HPAECs maintained in static culture conditions, the effects of BEC, BH4 and BEC+BH4 were investigated on the expression of cell stress related proteins (Proteome Profiler Human Cell Stress Array), the phosphorylation of protein kinases in the eNOS pathway (Human Phosphokinase Array) and on arginase activity (MAK384, Sigma-Aldrich kit). The effects of BEC and BH4 on the ratio of phospho-eNOS (S1177) to total eNOS, and on NO production (nitrite measured in culture medium after 48hr treatment using the Greiss reaction) were assessed in static conditions and in response to shear stress of 20dyn.cm2 (cells maintained under flow: Ibidi fluidic units). The effect of BEC on the fetoplacental vasculature of FGR was studied using the dual placental perfusion preparation. Baseline fetal inflow hydrostatic pressure (FIHP; a measure of fetoplacental vascular resistance), and the reduction in FIHP in response to incremental increases in flow rate (FMVD), were measured in NP and FGR. Initial studies to assess potential concentration-dependent effects of BEC and BH4 on HPAEC viability, showed that metabolism (MTT assay) was unaffected by BEC (0.125 µM -1250µM) but reduced by BH4 (at 0.2µM -20µM) (n=6 placentas: NP). BEC (12.5µM) reduced the expression of 15 pro-survival proteins, necessary for the regulation of cellular oxidative stress (expression <40% of control; pooled cell lysate from 3 placentas; NP); this effect was not reversed by BH4 (20 µM). BH4 alone increased the expression of proteins involved in cellular growth, angiogenesis, proliferation, activation of eNOS, regulation of inflammatory responses, and protection from reactive oxygen species (ROS). In general, neither BEC nor BH4 increased the expression of phosphorylated proteins involved in eNOS signalling. The ratio of active eNOS to total eNOS was also unaffected by BEC and BH4 in static culture conditions or under flow in NP (n=3-6) or FGR (n=2). Neither BEC nor BH4 increased NO production by HPAECs of NP (n=3-6) or FGR (n=2). However, BEC and BH4 failed to inhibit arginase enzyme activity in NP (n=6) or FGR (n=2). There was no evidence that BEC could lower fetoplacental vascular resistance or increase FMVD upon ex vivo placental perfusion in normal pregnancy (n=5). In the current study, reduced FMVD was not evident in FGR and BEC did not affect FIHP or FMVD (n=3) The limited number of samples used in this study precludes statistical analyses of some of the data and definitive conclusions cannot yet be made. The indication from the preliminary findings, that BEC failed to activate eNOS or stimulate NO production by HPAECs, could be related to its failure to inhibit arginase activity and/or its effect to lower the expression of anti-oxidant proteins. BH4 reduced cell metabolism but in general, increased the expression of proteins that have a cell protective effect. BH4 but did not elevate NO production alone or in concert with BEC. There was no evidence of the ability of the fetoplacental circulation to elicit a vasodilatory response upon perfusion with BEC arginase inhibitor in NP or FGR, arginase activity was not assessed in this preparation. Based on the preliminary findings, it seems that BEC might not be an effective therapeutic intervention for improving fetoplacental blood flow in cases of FGR. It is possible that BEC could increase oxidative stress through off-target actions, which could explain its lack of effect. Further studies are needed to determine whether BH4 could be a viable strategy for enhancing the ability of placental endothelial cells (ECs) to generate NO in FGR and reduce vascular resistance to improve blood flow.19 0Item Restricted Efficiency of Nitric Oxide and Peroxynitrite Release by Endothelial Nitric Oxide Synthase Variants - Implications for Cardiovascular Disease and Aging(Ohio University, 2024-05) Alsulami, Seham; Malinski, Tadeusz; Dewald, HowardThe cardiovascular system is mainly regulated by nitric oxide (NO). A reduction in its synthesis or bioavailability might underlie the impaired endothelium-dependent vasodilatation, which is observed in the blood vessels of individuals with cardiovascular disease (CVD). The dysfunction of endothelium, which is a main characteristic of vascular aging, has been associated with low NO production and high production of cytotoxic peroxynitrite (ONOO-). Thus, the ratio of NO to ONOO- is an indicator of endothelial dysfunction. Moreover, vascular NO is produced by an enzyme called (endothelial nitric oxide synthase (eNOS), and its gene exhibits high polymorphism. However, it is unclear whether polymorphisms or haplotypes in the eNOS gene affect the NO production, ONOO- production, and eNOS coupling, as well as how aging impacts these haplotypes. The influence of the eNOS haplotype (consisting of single nucleotide polymorphisms (SNP) in the promoter region (T-786C) and (C-665T) and exon 7 (Glu298Asp) and a variable number of tandem repeats (VNTR) in intron 4 (4a/4b/4c)) on the production of NO and ONOO- and eNOS coupling was investigated. Sanger sequencing and DNA electrophoresis were used to detect SNPs and VNTRs in the samples, respectively. To evaluate the production of NO and ONOO-, nanosensors were used to determine the maximal concentrations of NO and ONOO- and traditional and low-temperature SDS-PAGE to evaluate the expression of eNOS and the eNOS dimer/monomer ratio, respectively. Interestingly, these results indicated that the eNOS haplotype (H5) combining the “T T/C C 4b” of the G894T, T-786C, C-665T, and 27 bp VNTR a/b/c is more susceptible to endothelial dysfunction. Compared with other haplotype samples, it had lower [NO]/[ONOO-] and higher eNOS expression with reduced eNOS dimer/monomer (P < 0.005). These findings have important implications for understanding the genetic basis of cardiovascular disease and aging and may lead to new therapeutic approaches to these diseases.40 0