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Item Restricted Obesity is linked to remodelling of mitochondrial dynamics and the inflammasome in the heart(The University of Manchester, 2025) Albalawi, Zainab; Kitmitto, AshrafBackground: Obesity affects 890 million adults worldwide, with the number of obese people predicted to rise to 1 billion by 2030. Obesity is a common precursor to type 2 diabetes (T2DM) both of which are driving the prevalence of Heart Failure with preserved Ejection Fraction (HFpEF). HFpEF has few treatment options in part due to a limited understanding of the pathophysiological processes. This thesis took a combined in-vivo and in-vitro approach to characterise the intersection between changes to cardiac function, mitochondrial dynamics and the inflammasome resulting from diet-induced obesity (DIO) (a longitudinal study), and the effects of associated stressors. The experimental findings additionally led to investigations of the protein MIRO1 (a regulator of mitochondrial motility in neuronal cells), as little is known about the role of this protein in heart health. Methods and Results: 8-week-old C57BL/6J male mice were fed either a 60% High Fat Diet (HFD) or normal chow diet for 16 or 19 weeks. After 16 weeks, the HFD mice developed hyperglycaemia and hyperinsulinemia. Cardiac structure and function, as assessed by echocardiography and electrocardiography (ECG), revealed mild impairment of systolic function, associated with eccentric hypertrophy with no changes to the ECG. Western blotting and RT-qPCR showed a shift towards fission, with a reduction in mitochondrial fusion proteins MFN1 and MFN 2 (~0.6-fold, p = 0.04 and p = 0.05, respectively). Levels of MIRO1 also fell ~2-fold (p=0.0005). In contrast to expectations, extending the HFD protocol to 19-weeks did not affect cardiac function relative to control mice. Furthermore, at 19 weeks there was a shift towards increased fusion (up-regulation of the fusion proteins MFN1 and OPA1 and down-regulation of the fission protein DRP1). Interestingly, the mitophagy proteins PINK1 and PARKIN mirrored changes detected in the 16 week model (up and down-regulation respectively). Protein levels for the NLRP3 inflammasome components increased. Proteomics analysis of isolated cardiac mitochondria (19 weeks) identified increased expression of proteins regulating ketogenic activity. In-vitro (H9C2) cytokine treatments had mixed effects on cardiomyocytes. IL-1β treatment did not affect the mitochondrial and inflammasome proteins, whereas IL-6 and TNF-ɑ affected expression level changes to inflammasome related proteins (NLRP3 and Caspase 1) and proteins linked to mitophagy (PARKIN) and mitochondrial motility (MIRO1 and MIRO2). A cardiac specific MIRO1 knockout mouse (Cre-loxP) was next developed. Partial deletion of MIRO1, Cre+Het (heterozygous), mice exhibited mild diastolic dysfunction, which was exacerbated in the MIRO1cKO (homozygous) model. Mitochondrial function of MIRO1cKO mice was assessed using a high-resolution respirometer (Oxygraph) and displayed impaired oxidative phosphorylation and increased levels of mitochondrial reactive oxygen species (ROS) relative to wild type (WT) mice. Tissue from the apex of MIRO1cKO hearts was fixed for Transmission electron microscopy (EM) revealing more interfibrillar (IFM) and subsarcolemma (SSM) mitochondria with disrupted cristae, consistent with increased Cytochrome C expression compared to WT mice. Cre+Het mice when given a combination of a 60% HFD and L-NAME, when compared to control mice, exhibited a rapid onset of cardiac dysfunction consistent with HFpEF pathophysiology. Conclusion: This Thesis work developed and characterised a DIO mouse model that reiterated features of HFrEF, identifying a shift towards mitochondrial fission. Surprisingly, extending the HFD duration resulted in a reversion of the cardiac dysfunction to a healthy cardiac phenotype. This finding afforded the opportunity to compare the 16 and 19 week models at the molecular level to identify mitochondrial proteins that exhibited plasticity and thus may be linked to the pathogenesis of HF. For example, the shift from fission to fusion could suggest that promoting fusion improves cardiac outcomes. Additionally, upregulation of the ketogenesis pathway proteins was also identified at 19 weeks, suggesting HMGCS2 and BDH1 linked processes may represent intervention pathways. The link between obesity and inflammation status emerged as complex and further studies are required for clear stratification to HFrEF progression. This thesis also generated novel data revealing that the protein MIRO1 plays a crucial role in cardiac function and loss of MIRO1 leads to cardiac and mitochondrial dysfunction and altered the mitochondrial morphology. When combined, the novel results from this Thesis research have identified potential new target candidates/directions for developing novel approaches to preventing/delaying obesity-linked HF and serve as a platform for future investigation.19 0Item Restricted Benefits of Supplementation with LCn-3 PUFA during Diet-Induced Body Mass Loss and Maintenance Phases on Body Composition, Muscle Function, and Appetite(University Of Glasgow, 2025) Alblaji, Mansour Ghazi; Malkova, Dalia; Gray, StuartObesity is a complex medical condition that is associated with a range of comorbidities, including hypertension, type 2 diabetes, dyslipidaemia, gastrointestinal disorders, joint pain, and musculoskeletal complications. Current treatment approaches for obesity primarily involve lifestyle modifications, including diet-induced weight loss and physical exercise. However, evidence from previous research highlights a concern regarding diet-induced body mass loss: approximately 25–30% of the total body mass lost is derived from fat-free mass (FFM). This decline in FFM is associated with diminished muscle mass and function, reduced metabolic rate, and an elevated risk of body mass regain. Attenuating FFM loss during body mass loss is therefore critical for healthy body mass loss. Long-chain n-3 polyunsaturated fatty acids (LCn-3 PUFA) have been proposed as a potential strategy to mitigate these effects by influencing body composition, muscle mass and function, and inflammation during energy balance. Evidence suggests that LCn-3 PUFA can reduce fat mass while enhancing FFM, improving muscle mass, strength, and function, and mitigating inflammation. However, despite these potential benefits, the evidence supporting the efficacy of LCn-3 PUFA supplementation during diet-induced body mass loss on body composition, muscle function, and inflammatory markers remains limited and requires further exploration. The first aim of this thesis was to systematically investigate the effects of supplementation with LCn-3 PUFA during caloric restriction (CR) on body mass, fat mass and FFM loss (Chapter 2). Eleven studies were included in this systematic review and meta-analysis as they met the inclusion criteria of the systematic review, with a total of 637 participants. The participants’ age ranged between 18 and 61 years, with a mean BMI ranging between 27 and 36 kg/m2 . Pooled analyses showed that LCn-3 PUFA supplementation during CR had no additional effect on changes in body mass (SMD = -0.05: 95% CI -0.22 to 0.13; p = 0.62; I2 : 10%), BMI (SMD = -0.06, 95% CI -0.25 to 0.13; p = 0.55; I2 : 18%), fat mass (SMD = - 0.01; 95% CI -0.25 to 0.24; p = 0.96; I2 : 46%), or FFM (SMD = 0.12, 95%CI -0.14 to 0.37, p = 0.36; I2 :35%). The lack of impact of LCn-3 PUFA on body mass and composition observed in this systematic review (Chapter 2) may be attributed to some limitations in the iii included studies. Most of the studies assessed body composition using bioelectrical impedance analysis (BIA), applied low doses of LCn-3 PUFA, and also did not evaluate muscle strength during diet-induced body mass loss. To address the gaps identified in our systematic review, a double-blind, randomised, placebo-controlled trial (RCT) was conducted, including a 4-week preparation phase, an 8-week alternate-day fasting (ADF) phase, and an 8-week body mass maintenance phase, with participants taking 4 capsules/day of krill oil as a source of LCn-3 PUFA throughout (Chapter 4). Body composition was evaluated via the deuterium water (D2O) dilution method, and parameters of muscle function, and fasting blood samples were measured at the pre- and post body mass loss phase. Forty-one healthy adults completed this RCT. The two-way ANOVA revealed significant time and time*group interaction effects on FFM, handgrip strength, chair rising test, TNF-α, CRP, and systolic blood pressure (all p < 0.05). Post-intervention, there was a small, non-significant reduction in FFM (- 0.2 ± 0.9 kg, p > 0.05) and handgrip strength (-0.2 ± 0.5 kg, p > 0.05) in the krill oil group, whereas the placebo group experienced significant reductions in FFM (- 1.2 ± 2.0 kg, p < 0.05) and handgrip strength (-0.9 ± 0.7 kg, p < 0.05). The time to conduct the chair rising test decreased significantly in the krill oil group (-1.8 ± 0.9 s, p < 0.05), whereas the reduction in the placebo group was not significant (- 0.3 ± 1.2 s, p > 0.05). TNF-α levels decreased significantly in both groups (all p < 0.05), with a greater reduction in the krill oil group (-1.4 ± 0.2 pg/ml) compared to the placebo group (-0.9 ± 0.5 pg/ml). Similarly, CRP levels were significantly reduced in both groups (all p < 0.05), with a greater reduction in the krill oil group (-51.4 ± 25 ng/ml) than in the placebo group (-33.5 ± 12.6 ng/ml). Systolic blood pressure decreased significantly in both groups (all p < 0.05), with a greater reduction observed in the krill oil group (-9 ± 6 mmHg) compared to the placebo group (-4 ± 4 mmHg). No significant difference was observed in changes between groups in body mass, body fat, insulin, glucose HOMA-IR, TAG, or diastolic blood pressure (all p > 0.05). Therefore, from this RCT, it was concluded that supplementation with krill oil during diet-induced body mass loss via ADF helps to attenuate the associated decline of FFM and muscle function, improve functional capacity, and reduce TNF-α and CRP levels. Supplementation with LCn-3 PUFA, in the absence of CR, has been associated with appetite reduction and enhanced sensations of fullness and satiety in individuals iv living with overweight or obesity. However, the effects of LCn-3 PUFA supplementation during diet-induced body mass loss on appetite and gastrointestinal appetite hormones remain underexplored. In Chapter 5, the impact of LCn-3 PUFA during diet-induced body mass loss on changes in appetite and gastrointestinal appetite hormones was examined in a subset of the participants of the RCT (Chapter 4). This exploratory study included 28 adults (mean age: 39.4 ± 11.7 years; BMI: 27.9 ± 3.2 kg/m²) who participated in the RCT (Chapter 4). Body mass, body fat, and FFM were measured at baseline (week 4), at the end of the body mass loss phase (week 12), and at the end of the body mass maintenance phase (week 20). Fasting and postprandial subjective appetite scores, along with plasma concentrations of acylated ghrelin, Glucagon-Like Peptide-1 (GLP-1), and Peptide YY (PYY), were assessed before and after the body mass loss phase. The ANOVA revealed a significant time (p<0.05), but not group (p>0.05) or time*group interaction (p>0.05) effects for body mass, fat mass or FFM during the body mass loss phase. During the maintenance phase, no significant (p>0.05) time, group, or time*group interaction effects were found for body mass and FFM, but for fat mass, a significant time*group interaction effect was observed (p<0.05). During the maintenance phase, in the krill oil group, fat mass remained unchanged (p>0.05) but increased significantly (p< 0.05) in the placebo group. This coincided with the body mass loss-induced significant reduction (p<0.05) in the composite appetite score (CAS) in the krill oil but not the placebo group (p> 0.05). There was no significant (p>0.05) time, group, or time*group interaction effects for acylated ghrelin, GLP-1, and PYY during the body mass loss phase. Changes in body mass during the body mass loss and body mass maintenance phases were not correlated with acylated ghrelin, PYY, or GLP-1 (all p > 0.05). Body mass changes during the body mass loss phase showed a tendency toward a significant positive correlation with changes in CAS (r=0.36, p = 0.06). Therefore, krill oil supplementation during body mass maintenance may induce favourable changes in subjective appetite and prevent short-term fat mass regain. Overall, the current thesis demonstrates that supplementing with LCn-3 PUFA during diet-induced body mass loss is a promising strategy to attenuate the loss of FFM and muscle function. Beyond these benefits, LCn-3 PUFA supplementation also reduces inflammation and lowers blood pressure, underscoring its potential to enhance body composition, preserve muscle mass, and promote overall well- v being during body mass loss. Furthermore, LCn-3 PUFA supplementation may reduce subjective appetite and might help to prevent fat mass regain during the body mass maintenance phase, further supporting its role in long-term body mass management.19 0Item Restricted Using multi-modal PET and MRI to predict the site of tumour recurrence in high-grade glioma(The University of Manchester, 2024) Alfaifi, Bandar Q; Hinz, Rainer; Coope, David; Lewis, Daniel; Jackson, AlanBackground: High-grade gliomas (HGG) are highly aggressive and incurable brain tumours that often recur within 2 cm of the original site, even after complete oncological treatments. Advanced MRI and PET techniques hold promise for better tumour delineation and characterisation. This thesis investigates the spatial characterisation of translocator protein (TSPO) and amino acid PET in initially diagnosed HGG and at the point of post-treatment progression, with a view to identifying future sites of disease progression. Methods: HGG patients underwent prospective imaging with [11C](R)PK11195 and [11C]methionine PET alongside MRI including diffusion tensor imaging (DTI). [11C](R)PK11195 binding potential (BPND) and [11C]methionine tumour-to-background ratio were generated. The PET biomarkers were first used to characterise tumour regions defined on MRI as contrast-enhancing (CE) and peritumoral regions and diffusion connectivity map (N=12 initially diagnosed). Secondly, tumour biological volumes were delineated and compared among [11C](R)PK11195 and [11C]methionine PET and CE-MRI using overlap, Dice and Jaccard similarity coefficients (DSC and JSC), and discrepancy measures at baseline (12- initially diagnosed; 8 post-treatment). Disease progression was assessed using follow-up MRI (N=16) and registered with baseline PET biomarkers to explore overlap and similarity. Results: In newly diagnosed HGG, 67±22% of CE-MRI regions showed positive TSPO/methionine, while 36±15% of peritumoral regions showed positive TSPO but negative methionine. TSPO binding in CE-MRI and peritumoral regions was significantly higher than in the contra-lesional reference. [11C](R)PK11195 PET demonstrated a gradual decrease in TSPO binding along the DTI connectivity map compared to [11C]methionine. [11C](R)PK11195 and [11C]methionine biological volumes showed moderate spatial similarity (DSC=0.65±0.15, JSC=0.50±0.16) at initial diagnosis, which decreased for post-treatment HGG (DSC=0.35±0.15, JSC=0.22±0.11). Both PET volumes exhibited moderate overlap with CE-MRI at initial diagnosis, but post-treatment spatial similarity decreased, with substantial discrepancies. TSPO binding and methionine uptake show signals in areas where future disease progression occurred, with average overlaps of 0.51±0.25 and 0.43±0.26, respectively. Conclusions: This work provides the first detailed spatial characterisation of two PET biomarkers in newly diagnosed HGG and at the point of post-treatment progression. Elevated TSPO binding without increased methionine uptake may indicate inflammatory or discrete neoplastic populations not captured by standard imaging techniques. Both PET radiotracers demonstrated increased uptake beyond initial contrast enhancement, and although the exact site lacked specificity, disease progression almost universally occurred within this area. Notably, the discrepancy between TSPO and methionine binding increases post-treatment, with elevated TSPO expression likely reflecting late-stage inflammation contributing to symptomatic worsening in such patients.19 0Item Restricted The Role of OGG1- Mediated DNA Damage Signaling in Circadian Regulation of Pulmonary Cell Homeostasis: Implication for Chronic Lung Diseases(University of Liverpool, 2024-02-05) Al Duhneen, Abdulhadi; Iwanejko, Lesley; Pekovic-Vaughan, Vanja; Hamill, KevinCircadian rhythm disturbances have emerged as crucial factors underlying cellular dysfunction, compromised DNA repair mechanisms, and the development of a spectrum of chronic disorders, including lung diseases. While existing research has underscored the dependency of OGG1 activity on circadian timing, it remains unclear whether a malfunctioning circadian clock plays a pivotal role in the dysfunctional OGG1-BER signaling observed in chronic lung illnesses. Furthermore, the pro-inflammatory cytokine, TNF, is known to exert a pivotal role in the pathogenesis of various inflammatory conditions, attracting inflammatory cells, fueling the production of inflammatory mediators, enhancing oxidative stress, and triggering airway hyperresponsiveness. In this study, TNF stimulation was employed in human lung epithelial cell cultures (BEAS-2B and A549) to investigate the intricate relationship between DNA repair enzymes (Ogg1) and clock genes (Bmal1, Cry1, Per2, and NR1D1). Through the utilization of serum shock to synchronize the circadian clocks in both lung cell lines, we unequivocally establish that BEAS-2B and A549 cells conform to circadian cycles. Evidence suggests that OGG1 mRNA expression is intricately regulated by circadian time shifts in normal lung epithelial cells (BEAS-2B), where it oscillates in phase with the clock gene Per2 and antiphase with the clock gene Bmal1. Conversely, cancer lung epithelium (A549) exhibited an opposite pattern. Comparing A549 to BEAS-2B, it becomes evident that A549 displays an augmented amplitude of clock gene expression alongside a diminished OGG1 mRNA level. Intriguingly, silencing OGG1 with small interfering RNAs (siRNAs) leads to an upregulation of clock genes BMAL1 and NR1D1 in both cell lines. Furthermore, our findings indicate that OGG1 functions as a suppressor of pro-inflammatory cytokines in normal lung epithelial cells, but the inverse holds true for lung cancer. Lastly, the dynamic assessment of core clock gene expression, particularly Bmal1, in human lung epithelial cells was made possible through the development of two novel clock reporter cell lines stably transfected with the Bmal1::Luc clock reporter gene in BEAS-2B and A549. This research sheds light on the intricate interplay between circadian rhythms, DNA repair, and inflammation in lung diseases, offering valuable insights for future therapeutic interventions.19 0Item Restricted Assessing the effect of cisplatin on mitochondria in monocytes and macrophages(amnah alnami, 2022-09-15) Alhassan, Alnami Amnah; Mark, PaulCisplatin is a platinum based chemotherapeutic agent which is widely used to treat a variety of solid tumours. However, the use of cisplatin is limited because of its side effects such as nephrotoxicity, neurotoxicity, ototoxicity, and peripheral neuropathy. Clinically this has become more of a problem over the past 10 years as cancer survivorship has dramatically increased. The mechanism behind how cisplatin induces toxicity is still unclear. We aim to evaluate the effects of cisplatin on mitochondrial metabolism in monocytes and macrophages. THP-1 Blue, a human acute monocytic leukaemia cell line, was used in this study. THP- 1 Blue monocytes were differentiated into macrophages using 20 ng/ml PMA. Mitochondrial activity was determined by MTT assay, and cell viability was determined using Trypan blue assay. Mitochondrial function was assessed using Mito tracker red CMROS and nano live microscopy, and the change in mitochondrial respiration was assessed using the seahorse XF Cell Mito stress test kit. NF-KB activation was measured using Quanti-Blue assay, and IL-6 was measured using an ELISA. Cisplatin dose-dependently decreased mitochondrial activity in THP-1 Blue monocytes and macrophages. Cisplatin caused mitochondrial dysfunction and induced cell death in THP-1 Blue macrophages after exposure to high dose (30 μg/ml) and even at low dose (5 μg/ml) decreased mitochondrial respiration after 24 hours. However, cisplatin treatment did not significantly affect NF-KB signaling in monocytes or macrophages. Also, IL-6 levels were undetectable in THP-1 Blue macrophages after exposure to different concentrations of cisplatin. Therefore, there is a relationship between mitochondrial dysfunction and cisplatin induced toxicity in this cell line. However, further studies are needed to investigate more on macrophages to see if cisplatin triggers inflammation.11 0