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Guidelines published by the ACMG/AMP in 2015 and ACGS in 2020 provide a framework for the assessment and classification of novel variants identified through genetic testing; providing consistency and transparency to the variant classification process. Using variant datasets derived either from online databases, such as ClinVar or gnomAD, or novel variants identified through research and diagnostic panel and exome sequencing, a number of different aspects of the ACMG/ACGS guidelines are assessed for performance. Five key aspects are covered: (1) the use of is silico missense predictors (2) the use of co-localising alternative pathogenic variants (3) variants present in pseudo-population databases, and (4) the use of genetic constraint data (5) the use of multiple complementary in silico tools to assess the likely functional impact of a novel 67Kb duplication identified in 5 neonates; a variant type that is typically difficult to characterise and classify through standardised variant classification guidelines. A novel concept is introduced: the use of meta-positions, whereby the availability of data is increased through the use of information from regions considered to be functionally-equivalent to that being assessed. Internal inconsistencies in evidence weighting are identified and recommendations made over the usage and weighting of specific tools and algorithms; the application of evidence at different weighting based on algorithm score, and the use of LR+ to determine weighting within the framework. Together, this work seeks to improve the application of the variant classification guidelines through evidence-based weighting of criteria, and simplify the process by eliminating the burden of performing recursive variants assessments. The use of additional evidence to support the benignity of a variant, not currently implemented in the guidelines, can be used to exclude variants from the need for manual assessment.

Ingested choline is processed by gut commensal bacteria into trimethylamine (TMA). TMA is then enzymatically converted into trimethylamine N-oxide (TMAO) by members of Flavin-containing monooxygenases (FMOs) in liver. FMO3, TMA and TMAO are linked with metabolic syndrome, and cardiovascular diseases. We employed in vitro model of murine hepatocytes (AML12) treated with TMA and TMAO and C57BL/6 mice treated with A. mucinphila to investigate the health beneficial effect of this bacterium. Quantitative polymerase chain reaction, western blot, and lipid enzymatic assays were used to evaluate the changes of lipid genes expression, ER stress markers and inflammatory signalling molecules upon TMA, TMAO and A. mucinphila treatment. Our studies showed that TMA promotes lipogenic processes through upregulation of fmo genes expression (p<0.05). TMA treated hepatocytes had increased mRNA expression of cholesterol and triglycerides synthesis regulators: srebp2 and srebp1c (p<0.05) and their downstream target genes (p < 0.05) comparing to untreated controls. This promoted elevated triglyceride and cholesterol contents in cell media and in the treated cells (p < 0.05). These changes were associated with the upregulation of miR-125a and miR-125b (p<0.05). miR-125 has been shown to target mRNA of an anti-inflammatory protein A20. A20 protein level was significantly decreased in the TMA treated AML12 compared to untreated ones. TMAO induces lipogenesis (p<0.05) and increase ER stress (p < 0.05). A. mucinphila (Akk) treatment of mice upregulated A20 protein level in liver while reduced level of miR-125b (p<0.05). This can be associated with AMUC_1100 membrane protein of Akk which showed to prevent TMA mediated reduction in A20 in vitro. Conclusion: TMA has a pro-lipogenic properties associated with FMOs activity which mediate miR-125a/b dependent A20 downregulation. On the other hand, TMAO has pro-lipogenic properties, while inducing ER stress. TMA and TMAO effect on hepatocytes could be prevented by Akk treatment of mice or AMUC_1100 transfection of TMA treated AML12 cells.

Aicardi Goutieres Syndrome (AGS1-9) is a rare inflammatory mediated encephalopathy which occurs due to mutations in genes involved in the sensing, regulation and metabolism of self-nucleic acid species. Proteins implicated in AGS play an essential role in self-non-self-discrimination, which helps explain the excessive type I interferon (IFN) production and large upregulation of interferon stimulated genes (ISGs) that occurs in AGS patients. This strong antiviral response is thought to largely contribute to the devastating nature of the condition, which results in significant physical and neurological disability. AGS patients can also suffer from sub-type specific symptoms, such as cerebrovascular disease which only manifests in patients with mutations in SAMHD1 (AGS5). Indeed, AGS5 represents a monogenic form of inflammatory based stroke, and can be utilised to provide unique insight into the mechanisms and causes behind more common forms of stroke, such as viral infections as a risk factor, whereby excessive type I IFN signalling as an antiviral mechanism may be related to the onset of stroke. Moreover, antiviral signalling has also been attributed to cholesterol dysregulation, an additional clinical risk factor for both ischaemic and haemorrhagic stroke. To date, few mouse models successfully replicate the vast clinical phenotypes observed in AGS patients, with none phenocopying the neurological manifestations which are one of the hallmarks of the condition. Contrastingly, transient knockdown of the samhd1 gene in a zebrafish larval model appeared to recapitulate a number of clinically relevant phenotypes, including neurological aspects of the disease, as observed by the presence of intracerebral haemorrhage (ICH) in the larvae. Therefore, zebrafish may offer specific advantages over mice when modelling AGS pre-clinically. As such, the central aim of this PhD was to characterise a novel stable mutant zebrafish model of AGS5 to increase understanding of SAMHD1-induced AGS, and the associated cerebrovascular disease. Zebrafish disease modelling of AGS5 revealed the presence of a number of neurological phenotypes, not previously identified in the existing pre-clinical models, reinforcing the usefulness of zebrafish as a model organism to study AGS. Furthermore, the zebrafish exhibited cerebrovascular deficits and dysregulation of the cholesterol biosynthesis pathway, which was also observed in an AGS patient RNAseq dataset. Further investigation of the type I IFN signalling relationship with cholesterol in human brain endothelial cells, identified cholesterol dysregulation following treatment of the oxysterol 25 hydroxycholesterol (25HC), produced downstream of type I IFN. A reduction in cholesterol from the plasma membrane acted to alter the normal functioning properties of the brain endothelial cells. Taken together, we propose that these data in zebrafish and human cells provide new mechanistic insight into how cholesterol dysregulation, as a result of antiviral signalling, may lead to the generation of cerebrovascular deficits and stroke.

Atherosclerosis is the most common cause of ischaemic heart disease and stroke which represent a global health concern responsible for significant levels of morbidity and mortality worldwide. It is a chronic inflammatory disease characterised by plaque build-up within arterial walls initiated by low density lipoprotein cholesterol. Elevated plasma LDL is a major risk factor for atherosclerosis and its immunogenic oxidation creates neo-epitopes that drive inflammatory immune responses underlying the pathogenesis of atherosclerosis. The germinal centre is found within secondary lymphoid organs and is responsible for the production of high affinity antibody-producing plasma cells and long-lived memory B cells against antigens such as epitopes of oxidised LDL. Class-switched plasma cells and anti-oxLDL IgG antibodies have been detected within atherosclerotic plaques and sera of human and mice suggesting that the germinal centre response is pathogenically dysregulated in atherosclerosis. The tamoxifen-inducible AID-CreERT2-Rosa-EYFP lineage tracing mouse model was used and crossed with the atherosclerosis-prone LDLr-deficient mouse model. This model inducibly and specifically labels germinal centre B cells and their progeny using tamoxifen allowing for tracking of atherosclerosis-specific B cell clones comprising germinal centre, memory, and plasma cells. LDLr-/- and LDLr+/- ('WT') mice were fed chow or high fat diet for up to 8 weeks and upon tamoxifen dosing (timing varied throughout studies) via intra-peritoneal injection or oral gavage, AID-expressing cells (germinal centre B cells) were fluorescently labelled with EYFP. Cells were analysed using flow cytometry and FlowJo software. All graphs and statistics were created using the GraphPad Prism 7.0. In this thesis, I sought to characterise and understand the germinal centre B cell response in atherosclerosis using a lineage tracing mouse model. Firstly, the model was validated as EYFP cells were only detected after tamoxifen administration and were only present in germinal centre-derived cell populations. Furthermore, labelling efficiency was independent of time, diet, or genotype. The germinal centre response was characterised in both WT and LDLr-/- mice demonstrating an time-dependent increase in germinal centre responses. Germinal centre reactions were exacerbated, greater in magnitude and degree of class-switching, by increased plasma cholesterol levels consequent of both HFD and knock out of the LDL receptor. The combination of HFD and LDLr-/- genotype, replicating atherosclerotic conditions, synergistically exacerbated germinal centre responses biased towards pathogenic cellular output due to elevated levels of class switching. Due to the permanence of EYFP labelling, it was possible to track EYFP labelled cells over time and throughout the course of atherosclerosis. Tracking studies revealed that hyperlipidaemic conditions induced by HFD resulted in longer-lived EYFP clones which had increased propensity to undergo class-switching. Furthermore, there was a greater output of EYFP labelled memory and plasma cells. To investigate the mechanism by which HFD induced greater germinal centre responses, interventions were conducted to block the impact of cholesterol and inflammation separately. Use of a cholesterol inhibitor drug and cholesterol free diet revealed that dietary cholesterol is key in driving the exacerbated germinal centre responses observed at early stages i.e., 4 weeks of atherosclerosis. Use of a cytokine inhibitor to dampen inflammation limited germinal centre responses at later stages of atherosclerosis i.e., 8 weeks, suggesting that inflammation plays an important role in germinal centre responses at this later timepoint. Thus, the model proposed is that dietary cholesterol is critical early on in atherosclerosis as disrupted lipid homeostasis results in autoimmune B cell responses with germinal centres primarily reacting to accumulated oxidised LDL while at later stages, the inflammation associated with atherosclerosis fuels exacerbated and pathogenic germinal centre responses. To examine the role of IgG2c and its receptor FcgRIV in atherosclerosis, the impact of IgG2c antibody complexes was tested in vitro. IgG2c significantly enhanced TNF secretion, a marker of inflammation, in FcgRIV-expressing myeloid-derived monocytes. Thus, IgG2c has the potential to exacerbate inflammatory responses from plaque macrophages and dendritic cells. In conclusion, the AID lineage tracing mouse model has been validated for use in the atherosclerosis setting. It has been characterised showing that germinal centre responses are exacerbated during the course of atherosclerosis and are skewed towards production of class- switched longer-lived B cell clones. Dietary cholesterol is the main driver of this pathogenic response in tandem with the inflammatory conditions caused by atherosclerosis. Germinal centre-derived IgG2c antibodies could play an important role in exacerbating inflammation within the plaque through plaque macrophages and dendritic cells. This thesis presents a model whereby atherogenic dyslipidaemia, as a result of elevated serum LDL levels, breaks down B cell tolerance. This results in autoimmune pathology characterised by exacerbated germinal centre responses skewed towards pathogenic antibody isotype clone production in response to LDL-induced inflammation. This provides an opportunity to target B cell-related atherosclerosis-specific responses therapeutically.

Membrane proteins perform various important and wide-ranging functions. They exist within a lipid bilayer and their interactions with the components of this bilayer are of great scientific and pharmacological interest due to the ability of these components to regulate protein structure and function. Computational approaches such as multiscale molecular dynamics simulations are well suited to the study of these systems. As such, the research presented here uses an array of these approaches to examine and characterise the interactions between membrane proteins and lipids in two environments: nanodiscs and lipid bilayers. There has been exponential growth in the use of nanodiscs for solubilisation of membrane proteins in a native-like environment, where the membrane protein is embedded in a patch of lipid bilayer constrained by helical proteins. However, the impact of constraining the nanodisc components in this manner is currently not well understood. In this research molecular dynamics simulations are used to investigate these effects; the results presented here describe the changes in lipid properties which occur as a consequence of constraining the nanodisc components and subsequent variations in protein lipid interactions. One family of membrane proteins, the G-protein-coupled receptors, was selected for further study. A simulation approach was used to examine the binding of a membrane lipid, cholesterol, to potential allosteric modulatory sites on these proteins, including the previously identified TM345 site. Interactions with cholesterol at this site were found to be common to several members of this family despite limited sequence conservation. The results presented here demonstrate the importance of understanding the environment in which membrane proteins exist and the impact of changes to this environment upon protein behaviour, properties and interactions.

The programmable self-assembly facilitated by DNA nanotechnology provides unparalleled capabilities to construct functional devices at the nanoscale. Recently, DNA nanostructures have been developed to interact with biological membranes and serve as artificial counterparts of natural ion channels, or membrane bending, scaffolding, or fusion proteins. In this thesis, we design a synthetic DNA-built enzyme that facilitates rapid lipid mixing between the two leaflets of a lipid bilayer. It thereby mimics the function of yet another class of membrane proteins - lipid scramblases. Characterising this DNA nanostructure with gel electrophoresis, dynamic light scattering, atomic force and transmission electron microscopy, we find that the cholesterol groups required for membrane insertion, also induce clustering. Hence, we establish an easy-to-implement strategy to control hydrophobically mediated aggregation thereby introducing a solution to a common problem of amphiphilic DNA constructs. With a combination of fluorescence microscopy experiments and molecular dynamics simulations, we identify the mechanism behind the scrambling activity. The spontaneous membrane insertion of our DNA scramblase induces a toroidal pore that is lined by the lipid headgroups. This DNA-stabilised pore connects the inner and outer bilayer leaflet thereby facilitating diffusive lipid transport that rapidly equilibrates the membrane’s lipid composition. In good agreement between experiments and simulations, we find the scrambling rate catalysed by our DNA-made enzyme to exceed 10⁷ lipids per second, orders of magnitude faster than natural scramblase proteins. We thereby pioneer the use of self-assembled DNA nanostructures for controlling the lipid composition of biological membranes, opening new avenues for applications of membrane-interacting DNA systems in biophysical research and medicine.

Background: Phagocytes have diverse roles including the ingestion and clearance of microbes, foreign particulate matter and dead cells. The study of patients with phagocyte dysfunction, either because of young age or inborn errors of immunity, could help design novel immunomodulatory treatments. However, such insights have been limited by a lack of access to the relevant clinical samples. Methods: Phagocytes from three groups of infants and children with clinical features of phagocyte dysfunction, obtained by novel sample collection methods, were studied by in vitro functional and transcriptomic testing. Results: Analysis of the clinical phenotype combined with functional testing of patient phagocytes demonstrated: i) primary alveolar macrophages isolated from healthy infants exhibited poorer control of Mycobacterium tuberculosis replication, as well as reduced expression of genes involved in lysosomal function and mycobactericidal activity, as well as genes encoding chemokines, which may explain the observed clinical vulnerability of infants to severe tuberculosis; ii) impaired control of mycobacterial replication by monocyte-derived macrophages of children with deletion of genes involved in cholesterol metabolism (LIPA and CH25H) is associated with clinical susceptibility to infection caused by Bacillus Calmette-Guérin vaccination; and iii) neutrophils from children with glucose-6-phosphatase catalytic subunit 3 deficiency exhibited increased inflammatory responses, which drives inflammatory bowel disease and can be corrected by haematopoietic stem cell transplant. Conclusion: Age-dependent and genetic factors make clinically important contributions to phagocyte immunity.

The metabolic state of an immune cell directly influences its ability to function and differentiate, ultimately affecting immunity, inflammation and tolerance. Different immune cell subsets have differing metabolic requirements. Macrophages, as the frontline, tissue-resident cells of the innate immune system, undergo profound metabolic reprogramming in response to environmental stimuli. To date, there has been little consideration how macrophage metabolism might be affected by humoral immunity. IgG antibodies are the soluble effector molecules of the adaptive humoral immune system. Fcγ receptors (FcγRs) mediate the cellular functions of IgG antibodies and are expressed on most immune cells including macrophages. FcγR cross-linking induced by IgG immune complexes (ICs) is important for defence against some infections but can also play a pathogenic role in autoimmunity. Here, I studied the metabolic reprogramming induced in macrophages by IgG IC ligation of FcγRs. I first investigated how FcγRs cross-linking might impact glucose metabolism. We show that macrophages undergo a switch to glycolysis in response to IgG IC stimulation. FcγR-associated glycolysis was dependent on the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor (HIF)1α. Moreover, this glycolytic switch was required to generate a number of pro-inflammatory mediators and cytokines. Inhibition of glycolysis, or genetic depletion of HIF1α in macrophages resulted in the attenuation of IL1β and other inflammatory mediators produced in response to IgG IC in vitro. To determine the relevance of these observations to responses to IgG IC in vivo and, in particular, to IC-associated tissue inflammation in autoimmune diseases such as system lupus erythematosus (SLE), I developed three models to interrogate tissue macrophages. Following administration of IC to peritoneal macrophages, I observed IL1β-associated neutrophil recruitment that was abrogated by inhibiting glycolysis, or in the presence of HIF-1a deficiency. Similarly, following administration of intravenous IC, or nephrotoxic serum, kidney macrophage activation was abrogated by glycolysis inhibition or by myeloid HIF-1a deficiency. Together my data reveal the cellular molecular mechanisms required for FcγR-mediated metabolic reprogramming in macrophages and define a novel therapeutic strategy in autoantibody-induced inflammation. In the final part of the thesis I identified additional metabolic pathways that were altered by FcγR ligation, including cholesterol biosynthesis and fatty acid biosynthesis. This has important implications for protective immune responses and autoimmune susceptibility, since a number of intermediates in these pathways can directly regulate and contribute to immune responses. In summary, I have demonstrated the metabolic alterations triggered by FcγR ligation, reveal the cellular molecular mechanisms required for FcγR-mediated cellular respiration reprogramming in macrophages and define a potential therapeutic target in autoimmunity.

Chronic glucocorticoid (GC) excess (Cushing’s syndrome, pharmacotherapy) causes metabolic and cardiovascular disease. This might be predicted from the known metabolic (dyslipidaemia, insulin resistance/hyperglycaemia) and hypertensive effects of chronically elevated GC levels. Intracellular GC levels within target tissues are controlled by 11β-hydroxysteroid dehydrogenases. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an enzyme that, in intact cells and in vivo, converts inert GCs (cortisone in humans, and 11- dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively). Consequently, 11β-HSD1 amplifies intracellular GC levels. Additionally, 11β-HSD1 is also involved in the metabolism of 7-oxysterols; it catalyses the reduction of 7-ketocholesterol (7-KC) to 7β-hydroxycholesterol (7β- HC). 7-KC may inhibit cholesterol biosynthesis through its ability to inhibit cleavage/processing of sterol regulatory element binding protein-2 (SREBP-2), the key regulator of cholesterol synthesis. Alteration of cholesterol homeostasis is a major risk factor for cardiovascular disease. Improvement of metabolic syndrome and attenuation of atherosclerosis are observed in susceptible rodent models with 11β- HSD1 deficiency or inhibition. Conversely, pilot data showed decreased levels of 7- KC as well as increased levels of cleaved SREBP-2 protein (the transcriptionally active form) in liver of mice with hepatic 11β-HSD1 overexpression (LOE mice), suggesting increased cholesterol biosynthesis. It was hypothesised that hepatic 11β- HSD1 promotes cholesterol biosynthesis through hepatic induction of SREBP-2 target genes in the cholesterol biosynthetic pathway. The hypothesis was tested in adult, male LOE and wild-type C57BL/6 mice. In mice fed a standard chow diet, hepatic levels of mRNA encoding hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase and HMG-CoA synthase, SREBP-2 targets in the cholesterol biosynthetic pathway, did not differ between genotypes. Compared to chow, a cholesterol-rich ‘Western’ diet (WD) decreased hepatic levels of mRNA encoding SREBP-2, HMG-CoA reductase and HMG-CoA synthase in wild-type as well as in LOE mice. These data imply that LOE mice show a normal physiological response with respect to cholesterol synthesis when challenged with cholesterol-rich diet, and, contrary to the hypothesis, liver 11β-HSD1 does not increase cholesterol biosynthesis via elevated expression of mRNAs encoding hepatic cholesterol biosynthetic enzymes. The liver X receptors (LXR) are well-known as sensors of oxysterols and regulators of genes involved in processes that decrease total body cholesterol levels i.e. reverse cholesterol transport and cholesterol excretion into bile. Cholesterol is the precursor to oxysterol LXR ligands. It was predicted that liver overexpression of 11β-HSD1 leads to activation of LXRα (the isoform with dominant roles in reverse cholesterol transport and whole-body cholesterol homeostasis) and its downstream targets involved in cholesterol efflux and excretion, in response to increased intracellular cholesterol levels. Indeed, levels of Lxrα mRNA were increased in livers of WD-fed LOE mice compared to wild-type mice on the same diet. There was no evidence for increased cholesterol clearance through bile acid synthesis in LOE mice as indicated by unchanged levels of hepatic Cyp7a1 mRNA between LOE and wild-type mice. However, consistent with being direct targets of LXRα, increased Abcg5 and Abcg8 mRNA levels were observed in livers of WD-fed LOE mice compared to WD-fed wild-type mice. These results corroborate findings in chow-fed LOE mice. Moreover, these data suggest that LOE mice ‘sense’ intracellular cholesterol excess and respond to it by increasing cholesterol efflux into the biliary lumen for excretion, thereby supporting a role for hepatic 11β-HSD1 in promoting biliary cholesterol secretion. To assess the effect(s) of hepatic 11β-HSD1 deficiency on cholesterol homeostasis as well as evaluate the importance of liver 11β-HSD1 in metabolic syndrome, liver-specific 11β-HSD1 knockout (LKO) mice were generated by crossing “floxed” Hsd11b1 mice with Alb-Cre transgenic mice in which Cre expression is restricted to hepatocytes. In liver of LKO mice, 11β-HSD1 mRNA, protein and enzyme activity were reduced by >80%, with no differences in 11β-HSD1 protein levels in kidney, adipose tissue or muscle between LKO and floxed Hsd11b1 littermate controls. These results indicate liver-specificity of Hsd11b1 knockdown in these mice. Body weight and weights of liver, adipose tissue, adrenal, muscle, kidney and brain were unaltered by liver-specific 11β-HSD1 deficiency on a standard chow diet. These mice were subject to a 14-week high fat (HF) diet, which typically causes metabolic syndrome in control but not globally 11β-HSD1 deficient mice. In HF-fed LKO mice, weights of the subcutaneous and epididymal fat depots were decreased compared to HF-fed control mice, resulting in an overall decrease in total white adipose tissue weight. Although no differences were observed in subcutaneous adipocyte hypertrophy between HF-fed LKO and control mice in a small number of samples tested, the above finding suggests that liver 11β-HSD1 influences adiposity and that liver-specific deficiency of 11β-HSD1 may reduce diet-induced adiposity. In terms of cholesterol homeostasis, no differences were observed in hepatic levels of mRNAs encoding cholesterol biosynthetic enzymes as well as those encoding enzymes/transporters for cholesterol catabolism/excretion between LKO and control mice, on either chow or HF diet. In summary, these data do not support a role for hepatic 11β-HSD1 in cholesterol synthesis. However, my evidence suggests that increased hepatic 11β-HSD1 promotes hepatobiliary cholesterol secretion. Finally, knockdown of liver 11β-HSD1, combined with HF feeding, reduces adiposity, suggesting that hepatic 11β-HSD1 may play a key role in adipose tissue lipogenesis/lipolysis and/or lipid storage, and that liver-specific 11β-HSD1 inhibition (or deficiency) may be advantageous in diet-induced obesity. Data presented in this thesis contribute to the understanding of the role of hepatic 11β-HSD1 in cholesterol homeostasis and metabolic syndrome.

Cholesterol is essential for many life processes, including correct development, fluidity of cell membranes, production of steroid hormones and bile acids, and is a major component of myelin. Smith-Lemli-Opitz Syndrome (SLOS) and Niemann-Pick disease type C (NPC) are devastating diseases and both involve dysregulation of cholesterol homeostasis. SLOS is caused by a defect in 7-dehydrocholesterol reductase (DHCR7), resulting in increased levels of 7-dehydrocholesterol (7DHC) and decreased levels of cholesterol. On the other hand, NPC is caused by defects in NPC1 or NPC2. These genes encode two lysosomal proteins that are responsible for the transport of cholesterol and other lipids out of lysosomes. Consequently, defects in these proteins results in accumulation of unesterified cholesterol within late endosomes/lysosomes. The severity ranges of both disorders are broad, and no or limited therapeutic options are available. This thesis aimed to establish the incidence and mechanisms behind SLOS and NPC in order to aid development of novel therapeutic interventions. Using a bioinformatics approach, Chapter 2 showed that the estimated incidences of classical SLOS and NPC were similar to clinical reports. However, the analysis suggested that a late onset form of NPC1 could be more prevalent. Chapter 3 investigated the behavioural phenotype of the SLOS Dhcr7T93M/Δ3-5 mouse model and it was found that some parallels could be drawn to the behaviour observed in SLOS patients. It also highlighted some defects in neuro-anatomy that could potentially explain certain cognitive defects. Chapter 4 explored the suggestion of oxidative stress in the Dhcr7T93M/Δ3-5 mouse model. However, this study did not support a role for oxidative stress in this model. Genetic insights were generated via an RNAseq study on SLOS and NPC patient fibroblasts, which suggested that the WNT signalling pathway could play a role in the pathogenesis of SLOS. This pathway was also highlighted when the cells were treated with miglustat, the only approved therapy for NPC. However, the main pathway apparently affected by this drug in both SLOS and NPC was cell proliferation.

This PhD project studies the effects of cholesterol in phospholipid bilayer using molecular dynamics (MD) simulations. Dipalmitoylphosphatidylcholine(DPPC) bilayers with cholesterol concentrations between 0% and 40% were simulated using all-­‐atom CHARMM36 force filed. The main concerned aspects of the lipid bilayer with cholesterol include structural properties, the lipid dynamics and mechanical properties of the bilayer. It was found that the lipid bilayer was condensed when it was embedded with cholesterol molecules. Addition of cholesterol in bilayer systems induced a smaller average surface area occupied by DPPC molecules, a more ordered hydrocarbon chains of DPPC molecules, and a thickened bilayer. The effects of cholesterol on dynamics properties of lipid bilayer were also observed as a decrease in the lateral diffusion coefficients for both cholesterol and DPPC in DPPC-­‐cholesterol mixture bilayer. Furthermore, lateral pressure profiles were calculated to look into the local stress distribution along the bilayer norm. It was found that cholesterol introduced extra contraction troughs in the profiles at the position under the head groups of DPPC, which explained the structural changes observed in the study. Bending moduli of the bilayer with and without cholesterol were estimated by calculating the splay modulus of pairs of each bilayer components. It was found that the involvement of cholesterol reduced the splay angles of each pair of bilayer component, resulting in a higher bending modulus of the structure.

Cholesterol plays a critical role in the health and normal function of every mammalian cell. Cholesterol has multiple cellular functions including maintenance of cell membrane stability and fluidity, steroid hormone and bile acid synthesis, and membrane receptor signaling. As with any critical metabolite, cholesterol is highly regulated. I will detail my research focusing on the two prevalent disorders of cholesterol homeostasis. The first is a disorder of cholesterol biosynthesis, Smith-Lemli-Opitz syndrome (SLOS), and the second a disorder of cholesterol trafficking, Niemann-Pick disease, type C1 (NPC). I have identified a unique cellular phenotypic overlap between these two previously unrelated disorders. Specifically, increasing concentrations of the precursor sterol present in SLOS, 7-dehydrocholesterol, leads to the induction of an NPC-like cellular phenotype. Aspects of the NPC cellular phenotype that can be induced in SLOS fibroblasts include lysosomal storage of unesterified cholesterol, sphingosine, and glycosphingolipids. Also, similar to what is observed in NPC, dysregulation of acidic calcium stores is present in SLOS. Further research indicates that this cellular phenotype extends into both the mouse models of SLOS and the patients. The dysregulation of cholesterol trafficking in SLOS cells likely decreases cellular cholesterol bioavailabilty and thus potentially limits the therapeutic efficacy of dietary cholesterol supplementation in SLOS patients. I also show that treatment of SLOS cells with miglustat, an established therapy for NPC, ameliorates the NPC-like cellular phenotype and thus may have therapeutic benefit in SLOS. The incidence of both SLOS and NPC has been the subject of debate. Thus, using data generated from massively parallel sequencing of the human exome, I demonstrate that the previously predicted incidence of SLOS of 1:40,000 births is likely correct, but call into question the predicted NPC rate of 1:120,000 . In fact, NPC maybe closer to a rate of 1:40,000. The role of oxysterols in the teratogenicity of SLOS and the underlying secondary storage of lipids in the phenotypic presentation of SLOS has been investigated.

Coronary vascular disease (CVD) is the number one cause of death worldwide. According to a WHO report and global and regional projections of mortality and burden of disease, by 2030, the number of people dying from heart disease and stroke will increase to reach 23.3 million. Non-HDL cholesterol, determined by subtracting the high density lipoprotein cholesterol (HDL-C) concentration from the total cholesterol (TC) content, has been recommended as a target for preliminary CVD prevention. In recent times, single-step homogeneous assays have been developed which allow simple and selective measurement of cholesterol fractions. Electrochemical sensors have also been developed which are based on the electrocatalysis of hydrogen peroxide using low cost printed sensor methodologies and such platforms would have the potential to be used as the basis of fabricating cholesterol biosensors for point of care use. Here, the development of electrochemical biosensors for the selective measurement of HDL-C, TC, and by subtraction non-HDL-C was explored. A spectrophotometric assay for use at room temperature and with minimal sample dilution was first established in order to optimise the assay reagent components for the development of selective cholesterol assays. Assay chemistries based on polyoxyethylene tribenzylphenyl ethers (Emulgen B-66) and Triton X-100 for the selective measurement of HDL-C and TC, respectively, were developed. The impact of these reagents on the electrocatalytic reduction of hydrogen peroxide at silver paste screen printed electrodes was also evaluated and optimised. Electrochemical biosensors for HDL-C and TC using externally added assay reagents were developed by combining the homogeneous assay methodologies with the printed electrocatalytic electrodes. The effects of assay reagents such as surfactants, enzymes, HDL-C sample and delipidated serum on the electrode behaviour were assessed amperometrically in the presence of hydrogen peroxide solutions. The electrodes showed increases in their catalytic activity toward hydrogen peroxide in the presence of both selective and non-selective surfactant and decreases in the presence of cholesterol oxidase and HDL-C samples. Despite the negative effects of cholesterol oxidase and sample matrix on electrode behaviour, the electrode response was linear within the clinically relevant ranges of HDL-C and TC. The modified electrodes were evaluated for their ability to selectively measure HDL-C and TC in clinical serum samples. The resulting HDL-C biosensor yielded a sensitivity of 3.32×10-8 A/mM with a linear range of 0 to 4 mM (r2=0.999), LOD of 0.5 mM and average RSD of 9.5% (n=5) while the TC biosensor had a sensitivity of 2.24×10-8 A/mM and a linear range of 0 to 10 mM r2=0.984), LOD of 2 mM and average RSD of 10.8% (n=3). The correlation between the HDL-C sensor and a commercial laboratory assay in clinical serum samples had a slope of 0.87 and a Pearson correlation coefficient of 0.76 (n=13) while the correlation for TC measurement had a slope of 1.07 and a Pearson correlation coefficient of 0.87. Finally, in order to develop a disposable biosensor suitable for point of care testing, integrated biosensors for HDL-C and TC were fabricated by inkjet-print deposition of assay reagents on the electrode surface. Integrated biosensors for the measurement of HDL-C were optimised and yielded a sensitivity of 4.55×10-8 A/mM with a linear range of 0 to 4 mM (r2=0.993) with an LOD of 0.25 mM and average RSD of 6.6% (n=3). The integrated TC biosensor had a sensitivity of 9.38×10-9 A/mM and linear range of 0 to 9 mM (r2=0.982), LOD of 0.5 mM and average RSD of 9.5% (n=3).

Cholesterol equilibrium is meticulously orchestrated, and its proper regulation is pivotal inmitigating the risk of cardiovascular disorders. To preserve this delicate balance,cholesterol must activate sensing mechanisms at organelle membranes within the cell’sinterior. The recently discovered Aster protein family (Aster-A, -B, and -C) partakes innonvesicular cholesterol import to organelles from the plasma membrane (PM). Despitetheir high coding sequence homology, each Aster protein exhibits distinct tissue expressionpatterns, conferring unique functions. This work elucidates their nuanced roles inregulating whole-body cholesterol flux. It scrutinizes how existing therapeutics modulatecholesterol flux, emphasizing their effects on key proteins that are known regulators ofcholesterol homeostasis. The introductory chapter reviews the intended and off-targeteffects of these existing therapeutics. Aster proteins are then introduced as novelmodulatory nodes in cholesterol regulation. Aster-C, prominently expressed in sterol-regulatory tissues like the liver and testis, presents an intriguing target, especially inpotential therapeutic applications for dyslipidemia-associated disease pathology. The bodychapter delves into Aster-C’s role in whole-body homeostasis, unveiling its contribution tocholesterol balance and revealing modest effects on bile acid metabolism under low-cholesterol dietary conditions. Aster-C knockout prompts upregulation in the messengerribonucleic acid (mRNA) levels of paralogs Aster-A and -B in murine liver tissue,suggesting nuanced roles and potential redundancy with other Aster proteins. Comparative nalysis with existing literature indicates that Aster-C’s hepatic transcription is inducibleby FXR stimulation. These findings, intersected with our own research on the Aster-Cknockout-induced disruption of bile acid homeostasis, suggest an integral role for Aster-Cin mediating the rate of reverse cholesterol transport within hepatic tissue. Overall, wedeconstruct cholesterol metabolism, identify existing therapeutic interventions, and thenpropose potential benefits of targeting Aster proteins as novel nodes by which the rate ofreverse cholesterol transport can be stimulated. Ultimately, we aim to explore noveltherapeutic applications in Aster modulation by potentiating clinical relevance in our futuredirections section. Herein, we hope to broaden perspectives on inter-tissue cholesterolhomeostasis dynamics by emphasizing the need for further investigations into the role ofAster proteins in cholesterol trafficking.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.

Recently, cholesterol metabolism has been shown to modulate the infection of several viruses and there is growing evidence that inflammatory response to infection also modulates lipid metabolism. However little is known about the role of inflammatory processes in modulating lipid metabolism and their consequences for the viral infection. This study investigates host-lipid viral interaction pathways using mouse cytomegalovirus, a large double-stranded DNA genome, which represents one of the few models for a natural infection of its natural host. In this study, transcriptomic and lipidomic profiling of macrophages shows that there is a specific coordinated regulation of the sterol pathways upon viral infection or treatment with IFNγ or β (but not TNFα, IL1β or IL6) resulting in the decrease of free cellular cholesterol. Furthermore, we show that pharmacological and RNAi inhibition of the sterol pathway augments protection against infection in vitro and in vivo and we identified that the prenylation branch of the sterol metabolic network was involved in the protective response. Finally, we show that genetic knock out of IFNβ results in a partial reduction while genetic knock out of Ifnar1 completely abolishes the reduction of the sterol biosynthetic activity upon infection. Overall these results support a role for part of the sterol metabolic network in protective immunity and show that type 1 IFN signalling is both necessary and sufficient for reducing the sterol metabolic network upon infection; thereby linking the sterol pathway with IFN defence responses.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with one of the worst 5-year survival rates (

There is worldwide concern over the possible estrogenic effects of organic chemicals on a variety of wildlife and indeed on humankind. In the U.K., estrogenic compounds in sewage treatment works (STW) effluents have been implicated in causing the increases in egg yolk protein production observed in caged male trout and other fish species. At the initiation of the present study, few of the estrogenic compounds in STW effiuents had been recognised, although circumstantial evidence suggested that steroidal hormones were primary candidates. Cholesterol is abundant in STW effluents and is the precursor of all steroidal hormones biosynthesised in mammalian systems. Thus, the aim of the present study was to examine the hypothesis that cholesterol might undergo A-ring aromatisation, during sewage treatment, producing estrone and 17β-estradiol via, intermediates such as l9-norcholest-l,3,5(10)-trien-3-ol (NCT). To study this hypothesis NCT was first synthesised via a known route and several of its chromatographic and mass spectral properties established for the first time. NCT itself was found to possess some estrogenic potential determined using an established assay but this was rather weak compared to 17β-estradiol - about 200,000 times less active at the minimum concentration needed to invoke a response. NCT also proved to be a much more hydrophobic compound than, for example, 17β-estradiol with a computed log octanol-water partition coefficient (Kow) of over 9 compared with a log Kow of about 4 for l7β-estradiol. The established analytical properties of NCT were then used to investigate possible NCT formation in sewage. Radiolabelled 14C-cholesterol was incubated aerobically and anaerobically in Semi-Continuous Aaivated Sludge (SCAS), Die Away (DA) or simple stand alone STW simulation vessels. The products of incubation in both aqueous and solid fractions were examined by radio-high performance liquid chromatography (r-HPLC), radio-thin layer chromatography (r-TLC) and radio-gas chromatography (r-GC). Aerobic studies showed that side chain cleavage and A-ring rupture of cholesterol occurred rapidly (~25 % of added activity within 24 hrs) as measured by 14C02 evolution. Gaseous evolution was not monitored from the anaerobic experiments. Most remaining activity was associated with the solids fractions in all experiments. In the aqueous experiments both SCAS and DA systems, r-HPLC revealed rapid production of polar products which were not identified further. r-HPLC also revealed non-polar components of which choIest-3,5-diene, an unknown cholestadiene, a cholestadienol (other than 5,7-dienol), cholest-4-en-3-one and possibly NCT were identified by r-GC in the products of DA experiments. Whilst r-HPLC and r-TLC also revealed several products of anaerobic digestion of cholesterol, no compounds were detected by r-GC. STW effluents from two wastewater plants in the North London area were monitored over 7 months for A-ring steroids and other suspected estrogenic chemicals. Both effluents had previously proved estrogenic to caged fish. Liquid and SPM samples were taken, extracted and analysed by GC-MS. The two main estrogens, l7β-estradiol and estrone were identified from all liquid samples but not in SPM extracts. Generally the concentration of estrone (maximum ca 3 ng Lˉ¹) was significantly higher than that for 17β-estradiol (maximum 1 ng fˉ¹). The third natural estrogen, l6a,l7β-estriol, was identified in all Harpenden effluent samples analysed up to a concentration ca 0.5 ng Lˉ¹. However, estriol was not found in Deephams effluent extracts. The phytoestrogen, daidzein, found in soya based products was intermittently found in aqueous effluents samples from both sites >1 ng Lˉ¹. SPM made up <0.001 % of the effluent. Extracts showed that there was a high percentage of steroidal based material with coprostanol>cholesterol=β-sitosterol>>stigmasterol. NCT was twice identified from SPM taken from Deephams with a concentration of 39 and 136 ng 1ˉ¹ but

The oxidation of low density lipoprotein (LDL) is one mechanism by which serum cholesterol may deposit in the arterial wall causing coronary heart disease (CHD). Epidemiological studies have provided evidence to suggest an inverse relationship between the dietary intake of flavonoids and the risk of CHD. Of the many groups of flavonoids present in foods, chiefly in vegetables, fruit, tea and wine, the polyphenols are especially important since they have been shown to express potent antioxidant activities in vitro. They are particularly abundant in red wine, a high consumption of which has been implicated in the low mortality from CHD in countries such as France, giving rise to the ‘French paradox’. Despite intense interest in the potential health benefits of dietary polyphenols, the outcome of studies addressing this issue have been largely inconclusive. The overall aim of this study was to determine whether the putatively protective effects of red wine are mediated through an in vivo antioxidant action of extracted red wine polyphenols and related foods and whether alcohol affects their biological activity. The initial objective of this study was to develop methods for assessing the oxidative status of plasma and oxidative susceptibility of LDL. Once established, these assays were used to test a range of different polyphenols for their antioxidant potential in vitro for a human ‘intervention trial’. The selected polyphenol supplements were then used as dietary supplements in a 2 week dietary intervention in healthy male volunteers. Measures of oxidation status and polyphenol content in plasma and LDL were determined before and after dietary supplementation. Supplementation with red wine and extracted red wine polyphenols either alone or in combination with white wine, alcohol, or vitamin E was associated with a significant increase in total antioxidant status of plasma and decrease in the oxidative susceptibility of LDL and lipid peroxides. It was also shown to increase the polyphenol content of plasma and LDL, and total plasma TGF-β levels. From these data it can be concluded that the polyphenols contained within red wine and similar beverages are powerful inhibitors of LDL oxidation and that alcohol promotes pro-oxidation changes in these parameters.

The squalestatins are a new family of natural products which display potent cholesterol lowering effects. Common to all these natural products is the highly oxidised bicyclic core and the aim of this project was to achieve a concise synthetic route to this core unit. Initial studies were carried out using 2-benzyloxycyclohexanone as a model template. Following conversion to the 2-oxa-3-oxo-spiro<4.5>decan-6-one via addition of the dianion of 3-(para- tolylsulphonyl)propionic acid, coupling of a C(2) fragment was explored. Addition of carboethoxymethylenetriphenylphosphorane, followed by oxidation to the diol and protection as the acetonide led to the formation of 4- Ethoxycarbonyl-(2,2-dimethyl-5"-oxodispiro[perhydro[l,3]dioxolane-4,r- cyclohexane-2',2"-(5"-H-furan)]-5-yl. The alternative order of addition of the C(4) and C(2) units has also been undertaken. Manipulation of the ester group to a silyl ether afforded a less reactive functionality and C(4) was manipulated to allow for the coupling of the next fragment to form the spiro lactone. The addition of the dianion of 3-(parc-tolylsulphonyl)propionic acid to4-((^t)butyldimethylsilyloxymethyl)-2,2-dimethyl-l,3-dioxa-spiro<4.5>decan-6-one failed and another route to the spiro lactone was explored. Formation of 4 - ((^t)butyldimethy Isilyloxymethy l)-2,2-dimethyl-1,3,7-trioxa- dispiro<4.0.4.4>tetradecan-8-one (I) was achieved by allylation at C(4) followed by hydroboration of the double bond and subsequent oxidation. The C(l) side chain could be added to the spiro lactone using allyl magnesium bromide without compromising the other functionality present. Acid treatment of 4-((^t)butyldimethylsilyloxymethyl)-8-methoxy-2,2-dimethyl-8- propyl-l,3,7-trioxa-dispiro<4.0.4.4>tetradecane (II) promoted deprotection of the acetonide followed by concomitant cyclisation to the desired 6-hydroxy-9-propyl- 8,12-dioxatricyclo<7.2.1,0>dodec-7-yl-l-methanol (III). This showed the viability of the retrosynthetic analysis as a route to core analogues of the squalestatins. Studies to the fully substituted core were commenced using cis-cyclohexadiene diol. The diol was protected as its p-anisaldehyde acetal before the formation of the Diels Alder adduct (IV) using 4-phenyl-l,3,5-triazolinone. However a lack of time prevented its manipulation to the a-alkoxy ketone species through Lewis Acid mediated cleavage of the acetal. In a second retrosynthetic plan 2-benzyloxycyclohexanone was coupled with methyl tetronate prepared following the procedure of Pelter. Preliminary studies towards the addition of the C(l) side chain have been undertaken and initial results seem promising

This thesis sought to investigate the relationship between structural and metabolic heterogeneity in LDL and examine the influence of plasma triglyceride on the properties of this lipoprotein. A technique to quantify individual LDL subfractions was developed which was superior to previously published methods and was employed to characterise three LDL subfractions in normals and in subjects with coronary disease, primary hyperlipidaemia and the secondary hyperlipidaemia observed in non insulin dependent diabetes mellitus and the nephrotic syndrome. In a case controlled study, a sevenfold increased risk of myocardial infarction was found to be associated with a concentration of small dense LDL III > 100mg/100ml. Subjects with hypertriglyceridaemia, combined hyperlipidaemia and the secondary hyperlipidaemia were found to exhibit an atherogenic lipoprotein phenotype of raised plasma triglyceride, low HDL cholesterol and a preponderance of small dense LDL. The data in the entire cohort was pooled and multivariate analysis revealed that plasma triglyceride was the most important independent predictor of LDL III. Pharmacological modulation of the LDL profile was investigated in normals and in a variety of hyperlipidaemic states using the main classes of lipid lowering drugs- resins, nicotinic acid derivatives, fibrates and statins. Those drugs which act through the lowering of plasma triglyceride reduced the concentrations of LDL III and those whose mode of action is by activation of the LDL receptor, reduced the concentrations of the larger LDL I and LDL II species. An important finding was in the unexpected beneficial effect of statins in combined hyperlipidaemia when LDL III concentrations were lower than expected for the plasma triglyceride level. Kinetic heterogeneity of LDL was studied by undertaking radioactive turnovers in normals and hypercholesterolaemic subjects who were given triglyceride lowering therapy. A two pool model for plasma LDL was developed to explain the radioactive decay curves of plasma and urine. One pool (pool A) was rapidly removed by the LDL receptor while the other (pool B) was more slowly removed and its synthesis was positively related to plasma triglyceride. The kinetics of apo-LDL were further examined in subjects with moderate hypertriglyceridaemia. Here it was necessary to expand the model to a third plasma pool (pool C) to account for an LDL species which was rapidly catabolised by receptor- independent mechanisms. A high level of receptor-independent catabolism was associated with an increased ratio of LDL III to LDL II. Pool C was seen in subjects with plasma triglyceride >2.5mmol/l in whom there was the presence of an abnormally small, dense LDL. On the basis of these observations and published VLDL metabolic studies, a model was postulated in which pool A LDL was produced from the delipidation of hepatic lipoproteins secreted in the Sf 0-60 density range and circulated m the form LDL I and LDL II. Pool B LDL, on the other hand, was derived from large triglyceride-rich VLDL of Sf 60-400 and circulates in most subjects in the form of LDL II. Neutral lipid exchange and lipolysis via the agency of hepatic lipase converts a proportion of pool B LDL II to LDL III, depending on the level of plasma triglyceride and the activity of the lipase enzyme. The data presented in this thesis are in line with current concepts of the atherogenicity of small dense LDL and provide evidence that it is an independent risk factor for coronary disease. It demonstrates that the properties of LDL are strongly influenced by its pedigree. Furthermore it shows that there are currently available drugs that are capable of altering not only the quantity of LDL but also its quality and offer hope in the continuing problem of tackling coronary heart disease.

The effect of bilayer composition, specifically the presence of cholesterol, upon the function of the reconstituted nicotinic acetylcholine receptor (nAcChoR) was investigated using stopped-flow fluorescence. The nAcChoR was purified and reconstituted from the electroplaques of Torpedo nobiliana, using affinity column chromatography, into bilayers of defined composition and the function of each sample assessed and compared with those of the native receptor. Investigation of the effect of bilayer composition upon the kinetics of agonist binding to the nAcChoR, using the fluorescent acetylcholine analogue, Dns-C6-Cho, established that the receptor pre-existed in equilibrium between the resting and two desensitised states. However, Dns-C6-Cho inhibited channel gating at high concentrations and another fluorescent probe was sought. The kinetics of carbachol induced nAcChoR conformational changes, reported by ethidium bromide (a non-competitive inhibitor) fluorescence, in native membranes were characterised and an assay developed to investigate whether cholesterol mediated rapid conformational changes in reconstituted samples. It was found that ethidium bromide reported on the carbachol-induced development of the fast desensitised state from the open channel state, and that this conformational change was sensitive to changes in bilayer composition. The onset of fast desensitisation from the open channel state was not observed when the receptor was reconstituted into DOPC or DOPC-DOPA bilayers. However, increasing the cholesterol content in these bilayers increased the amplitude of the component reporting this conformational change, while the observed rate at which it occurred was independent of bilayer cholesterol content. This result agrees with the suggestion that cholesterol facilitates channel opening and the onset of fast desensitisation by binding to specific sites on the nAcChoR and that these must be occupied for a functionally viable receptor (Jones and McNamee, 1988).

Two hundred patients with familial hypercholesterolaemia (FH) were examined for known mutations in the 3 part of exon 4 of the LDL receptor gene by electrophoresis of a Ddel digested PCR amplified fragment. Fifteen individuals (7.5%) were identified with 4 different mutations (of which 2 were novel) in this region of the gene. DNA from 311 patients with FH were then subjected to analysis of the 3'part of exon 4 by single strand conformation polymorphism which had been specifically adapted for sensitive detection of sequence variation in this region of the LDL receptor gene. A total of 29 patients or 9.3% (including the previous 15 patients) were identified, with a total of 6 mutations (additional two novel ones) in a region of 50 bases in the 3'part of exon 4. To examine the impact of the mutations on plasma cholesterol concentration, patients were grouped according to the localisation and nature of the mutations they carried. Four groups were compared, those with a defective protein and a mutation in repeat 5 of the ligand binding domain, those with a defective protein due to mutations elsewhere in the gene, those with a null mutation producing no protein at all and those with no detected mutation. Those individuals with null mutations had the highest plasma cholesterol concentration (11.31 mmol/l) which was similar to that in patients with any mutation in repeat 5 producing defective protein (11.23 mmol/l). These levels were both statistically significantly higher than in patients with mutations elsewhere in the gene producing a defective protein (9.64 mmol/l), or in the whole group (9.37 mmol/l). This suggests that the nature of the mutation in the LDL receptor gene is to some extent determining the phenotype of patients with FH. In addition to mutations in the LDL receptor gene that cause FH there may be mutations affecting plasma cholesterol concentration without causing FH; one such mutation is the common amino acid change A370T. The effect of this amino acid change on the uptake and degradation of LDL was investigated by analysis in CHO cells transfected with the human LDL receptor cDNA with either the T370 or the A3 70 allele. There was no detectable difference in receptor activity between the two alleles within the limits of the sensitivity of these assays. The effect of this polymorphism on plasma cholesterol concentration was also examined in three samples. In an Icelandic sample of 318 men and women the T370 allele was associated with 8.9% higher mean plasma cholesterol concentration in the men which was statistically significant. In contrast, women carrying the T370 allele, had 6% lower mean plasma cholesterol concentration. In a sample of offspring of men that had had a myocardial infarction before 55 years of age, the same trend was seen. Female offspring carrying the T370 allele had 8.8% lower, and male offspring carrying the T370 allele had 5.6% higher mean plasma cholesterol concentration, though this difference only reached statistical significance in the female. In addition, the frequency of the rare T370 allele was statistically significantly higher in the offspring of cases than in the controls, 0.06 vs 0.042 respectively (p<0.05). In the third sample examined, which consisted of 706 British healthy men that were followed for 4 years, the T370 allele was associated with 7.7% higher mean plasma cholesterol concentration at baseline. When only those for whom data were available at all visits were examined, those with the T370 allele had 2.8% lower mean plasma cholesterol concentration. Those individuals carrying the T370 allele who were lost on follow up had 0.5 mmol/1 higher mean plasma cholesterol concentration than those with an A3 70 allele only. These results suggest that the A370T polymorphism may have a small impact on the development of hypercholesterolaemia and the risk of coronary artery disease, but the mechanisms of these effect is yet to be determined.

Familial combined hyperlipidaemia (FCHL) is a common inherited disorder of lipid metabolism, with a prevalence of 0.5%-2% in Caucasians. It was first described in 1973 by Goldstein et al. and is characterised by raised plasma cholesterol and triglyceride levels and by small dense low density lipoprotein (LDL) particles. The principal strategy to identify the underlying defects in FCHL in this study was that of reverse genetics. A total of 43 pedigrees where the proband had combined hyperlipidaemia were assembled. Thirteen of these pedigrees were defined as having FCHL according to the criteria of Goldstein et al, and by small dense LDL. Population association was performed using twelve of these, ascertained without prior knowledge of their genotype. Linkage analysis was performed in all thirteen pedigrees. Population association demonstrated linkage disequilibrium between FCHL and the 6.6 kilobase (X2) allele of the XmnI RFLP in the apo AI-CIII-AIV cluster (p< 0.021). Linkage analysis was carried out in seven FCHL families in which the proband carried this X2 allele. The peak lod score was 5.87 at 0 = 0.00, demonstrating that FCHL was linked to the apo AI-CIII-AIV locus in this pedigree subset. Furthermore, apo CIII levels in this subset were significantly elevated in affected members (p < 0.0001), suggesting that the defect is associated with increased levels of apo CIII. Subsequent analysis in the six FCHL pedigrees in which the proband did not carry the X2 allele demonstrated a negative lod score of -5.67, indicating that a major subset of FCHL pedigrees are not linked to the apo AI-CIII-AIV locus, and suggesting that FCHL is genetically heterogeneous. The admixture test at this locus using the lod scores from all thirteen pedigrees verified the presence of genetic heterogeneity (p< 0.001), and indicated linkage in the subset of pedigrees carrying the X2 allele (p< 0.0001). These results demonstrate that combined hyperlipidaemia in the X2 FCHL pedigree subset is caused by a defect in or close to the apo AI-CIII-AIV locus on chromosome 11q23-q24. In the remaining pedigrees, FCHL is linked to one or more further gene loci. At 8 further candidate loci, linkage analysis failed to detect other major genes for FCHL. However, the detection of a mutation in the lipoprotein lipase gene in a single additional FCHL pedigree indicates that defects in this gene may also cause FCHL, and further confirms genetic heterogeneity in this disorder. The principal findings of this study suggest that FCHL is genetically heterogeneous and is caused by a defect in at least two major gene loci. These are predominantly the apo AI-CIII-AIV cluster (or a nearby gene) and the lipoprotein lipase gene.

Parabens are commonly used as preservatives in regularly used topical products, but their safety is under discussion since small amounts of paraben have been found in tumor tissue. Mononitroparaben causes cell death in melanoma cells with an LC50 value of 7.02mM after twelve hours of treatment. This study focused on how mononitroparaben affects the cellular lipid content during induced cell death. The experiment was conducted by growing the M624 melanoma cells, dissolving the paraben in methanol, and then leaving 0 mM, 5 mM, and 10 mM concentrations of paraben on the melanoma cancer cells for twelve hours. The paraben was then removed from the cells, and the cells were lysed. Colorimetric cholesterol assays and ceramide assays were completed to determine the changes in cellular cholesterol and ceramide content and the role of cellular lipids in cell death signaling. The results showed that cholesterol concentrations did not significantly change among cells treated with mononitroparaben. However, ceramide significantly increased for cells treated with 10 mM mononitroparaben, which indicated that apoptosis occurred.

Hyperlipidemia is a key risk factor in cardiovascular mortality, and is prevalent in approximately 38% of American adults (CDC, 2022b). Cholesterol levels are intensified by unhealthy lifestyle choices, which means a change in lifestyle behaviors could prevent cardiovascular related deaths (WHO, 2022). The PICOT question for this project was: In adults aged 20 years or older in the primary care setting who have elevated low-density lipoprotein (LDL) levels (P), does the implementation of behavioral counseling on lifestyle modifications (I) compared to current practice (C) decrease LDL levels (O) over a 10- to 12- week period (T)? Fourteen participants from a small direct primary care clinic in Indiana completed the entirety of the project. LDL levels were measured pre-intervention, along with a rapid assessment of physical activity (RAPA) and the rate your plate (RYP) tool, blood pressure (BP), body mass index (BMI), weight, and atherosclerotic cardiovascular disease (ASCVD) risk scores. The nurse practitioner initiated a behavioral counseling session on lifestyle modifications, assisted by educational handouts, and created three healthy goals with the participant. A follow-up telephone counseling session was scheduled at five weeks to review those goals, followed by an in-person counseling session at 10- to 12- weeks. LDL levels were redrawn and the RYP and RAPA tools, weight, BMI, BP and ASCVD scores were completed once more to show a within-group evaluation of pre- and post-intervention outcomes. A paired t-test was used for analysis, and statistically significant data was found by increased RYP scores (p = .001), increased RAPA scores (p = .004), weight reduction (p = .035), BMI reduction (p = .026), systolic BP reduction (p = .025), and ASCVD score reduction (p = .002). There was no statistical significance in LDL reduction (p = .051); however, there was still a decrease in mean scores pre- (137.36) and post- (114.43) intervention. These findings support the use of behavioral counseling for lifestyle modifications in patients with elevated LDL levels.

Voltage-gated and Ca2+ activated K+ channels of large conductance (BK) channels are essential for many key physiological processes, such as controlling the contraction of smooth muscle, including cerebrovascular smooth muscle. Importantly, BK channels are made up of a homotetramer of α subunits which associate with accessory β subunits. These β subunits alter the activity of the channel; this modulation is subunit-specific. Moreover, these accessory subunits have tissue-specific expression, such that the β1 subunit is highly expressed within cerebrovascular smooth muscle and greatly increases the open probability of BK channels. Increased BK channel open probability is linked to increased dilation of cerebral arterial smooth muscle. The tissue specificity and functional effects of the β1 subunit offer a unique opportunity for the identification of pharmacological activators to be developed with few off-target effects. While many steroids have been implicated to have vasoactive effects, including but not limited to, pregnenolone and progesterone, these effects have been mainly attributed to the actions produced by the activation of traditional steroid receptors. Importantly, both pregnenolone and progesterone are currently under investigation for clinical applications, including substance- and alcohol-use disorders, and treatments for ischemic-related brain injury, respectively. Additionally, cholesterol maintains clinical importance as the western diet maintains high levels of cholesterol which are known to increase the risk of heart disease and stroke through maladaptive vasoactive effects. Since the precursor to, and the neurosteroids all produce vasoactive effects, it is important to identify the molecular mediators modulating these effects. This body of work examines the cerebrovascular effects of and determines the mechanisms of action by which the neurosteroids pregnenolone, progesterone, and the precursor cholesterol, modulate the BK channel activity while investigating pre-clinical implications for the K+ channel activators. Through functional, biochemical, and molecular analysis we are able to determine: 1. the physiological implications of; 2. the subunit-specificity for; 3. binding site(s) of; and 4. the gating parameters affected by, the direct binding of neurosteroids to the BK channel complex.

The lipid membranes of cells are complex structural and functional landscapes. Beyond being a selective barrier separating the cell from its surroundings, the membrane serves also as a two-dimensional solvent dictating the thermodynamic environment in which membrane protein biochemistry takes place, and as a platform that facilitates and responds to the organization of membrane proteins into functional domains. Membranes including vesicles derived from eukaryotic plasma membranes also exhibit liquid-liquid phase coexistence. This dissertation aims to link the biochemical and organizational properties of membranes to their phase behavior. The membrane's role as a thermodynamic platform is addressed in a chapter on the availability of cholesterol, specifically its chemical potential (Chapter 3). This work consists of measurements of the chemical potential of cholesterol in a family of synthetic lipid membrane compositions. This chemical potential describes the availability of cholesterol, and is a primary determinant of the occupancy of protein binding sites for cholesterol. The synthetic membranes used in this study are similar to mammalian plasma membranes in phase behavior and cholesterol concentration. The measurements show a close connection between the role of cholesterol in phase separation of these membranes and its availability. This finding suggests that treatments that modify the phase behavior of the membrane, of which many are known, may act through their effect on the availability of cholesterol. In addition, this study provides a framework for how to approach other questions about the biochemistry of cholesterol. The remaining chapters describe methods that will enable more precise and robust measurement and analysis of the organization of membrane proteins using single molecule localization microscopy (SMLM). SMLM techniques produce location information of target molecules with precisions on the order of 10 nm, and so have been invaluable for characterizing protein organization in membranes. The methods contributions include direct improvements to the precision of these datasets through improved sample drift correction (Chapter 4), a novel method for characterizing SMLM measurement precision (Chapter 5), and a method for correcting spatially non-uniform labeling or detection artifacts in measurements of colocalization (Chapters 6 and 7). These methods extend the usefulness of SMLM so that it can detect more detailed and subtler structure in the organization of proteins on membranes. In particular, they will enable future experiments to measure the role of membrane phase behavior in biological systems where it has been too subtle to detect using past methods. Overall, the developments described in this dissertation strengthen the connections between membrane phase behavior and biological function, by linking phase behavior to a new biochemical property of the membrane, and by enabling future investigations into how it organizes membrane proteins.

Cholesterol is vital for membrane function, yet toxic in excess and associated with cardiovascular disease and cancer. Squalene monooxygenase (SM) catalyses the rate-limiting and first oxygen-dependent step of the committed cholesterol synthesis pathway, and past research has shown that cholesterol modulates its protein levels to ensure an appropriate amount of pathway flux according to supply and demand. This is mediated by the N‑terminal regulatory domain of SM (SM‑N100), which senses high cholesterol levels and accelerates entry into the endoplasmic reticulum-associated degradation (ERAD) pathway. A mechanistic understanding of how SM is regulated by such stimuli is critical, as aberrant SM activity is oncogenic in a broad range of cancers. Thus, this thesis sought to identify additional metabolic factors controlling SM degradation. Using a chemical genetics screen and SM‑N100 reporter constructs, we found that SM is protected from degradation by the accumulation of its substrate, squalene. This feedforward regulation involves allosteric binding of squalene to the SM‑N100 domain, preventing its recognition by ERAD effectors. We next studied a lower molecular-weight form of SM routinely detected by immunoblotting, and through SM mutagenesis and targeting of the ERAD pathway found that it arises through the rare phenomenon of partial proteasomal degradation. This disrupts the cholesterol-sensing SM‑N100 domain but not the catalytic domain, rendering truncated SM constitutively active. Truncated SM, but not full-length SM, is also capable of localising to lipid droplets. Finally, we identified hypoxia as a physiological trigger for truncation through a combination of accelerated entry into ERAD and the accumulation of squalene, which prevents complete degradation of SM at the proteasome. Analysis of endometrial cancer tissues revealed a marked upregulation of SM truncation that was well-correlated with a hypoxic marker protein, suggesting that hypoxia-induced truncation occurs in vivo and plays a role in the oncogenic functions of SM. In summary, this thesis identifies dual mechanisms of substrate-induced SM regulation that impair its proteasomal degradation and preserve catalytic capacity. Beyond these fundamental insights into the control of cholesterol synthesis, our data highlight the significance of SM feedforward regulation under both physiological and pathophysiological conditions.

Cardiovascular disease remains the leading cause of morbidity and mortality, and high blood pressure (BP) and low-density lipoprotein cholesterol (LDL-C) are two major modifiable risk factors. This thesis explores strategies to improve cardiovascular prevention through BP and LDL-C lowering. First this thesis highlights discrepancies between guideline recommendations for BP and LDL-C lowering, and the current evidence generated from randomized clinical trials. We show that more intensive BP and LDL-C lowering beyond traditional targets are associated with further reductions in adverse cardiovascular events. We also show that the benefits of BP and LDL-C lowering appear to be additive to one another, and combination BP and LDL-C lowering can effectively reduce cardiovascular disease by more than half. This thesis also explores the cumulative risk factor hypothesis and shows that cardiovascular risk from exposure to elevated BP and LDL-C accrues over time. These findings emphasise the need for earlier and sustained risk factor control. We introduce a novel metric to quantify risk factor exposure, namely cumulative BP load, which was shown to be a superior predictor of cardiovascular events. The thesis also shows that low dose combination antihypertensives are highly efficacious and well tolerated, and have an important role in the initial management of hypertension. We highlight the alarming rates of therapeutic inertia in hypertension management, and identify two main reasons for treatment inertia; large reductions in BP from one visit to the next, and satisfaction with BP readings that are close to target. Finally, we highlight the need for a major shift in the current prescribing paradigm for hypertension management and show that BP lowering efficacy cannot be accurately measured on an individual basis because BP readings have a poor signal:noise ratio. We conduct the largest meta-analysis of BP lowering clinical trials to date to provide estimates of average BP lowering efficacy and developed the first ever BP prediction model that accounts for both regression to the mean and estimates of BP lowering efficacy for a given antihypertensive regimen. In conclusion, the present thesis offers novel strategies to improve BP and LDL-C lowering, and demonstrates the potential for these strategies to achieve major reductions in cardiovascular disease.

ABSTRACTEstablishing mitochondrial cholesterol enrichment as a fundamental causal mechanism underlying the dysfunctional clearance of neurotoxic substances by microglia would help pave the way toward the discovery of potential new therapeutics for Alzheimer’s disease (AD). The dissertation is divided into four chapters with the corresponding objectives:1. The first chapter is a literature review describing the mechanisms by which cholesterol has been implicated in influencing microglia phenotype and function, and the implications for a role in contributing to the pathophysiology of AD. We describe both what is known and the research gaps that exist and should be explored for the discovery of novel therapies for AD 2. The second chapter is an investigation on the effects of a 36h fast on HDL particle size distribution by transmission electron microscopy (TEM), and ability to efflux cholesterol from activated microglia. Importantly, HDL from 36h fasted individuals effluxed cholesteryl esters from immortalized human microglia (HMC3) loaded with cholesterol + amyloid beta oligomers (AβO) 10-fold more effectively than HDL from postprandial individuals, and microglia treated with cholesterol+AβO had reduced apolipoprotein E (ApoE) secretion, which was attenuated more effectively by HDL from 36h fasted individuals than HDL from postprandial individuals. 3. In the third chapter we use a multi-stimulant approach to test the usefulness of the HMC3 microglia cell line in duplicating critical aspects of the dysfunctional microglia phenotype and to better mimic the multi-factorial nature of AD, which is associated with multiple comorbidities, including diabetes and cardiovascular disease. HMC3 microglia were treated with cholesterol (Chol), AβO, lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol+AβO+fructose+LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol+AβO+ fructose+LPS increased mitochondrial Chol content. Microglia treated with combinations containing cholesterol+AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol+AβO+fructose+LPS having the strongest effect. Combination treatment with Chol+AβO+fructose+LPS also induced APOE and TNF-alpha expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events.

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by the expansion of the polyglutamine (polyQ) domain of the huntingtin protein (htt). The expansion of the polyQ domain beyond a threshold of approximately 35 repeats triggers complex toxic aggregation mechanisms and results in altered interactions between htt and lipid membranes. Many factors modulate these processes. One such modulator includes sequences flanking the polyQ domain, most notably the first 17 amino acids at the N-terminus of the protein (Nt17), and environmental factors including the presence of membranous structures. Nt17 has the propensity to form an amphipathic a-helix in the presence of binding partners, which can facilitate aggregation and lipid binding. These processes can be further modulated by overall membrane composition and physiochemical properties. Considering the influence of membrane composition and the known dysregulation of cholesterol homeostasis in HD, cholesterol may be a crucial membrane component that can modulate early htt interactions through influencing membrane properties such as permeability, fluidity, and overall organization. Early interactions may also be modulated through altered electrostatics, hydrophobicity, and hydrogen bonding introduced through mutations to residues within the Nt17 domain. A mechanistic understanding of such modulating factors and their impacts on early htt interactions can provide crucial insights into the toxic mechanism of HD. Early htt interactions were further explored in the studies presented here. Thioflavin T (ThT) aggregation assays, atomic force microscopy (AFM), polydiacetylene (PDA) lipid binding assays, and mass spectrometry (MS) were used to identify changes to aggregation, aggregate morphologies, htt/lipid binding, and htt/lipid complexation respectively. When htt was exposed to lipid systems composed of pure POPC, DOPC, and POPG distinctly different htt interactions with increasing amounts of exogenously added cholesterol were observed. Increasing cholesterol content increased aggregation for the DOPC systems, but reduced aggregation for the POPC and POPG systems. Htt/lipid binding decreased with increasing cholesterol for the POPC systems, while binding increased for the DOPC and POPG systems. Differences in htt/lipid complexation were also observed for each pure lipid system with increasing cholesterol content. When htt was incubated with Nt17 peptides with mutations that altered residue charges aggregation was significantly reduced, though the extent was dependent on the type of modification. Incorporation of Nt17 peptides into oligomeric structures resulted in minimal changes to oligomer/lipid interactions, though monomeric peptide/lipid complexation was altered with the introduction of modifications.