Alzheimer’s Disease and other neurodegenerative diseases have been linked to dysfunction in proteostasis in the endoplasmic reticulum (ER). The ER provides an exclusive environment for protein synthesis and folding, which is vital to the cellular function. Under normal conditions, the synthesis and degradation of proteins remain in balance. During aging or during pathological states, disturbances of ER occur and consequently the failure of protein homeostasis. The cells rely on a system, the unfolded protein response (UPR), which regulates the homeostasis by three ER sensors: PERK, ATF6, and IRE-1. Perturbations of ER function result in UPR. In physiological condition, the cell may overcome the insult and regain homeostasis. However, prolonged or chronic UPR activates apoptotic pathways and may cause cell death. The sigma-1 receptor (Sig-1R) is a 25 kD polypeptide and a chaperone protein concentrated at the mitochondria-associated ER membrane domain (MAM). The Sig-1R plays significant roles governing calcium signalling, mitochondrial function, oxidative stress, protein chaperoning and ER stress. Results of this investigation demonstrate that immortalized mouse embryonic fibroblasts (MEFs) derived from Sig-1R-/—(KO) mice have higher baseline activation in all three branches of the UPR in the absence of ER stress compared to MEFs derived from Wild-type mice. Despite this increase in baseline activation, the PERK and ATF6 pathways have a significantly blunted response to acute stress. Rescue experiments by expressing the Sig-1R in KO MEFs did not recover the WT MEFs phenotype. Primary Sig-1R KO MEFs did not show baseline ER stress, but did show inhibited recovery following treatment with the acute ER stressor DTT. Overall, our data suggests that Sig-1R is important for the reestablishment of proteostasis following acute stress.

Chronic kidney disease (CKD) is a common health problem with a high prevalence in the elderly and is associated with high mortality rates and co-morbidity. CKD guidelines recommend that diagnosis and staging of CKD be based on estimated glomerular filtration rate (eGFR). Estimating GFR requires estimating equations using the variables gender, race and age and body surface area based on serum creatinine levels. The commonly recommended and used equations are the Modification of Diet in Renal Disease (MDRD) study and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations but these have not yet been validated in elderly people, who are at significant risk of developing CKD. The numbers of patients with progressive CKD is reportedly low with only a small proportion of patients reaching end-stage renal disease (ESRD). This study set out to find out why there is such a disproportion in the high prevalence of CKD and the low incidence of ESRD patients. Many patients die before they reach ESRD but prevalence studies have shown that mortality rates alone do not account for these numbers. I hypothesised that the methods used to estimate GFR underestimate renal function in elderly people causing an overestimate in CKD prevalence. This study firstly set out to assess the accuracy of the MDRD and CKD-EPI equations in an elderly Caucasian population against measured GFR across a wide range of renal function. The study demonstrated both equations perform fairly accurately in the elderly population with a tendency to slightly over-estimate GFR. This study has validated the use of these estimating equations in an elderly Caucasian population disproving my first hypothesis. If the CKD prevalence data is a fair estimate and only a small proportion progress then the answer may lie in how CKD progresses. There are several known factors that influence CKD progression including GFR and albuminuria category, cause of renal disease and hypertension. Some of these risk factors are modifiable and need to be identified and managed in order to impact on long term outcomes including death, cardiovascular events and disease progression. Acute kidney injury (AKI) is also rising in incidence and is complicated by high mortality rates, increased risk of cardiovascular events and more recently CKD progression. Little is known about the impact of more minor episodes occurring in the community on renal outcome. The second part of this study examined the relationship of multiple episodes of community AKI with CKD progression in a population of patients with CKD stage 3-5 referred to renal services. In this observational study, patterns of CKD progression were assessed and multiple AKI events were recorded. This study demonstrated a clear relation between multiple AKI events and CKD progression however only low eGFR at referral, diabetes and albuminuria were independent risk factors associated with disease progression. During the study it emerged that there were two patterns of CKD progression. In comparison to the more commonly assumed linear decline, the more common pattern was a stepwise progressive pattern characterised by accelerated rates of decline followed by a period of stability. Multiple AKI events were significantly more common in the stepwise progressive group suggesting AKI may have an important role as a promoter of CKD progression. This study suggests that community AKI is a modifiable risk factor that needs identifying at early stages in order to minimise risk of poor outcomes including CKD progression.

Coronary heart disease (CHD) is the most common cause of death in Western societies. This disease affects both men and women and accounts for approximately 500,000 deaths annually in the U.S.A. alone. For a number of years, plasma concentration of HDL cholesterol (HDL-C) was found to correlate inversely with the incidence of coronary heart disease and atherosclerosis. In addition, other studies in humans showed that higher plasma HDL-C levels correlates with slower progression of atherosclerotic lesions and possible stabilization of unstable atherosclerotic plaque. These findings have led to the suggestion that raising HDL-C will protect from the disease. One of the first trials demonstrating the potential benefit of raising HDL-C levels was the Helsinki Heart Study. In this randomized trial, 4081 men with dyslipidemia received gemfibrozil or placebo and five years later it was seen that the drug reduced CHD risk. Gemfibrozil treatment increased HDL-C levels by 11% but also reduced total plasma cholesterol levels by 10%, LDL-C cholesterol levels by 11% and triglyceride levels by 35%. Although all fibrates have been revealed to raise HDL-C significantly, their overall effect on all-cause mortality and cardiac mortality remains debatable. More recent studies have suggested that the relationship between HDL and cardiovascular risk is more complex than first thought and extends beyond consideration of levels of total HDL-C in plasma. In particular, Mendelian randomization studies challenged the existing view on HDL-C and cardiovascular risk and prompted a discussion as to whether low HDL-C is a causal risk factor for the development of heart disease. In parallel, research interest has intensified in studies aimed at better understanding the many biological functions of HDL and the partner proteins and receptors with which it interacts. There have been a number of studies over recent years indicating that HDL can fail to function effectively in subjects at risk for coronary diseases. It is important therefore to attempt to understand if abnormalities in HDL function are associated with variation in CVD risk. This is especially true in light of the failure of recent trials that raise total HDL to reduce risk of myocardial infarction and CVD. A feature that appears to be related to the atheroprotective functions of HDL is the relative level and distribution of HDL subpopulations in different individuals. Although HDL is unusually regarded as a single entity in clinical settings, studies using non- denaturing two-dimensional electrophoresis have revealed a number of HDL particles with distinct shape, size and composition. Along the same lines, administration of statins - a medication proven to reduce CHD risk - to patients increases specific subpopulation of HDL suggesting that some of the atheroprotective properties of statins may be mediated by increasing selected HDL subpopulations. The overall objective of the present work was to examine in details the relationship of HDL oxidation potential, the ability of HDL to protect LDL from oxidation, and the abundance of the major antioxidant enzyme, PON1, to atherosclerosis in a cross section of subjects recruited from across the social economic spectrum in the West of Scotland (the pSoBid study). PSoBid is valuable as a means of testing these properties of HDL because of its mix of males and females, wide age range, and the fact that it focused in recruiting people at extremes of social deprivation with widely varying lifestyles. The population has been well characterized in terms of classical risk factors and this thesis takes the investigation to a new level of detail with respect to HDL. The major questions addressed were: 1. Is HDL oxidation (measured by three factors; time at half maximum (T1/2max), maximum velocity of oxidation (Vmax), or maximum amount of oxidized HDL measured by optical density) related to a commonly used index of atherosclerosis? 2. Is HDL antioxidant potency to protect LDL from oxidation related to its protective effect in atherosclerosis risk? 3. Is HDL PON1 activity related to atherosclerotic marker? 4. If some HDL subclasses, rather than HDL-C, are particularly related to atherosclerosis? Our major findings revealed that: 1. HDL is more readily oxidized in those subgroups associated with less atherosclerosis. 2. The susceptibility of HDL to oxidation was also related to lifestyle factors associated with less atherosclerotic disease such as moderate alcohol intake, not smoking, active exercise habit and high intake of fruits and vegetables. 3. For HDL structure, %HDL2b was inversely associated with atherosclerosis while %HDL3b was directly associated with atherosclerosis. 4. Comparing the two factors which were inversely associated with atherosclerosis, HDL oxidation and the distribution of HDL subpopulations, our results revealed that HDL oxidation potential was more important than the percentage HDL subfraction distribution in relation to atherosclerosis. 5. HDL mediated inhibition of LDL oxidation was not found to be associated with carotid atherosclerosis nor did it appear to be related to major risk factors. Our interpretation for atherosclerosis is that HDL particles, which are very abundant in the circulation, might play a sacrificial role in that they are oxidized first and therefore have the capacity to prevent LDL oxidation in vivo.

Polyhydroxyalkanoates (PHA) are biopolymer molecules that have shown increasing evidence of suitability for use as biomaterials. Many different cell types from a range of species have been shown to adhere and develop on PHA scaffolds, with some preliminary in vivo studies having been performed and showing promising results. Several cell types have shown potential for use in tendon tissue engineering. Primary tendon cells (tenocytes) isolated from a rat Achilles tendon (RaT), human Mesenchymal Stem Cells (hMSCs) and human Embryonic Stem Cells (hESCs) have all been utilised throughout this investigation in many different experimental models. To date, research has focused on scaffold design and manufacture, suitability of the polymer for cell culture, in vitro testing in both static and dynamic environments and a pilot in vivo study. The aims of this study were to find if the PHA molecule Poly (hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) is able to support tendon cell culture, to design a scaffold using the polymer that could replicate tendon in vitro and in vivo, to mechanically stimulate cells seeded in scaffolds to encourage extracellular matrix remodelling into tendon like structures and to monitor the construct when placed in situ in a pilot in vivo model. Results have shown that PHBHHx can support RaT, hMSC and hESC development. Mechanical testing revealed a design with similar properties to those of a rat Achilles tendon. Bioreactor studies have demonstrated how hMSCs, hESCs and rat tenocytes can remodel collagen gels incorporated into the scaffold towards morphologies resembling tendon. Preliminary in vivo studies have found that a PHBHHx and PHBHHx/collagen hybrid scaffold show little immune response when placed in situ. In conclusion, this study has demonstrated the effectiveness of PHBHHx and PHBHHx/collagen hybrid scaffolds for use in tendon tissue engineering, providing scope for future breakthrough products and innovations in both scientific and clinical arenas.

The research described here focuses on the molecular variations, clinical significance and structural interactions of the innate immune protein, C-reactive protein (CRP). Monomeric CRP (mCRP) was produced through urea-induced dissociation, at an optimum concentration of 3M urea over a 10-week period. Dissociated samples were purified via size exclusion chromatography and confirmation was provided via Western Blot analysis. In contrast with previous published works, this mCRP retained the ability to reversibly bind to phosphocholine. Furthermore, the binding of mCRP with cell wall polysaccharide does not result in the generic precipitation trend as that seen with pentameric CRP (pCRP). Human serum samples with raised CRP levels ( > 100mg/L) were analysed (n =40) to determine whether circulating mCRP could be detected in vivo. All 40 samples tested positive for pCRP via Western Blot and ELISA analysis, with further quantification via the UV Spectrophotometer supporting the calculated pCRP levels. Monomeric CRP was identified in all the 40 patient samples tested, with an average value recorded of 0.88mg/L (SE = ±0.142). Informed by the results from in vitro mCRP studies, the mCRP displayed a molecular weight of approximately 25kDa based on size exclusion chromatography and Western Blot analysis and demonstrated an ability to reversibly bind phosphocholine. To our knowledge, we are the first to successfully purify and identify a monomeric form of CRP from human serum through these procedures. Crystallisation trials of in vitro produced mCRP and the complex of pCRP with C1qGHR and CWPS did not result in the growth of any protein crystals of sufficient diffraction quality. One crystal from the complex crystallisation trials diffracted to 9Å with follow up conditions currently being pursued. Furthermore, additional crystallisation trials from both experiments have generated potential conditions which may be refined in future research to produce high-quality diffraction grade crystals.

No description available.

The renin angiotensin system (RAS), a homeostatic system involved in blood pressure and volume control, is implicated in the pathology of several risk factors for ischaemic stroke. Mounting evidence now suggests that the RAS may play a role in the pathophysiology of ischaemic stroke. This is thought to be due to an imbalance between the classical RAS axis, Angiotensin converting enzyme/Angiotensin II/Angiotensin II receptor type I (ACE/Ang II/AT1R), and the counter-regulatory RAS axis, Angiotensin converting enzyme 2/Angiotensin-(1-7)/Mas receptor [ACE2/Ang-(1-7)/MasR]. The counter- regulatory axis has been shown to provide neuroprotection in ischaemic stroke animal models. Therefore, the studies conducted in this thesis aimed to test the neuroprotective potential of Ang-(1-7) as a post-stroke therapy following transient focal cerebral ischaemia. Furthermore, experiments were conducted to test a potential synergistic effect between MasR and alternative Ang II receptor, Angiotensin II receptor type II (AT2R), agonism following stroke.

Non-communicable disease (NCD) represent a modern global health challenge, with decreased life expectancy and quality of life, and a greater increase in health costs. Overweight and obesity are recognized as key risk factors for NCD, and the major link between these states is low-grade inflammation (LGI). Adipose tissues are now established as major contributors of pro-inflammatory molecules. One driver for low-grade inflammation is endotoxaemia (characterised by an increase in circulating endotoxin, or lipopolysaccharide (LPS)). Endotoxin is released in the gut lumen as part of the cell wall of Gram-negative bacteria, and endotoxaemia has been associated with high-fat high-calorie intake, one of the factors contributing to obesity. Thus high-fat high-calorie intake, endotoxaemia, LGI, obesity and NCD may all be linked and dietary approaches, such as functional foods with anti-inflammatory properties, may modulate this interaction. Curcuminoids (polyphenols found in the turmeric root) have anti-inflammatory and anti-bacterial effects and are potential dietary agents for preventing LGI and NCD. Little is known about the potential preventive action of curcuminoids on LGI. Therefore, this thesis aims to investigate the potential for curcuminoids to reduce endotoxaemia and LGI in apparently healthy overweight and obese people. A systematic review of the evidence for the anti-inflammatory effect of curcuminoids in healthy people and people at risk of NCD was performed. Nine papers describing five intervention studies published in this context have been assessed. Promising effects were seen in reducing CRP level (mean difference -0.71 mg/dl; 95% CI: -1.31 to -0.12; p < 0.05) and increasing adiponectin level (mean difference 5.12 ng/ml; 95% CI: 3.78 to 6.45; p < 0.01). However, there was insufficient data to make a clear conclusion that curcuminoids reduce LGI in those groups, thereby justifying the need for further work on the topic. In the second study, the phenolics and curcuminoids content in fresh turmeric root, turmeric powder, and four types of supplements were characterised to choose the supplement for a human intervention. Amongst all turmeric extracts assessed, the supplement BCM-95® (Dolcas-Biotech, Landing, NJ, USA) had the highest total phenolics (7.11 vs 1.94 mg GAE/g in average) and curcumin content (191.75 vs 13.7 mg/g in average). Therefore, BCM-95® was used in the later studies. The fate and effect of curcuminoids in the human gut were investigated further using established in vitro models for digestion and fermentation. Phenolic compounds were released after gastric and intestinal digestion. The presence of oil during digestion increased the release of phenolics from turmeric extracts. The presence of curcuminoids in a 24 h fermentation by faecal bacteria in vitro did not affect the production of short-chain fatty acids from Raftiline, which was used as the substrate for fermentation. The third study investigated the effect of curcuminoids on the intestinal permeability and endotoxin translocation using Caco-2 cell monolayers intestinal model. Administration of 115 nM of curcuminoids (in BCM-95®) for 3 day (3 h each day) unexpectedly resulted in increased monolayer permeability (by 30%) and endotoxin translocation (by 6-fold). The final study was a double-blind, placebo-controlled, crossover trial of curcuminoids supplementation (BCM-95®) for 21 days in healthy overweight and obese adults (n =14). Due to ongoing work in other parts of this project, the treatment code was not broken at the time of submission. In conclusion, there is very little published data on curcuminoids and LGI, however the Caco-2 cell monolayers study in this thesis suggest that turmeric may impair gut permeability under the experimental conditions tested. The effect may be dependent on the type of curcuminoids, the dose, and the associated oils. From the digestion study, it would appear that it is better to consume supplements associated with oil in order to optimize the release of phenolics. Although we cannot determine which of the two treatments in the human study was the active compound, the study was feasible, no side effects were reported and there was a difference between treatments. The relevance of this will be known when the code is broken.

As protozoan organisms adapted to parasitism, they became unable to synthesise the purine ring using the de novo pathway, relying exclusively on the uptake of these nutrients from their hosts, using high affinity transporters. To date, only genes encoding for members of the Equilibrative Nucleoside Transporter (ENT) family have been associated with purine or pyrimidine uptake in these organisms, and are believed to play a major role in nucleobase and nucleoside transport in protozoan parasites. Given the requirement of these organisms to transport purines with high efficiency, the ENT transporters became potential carriers for chemotherapeutic molecules. T. brucei, the causative agent of sleeping sickness, is the best studied protozoan parasite in terms of purine and pyrimidine transport functions, and although a dozen genes have been isolated and characterized as members of the ENT family, the susceptibility of these parasites against melamine-containing molecules, even after the genetic knockout of its target carrier, the P2 transporter, suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Using a combination of gene knockouts, we discovered that bloodstream forms of T. brucei possess two previously undescribed transporters: HXT1 and ADET1, with exclusive affinity for hypoxanthine or adenine, respectively. HXT1 was characterised as a medium-affinity hypoxanthine transporter, with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol.(107 cells)-1s-1 for this nucleobase, whereas ADET1 is a high-affinity adenine transporter, showing a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol.(107 cells)-1s-1. Neither HXT1 nor ADET1 showed any affinity for any other natural purine or pyrimidine, and could not be completely inhibited by hypoxanthine or adenine analogues either. Given the unprecedented specificity for their substrates, and the fact that all T. brucei ENT transporters have previously been studied, it is likely that HXT1 and ADET1 are not part of the ENT family and might indicate the presence of other nucleoside transporter family in these parasites. Different from T. brucei, and despite being the aetiological agent of Chagas’ disease, no systematic characterisation of T. cruzi ENT transporters has been done. We therefore decided to use a genetically adapted T. brucei procyclic cell line (TbNBT-KO) with reduced (~86%) [3H]-Hypoxanthine uptake as a surrogate system to express and characterise T. cruzi transporters. We successfully expressed and characterized three out of four ENT genes from the T. cruzi Y strain and showed that its transporters have higher affinity for oxopurines than for aminopurines. TcrNBT1 was shown to be a very high-affinity hypoxanthine/guanine transporter (Km of 93.8 ± 4.7 nM for hypoxanthine and Ki of 121.9 ± 22.4 nM for guanine), with a Ki of 3.7 ± 0.5 μM for adenine. TcrNBT1 harboured lower affinity for purine nucleosides and poor affinity for pyrimidines. In contrast, TcrNT1 was found to be a high-affinity inosine/guanosine transporter with a Km of 1.0 ± 0.03 μM for inosine and Ki of 0.92 ± 0.2 μM for guanosine. Interestingly, TcrNT1 showed higher affinity for hypoxanthine (Ki = 23.9 ± 5.5 μM) than for adenosine (Ki = 38.9 ± 5.8 μM) and virtually no affinity for other purines or pyrimidines. Different from the other two, TcrNT2 turned out to be a high-affinity thymidine transporter (Km = 223.5 ± 7.1 nM), also displaying some affinity for uridine and cytidine (Ki values of 66 ± 6 and 728 ± 70 μM), but barely sensitive to inhibition by pyrimidine nucleobases or purines. The fourth ENT gene cloned from T. cruzi (TcrNB2) could not be characterised despite our efforts, but its genomic and structural similarity to Leishmania NT4 transporters indicate it might be a low-affinity purine nucleobase transporter. We further used TbNBT-KO to characterise a codon-adapted Plasmodium falciparum ENT1 transporter and, different from previous reports, found it to be a medium-affinity guanine/hypoxanthine transporter, with a Km of 11.22 ± 1.18 μM for guanine. The existence of high-affinity purine transport mechanisms in trypanosomatids also led us to test the potential of C7-substituted tubercidin analogues against African trypanosomes (T. brucei and Trypanosoma congolense). We verified that, although the size of the substituent is not a determinant for transport into the cell, analogues containing small substitutions yielded the highest trypanocidal effects against wildtype and drug-resistant T. brucei strains, as well as against wildtype T. congolense, with special regard to 3’-deoxyanalogues. We verified that the 3’-deoxytubercidin analogues are likely to exert their trypanocide effect by inhibiting the synthesis of nucleic acids, whereas their ribonucleoside counterparts probably act like tubercidin, by inhibiting the glycolytic pathway. Our findings contribute to the understanding of purine and pyrimidine transport mechanisms in trypanosomes, reports the potential existence of an as yet unreported nucleobase transporter family in trypanosomatids, and shows the potential of TbNBT-KO as a model cell line for the systematic characterisation of ENT genes from other protozoans. Moreover, we show the potential exploitation of the purine transport mechanisms as carriers of drugs against trypanosomatids.

Falls are a common occurrence in older people and multisite pain has been identified as a potential falls risk factor in this age group. This thesis aims to describe the relationship between multisite pain and falls in community-dwelling older people. A systematic review identified 20 studies investigating multisite pain and falls. Meta-analysis showed multisite pain increased the odds of falling. Data from the North Staffordshire Osteoarthritis Project, a prospective cohort study of community-dwelling adults aged ≥50 years with follow ups at three and six years, was used. Survey data was linked with general practice (GP) records, Hospital Episode Statistics (HES) and Office for National Statistics mortality data. Logistic regression tested the relationship between multisite pain and risk of self-reported falls in 4386 participants with complete data. Survival analysis tested the relationship between multisite pain and risk of GP or HES recorded falls in 11,375 participants. Analyses were adjusted for confounders and putative influencers of the pain-falls relationship. Multisite pain most strongly predicted future self-reported falls, followed by GP recorded falls. Multisite pain was not associated with HES recorded falls. Increasing age, being female, increasing number of medications used and strong analgesic use predicted all future falls; increasing cognitive complaint and previous self-reported fall additionally predicted GP recorded falls and all confounders and putative influencers predicted self-reported falls and had a significant association with multisite pain. These data suggest that multisite pain is an independent risk factor for self-reported falls. In addition, multisite pain is a likely influencer of the relationship between other risk factors and future falls. Primary care should proactively identify older adults with multisite pain due to their increased risk of falling and instigate falls prevention management according to current guidelines. Future research will establish the impact of pain management interventions on future risk of falls.