Zinc finger nuclease gene repair as a treatment for cystinosis

Cystinosis is a multi-system autosomal recessive disorder caused by mutations and/or deletions in both alleles of CTNS, a gene encoding for the low pH dependent lysosomal cystine exporter cystinosin. Cystinosis occurs in approximately 1:200,000 newborns worldwide and is characterised by an accumulation of cystine in the lysosomes. The most severe form of the disorder is nephropathic cystinosis presenting Fanconi syndrome and leads without treatment to an end-stage renal failure before the age of ten. The only treatment available so far is cysteamine therapy, which delays disease progression by five years, but does not provide a cure for cystinosis patients. Current gene and cell based therapeutic approaches have not yet provided a suitable alternative. A potentially approach for a long-term treatment could be to generate autologous gene–modified stem cells by repairing the gene. Zinc Finger Nucleases (ZFNs) serve as a tool to increase HDR up to a 200,000-fold by introducing a double-stranded break (DSB). Thus, simple mutations in the CTNS gene could be corrected by introduction of a double-stranded break using ZFNs to boost the process of HDR with a suitable donor DNA sequence. A permanent repair of the most common lesion CTNS, a 57 kb deletion, could be achieved by ZFN-mediated HDR using a minigene CTNS promoter/cDNA construct. The thesis describes the design and testing of seven zinc finger nuclease pairs for their cleavage activity in vitro and in cellulo.. A highly sensitive assay to detect even low levels of ZFN-mediated HDR was also developed. Finally, to further investigate the role of autophagy in tissue injury in cystinotic cells an assay to monitor autophagy levels in the cells was successfully developed. This assay provides the opportunity to demonstrate functional restoration of CTNS after successful ZFN-HDR in cystinotic cells.

Fas ligand expression and tumour immune evasion; the role of prostaglandin E2 and the EP1 receptor

Background and Aim: During carcinogenesis, tumours develop multiple mechanisms to evade the immune system and suppress the anti-tumour immune response. Upregulation of Fas Ligand (FasL/CD95L) expression may represent one such mechanism. FasL is a member of the tumour necrosis factor superfamily that triggers apoptotic cell death following ligation to its receptor Fas. Numerous studies have demonstrated upregulated FasL expression in tumor cells, with FasL expression associated with numerous pro-tumorigenic effects. However, little is known about the mechanisms that regulate FasL expression in tumours. The cyclooxgenase (COX) signalling pathway may play an important role in colon carcinogenesis, via the production of prostaglandins, in particular PGE2. PGE2 signals through four different receptor subtypes, EP1 – EP4. Thus, the aim of this study was to investigate the effect of targeting the PGE2-FasL signaling pathway. Results: (i) PGE2 induces FasL expression via the EP1 receptor in colon cancer cells. (ii) Suppression of FasL expression in colon tumour cells in vivo significantly delays and reduces tumour growth. (iii) Blocking EP1 receptor signaling, or suppression of the EP1 receptor in colon tumour cells, reduces tumour growth in vivo. Suppression of tumour growth correlates in part with suppression of FasL expression. (iv) The reduction in tumour growth is associated with an improved anti-tumour immune response. Tumour infiltration by Treg cells and macrophages was reduced, and the cytotoxic activity of CTL generated from splenocytes isolated from these mice increased. Conclusion: 1) Targeting FasL expression by blocking PGE2-EP1 receptor signalling reduces tumour development in vivo. 2) The mechanism is indirect but is associated with an increased anti-tumour immune response. Thus, unraveling the mechanisms regulating FasL expression and the pro-tumorigenic effects of the EP1 receptor may aid in the search for new therapeutic targets against colon cancer.

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

Vitamin D status and requirements during pregnancy and lactation

Vitamin D deficiency during pregnancy, lactation, and early infancy has been widely reported. Current understanding of vitamin D metabolism during pregnancy and lactation is incomplete, and to date, experimental data to support vitamin D requirements for these life stages are scarce. There is a shortage of nationally representative data and appropriate reference ranges for serum 25-hydroxyvitamin D (25OHD) during pregnancy, lactation and infancy, including in umbilical cord blood. This thesis described concentrations of total 25OHD and individual metabolites including 25OHD3, 25OHD2, and 3-epi-25OHD3 at 15 weeks’ gestation in a large seasonally balanced pregnancy cohort study (n 1768), carried out in Cork, Ireland (52oN). The prevalence of low 25OHD concentrations in pregnant women was higher than published reports in other Caucasian women, and was highest among non-users of vitamin D-containing supplements during winter. A longitudinal pregnancy study was included which suggested gestational stages had an impact on the total serum 25OHD concentration. This thesis incorporated a randomized controlled trial carried out among 100 women across 3 intervention groups using 20 μg/day of vitamin D3 with or without 500 mg calcium, or placebo, over 12-weeks of lactation to investigate the vitamin D requirement for lactating mothers and the vitamin D content of human milk. A daily intake of 25 μg/day was suggested to meet the requirement of lactating women to maintain a 25OHD levels above 50 nmol/L in 97.5% of the population at 52oN all year around. However, vitamin D content in human milk did not increase in response to supplementation. Serum 25OHD concentration has been used as a predictor of a number of health outcomes. This thesis reported large differences in serum 25OHD concentrations using different methods in 86 umbilical cord samples. The need for international standardization of serum 25OHD measurements was re-emphasized in this thesis.

The use of atraumatic restorative treatment in elderly patients: a randomised controlled clinical trial

Objectives: to assess elderly patients’ dental status and dental habits and compare the survival rates, impact on patients’ quality of life and cost-effectiveness of Atraumatic Restorative Treatment (ART) and a conventional treatment (CT) to restore carious lesions in an elderly population. Methods: In this randomised clinical trial, 99 independently living adults (65-90 yrs) with carious lesions were randomly allocated to receive either ART or CT. Details of restored, missing and carious teeth were recorded and patients answered some questions about their oral hygiene and dental attendance habits. Direct and indirect costs were measured based on treatment time, materials and labour. Effectiveness was measured using restoration survival percentage after one year. The survival of restorations was assessed 6 months and one year after restoration placement by an independent examiner. Oralhealth related quality of life (OHRQoL) was assessed using the OHIP-14 at baseline and 2 months after treatment together with a global transition statement. Results: The patient sample comprised 46 (46.46%) male and 53 (53.54) female participants at baseline, with a mean age of 73.18 (SD=6.76). The mean DMFT of the entire sample was 27.10. Ninety patients and 268 restorations could be assessed after one year, 127 ART (46 patients) and 141 conventional restorations (44 patients). 93.7% and 97.2% of the restorations placed were considered successful in the ART and CT groups, respectively. The OHIP scores did not change dramatically 2 months after treatment, in either group. The global transition scale showed an improvement in overall oral health after treatment for the majority of patients. The ART were more cost-effective compared to the CT restorations. Conclusions: ART presented survival rates similar to CT after 1 year and was a more cost-effective alternative to treat the elderly.

Impact of the smoke-free workplace legislation on smoking behaviour, risk perception & stigmatisation

The Republic of Ireland became the first European country to implement nationwide smoke-free workplace legislation. Aims: To determine prevalence of smoking among bar workers and estimate the impact of the smoke-free workplace legislation on their smoking behaviour to that of a comparable general population sample. To approximate the influence of tobacco control measures on risk perception of second-hand smoke (SHS) among the general population. To explore the de-normalisation of smoking behaviour and the potential increased stigmatisation of smokers and their smoking. Methods: Prevalence estimates and behavioural changes were examined among a random sample of bar workers before and 1 year after the smoke-free legislation; comparisons made with a general population sub-sample. Changes in risk knowledge related to SHS exposure were based on general population data. Qualitative interviews were conducted among a purposive sample of smokers and non-smokers four years after the implementation of the legislation. Results: Smoking prevalence was extremely high among bar workers. Smoking prevalence dropped in bar workers and significantly among the general population 1 year post ban while cigarette consumption dropped significantly among bar workers. Disparity in knowledge between smokers and non-smoker of risk associated with SHS exposure reduced. Lack of understanding of the risk of ear infections in children posed by SHS exposure was notable. Evidence for advanced de-normalisation of smoking behaviour and intensification of stigma because of the introduction of the legislation was dependent on many factors, quality of smoking facilities played a key role. Conclusions: Ireland’s smoke-free legislation was associated with a drop in prevalence and cigarette consumption. Disparity in knowledge between smokers and non-smokers of the risk posed by SHS exposure reduced however the risk of ear infections in children needs to be effectively disseminated. The proliferation of ‘good’ smoking areas may diminish the potential to reduce smoking behaviour and de-normalise smoking.

National food consumption surveys: micronutrient intakes and the role of fortified foods in the diets of Irish pre-school children and adults

This research investigated the micronutrient intakes of Irish pre-school children (1-4 years) and adults (18-64 years) and the role that fortified foods (FFs) play in the diets of these population groups. Dietary intake data were collected as part of the National Pre-school Nutrition Survey (NPNS) (2010-2011) and the National Adult Nutrition Survey (NANS) (2008-2010) using 4-day food and beverage records. Nutrient intakes were estimated using WISP©, which encompasses McCance and Widdowson’s The Composition of Foods and the Irish Food Composition Database. A FF is one in which one or more micronutrients are added. Key dietary sources of micronutrients in NPNS and NANS were “milk”, “meat & meat products”, “breakfast cereals”, “fruit & fruit juices” and “breads”. In general, intakes of most micronutrients were adequate with the exception of iron (1 year old children and adult women) and vitamin D (in all population groups). Small proportions of the pre-school population had intakes which exceeded the upper level (UL) (zinc: 11%, folic acid: 5%, retinol: 4%, copper: 2%). Less than 2% of adults had intakes of iron, copper, zinc and vitamin B6 which exceeded the UL. FFs were consumed by 97% of pre-school children and 82% of adults, representing 17% and 9% of mean daily energy intake respectively. Relative to energy intake, FFs contributed substantially greater proportions to intakes of key micronutrients, such as iron and vitamin D. FFs were effective in reducing the prevalence of inadequate micronutrient intakes in these population groups, particularly for iron in women and 1 year old children. FFs made a significant contribution to folate intake in women of childbearing age (72µg). FFs contributed greater proportions of carbohydrate and lower proportions of fat to the diets of consumers. Voluntary addition of nutrients to foods did not contribute appreciably to intakes exceeding the UL in these population groups.

Defining metabolically healthy obesity: role of dietary and lifestyle factors

Background: There is a current lack of consensus on defining metabolically healthy obesity (MHO). Limited data on dietary and lifestyle factors and MHO exist. The aim of this study is to compare the prevalence, dietary factors and lifestyle behaviours of metabolically healthy and unhealthy obese and non-obese subjects according to different metabolic health criteria. Method: Cross-sectional sample of 1,008 men and 1,039 women aged 45-74 years participated in the study. Participants were classified as obese (BMI ≥30kg/m2) and non-obese (BMI <30kg/m2). Metabolic health status was defined using five existing MH definitions based on a range of cardiometabolic abnormalities. Dietary composition and quality, food pyramid servings, physical activity, alcohol and smoking behaviours were examined. Results: The prevalence of MHO varied considerably between definitions (2.2% to 11.9%), was higher among females and generally increased with age. Agreement between MHO classifications was poor. Among the obese, prevalence of MH was 6.8% to 36.6%. Among the non-obese, prevalence of metabolically unhealthy subjects was 21.8% to 87%. Calorie intake, dietary macronutrient composition, physical activity, alcohol and smoking behaviours were similar between the metabolically healthy and unhealthy regardless of BMI. Greater compliance with food pyramid recommendations and higher dietary quality were positively associated with metabolic health in obese (OR 1.45-1.53 unadjusted model) and non-obese subjects (OR 1.37-1.39 unadjusted model), respectively. Physical activity was associated with MHO defined by insulin resistance (OR 1.87, 95% CI 1.19-2.92, p = 0.006).

Renal failure and the neural control of the kidney

Renal failure (RF) is associated with an over activation of the sympathetic nervous system. The aim of this thesis was to investigate the hypothesis that as the kidney progresses into RF there is an inappropriate and sustained activation of renal afferent nerves which results in a dysregulation of basal RSNA and reflexly controlled RSNA by the high and low pressure baroreceptors. Baroreflex gain curves for both RSNA and HR were generated in control and RF rats. This study clearly showed a blunted high-pressure baroreflex in RF rats, an impairment which was almost completely corrected by bilateral renal denervation. The integrity of the low-pressure cardiopulmonary receptors to inhibit RSNA was investigated using acute saline volume. Again, a blunted reflex sympatho-inhibition of RSNA was observed, which was corrected by renal denervation. Finally a functional study to examine how the renal excretory response to volume expansion differed in RF was carried out. This study revealed an impairment of the low-pressure baroreflex control of the sympathetic outflow. The result of these studies suggest that cisplatin induced RF initiates a neural signal from within the kidney, which over rides the normal reflex regulation of RSNA by the high and low – pressure baroreceptors and that this impairment in function can be normalised by renal denervation. This raises further questions as to the mechanisms involved in the afferent over activation arising from the diseased kidneys.

Investigation of p75 neurotrophin receptor and the role of its post-translational modifications in tumour cell motility and invasiveness

The p75 neurotrophin receptor (p75NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners - including the TNF receptor associated factor 6 (TRAF6) E3 ubiquitin ligase - to produce cellular responses such as apoptosis, survival, and inhibition of neurite outgrowth. Recently,p75NTR was also shown to undergo γ-secretase-mediated regulated intramembrane proteolysis, and the receptor ICD was found to migrate to the nucleus where it regulates gene transcription. Moreover, γ-secretase-mediated proteolysis was shown to be involved in glioblastoma cell migration and invasion. In this study we report that TRAF6-mediated K63-linked polyubiquitination at multiple or alternative lysine residues influences p75NTR-ICD stability in vitro. In addition, we found that TRAF6-mediated ubiquitination of p75NTR is not influenced by inhibition of dynamin. Moreover, we report beta-transducin repeats-containing protein (β-TrCP) as a novel E3- ligase that ubiquitinates p75NTR, which is independent of serine phosphorylation of the p75NTR destruction motif. In contrast to its influence on other substrates, co-expression of β-TrCP did not reduce p75NTR stability. We created U87-MG glioblastoma cell lines stably expressing wild type, γ-secretaseresistant and constitutively cleaved receptor, as well as the ICD-stabilized mutant K301R. Interestingly, only wild-type p75NTR induces increased glioblastoma cell migration, which could be reversed by application of γ-secretase inhibitor. Microarray and qRT-PCR analysis of mRNA transcripts in these cell lines yielded several promising genes that might be involved in glioblastoma cell migration and invasion, such as cadherin 11 and matrix metalloproteinase 12. Analysis of potential transcription factor binding sites revealed that transcription of these genes might be regulated by well known p75NTR signalling cascades such as NF-κB or JNK signalling, which are independent of γ-secretase-mediated cleavage of the receptor. In contrast, while p75NTR overexpression was confirmed in melanoma cell lines and a patient sample of melanoma metastasis to the brain, inhibition of γ-secretase did not influence melanoma cell migration. Collectively, this study provides several avenues to better understand the physiological importance of posttranslational modifications of p75NTR and the significance of the receptor in glioblastoma cell migration and invasion.

Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

The ELDERMET biobank: Isolation and characterization of the intestinal microbiota from elderly Irish subjects

The human gastrointestinal (GI) tract is colonized by a dense and diverse bacterial community, the commensal microbiota, which plays an important role in the overall health of individuals. This microbiota is relatively stable throughout adult life, but may fluctuate over time with aging and disease. The adaptation of the gut microbiota to our changing life-style is probably the reason for the large inter-individual variation observed among different people. Since the gut microbiota plays an essential role in interactions with host metabolism, it is of utmost importance to explore this relationship. The elderly intestinal microbiota has been the subject of a number of studies in recent years. The results presented in this thesis have further contributed to the expansion of knowledge related to gut microbiota research highlighting the combined effect of culture based and molecular methods as powerful tools for understanding the true impact of microbes. The degree of correlation between measurements from both methods suggested that a single method is capable of profiling intestinal Bifidobacterium spp., Lactobacillus spp. and Enterobacteriaceae populations. Bacteriocins have shown great promise as alternatives to traditional antibiotics. In this respect, the isolation and characterisation of bacteriocinogenic strains are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine. The selection pressure applied on the bacterial population during antibiotic usage is the driving force for the emergence of antibiotic resistant bacteria. Identification of antibiotic resistant isolates opens up the possibility of using such probiotics to offset the problems caused by antibiotics to the gut microbiota and to improve the intestinal microbial environment. Future work is required to explore the culture collection housing thousands of bacterial isolates as a valuable source of potential probiotics for use for the elderly Irish community.

Lower extremity amputation in people with diabetes: Trends and determinants

Introduction: The prevalence of diabetes is rising rapidly. Assessing quality of diabetes care is difficult. Lower Extremity Amputation (LEA) is recognised as a marker of the quality of diabetes care. The focus of this thesis was first to describe the trends in LEA rates in people with and without diabetes in the Republic of Ireland (RoI) in recent years and then, to explore the determinants of LEA in people with diabetes. While clinical and socio-demographic determinants have been well-established, the role of service-related factors has been less well-explored. Methods: Using hospital discharge data, trends in LEA rates in people with and without diabetes were described and compared to other countries. Background work included concordance studies exploring the reliability of hospital discharge data for recording LEA and diabetes and estimation of diabetes prevalence rates in the RoI from a nationally representative study (SLAN 2007). To explore determinants, a systematic review and meta-analysis assessed the effect of contact with a podiatrist on the outcome of LEA in people with diabetes. Finally, a case-control study using hospital discharge data explored determinants of LEA in people with diabetes with a particular focus on the timing of access to secondary healthcare services as a risk factor. Results: There are high levels of agreement between hospital discharge data and medical records for LEA and diabetes. Thus, hospital discharge data was deemed sufficiently reliable for use in this PhD thesis. A decrease in major diabetes-related LEA rates in people with diabetes was observed in the RoI from 2005-2012. In 2012, the relative risk of a person with diabetes undergoing a major LEA was 6.2 times (95% CI 4.8-8.1) that of a person without diabetes. Based on the systematic review and meta-analysis, contact with a podiatrist did not significantly affect the relative risk (RR) of LEA in people with diabetes. Results from the case-control study identified being single, documented CKD and documented hypertension as significant risk factors for LEA in people with diabetes whilst documented retinopathy was protective. Within the seven year time window included in the study, no association was detected between LEA in patients with diabetes and timing of patient access to secondary healthcare for diabetes management. Discussion: Many countries have reported reduced major LEA rates in people with diabetes coinciding with improved organisation of healthcare systems. Reassuringly, these first national estimates in people with diabetes in the RoI from 2005 to 2012 demonstrated reducing trends in major LEA rates. This may be attributable to changes in diabetes care and also, secular trends in smoking, dyslipidaemia and hypertension. Consistent with international practice, LEA trends data in Ireland can be used to monitor quality of care. Quantifying this improvement precisely, though, is problematic without robust denominator data on the prevalence of diabetes. However, a reduction in major diabetes-related LEA rates suggests improved quality of diabetes care. Much controversy exists around the reliability of hospital discharge data in the RoI. This thesis includes the first multi-site study to explore this issue and found hospital discharge data reliable for the reporting of the procedure of LEA and diagnosis of diabetes. This project did not detect protective effects of access to services including podiatry and secondary healthcare for LEA in people with diabetes. A major limitation of the systematic review and meta-analysis was the design and quality of the included studies. The data available in the area of effect of contact with a podiatrist on LEA risk are too sparse to say anything definitive about the efficacy of podiatry on LEA. Limitations of the case-control study include lack of a diabetes register in Ireland, restricted information from secondary healthcare and lack of data available from primary healthcare. Due to these issues, duration of disease could not be accounted for in the study which limits the conclusions that can be drawn from the results. The model of diabetes care in the RoI is currently undergoing a re-configuration with plans to introduce integrated care. In the future, trends in LEA rates should be continuously monitored to evaluate the effectiveness of changes to the healthcare system. Efforts are already underway to improve the availability of routine data from primary healthcare with the recent development of the iPCRN (Irish Primary Care Research Network). Linkage of primary and secondary healthcare records with a unique patient identifier should be the goal for the future.

Molecular and cellular aspects of the SREBP pathway

The SREBP (sterol response element binding proteins) transcription factors are central to regulating de novo biosynthesis of cholesterol and fatty acids. The SREBPs are regulated by retention or escape from the ER to the Golgi where they are proteolytically cleaved into active forms. The SREBP cleavage activating protein (SCAP) and the INSIG proteins are essential in this regulatory process. The aim of this thesis is to further characterise the molecular and cellular aspects surrounding regulation of SREBP processing. SREBP and SCAP are known to interact via their carboxy-terminal regulatory domains (CTDs) but this interaction is poorly characterised. Significant steps were achieved in this thesis towards specific mapping of the interaction site. These included cloning and over expression and partial purification of tagged SREBP1 and SREBP2 CTDs and probing of a SCAP peptide array with the CTDs. Results from the SREBP2 probing were difficult to interpret due to insolubility issues with the protein, however, probing with SREBP1 revealed five potential binding sites which were detected reproducibly. Further research is necessary to overcome SREBP2 insolubility issues and to confirm the identified SREBP1 interaction site(s) on SCAP. INSIG1 has a central role in regulating SREBP processing and in regulating stability of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a rate limiting enzyme in cholesterol biosynthesis. There are two protein isoforms of human INSIG1 produced through the use of two in-frame alternative start sites. Bioinformatic analysis indicated that the presence of two in-frame start sites within the 5-prime region of INSIG1 mRNA is highly conserved and that production of two isoforms of INSIG1is likely a conserved event. Functional differences between these two isoforms were explored. No difference in either the regulation of SREBP processing or HMGCR degradation between the INSIG1 isoforms was observed and the functional significance of the two isoforms is as yet unclear. The final part of this thesis focused on enhancing the cytotoxicity of statins by targeted inhibition of SREBP processing by oxysterols. Statins have significant potential as anti-cancer agents as they inhibit the activity of HMGCR leading to a deficiency in mevalonate which is essential for cell survival. The levels of HMGCR fluctuate widely due to cholesterol feedback of SREBP processing. The relationship between sterol feedback and statin mediated cell death was investigated in depth in HeLa cells. Down regulation of SREBP processing by sterols significantly enhanced the efficacy of statin mediated cell death. Investigation of sterol feedback in additional cancer cell lines showed that sterol feedback was absent in cell lines A- 498, DU-145, MCF-7 and MeWo but was present in cell lines HT-29, HepG2 and KYSE-70. In the latter inhibition of SREBP processing using oxysterols significantly enhanced statin cytotoxicity. The results indicate that this approach is valid to enhance statin cytotoxicity in cancer cells, but may be limited by deregulation of SREBP processing and off target effects of statins, which were observed for some of the cancer cell lines screened.

Towards a cognitive neurobiology of brain-gut axis disorders

The past two decades have seen substantial gains in our understanding of the complex processes underlying disturbed brain-gut communication in disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Despite a growing understanding of the neurobiology of brain-gut axis dysfunction, there is a relative paucity of investigations into how the various factors involved in dysregulating the brain-gut axis, including stress, immune activation and pain, could impact on fundamental brain processes such as cognitive performance. To this end, we proposed a cognitive neurobiology of brain-gut axis dysfunction and took a novel approach to examine how disturbed brain-gut interactions may manifest as altered cognitive performance in IBS and IBD, both cross-sectionally and prospectively. We have demonstrated that, disorders of the brain-gut axis are characterised by stable deficits in specific cognitive domains. Specifically, patients with IBS exhibit a consistent hippocampal mediated visuospatial memory impairment. In addition we have found evidence to suggest a similar visuospatial impairment in IBD. However, our most consistent finding within this population was that patients with Crohn’s disease exhibit impaired selective attention/ response inhibition on the classic Stroop interference test. These cognitive deficits may serve to perpetuate and sustain brain-gut axis dysfunction. Furthermore, this research has shed light on some of the underlying neurobiological mechanisms that may be mediating cognitive dysfunction in IBS. Our findings may have significant implications for the individual who suffers from a brain-gut axis disorder and may also inform future treatment strategies. Taken together, these findings can be incorporated into existing neurobiological models of brain-gut axis dysfunction, to develop a more comprehensive model accounting for the cognitive-neurobiology of brain-gut axis disorders. This has furthered our understanding of disease pathophysiology and may ultimately aid in both the diagnosis and treatment of these highly prevalent, but poorly understood disorders.