The aim of this study was to investigate the potential use of ELISA and LC-MS-MS in combination and as individual techniques, for the detection of drugs of abuse in biological matrices. Overall the LC-MS-MS method showed good correlation results for opiates compared to the GC-MS method. 6-MAM was however detected in more root segments and segments excluding roots by LC-MS-MS. Morphine was detected in a greater number of root segments by LC-MS-MS compared to GC-MS. However, morphine was detected in a greater number of segments excluding roots by GC-MS. Codeine and dihydrocodeine were also detected in a greater number of root segments and segments excluding roots by GC-MS. The cocaine results showed excellent qualitative correlation between the LC-MS-MS and GC-MS methods for cocaine and benzoylecgonine. The GC-MS method did not however extract greater concentrations of cocaine and its metabolites compared to LC-MS-MS due to the higher recovery of the drug group specific GC-MS method. Cocaethylene and EME were detected in some samples by LC-MS-MS method for opiates and cocaine and its metabolites compared to the GC-MS method; there may be some cases where the GC-MS method would detect the analytes where the LC-MS-MS method would not. This has been demonstrated in 3 samples for morphine and in 6 samples for codeine. The LC-MS-MS method analysed for and detected amphetamines in samples that were not tested for amphetamines by GC-MS. In one sample that was tested by both methods, amphetamine was detected in the root sample by LC-MS-MS where GC-MS failed to detect it. Also a greater concentration of amphetamine was extracted using the LC-MS-MS method in the segment without roots. The LC-MS-MS method was useful for the analysis of 17 drugs of abuse in post-mortem hair samples in forensic toxicology cases. Using this method, it is possible to obtain maximum information from one hair sample which is extremely useful when the sample weight is limited. The ability of the LC-MS-MS method to extract and analyse a greater number of drug groups from one hair sample highlights the advantages of using this method over GC-MS which targets individual drug groups and requires splitting of the sample. This method is particularly applicable for implementation in the forensic toxicology laboratory at the University of Glasgow where currently GC-MS methods that target individual drug groups are used for routine hair screening and confirmation.

Multiple sclerosis (MS) is a chronic disease of the human central nervous system (CNS) in which repeated episodes of inflammatory demyelination result in formation of persistently demyelinated plaques of gliotic scar tissue associated with varying degrees of axonal loss. MS is now considered a “complex trait” that is triggered in genetically susceptible individuals by environmental factors. The disease is also considered to contain an autoimmune component where both the adaptive and innate immune systems have been implicated in disease pathogenesis. There has been a steady accumulation of circumstantial evidence from both clinical and experimental studies that implicate a role for autoantibody dependent mechanisms. However this issue remains controversial in the absence of formal evidence that patients actually develop a pathogenic autoantibody response. The aim of this thesis was to resolve this question. To do this we developed an in vitro bioassay based on a dissociated myelinating culture system from embryonic rat spinal cord. We demonstrated that this in vitro system could reproduce many features of in vivo myelinated axons. To validate this model as a viable screening assay characterised complement mediated autoantibody responses using a series of monoclonal antibodies and anti-sera. Due to their significance in the literature we focussed in particular on the MOG specific and Nfasc specific responses and comprehensively demonstrated that our bioassay offered a robust screening strategy in which to detect pathogenic antibody responses in the presence and absence of exogenous complement. To determine whether we could use our model to detect pathogenic autoantibody responses in MS patients, we purified the IgG fraction from a cohort of MS patients (n=20), OND (n=10) and healthy controls (n=13). Using this patient purified IgG we demonstrated a MS specific demyelinating activity, which was present in ~50% of samples screened. However in 10% of patients demyelination occurred secondary to pronounced axonal injury. These effects were dependent on exogenous complement and were unique to the MS cohort. Pathogenic antibody responses tended to be most prevalent in those patients with an aggressive disease course. In addition to complement mediated CNS injury we also demonstrated that this pathogenic MS IgG could disrupt myelin formation in developing myelinating cultures. Attempts to define the specificity revealed that this was heterogeneous, however in one MS patient we discovered that Nfasc155 provided a dominant antigen for pathogenic autoantibody responses. Together these data provide formal demonstration that MS is associated with pathogenic autoantibody responses. This has significant long term consequences for the clinical management of the disease

The work in this thesis describes a series of studies utilising diverse data sources which were analysed using a number of regression methods (logistic, linear, Cox, Poisson), to address the factors associated with normal and abnormal pregnancy outcome. A series of maternal characteristics were related to adverse pregnancy outcome. Teenage pregnancy was not associated with an increased risk of any adverse outcome among first births, but was strongly associated with adverse outcome among second births [8]. Parity also interacted with body mass index: maternal obesity was associated with an increased risk of preterm birth among nulliparous but not multiparous women. This was explained by higher rates of elective preterm deliveries among obese nullipara [31]. There was a linear relationship between maternal age and the duration of labour, and the risk of operative vaginal and caesarean delivery [37]. It was hypothesised that age-related deterioration in obstetric performance was due to prolonged hormonal stimulation prior to the first birth. This hypothesis was supported by the observation that later menarche was independently associated with a decreased risk of operative delivery [38]. A short inter-pregnancy interval was associated with an increased risk of spontaneous preterm birth, but not stillbirth or intra-uterine growth restriction [16]. The risk of unexplained stillbirth at term was increased among nulliparous women [5] and nulliparous women also had slightly longer pregnancies [7]. A U-shaped relationship between birth weight and caesarean risk was observed at term. There was an interaction between fetal sex and caesarean risk: small boys were at increased risk of emergency caesarean [3]. The same interaction was observed for antepartum stillbirth [4]. Previous pregnancy outcome was predictive of the outcome of subsequent pregnancies. Women who were delivered by caesarean section in their first pregnancy had an increased risk of unexplained stillbirth in their second [17]. This finding was confirmed in a separate cohort and associations were also observed between previous complicated livebirths and the subsequent risk of unexplained stillbirth [32]. Some specific situations were also studied (vaginal birth after caesarean section (VBAC) and twins). Among women attempting VBAC, the absolute risk of delivery-related perinatal death was comparable to primiparous women but was significantly higher than women delivered by elective caesarean section [11]. The risk of perinatal death associated with uterine rupture was increased in low throughput obstetric units and among women induced with prostaglandins [19]. Using simple maternal characteristics, approximately 50% of women attempting VBAC could be classified into having a high (>40%) or low (<10%) risk of emergency caesarean [24]. This was better discrimination than could be achieved using similar characteristics among nulliparous women being induced at term [21]. The risk of delivery related perinatal death was increased among second twins, although this was only evident among births at term [13]. The association was observed among sex discordant twins, but was not observed among twins delivered by elective caesarean section [23]. The association between birth order and the risk of death due to anoxia was confirmed in data from England and Wales [33]. Ultrasonic measurements of the fetus were related to eventual birth weight. The range of error associated with such estimates was quantified and abdominal circumference on its own was as predictive as models using abdominal circumference and femur length [1]. Estimating fetal weight using ultrasound was not found to be a better measure of human fetal blood volume than simply using gestational age [10]. A series of ultrasonic measurements in the first and second trimester were predictive of pregnancy outcome, including smaller than expected crown rump length and intra-uterine growth restriction, preterm birth and low birth weight [2]; a long cervix in mid gestation and caesarean section [36]; and, high resistance patterns of uterine artery Doppler flow velocimetry and stillbirth [30]. Biochemical measurements performed in early pregnancy were also predictive of later adverse outcome: low maternal levels of pregnancy-associated plasma protein A (PAPP-A) were associated with an increased of pre-eclampsia, preterm birth and growth restriction [9]; low PAPP-A prior to 13 weeks was associated with birth weight at term in healthy pregnancies [12] and with a dramatically increased risk of stillbirth due to placental dysfunction [22]. Low first trimester levels of placenta growth factor were associated with increased risks of pre-eclampsia and growth restriction, whereas there was no association between elevated levels of the soluble fms-like receptor and adverse outcome [35]. Measurements of biochemical variables in the second trimester were also predictive of outcome, with elevated maternal serum alphafetoprotein (AFP) being associated with an increased risk of stillbirth [34] and spontaneous preterm birth [29]. Women with the combination of low first trimester PAPP-A and high second trimester AFP were at particularly high risk of complications, reflecting the synergistic predictive ability of the two measures [27]. Given proposed similarities between stillbirth and sudden infant death syndrome (SIDS), this outcome was also studied. Elevated second trimester levels of AFP were also associated with an increased subsequent risk of SIDS [20]. Women with a pregnancy resulting ultimately in SIDS were found to be more likely to have had complications in past and future pregnancies [25]. The risk of SIDS declined with advancing gestational age at term following spontaneous, but not elective birth [15]. Obstetric characteristics were used to generate a predictive model for SIDS [26]. Pregnancy outcome was also predictive of other aspects of child health, specifically, respiratory morbidity following birth at term was associated with an increased risk of hospital admission for asthma [18]. Pregnancy complications were also related to long term maternal health. Elective caesarean delivery for breech presentation did not appear to have an independent effect on fertility [28]. However, pregnancy complications were associated with the mother’s subsequent experience of cardiovascular disease. Women experiencing growth restriction, preterm birth or pre-eclampsia were at increased risk of subsequent ischaemic heart disease (IHD) [6] and the risk of this was also related to the number of miscarriages experienced prior to the first birth [14]. The parents of women who had experienced pregnancy complications or recurrent miscarriage had an increased incidence of IHD [39 & 40, respectively].

The Apc gene encodes the Adenomatous polyposis coli tumour suppressor protein, the germ line mutation of which characterizes Familial Adenomatous Polyposis (FAP), an autosomal syndrome characterized by multiple colorectal lesions. Inactivation of the Apc gene is recognized as a key early event in the development of colorectal cancers and leads to the deregulation of the Wnt pathway and the activation of TCF/LEF target genes. This project focuses on the proto-oncogene c-Myc as it is a key Wnt target gene which is activated following loss of Apc in vivo. This upregulation is noteworthy as c-Myc is implicated in stem cell survival, proliferation, apoptosis and tumourigenesis. Previous studies have shown c-Myc dependency for both apoptosis and proliferation following activation of the Wnt pathway, however little is known about the role c-Myc plays in inducing apoptosis following DNA damage in vivo. To study this I have conditionally deleted c-Myc from the intestinal epithelium and examined the response of intestinal enterocytes following DNA damage. Remarkably, following DNA damage, c-Myc deficient enterocytes were unable to upregulate p53 and induce apoptosis, which was mechanistically due to an upregulation of MDM2. Taken together, results from this study showed for the first time in vivo, a key role for c-Myc in inducing apoptosis following DNA damage through control of p53. Previous studies from this lab have shown that within the intestinal epithelium, c-Myc is absolutely required for the hyper-proliferative phenotype that is observed following loss of Apc. Therefore one of the key aims of this thesis is to look downstream of c-Myc in order to delineate how c-Myc induces and controls this proliferation. Given that one of the key postulated functions of c-Myc is the transcriptional repression of p21, this thesis examines this hypothesis by investigating the significance of the upregulation of p21 following c-Myc deletion in Apc deficient intestinal enterocytes. To do this, I have generated triple knockout (TKO) intestines by intercrossing p21 knockout mice to mice where we can conditionally delete both Apc and c-Myc within the murine intestinal epithelium. Surprisingly, the levels of proliferation were the same between double knockout Apc Myc and TKO intestines, which had markedly less proliferation than Apc deficient intestines. However, unlike double knockout enterocytes, TKO intestinal enterocytes no longer moved up the crypt-villus axis and failed to generate villus. To examine which of these phenomena were key to tumourigenesis (differentiation or proliferation), we investigated whether TKO intestines could form intestinal adenomas and found that even in the absence of p21, c-Myc deficient cells were unable to form tumours. Taken together we have identified a novel role for p21 in driving differentiation following Apc and Myc deletion. This is consistent with the expression of p21 in the normal crypt at the crypt villus junction. Remarkably this function of p21 is independent of its key role as a cell cycle inhibitor. Moreover, this study also examined the importance of the upregulation of the Cyclin D/CDK4 complexes following Apc loss and their role in c-Myc dependent proliferation. Results from these studies showed that Cyclin D2 is required for efficient proliferation immediately following loss of Apc as well as for tumourigeneis in the Apc Min/+ mouse. Taken together, results from these studies showed that the upregulation of Cyclin D2 and CDK4 are c-Myc dependent and that the upregulation of these complexes are key for Wnt driven proliferation and tumourigenesis. Lastly, in this study I have examined whether Apc loss within the intestinal epithelium, where it is a bona fide tumour suppressor gene, can provoke senescence, and compared this to the ability of Apc gene deletion to trigger senescence in the renal epithelium, where it is not mutated in human cancer. This study showed that deletion of Apc within the renal epithelium invoked a p21 dependent senescence response, and Apc deficient renal epithelial cells were cleared and very rarely initiated tumourigenesis. However, combined Apc and p21 gene deletion rapidly initiated tumourigenesis, with all mice developing renal carcinoma by 2 months of age. In contrast to Apc deficient intestinal epithelium, this process was unaffected by loss of c-Myc. However within the intestinal epithelium, deletion of Apc did not invoke senescence, but lead to a highly proliferative, p21 independent response. Combined Apc and p21 gene loss had no impact on either the short term phenotypes of Apc loss or upon tumourigenesis. Taken together these results show for the first time that Apc loss in vivo can invoke a senescence program but in a context dependent fashion. This implies escape from senescence is not a crucial pathway in colorectal cancers that are initiated by Apc loss, and goes to explain why renal carcinoma is not observed in FAP patients who are germline heterozygous for APC. Therefore the aims for this thesis are: • To investigate the role of c-Myc in inducing apoptosis within the intestinal crypt, and whether this is p21 dependent? • To investigate the role of p21 in causing senescence of Apc deficient cells, and whether this is c-Myc dependent? • To determine the functional importance of repression of p21 by c-Myc in Apc deficient cells. • To determine the significance of Cyclin D2 upregulation within Apc deficient cells.

The focus of this study is the potential use of FTIR imaging as a tool for objective automated histopathology. The Thesis also reports the use of multivariate statistical techniques to analyse the FTIR imaging data. These include Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), Multivariate Curve Resolution (MCR) and Fuzzy C-Means Clustering (FCM). The development of a new PCA-FCM Clustering hybrid that can automatically detect the optimum clustering structure is also reported. Chapter 1 provides a brief introduction to the use of vibrational spectroscopy to characterise biomolecules in tissues and cells for medical diagnosis. Chapter 2 details the basic histology of a lymph node before proceeding to present imaging results gained from the analysis of both healthy and diseased lymph node tissue sections. The ability of each multivariate technique to discriminate different tissue types is discussed. In addition, the spectral features that are characteristic for each tissue type are reported. The development and application of a new PCA-FCM Clustering algorithm that can automatically determine the best clustering structure is also described in full. The results indicate that cellular abnormality provides changes to both the protein and nucleic acid vibrations. However, similar spectral profiles were identified for highly proliferating cells that were contained within reactive germinal centres of the lymph node. Chapter 3 provides a short introduction to the histology of the cervlx before presenting imaging results that were gained from the analysis of both healthy and diseased cervical tissue sections. The ability of each multivariate technique to discriminate different tissue types is discussed. In addition, the spectral features that are characteristic for each tissue type are described in detail. Novel imaging experiments upon exfoliated cervical cells are also presented. It would appear that cellular abnormality in cervical tissues and cells affects both the protein and nucleic acid features of the spectra. Glycogen and glycoprotein contributions that are prevalent in healthy tissues are also absent. Chapter 4 details sample preparation methods, the instrumentation and procedures used for data acquisition, and the subsequent data processing and multivariate techniques applied to analyse the collected spectral datasets.

Campylobacter spp. are the most common causes of bacterial gastroenteritis in humans worldwide, and although poultry and cattle are considered major sources of Campylobacter spp., infection has also been associated with dogs. In order to investigate the potential zoonotic risk to humans, dog faeces were examined for the presence of Campylobacter spp. from several different dog populations including; vet-visiting, boarding, rescue and hunt dogs. The Campylobacter spp. prevalence, and species distribution was determined for all studies, and some studies were analysed for possible risk factors for Campylobacter spp. carriage in dogs. Longitudinal studies were carried out on kennelled dogs to investigate shedding patterns, and possible transmission. All C. jejuni, and 41 C. upsaliensis isolates from these studies underwent multilocus sequence typing (MLST), along with nine C. upsaliensis isolates originating from human clinical cases, in order to identify possible sources of infection, and assess the potential zoonotic risk to humans. Additionally a pilot study was performed to annotate a plasmid as part of a C. upsaliensis genome project. The findings of this thesis found that the overall prevalence of Campylobacter spp. ranged from 0-73%, although the majority of studies had a prevalence greater than 30%. The prevalence and species distribution differed depending upon the dog population. Kennelled dogs generally demonstrated the highest overall Campylobacter spp. prevalence, whilst the greatest species diversity was found in hunt dogs. C. upsaliensis dominated in most of the populations sampled, except for two hunt kennels where C. lari and C. jejuni dominated. The prevalence of C. jejuni was relatively high in some of the rescue and hunt kennels, reaching 20% and 26% respectively, whereas in vet-visiting and boarding dogs it was relatively low, 1.2-9%. Longitudinal studies indicated that the majority of dogs entered the kennels already carrying Campylobacter spp. but when possible transmission events occurred they often involved C. jejuni. Rescue dogs appeared to be exposed to sources of C. jejuni before and after entry to the kennel, but boarding dogs were only exposed after entry. The shedding of C. jejuni in dogs appeared to be over short durations, whereas dogs that carried C. upsaliensis shed the bacterium in nearly every sample. Data suggested that dogs carried the same C. upsaliensis strain throughout the study, providing further evidence that the species may act as a commensal in dogs. Further to this no associations could be made between Campylobacter spp. carriage, specifically C. upsaliensis, and disease in dogs in any of the studies. Younger dogs were significantly more likely to carry C. upsaliensis than older dogs in the vet-visiting study (OR for every additional month 0.99) and living with another dog carrying Campylobacter spp., was significantly associated with Campylobacter spp. carriage in dogs. A considerable amount of genetic diversity was observed within the C. jejuni and C. upsaliensis isolates originating from dogs, and MLST results suggested that strains of both species were the same, or highly similar to strains found in humans. This suggests that there may be common sources of infection for both humans and dogs and that dogs remain a potential zoonotic risk to humans. Although only a small number of household dogs carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel populations where the prevalence may be higher. Dogs remain a significant reservoir of C. upsaliensis, but the relationship between the presence of C. upsaliensis and gastroenteritis in both dogs and humans is still unclear.

Una de las opciones para el control de la enfermedad más devastadora del cultivo de papa (Solanum tuberosum), el tizón tardío, producido por Phytophthora infestans, es el desarrollo de variedades resistentes a este patógeno, mediante la transferencia directa de genes de resistencia R por ingeniería genética. En el siguiente trabajo de investigación, se usó el gen RB de Solanum bulbocastanum, que otorga un amplio espectro de resistencia a razas de P. infestans. Para dicho fin, se transformó genéticamente vía Agrobacterium tumefaciens la variedad susceptible de papa Desiree (Solanum tuberosum) con el vector binario pCIP68 que contiene el gen RB. Como resultado, se obtuvieron 19 plantas transformadas con el gen RB, confirmadas por la prueba de resistencia a kanamicina y por la prueba de reacción en cadena de la polimerasa (PCR). Las 19 plantas transgénicas fueron sometidas a infección en invernadero bajo condiciones de bioseguridad con el aislamiento POX067 de P. infestans perteneciente al linaje clonal EC-1 que es dominante en el Perú. Tres de las 19 plantas ([RB]54, [RB]56 y [RB]70) presentaron un alto nivel de resistencia al aislamiento POX067 de P. infestans.

There is growing evidence that suggests Traumatic brain injury (TBI) may initiate long-term neurodegenerative processes. Exposure to a single moderate or severe TBI, or to repetitive TBI, reveals a complex of pathologies including abnormalities of tau, amyloid-β and TDP-43; neuronal loss; neuroinflammation; and white matter degradation. The mechanisms driving these late post-TBI neurodegenerative pathologies remain elusive. Firstly, a potential association between blood-brain barrier (BBB) disruption and TBI was investigated. Results showed that increased and widespread BBB disruption was observed in material from patients dying in the acute phase following a single, moderate to severe TBI and persisted in a high proportion of patients surviving years following injury. Furthermore, there was preferential distribution to the deep layers of the cortex and to the crests of the gyri rather than the depths of the sulci. This post-TBI BBB disruption was investigated further within a paediatric TBI cohort. BBB disruption was noted in both paediatric and adult TBI in a similar pattern and distribution, however, interestingly, in sharp contrast to adult TBI cases, BBB disruption in paediatric cases appears preferentially distributed to capillary sized vessels. This vulnerability of the small vessels was rarely observed in adult material. In addition to the post-TBI vascular change observed, the cellular response was investigated, which interestingly, demonstrated regional differences. Specifically, in the grey matter, reactive astrogliosis was observed subpially, around cortical vessels, at the grey and white matter boundaries and subependymally. This astrogliosis was evident in a proportion of acute and continued into the late phase following TBI. In contrast, microglial activation was observed as a delayed response and localised to the white matter tracts. In addition, this delayed microglial response expressed an M2-like phenotype. Furthermore, there was an increased population of inactivated perivascular microglia beyond the perivascular space in the grey matter regions, observed in the acute phase and persisted in a proportion of patients surviving years following injury. Collectively these findings are interesting and indicate TBI induces both a vascular and cellular responses which may contribute to the long-term post-TBI neurodegenerative processes.

Diffuse, multi vessel coronary artery disease (CAD) affects about one third of patients with CAD and is associated with worse outcomes. Abnormal vascular stiffness and function (e.g., reflected by increased endothelial microparticles and diminished microvascular endothelial-mediated responses), cell mediated pro-inflammatory status (e.g., reflected by levels of specific monocyte subsets), and platelet function (e.g., increased monocyte-platelet aggregates (MPAs) and platelet microparticles) have established roles in CAD pathogenesis but their contribution to the unfavourable diffuse CAD form is unclear. The aim of this study was to compare measures of vascular function, monocyte subsets, MP As, and endothelial and platelet microparticles in patients with diffuse and focal CAD and subjects without CAD. Additionally, prospective changes in these characteristics were analysed over one year. I found increased counts of aggregates of Mon2 monocyte subset with platelets and apoptotic endothelial microparticles in patients with diffuse CAD and I identified a negative correlation between Mon2 MPAs and microvascular endothelial function and increased diastolic elastance. My findings suggest that excessive levels of Mon2 aggregates with platelets and apoptotic endothelial micropa1iicles could be important contributors to the diffuse type of CAD by a mechanism involving microvascular endothelial dysfunction and abnonnal cardio-vascular interactions.

Type 1 diabetes (T1D) is characterised by pancreatic cell autoimmunity and inflammation, resulting in cell islet destruction and insulin deficiency. Prospective studies from different continents have shown that insulin resistance is independently associated with risk for the development of T1D. We wanted to investigate the role of adiponectin in mediating this link. Adiponectin is a circulating adipokine whose anti-inflammatory and insulin sensitising actions appear to be mediated via two related receptors, AdipoR1 and AdipoR2. We began by characterising adiponectin receptor expression on PBMC by flow cytometry. We showed that monocytes express both receptors abundantly, that this expression correlates with insulin sensitivity in both health and diabetes. Furthermore, expression can be increased with lifestyle intervention. Adiponectin receptor expression on monocytes is reduced in T1D, and we demonstrate this leads to an apparent resistance in the ability of adiponectin to inhibit the stimulatory capacity of antigen presenting cells (APC). Specifically, we show that adiponectin inhibits the stimulatory capacity of APCs through down-regulation of CD86 expression, and that this effect is decreased in T1D. In this way, the release from the regulatory effects of adiponectin is one potential mechanism by which immune tolerance is lost in T1D.