Much is known about the disease pathology related to schizophrenia, however, little is known with regards to its aetiology. Recent evidences suggest a neurodevelopmental hypothesis for schizophrenia where environmental factors including: infection, stress and malnutrition, can adversely affect the pregnant mother thereby elevating the risk for schizophrenia developing in the offspring during adulthood (Meyer et al., 2008d; Meyer and Feldon, 2009; 2012; Forrest et al., 2012; Meyer, 2013). Since a variety of viral and bacterial infections in animal models have demonstrated to increase the risk in schizophrenia, it is proposed that factors common to the immune response may mediate this link. While many laboratories have reported several behavioural abnormalities following maternal immune activation, we sought to examine molecular changes following poly(I:C) exposure, a synthetic viral mimetic, in the pregnant mother and assessed a range of protein markers with known developmental roles, since an appreciable understanding of the molecular alterations taking place would permit suitable therapies to follow. Interestingly, poly(I:C) was able to induce a range of changes resembling those observed during schizophrenia, where the major NMDA receptor subunit GluN1 and α-Synuclein was reduced in postnatal day 21 animals born to mothers treated with poly(I:C) during gestation days 14, 16 and 18. Furthermore, these changes suggest a mechanism by which maternal immune activation may lead to the subsequent emergence of schizophrenia. Another aspect of this work examined the role of the kynurenine pathway on brain development. There is increasing evidence suggesting the involvement of the kynurenine pathway, a biochemical pathway responsible for the oxidative metabolism of tryptophan, in the disease pathology of schizophrenia, including neurodegenerative disorders such as Parkinson’s, Alzheimer’s and Huntington’s disease (Giorgini et al., 2005; Ting et al., 2009; Bonda et al., 2010). Since immune activation induces the activation of the kynurenine pathway, it was hypothesised that alterations in central kynurenine concentrations during development may be involved in mediating the subsequent increased risk for schizophrenia (Forrest et al., 2013, Khalil et al., 2013, 2014). As very little is known about the physiological activity of the kynurenine pathway during development, we sought to examine the potential consequence of disrupting this pathway and examining its effects upon brain development. Therefore, a kynurenine monooxygenase inhibitor, Ro61-8048, was administered to pregnant rats during gestation day 14, 16, and 18, that would inhibit the synthesis of the neurotoxic metabolite quinolinic acid, while redirecting the pathway to increase the neuroprotectant kynurenic acid. Brain development was assessed by examining changes in protein expression of markers intimately involved in synaptic transmitter release machinery, neurogenesis and many aspects of neuronal development. Interestingly, we found the kynurenine pathway is highly active during brain development, and induces a variety of changes in protein markers that may be involved in precipitating a range of neuronal and cognitive deficits. While Ro61-8048 induced no changes in the embryo brains at 5 and 24 h following treatment, delayed changes were seen in postnatal day 21 animals displaying a decrease in RhoB expression as examined in the western blots. Since the full blow symptoms of schizophrenia become apparent during early adulthood, we sought to examine any changes in protein expression in postnatal day 60 animals in regions of the cortex, hippocampus, midbrain and cerebellum. Interestingly, profound alterations were seen in doublecortin and the netrin receptors responsible for axonal guidance. Perhaps the most striking protein change in the postnatal day 60 animals is the significant alteration induced in the expression of disrupted in schizophrenia (DISC)-1, a protein strongly linked with schizophrenia. Glutamate function was assessed as indicated by the density of glutamate transporters, VGLUT-1 and VGLUT-2, in the CA1 region of the hippocampus of postnatal day 60 animals using immunocytochemistry. While the relative density of glutamate transporters were substantially increased, there were no changes in the GABA transporters, indicating that while GABA transmission remained the same, glutamate function may have increased in the absence of an increase in synaptic connections. Spine densities of pyramidal neurons in the hippocampus were also examined, using the golgi-impregnation method, to reveal a significant loss in spines of the apical and basal dendrites, consistent with reports in schizophrenia. To conclude, the kynurenine pathway is highly active during development, and alterations in central kynurenines during pregnancy, as induced by environmental factors such as stress and infection, may be involved in the subsequent emergence of neurodevelopmental disorders.

Non-compressible haemorrhage from within the torso and junctional regions constitutes the leading cause of potentially preventable death on the battlefield. It can be defined as haemorrhagic shock arising from injury to named torso vessels, pulmonary parenchyma, high grade solid organ injury and/or disruption of the bony pelvis. Data from the US Department of Defence Trauma Registry demonstrate a torso injury rate of 12.7% with 17.1% of casualties exhibiting torso injury and shock. The overall mortality is 18.7%, with major arterial injury and pulmonary injury identified as independent predictors of mortality on multivariate analysis. The UK Joint Theatre Trauma Registry reports similar findings with the greatest burden of mortality occurring prior to hospital admission (75.0%), a rate that has remained unchanged over a decade of war. Injury from improvised explosive devices (IEDs) in particular are associated with non-compressible haemorrhage, frequently causing traumatic lower extremity amputation in combination with torso injury. Contemporary surgical strategy relates to early operative haemorrhage control in patients presenting with shock. In patients sustaining a circulatory arrest, resuscitative thoracotomy and aortic cross clamping can be used to control inflow and increase cardiac afterload. The UK experience over 5 years at Camp Bastion demonstrated a mortality of 78.5%, with greatest survival observed in patients with the shortest time to thoracotomy. In patients sustaining lower extremity amputation following IED injury, 1 in 5 require a laparotomy for proximal vascular control, with less than half requiring further intra-abdominal intervention. There is a pressing need for a haemorrhage control and resuscitation adjunct in non-compressible haemorrhage that can be deployed prior to or as an adjunct to operative haemorrhage control. Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a technique that can occlude the aorta without the need for an operating theatre. It is an experimental technique, so its effect on survival and physiology is unknown. In a porcine model of uncontrolled pelvic haemorrhage, infra-renal REBOA was shown to be as effective as chitosan gauze in the setting of normal coagulation. However, REBOA was associated with a significantly greater survival in a coagulopathic setting. Similar results were obtained when using a porcine model of abdominal haemorrhage in conjunction with thoracic REBOA. In both studies, balloon occlusion demonstrated a significant improvement in systolic blood pressure and other haemodynamic measures compared to the no-occlusion control groups. Having demonstrated a survival and haemodynamic benefit in uncontrolled haemorrhage models, the metabolic and inflammatory consequences of thoracic REBOA were characterised in further detail using a porcine model of controlled hypovolaemic shock. Occlusion for 30 and 90 minutes was associated with a significant lactate burden when compared to animals undergoing shock alone. However, following resuscitation with blood and intravenous fluid, normal physiology was restored within 6 hours. The inflammatory sequelae were studied following 30, 60 and 90 minutes of shock and occlusion. Increasing occlusion time resulted in an escalating release of interleukin-6 which manifest clinically as an increase in ARDS and need for vassopressor support. In order to develop a fluoroscopy free REBOA system, a series of human studies were undertaken to examine the relationship between an external measure of torso height and aortic length in order to guide insertion length. A retrospective examination of computed tomography in male trauma patients demonstrated a correlation between torso height and aortic length. This was confirmed by a prospective study which was also used linear regression to develop equations predictive of insertion length. Finally, the UK Joint Theatre Trauma Registry was used to determine the need for REBOA in a population of UK military personnel injured over 10 years of conflict. Of 1317 severely injured patients 70.2% had no indication, 11.2% had a contra-indication and 18.5% had an injury pattern indication for REBOA. Of those with an indication for REBOA, 66 (27.0%) patients died en-route to hospital and 29 (11.9%) died in-hospital. In conclusion, non-compressible haemorrhage constitutes a significant burden of potentially preventable battlefield mortality. REBOA is a technique that can be used in the thoracic or infra-renal aorta as a haemorrhage control and resuscitation adjunct, prior to operative haemorrhage control. While associated with a significant survival advantage in models of uncontrolled haemorrhage, it is associated with a significant metabolic penalty, although with resuscitation this can be ameliorated successfully.

Malignant glioma is the most common and aggressive form of tumours and is usually refractory to therapy. Telomerase and its altered activity, distinguishing cancer cells, is an attractive molecular target in glioma therapeutics. The aim of this thesis was to silence telomerase at the genetic level with a view to highlight the changes caused in the cancer proteome and identify the potential downstream pathways controlled by telomerase in tumour progression and maintenance. A comprehensive proteomic study utilizing 2D-DIGE and MALDI-TOF were used to assess the effect of inhibiting two different regulatory mechanisms of telomerase in glioma. RNAi was used to target hTERT and Hsp90α. Inhibition of telomerase activity resulted in down regulation of various cytoskeletal proteins with correlative evidence of the involvement of telomerase in regulating the expression of vimentin. Vimentin plays an important role in tumour metastasis and is used as an indicator of glioma metastasis. Inhibition of telomerase via sihTERT results in the down regulation of vimentin expression in glioma cell lines in a grade specific manner. While, 9 of 12 glioblastoma tissues (grade IV) showed vimentin to be highly expressed, its expression was absent in lower grades and normal tissues. This suggests that vimentin can be potentially used as a glioma progressive marker. This is the first study to report the potential involvement of telomerase in the regulation of vimentin expression. This study also identified that combination therapy, comprising siRNA targeted towards telomerase regulatory mechanisms and the natural product Epigallocatechin-3-gallate (ECGC), results in decreased cell viability producing comparable results to that of other chemotherapeutic drugs.

This thesis presents the findings of investigations carried out relating to the creation of full joint contact patient specific finite element models for correlation with biological studies in the study of Osteoarthritis (OA) development. To understand the relationship between altered loading and biological changes in articular cartilage (AC), a method for predicting stresses and strains experienced inside the tissues is required. An in-vitro study was conducted to explore the possibility of correlating finite element (FE) and gene expression study results. FE models were used to predict the stresses and strains inside the AC for explants subjected to different loading conditions. The study demonstrated that the accurate representation of AC surface geometry is crucial and current flat surface axisymmetric cylinder representations used in AC explant modelling introduces significant error in the prediction of tissue mechanical behaviour. Cutting of the AC explant to achieve a flat surface can affect the biological, mechanical and tribology behaviour of the tissue. Thus, a method for creating explant specific finite element models with the use of digital image correlation (DIC) was developed and is presented, allowing for surface layer preservation in AC explants for correlated gene expression and inverse FE. Reconstruction of tissue geometries from magnetic resonance (MR) imaging scan data of the knee was explored. It was possible to segment both hard and soft tissues from the same set of MR imaging scan data. Meshing of the geometries using a fundamentally voxel based algorithm proved to cause significant error in the segmented volume. An alternative contour based algorithm needs to be explored. Uncertainties concerning the presence and modelling of meniscotibial ligaments (MTLs) in full joint contact FE models found in literature were addressed. An anatomy study revealed that the MTLs are found in both the medial and lateral side of the joint around the periphery of the anterior, middle and posterior portion of the menisci. With the use of cross polarised light microscopy, it was established Page | VII that MTLs consist of Type I collagen orientated in the circumferential direction around the menisci. As a result, the MTLs were modelled as an anisotropic membrane. Using the full joint contact finite element model, the influence of MTLs on knee joint kinematics was investigated. It was found that the MTLs reinforce the function of the meniscal horns and circumferential fibres in the meniscus and help constrain the meniscus. Therefore, it was concluded that the MTLs are mechanically significant in the stabilisation of knee joints and should be included in knee models for accurate prediction of knee joint behaviour.

1. The hypothalamic arcuate nucleus (Arc) is a key integrative centre of the central nervous system (CNS) involved in the control and maintenance of energy balance. Whole-cell patch clamp recording techniques were utilised, in isolated hypothalamic brain slice preparations, to investigate the electrophysiological and morphological properties of Arc neurones. Differential expression of subthreshold active conductances were identified and used to functionally classify Arc neurones into 8 electrophysiological clusters. This classification was based based upon differential expression of the following conductances: anomalous inward rectification (Ian); hyperpolarisation-activated non-selective cation conductance (Ih); transient outward rectification (Ia); T-type-like calcium conductance. Morphological analysis of recorded neurones, revealed retrospectively with biocytin staining, showed four populations based upon the orientation and number of primary dendrites. There were no obvious direct correlations between morphology and electrophysiological properties, suggesting considerable functional diversity of neurones and their associated circuits at the level of the Arc. 2. The physiological levels of glucose to which the brain is exposed are believed to be around 1-2.5 mM, and glucose-sensing neurones have been identified in the Arc. However, in vitro slice studies routinely use glucose around 10 mM in aCSF. The impact of this high level of glucose on fundamental properties and operation of hypothalamic circuits remains unclear. Here the effect of different ambient glucose levels (10 mM, hyperglycaemic and 2 mM, euglycaemic) on electrophysiological properties of Arc neurones was compared. Significant differences in passive and active subthreshold membrane properties of Arc neurones were observed, including: changes in neuronal input resistance, spontaneous activity and magnitude of Ih and Ia. Data from this study suggests a need to re-evaluate studies previously conducted in non-physiological levels of glucose. 3. The effects of noradrenaline (NA) on the neuronal excitability of hypothalamic Arc neurones were studied. Application of NA induced a membrane depolarisation and increase in electrical excitability in 51% of Arc neurones, including orexigenic NPY/AgRP neurones, a response that persisted in the presence of TTX indicating a direct effect. NA-induced depolarisation was mediated through α1-ARs, in particular through α1A-ARs, and associated with multiple ionic mechanisms including: closure of a potassium conductance, activation of a non-selective cation conductance, or a combination of the two. 4. NA also induced a membrane hyperpolarisation in a sub-population of Arc neurones (15%) including 4/9 putative anorexigenic CART-expressing neurones, the remaining CART neurones responded with a NA-induced excitation. NA-induced hyperpolarisation, mediated via α2-ARs and activation of one or more potassium conductances, persisted in the presence of TTX indicating a direct effect on Arc neurones. 7.5% of neurones responded to NA with biphasic inhibitory/excitatory responses. Taken together, these data suggest that NA, at least in part, excites a subpopulation of NPY/AgRP neurones and inhibits a population of CART expressing neurones which may serve an orexigenic role at the level of the Arc. 5. Histamine induced membrane depolarisation in a population of Arc neurones (65%), most likely through H1 receptors, via a direct effect on the postsysnaptic membrane. Histamine induced depolarisation through multiple ionic mechanisms, including closure of a potassium conductance or activation of an electrogenic pump.

1. The effects of glucose on the electrophysiological properties of rat hypothalamic arcuate nucleus (ARC) neurones were investigated. Neurones were recorded in 10 mM (hyperglycaemic) and 2 mM (euglycaemic) glucose-containing aCSF. The major findings were that input resistance increased in 10 mM glucose, there was an increase in the activity of neurones in 2 mM glucose and there were a greater percentage of neurones expressing lh in 10 mM glucose. Subthreshold active conductances were differentially expressed in ARC neurones including: anomalous inward rectification Q. ), time- and voltage-dependent inward rectification 00, A-like transient outward rectification (IA) and T-type calcium-like conductance. Characterisation of the differential expression of these conductances may represent one way of functionally classifying ARC neurones. 2. Whole-cell patch clamp recording techniques were used in isolated hypothalamic brain slice preparations to investigate the effects of 5-HT on ARC neurones. Bath application of 5-HT induced a membrane depolarisation in a sub-population of ARC neurones (30%), a response that persisted in the presence of TTX indicating a direct effect. 5-HT excited ARC neurones through three potential mechanisms: closure of one or more resting potassium conductances; activation of a non-selective cation channel, or a combination of the two; or activation of a pump in the membrane. This response was mediated through the 5-HT2A. 5-HT2B and/or 5-HT2C receptors revealed using a range of 5-HT receptor agonists and antagonists. 5-HT was shown to excite CART-expressing neurones suggesting an anorexigenic role for 5-HT, via 5-HT2 receptors at the level of the ARC. 3.5-HT induced a membrane hyperpolarisation in a sub-population of ARC neurones (37%). The 5-HT-induced hyperpolarisation persisted in the presence of TTX indicating a direct effect on ARC neurones. 5-HT inhibited ARC neurones most likely through the activation of one or more potassium conductances,including an inwardly rectifying potassium conductance. Potential roles for 5-HTIA, 5-HTIB and 5-HT7 receptors were suggested from studies utilising 5-HT receptor agonists and antagonists. 5-HT inhibited orexigenic NPY/AgRP neurones, identified by their response to ghrelin and by their electrophysiological properties, suggesting an anorexigenic role for 5-HT, acting via 5-HTI and 5-HT7 receptors on NPY/AgRP neurones at the level of the ARC. 4. The effects of feeding-related signals on hypothalamic neuropeptide expression were investigated using real-time-PCR. A new protocol measuring gene expression from hypothalamic explants was developed. Effects of GABA and AMPA on c-fos expression were investigated and subsequent studies showed leptin and glucose modulated the expression of NPY, POMC and AgRP, in fed and fasted animals. Further work is required to validate this novel approach to studying the central control of energy balance.

Research investigating the regulation of muscle fibre type has traditionally been conducted in vivo, analyzing global changes at a whole muscle level. Broadly, this thesis aimed to explore more “molecular” approaches, utilizing molecular and cell biology to understand the expression and regulation of myosin heavy chain (MyHC) isoforms as an indicator of muscle fibre composition. The mRNA expression profile of six MyHC isoform genes during C2C12 myogenesis was elucidated to reveal that the C2C12 cell line mimics developing fast-twitch muscle fibres. Additional characterization of the C2C12 cell line revealed a dramatic restructuring of metabolic gene expression during the switch from proliferating to fully differentiated C2C12 muscle cells. Post-mitotic muscle cells exhibited increased glycolytic gene expression and reduced oxidative gene expression and an increase in gene expression of enzymes involved in redirecting glucose carbons into ATP generating pathways and away from macromolecule biosynthesis (p<0.01 for all genes). The dynamic plasticity of MyHC isoform gene expression was compared between C2C12 muscle cells and fully differentiated adult muscle. Exposure of adult muscle to the beta-adrenergic agonist, Ractopamine, induced dynamic transitions in MyHC isoform expression, from the IIA/IIX isoforms to increased IIB isoform expression (p<0.05 for all genes). An acute exposure of C2C12 muscle cells to Ractopamine was capable of inducing an exclusive and rapid induction of the MyHC IIB isoform gene expression during myogenesis (p<0.001). The C2C12 cell line was utilized as a host environment for a molecular-based approach to understand the role of the promoter sequence in regulating the species-differential induction of the MyHC IIB gene during myogenesis. A 3bp miss-match in the CArG-Ebox region (at -74bp, -68bp and -48bp) of the proximal MyHC IIB promoter was identified that dictates the differential expression of MyHC IIB in pigs and humans.

Phosphoinositides are lipid signals that control cellular processes and are particularly closely associated with the control of membrane trafficking. PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is the most recently identified phosphoinositide and was previously recognised as controlling events in the late endocytic system between the late endosome and the vacuole/lysosome. Primarily associated with retrograde trafficking from the vacuole/lysosome to the late endosome/MVB, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is generated by the kinase Fab1p (PIKfyve in animals). In mammalian cells, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) has also been implicated in control of ill-defined trafficking pathways close to the Golgi; for example, the recycling of mannose-6-phosphate receptor (M6R) back to the Golgi and also the trafficking of some types of ion and nutrient channels from the Golgi to the cell surface. This thesis describes attempts to study putative PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) dependent trafficking in the early endocytic system of \(\char{cmmi10}{0x53}\). \(\char{cmmi10}{0x63}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x72}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x76}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x73}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x61}\)\(\char{cmmi10}{0x65}\) using two model proteins; the recycling of Vps10p from late endosome to Golgi and of Chs3p from recycling endosome to Golgi.

The introduction of electron capture dissociation mass spectrometry in 1998 has provided a unique technique for the analysis of peptides and proteins, especially for the identification and localisation of posttranslational modifications. Despite many successes debate continues on the radical-based mechanism of ECD. This thesis explores ECD behaviour in a wide range of PTMs with the intention of furthering our knowledge of the radical-based mechanism. Studies were undertaken on the effect of 3-nitrotyrosine, which is an electron withdrawing group, on ECD. The presence of nitration dramatically decreases peptide backbone sequence coverage but results in the presence of abundant small neutral losses. The key finding is the insight provided into the hierarchy of the various proposed ECD mechanisms. ECD of cysteine bound modifications is shown to result in the fragmentation of the sulfur-modification bond and backbone sequence coverage is highly diminished when analysing S-nitrosopeptides. ECD behaviour of hydrogen-deficient radical peptides is highly dependent on gas-phase peptide structure, with electron capture typically resulting in an increase in charge-reduced precursor intensity. Comparisons of the intermolecular phospho-guanidinium bond strengths between phospho-serine, -threonine and -tyrosine were undertaken. ECD of these complexes results in the retention of the noncovalent bond allowing backbone sequence coverage.

The principal aim of this project is to examine posture and muscle activity when using an ergonomically designed saddle seat compared with a conventional seat during common dental procedures with the dental students and practising dentists. The study was conducted with practising dentists across the West Midlands and the dental students in the School of Dentistry – University of Birmingham. The study is mainly divided into a questionnaire survey of practising dentists, a questionnaire survey of dental student posture in the dental schools across the U.K, postural analysis, and a daily symptom survey of practising dentists and dental students, and finally the EMG analysis of practising dentists and dental students working posture. This thesis has established the relationship between posture, seating and muscle activity and indicates that use of an ergonomic aid (dental operator stool) may improve posture, decrease pain and muscle activity and may decrease the development of musculoskeletal disorders among dental students and dentists.