The interior homeostasis of a solid organ, such as a kidney, is greatly altered during the surgical removal, storage and transplantation process from donor to recipient. Consequently, normal organ function may be delayed or prevented following transplantation. The main injury that organs have to withstand often occurs subsequent to the restoration of blood flow; this injury is known as ischaemia reperfusion-injury (I/R injury) and is associated with hypoxia, free radical formation and organ failure. Viral gene therapy, such as BacMam gene transfer technology (Baculovirus-based expression in mammalian cells) is an attractive option for reducing I/R injury due to its high safety profile compared to mammalian viruses. However, limiting factors, including the requirement of high-doses of viral vector and low transgene expression levels are still a hurdle for the successful use of this technology. Therefore, the initial objectives of this study were to develop and validate a number of methods for concentrating BacMam along with the use of a number of chemical compounds to improve BacMam gene delivery in kidney cells. Amongst all the approaches screened, high-speed (for small volumes) and low-speed (for large volumes) centrifugation methods were the most effective for concentrating BacMam virus (4x109 and 1x1010 pfu/ml, respectively). Transduction of HEK cells was improved between two- to 10-fold in the presence of sodium butyrate or hydroxyurea alone and significantly improved egfp (enhanced green fluorescence protein) expression (~40-fold) when both chemicals were used in combination. Subsequently, in the second part of this study an in vitro culture model was developed and evaluated for assessing ischaemic injury in kidney cells using antimycin A in combination with 2-deoxyglucose. It was shown that sod-2 and bcl-2 overexpression in injured kidney cells improved the effect of I/R injury by enhancing the recovery of ATP post-reperfusion Furthermore, to examine the possibility of reducing injury associated with cold preservation, an ex vivo hypothermic perfusion system of porcine kidney was established and validated using reporter and protective genes. Importantly, for the first time, BacMam has been used to transfer ex vivo a protective gene (sod-2), into kidneys which appeared to provide protection against hypothermic preservation-associated injury by improving the recovery of cellular ATP.

No description available.

Background: Stratified medicine aims to use a patient's genotype and other individual characteristics to predict their treatment outcomes. Several treatments have been developed which may potentially offer patients an increased response to treatment. For instance 5% of patients with cystic fibrosis can be prescribed Ivacaftor based on a specific genetic mutation. However, it is unclear about how a patient's genotype may influence particular aspects of medical decision-making, despite the relevance that this may have in routine clinical practice. Methods: Medical decision-making was investigated using a series of discrete-choice experiments (DCEs) in which participants were asked to consider and choose one of the presented scenarios. Regarding profession decision-making, in particular, the PhD research investigated extent to which a patient's genotype influenced the treatment judgements and recommendations of psychiatrists (n=68). Patient decision-making was investigated by using DCEs to assess how people with cystic fibrosis (n=80) 'traded-off the risks and benefits that were associated with each treatment option. In the final study of the thesis, I investigated whether or not members of the public (n=2804) would be willing to incur an increase in tax to help fund stratified medicine treatments. Results: The main findings of these studies suggest that clinicians may be unduly influenced by a patient's genotype when judging a patient's response to treatment and in their treatment recommendations. Cystic fibrosis patients may not be willing to tolerate some of the increased risks associated with their treatment options. Thus clinicians should discuss the risks and benefits associated with treatments with their patients. The PhD research highlighted that members of the public may not be willing to pay an increase in taxation unless the majority of people were eligible for the stratified medicine treatment, a result that poses a challenge for stratified medicine because only few people are eligible for potentially more effective treatments. Conclusions: Clinicians need to be cautious about being unduly influenced by a patient's genotype and should discuss the risks and benefits associated with different treatment options. Further research is needed to understand how a patient's genotype may influence the decisions that are made at the clinician, patient and policy level.

No description available.

Bacterial cell envelope glycans are a valuable resource industrially and pharmacologically. Their production enables bacteria to withstand harsh environmental stresses including protection against antimicrobial agents, can be crucial in the formation of biofilms and, can be catastrophic to the host, causing an overwhelming inflammatory response that can ultimately lead to sepsis. Synthesis of these glycostructures is initiated by one of two enzyme families: the polyisoprenyl-phosphate N- acetylamino sugar-1-phosphate transferases (PNPTs) or the polyisoprenyl-phosphate hexose-1- phosphate transferases (PHPTs) that transfer the initial sugar-1-phosphate from a nucleotide-sugar precursor onto a universal polyisoprenoid lipid carrier. Unlike PNPT, PHPTs are unique to prokaryotes and are priming enzymes in the synthesis of surface polysaccharides of varying complexity. WcaJ is a PHPT protein that initiates the synthesis of colanic acid in the Enterobacteriaceae. Colanic acid is a high molecular weight capsular polysaccharide that protects against environmental stresses such as low temperature conditions, resistance to desiccation and antimicrobial compounds. In this study, WcaJ was utilised as a model PHPT member to investigate the structure and functional mechanisms of this family of proteins. The topology of WcaJ experimentally validated identifying a novel topological arrangement whereby the C-terminal tail and the soluble loop domain both reside in the cytoplasm. This arrangement is the result of ‘horseshoe’ structure formed by the terminal transmembrane helix; a model that is proposed extends to all PHPT members. It was further identified that WcaJ forms homoologomers mediated by self-interaction of this helix and that conserved charged residues may play a role in this interaction. Finally, in silico modelling of the cytoplasmic loop domain suggests that it is structurally analogous to the Rossmann fold nucleotide binding domains of UDP-sugar dehydrogenases. Biochemical analysis of WcaJ and other PHPT proteins suggests that the cytoplasmic loop domain could provide a site for NAD reduction that could allow PHPT to perform dual enzymatic functions. It is proposed that this could provide a mechanism for self-regulation and/or regulation of the biosynthetic pathway. Together, the data gathered within this study have provided insights into the structural and functional complexity of this family of proteins that will be a valuable resource for future study.

This thesis entails the synthesis and in vitro evaluation of a number of cationic lipids. These lipids include those with a dimethylamine (DMA) headgroup or trimethylamine (TMA) headgroup, a seven or eleven carbon chain length or a mixed carbon chain length within the hydrophobic domain. The lipids are characterised by acyclic or macrocyclic hydrophobic regions that are either saturated or unsaturated. The work describes the successful synthetic routes to these various different cationic lipids and looks at the biological effects that are had on toxicity, transfection and DNA binding, when the various different structural aspects of the cationic lipids are changed.

No description available.

Background: Frustrations are noted in the management of CFS/ME as it’s a complex and individual condition with no known cure. Despite being a Long-term Condition (LTC) limited research has focused on long-term experiences. This study aims to extend the knowledge of long-term experiences of CFS/ME specifically focusing on management of the condition. Thinking about therapeutic moderators and mechanisms of change, whether management changes throughout the course of the illness and what support people might benefit from. Methods: A qualitative research design, using semi-structured interviews was adopted. Nine participants' were recruited from a specialist CFS/ME Service in the UK who were over 18 years old, had a diagnosis of CFS/ME and reported experiencing fatigue related symptoms for over 5 years. Interviews were audio recorded, transcribed verbatim and analysed using Thematic Analysis. Findings: Three themes; Awareness, Acceptance, Connection and two subthemes; connection with self and connection with others were constructed from the data. An overarching theme of Awareness appeared to facilitate the ability to accept and connect with what was important for people with CFS/ME, enabling people to adapt to living with the condition and achieving a standard of living. Commonalities occurred across all themes of development over time, individuality and ongoing balance or monitoring. Discussion: Findings suggest supporting adults with CFS/ME to become more self-aware of their illness experience and identifying their values will be beneficial at any stage of the illness duration, but particularly important for long-term management. These findings provide further support for tailored treatment plans (NICE, 2007) with some individuals' needing more, or occasional contact with understanding professionals to achieve. The results support the use of acceptance-based interventions in LTC management; specifically Acceptance and Commitment Therapy (ACT) and Focused ACT. Further research into outcomes and experiences of ACT in CFS/ME, and interdisciplinary approaches is advocated.

Obesity and type 2 diabetes mellitus (T2DM) are positively associated with the destructive, chronic inflammatory disease periodontitis. The adipokine leptin is elevated in obesity and T2DM, and promotes inflammatory responses. Gingival fibroblasts are implicated in the pathogenesis of periodontitis because inflammatory stimuli can drive these cells towards destructive, inflammatory responses. The aim of this study was to identify whether leptin promotes inflammatory responses in gingival fibroblasts, focussing on the extracellular matrix-degrading matrix metalloproteinases (MMPs), and their inhibitors (TIMPs). Primary human gingival fibroblasts (hGFs) were isolated from gingival tissue and cultured in vitro. RT-PCR, ELISA and flow cytometry were used to assess MMP, TIMP and TLR expression; hGF signalling (±chemical pathway inhibitors) was assessed by Western blotting. hGFs constitutively expressed numerous MMPs and TIMPs. Leptin increased the expression of MMP-1, MMP-3, MMP-8 and MMP-14, but not TIMPs, in hGFs. Leptin and interleukin-1 or the TLR2 agonist pam2CSK4 synergistically increased MMP-1 and MMP-3 production by hGFs; in contrast, leptin and Escherichia coli LPS regulated MMP production in a donor-dependent manner. TLR4 was detected on the surface of hGFs from all donors tested, suggesting that differential responses to E. coli LPS were not due to absent cell surface TLR4. Overall, these results suggest that leptin promotes an ECM-degrading hGF response, which is further enhanced under inflammatory conditions. Leptin activated multiple intracellular signalling pathways, but the MAPK pathway and ERK in particular, regulated leptin-stimulated MMP-1 expression in hGFs. Genome-wide expression profiling revealed that leptin enhanced the expression of genes in hGFs whose products function in inflammation; similarly, leptin enhanced the inflammatory gene expression profile induced by IL-1 in hGFs. Leptin+interleukin-1 did not promote collagen degradation in human gingival connective tissue explants. In conclusion, leptin enhances wide-ranging inflammatory responses in hGFs in a context-dependent manner. This response could be a mechanistic link between obesity, T2DM and periodontitis.

The treatment and management of pain continues to reflect the complex nature of pain itself. At present, pain cannot be definitively tested for, only inferred through a combination of sensory and self-report measures, such as quantitative sensory testing procedures and validated questionnaires, which survey various aspects of the pain experience in more detail, as well as the impact of pain on daily life at home and in the workplace. Whilst clinical sensory testing outcomes are analogous to in vivo evoked sensory data, modelling the impact of pain on quality of life is more challenging. One approach is to use an ethological paradigm, i.e. one that measures a behaviour that reflects an innate environmental response. Tests such as the open field and burrowing measure how pain affects threat avoidance (thigmotaxis in the open field) and tunnel maintenance (burrowing) in laboratory rats. Studies have shown their efficacy in detecting subtle behavioural deficits in experimental models of pain, including response to analgesics. Using ethological outcome measures also avoids applying clinical terminology to animal behaviour, and should be used in conjunction with classical sensory reflex-driven assays, such as hotplate and von Frey filaments, to yield a global picture of how experimental pain states affect wellbeing and quality of life. This study examines how acute visceral inflammation, persistent visceral inflammation, and drug-induced neuropathy (d4T-antiretroviral nucleoside) alter the behaviour of adult female Wistar rats in the open field paradigm. It also investigates how the behavioural phenotype of naïve male Wistar rats in the open field paradigm. It also investigates how the behavioural phenotype of naïve male Wistar rats responds to repeated open field exposure. Significant increases in thigmotaxis were observed following acute bladder inflammation, and repeated exposure to open field in naïve rats. There was no effect on either open field behaviour or evoked sensory measures in female rats treated with d4T, and acute bladder inflammation failed to alter burrowing behaviours. As d4T has been previously shown to alter thigmotactic behaviour, this data suggests that females show different sensitivity compared to males. To determine whether visceral inflammation influences behaviour in a dose-dependent manner, bladder tissue from the visceral inflammation groups was assayed for levels of cytokines using an RNA microarray featuring 92 inflammatory cytokines and 4 housekeeping genes. The greatest effect was detected following acute inflammation, with both models showing increased levels of CCL12, CCL7, and IL-1β when compared to naive tissue. To correlate the changes in behaviour with a neural substrate, c-Fos immunoreactivity in the amygdala was measured in both visceral inflammation groups. No significant activational differences were seen following acute bladder inflammation, whereas persistent visceral inflammation significantly increased c-Fos immunoreactivity in the caudal regions of the capsular central amygdala. Visceral inflammation is associated with peripheral increases in inflammatory cytokines, and thigmotactic behaviour in the open field. Differences in activation in the amygdala were lower than expected due to high levels of variation associated with inflammatory stimuli at the time-point tested. Significant variation was particularly seen in repeated visceral inflammation, suggestive of the biphasic behavioural response to stress e.g. attack or defend phenotypes. Further examination of these differences at the individual level could shed light on the process of pain chronification, and ultimately help understand why only some individuals develop chronic pain.