This thesis demonstrates the relevance of advanced transmission electron microscopy (TEM) techniques such as aberration corrected scanning transmission electron microscopy (STEM) to the study of biological samples. By developing the application of these techniques to biologically relevant systems, this study shows how advanced EM can be an effective tool by providing insight into the structure of biological systems at the highest (i.e. atomic) resolutions. High angle annular dark field (HAADF) STEM has been used to gain insight into the core structure and iron loading mechanisms of the iron storage protein, ferritin. The iron content of ferritin was quantified using size-selected gold clusters as a mass balance, the first application of this technique to a biological sample. Preliminary structural studies of a novel colorectal cancer therapy have been undertaken, where polymeric alginate molecules chelate chemotoxic luminal iron in the colon. In particular the nano-structures built when alginates bind iron under physiological conditions have been established and investigated for the first time, using HAADF-STEM. Finally using TEM, it has been revealed for the first time the structures formed, and morphology changes induced, when proteins are encapsulated by membrane mimicking nano-discs.

Cytomegalovirus (CMV) infection is a major complication after kidney transplantation. Despite antiviral therapy it contributes significantly to high morbidity. This study was aimed at (a) detecting· a CMV specific antigen pp65 in CMV -infected fibroblast cells and in leukocytes of kidney transplant recipients by flow cytometric assay (FCA) (b) determining the stimulation index (S.I.) of phytohaemagglutinin (PHA) and CMV-stimulated peripheral blood mononuclear cells (PBMC), (c) determining the levels of Th1- and Th-2 related cytokines in the supernatant of stimulated PBMC from kidney transplant recipients with and without active CMV infection (d) determining immunophenotyping of cells found in the peripheral blood of CMV -infected and CMV -uninfected kidney transplant recipients by flow cytometry using antibodies specific to CD2+ (pan T), CD3+ (mature T), CD4+ (T helper), CD8+ (T suppressor), CD26+(T activated), CDI6+/CDS6+ (NK cell), CDI9+(pan B), CDIS+ (granulocytes). Thirty-five patients with, and 44 without active CMV infections, as diagnosed by a CMV antigenemia assay (AA), were inducted into this study. FCA distinguished clearly between the infected and uninfected fibroblast cells. Regarding kidney transplant recipients, the FCA was positive when the number of AA positive cells was five or more per 5x10(4). Moreover, the percentage of antigenemia-positive cells by FCA correlated well with symptomatic CMV infections. After PHA and CMV stimulation of PBMC from patients, S.I. was determined by radioactive thymidine uptake while the production of Th1-type cytokines [interleukin-2 (iL-2), interferon-y (IFN-y) and tumor necrosis factor-a (TNFex)] and Th2-type cytokines (IL-4, IL-1O) were measured by ELISA. PBMC of patients with active CMV infection showed significantly lower S.I. values than patients without an ongoing CMV infection (p<O.OOO1). Levels of Th2- type cytokines in CMV -infected and uninfected kidney recipients were similar; however, the levels of the Th1-type cytokines were significantly lower in CMV -infected patients (p<O.O5). Low levels of Th1-type cytokines seem to correlate well with active CMV infection in kidney recipients. The percentage of CD3+ immunocompetent T lymphocytes and CD4+ T lymphocytes were consistently higher in kidney transplant recipients without an active CMV infection than in the group of recipients with an active CMV infection. These differences were statistically significant in the case of CD3+ (p<O.O5) and CD4+ (p<O.OO5). On the other hand the difference in percentage CD2+, CD8+, CD16++CD56+, CD19+, CD15+ cells were statistically insignificant. Therefore, these data suggest that active CMV infection in kidney transplant recipients is associated with a significant alteration in the lymphocyte proliferative responses, the levels of Th1-type cytokines (IFN-y, TNF-ex, IL-2), and the percentage of CD3+, CD4+ when compared to kidney transplant recipients without active CMV infection.

No description available.

In this project the influences on human adult mesenchymal stem cells using nanomechanical stimulation techniques has been explored. It is expected that human mesenchymal stem cells will find use in many autologous regenerative therapies and in tissue engineering. However, the ability to control stem cell growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, unpredictable number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, etc.) culture substrates. According to known cellular reactions to environmental stimuli, it was expected that human cells show some reactions to nanoscale vibration that in the case of stem cells it could be a differentiation response. This thesis gives a first demonstration of using nanoscale mechanotransductive protocols (10-14 nm vertical displacements at 1 kHz frequency), “nanokicking”, to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, laser interferometry was used to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of biological techniques has then been used to demonstrate osteoblastogenesis. Furthermore, RhoA has been implicated as being central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol.

The use of haemostatic materials that could be used to mitigate against the effects of the chemical warfare agent soman (GD) on contaminated personnel that may also present with wounds were investigated. To support the in vitro diffusion cell component of this work, the penetration rate of \(^1\)\(^4\)C-GD into different receptor fluids was evaluated to enable determination of the most appropriate receptor fluid to use as a sink for GD. Of the receptor media evaluated only 50% aqueous ethanol was able to maintain sink conditions. A number of haemostatic materials were shown to retain haemostatic efficacy in the presence of blood contaminated with GD, and were also shown to irreversibly sequester GD. The lead candidate, WoundStat™, was shown to be as effective a decontaminant as the current in service countermeasure fullers’ earth. Complementary in vivo studies using damaged ear skin in a terminally anaesthetised large white pig model showed that whilst use of WoundStat™ was not 100% effective in the prevention of mortality after GD poisoning, it did increase the therapeutic window where further nerve agent-specific medical countermeasures could be employed. Perhaps most importantly, application of WoundStat™ onto GD contaminated damaged skin did not increase the toxicity of GD.

Electrospinning is a versatile method of producing nanofibrous polymeric material with potential applications as tissue engineering scaffolds. The main aim of this project was to produce and characterise electrospun polymeric scaffolds based on chitosan and bacterial polyhydroxybutyrate. The effect of the parameters used in the electrospinning process were studied and optimised by electrospinning polyvinyl alcohol from 8 wt% and 10 wt% solutions under a variable applied voltage from 10-25 kV. Attempts were made to electrospun chitosan however it was found that the creation of a polymer blend was necessary to facilitate fibre formation. PVA-chitosan blends were successfully electrospun at blend ratios of up to 80:20. A chitosan-hydroxybenzotriazole-PVA aqueous solution was successfully prepared enabling the production of chitosan/PVA nanofibres without the need for the use of an organic solvent. Polyhydroxybutyrate produced by bacterial synthesis from R. Eutropha using three different carbon sources; olive oil, rapeseed oil and glucose were electrospun and characterized. The choice of carbon source did not have a significant effect on the morphology or crystallinity of the produced fibres. PHB fibre diameters were reduced by 30% through the addition of the salt Benzyl tributylammonium chloride to the electrospinning solution.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases associated with damage and proteolytic loss of protein from affected joints. The types and amounts of protein damage and related proteolytic debris in synovial fluid and released into the circulation has not been studied comprehensively. The aim of this study is to quantify levels of protein damage in a cross-sectional pilot study of synovial fluid and plasma of patients with early-stage OA (eOA) and RA (eRA), and self-resolving arthritis (non-RA), and compare these with patients with advanced stage OA (aOA) and RA (aRA), and with plasma of healthy subjects. Patients with eOA, aOA, eRA and non-RA were recruited by collaborating clinicians from rheumatology and orthopaedic clinics in hospitals in Coventry, Birmingham, Ipswich and Exeter and synovial fluid and plasma samples collected from patients with consent. Major chemically-defined markers of protein damage by glycation, oxidation, nitration and citrullination were quantified in sample protein and in ultrafiltrate (glycated, oxidized and nitrated amino acids) by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Hydroxyproline was also determined. Our results demonstrated varying degrees of changes within protein damage marker concentrations across all subject groups, however there were many significant changes. Protein oxidation and advanced glycation endproducts were noted to be elevated both synovial fluid and plasma of patients with aRA and aOA with elevated levels also noted in eRA and eOA synovial fluid. Citrullinated proteins were noted to be markedly increased in plasma protein in both eOA and eRA. Markers of nitration were also elevated in non-RA plasma there was a decrease in nitration despite increases in glycation and oxidation. Changes in damaged amino acids in synovial fluid and plasma were similar across all patients. In aRA and aOA there was increased amino acid oxidation and advanced glycation and decreased amino acid nitration. There was also increased hydroxyproline in plasma. In eRA and eOA there was increased amino acid oxidation and increased advanced glycation in eRA (Nω-carboxymethylarginine). Changes in amino acid oxidation and advanced glycation were restricted to plasma in non-RA. The changes in protein damage and citrullination were characteristic “signatures” that allowed production of data trained algorithms with over ≥ 97% specificity and sensitivity for diagnosis and discrimination of eOA, eRA and non-RA. This study provides the first comprehensive attempt at quantification of protein damage markers in joint disease using LC-MS/MS techniques – a gold standard. The improved characterisation of protein damage and related metabolites will likely advance understanding of early-stage processes in joint degeneration which is still poorly understood and may provide novel plasma biomarkers for diagnosis and risk of disease progression.

Porphyrins, a substance class that can be found in diverse materials including green leaves and red blood cells, have been studied extensively over many years due to their potential industrial applications, e.g. in making optical electronic devices, in artificial photosynthesis or in sensors. In contrast, DNA has only recently been found to be a good scaffold for the construction of functional molecules. Combining the chemical properties of porphyrins and DNA could open the door to the production of multiporphyrin arrays using DNA as a scaffold; such materials could have multiple applications, an example being molecular electronic devices. The work described in this thesis reports on further investigations of the synthesis of the porphyrin‐nucleotide and its incorporation into DNA sequences, in order to study the structural, chemical and electronic properties of the porphyrin‐modified DNA. The analytical results, obtained from employing a variety of techniques such as UV‐vis and fluorescence spectroscopy, UV‐vis and fluorescence melting studies, circular dichroism spectroscopy and EPR (electron paramagnetic resonance) spectroscopy showed electronic interaction between porphyrins stacked on DNA. Metallation with zinc or copper of different porphyrins (namely diphenyl porphyrins and tetraphenyl porphyrins) was also successfully achieved, after they had been attached onto DNA. Based on EPR measurements, evidence was found for intermolecular stacking of the porphyrin‐DNA which leads to self‐assembled higher order structures. The EPR investigation also demonstrated that different oxidation states of metals held inside porphyrin‐DNA could be monitored. Using CD spectroscopy based on a synchrotron light source, the first measurements were made of DNA in the far UV region (< 200 nm). All the results obtained in this work and elsewhere during the past few years show that DNA based materials are highly promising for future applications in many areas of science, but especially in electronics and health care.

Direct Infusion (DI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) is becoming a popular measurement platform in metabolomics. This thesis aims to advance the data processing and analysis pipeline of the DI FT-ICR based metabolomics, and broaden its applicability to a clinical research. To meet the first objective, the issue of missing data that occur in a final data matrix containing metabolite relative abundances measured for each sample analysed, is addressed. The nature of these data and their effect on the subsequent data analyses are investigated. Eight common and/or easily accessible missing data estimation algorithms are examined and a three stage approach is proposed to aid the identification of the optimal one. Finally, a novel survival analysis approach is introduced and assessed as an alternative way of missing data treatment prior univariate analysis. To address the second objective, DI FT-ICR MS based metabolomics is assessed in terms of its applicability to research investigating metabolomic changes occurring in liver grafts throughout the human orthotopic liver transplantation (OLT). The feasibility of this approach to a clinical setting is validated and its potential to provide a wealth of novel metabolic information associated with OLT is demonstrated.

Prostate cancer is one of the most prevalent cancers worldwide. The early stages of prostate cancer (PCa) are highly dependent on the androgen receptor (AR) pathway and hence therapies target this signalling axis. This approach is successful initially but invariably fails and the tumours progress to castration resistant prostate cancer (CRPC), for which few therapeutic options exist. Therefore, there is a great need to identify and characterize novel therapeutic targets for this stage of the disease. Cancer cells undergo alterations that allow them to survive and proliferate, and metabolic reprogramming is one of the most important manifestations in cancer progression. Therefore, targeting tumour metabolism is an attractive approach to treat cancer. Screening for novel metabolic targets was performed using an siRNA library. 26 metabolic factors were identified to affect proliferation and/or migration, and these were found to be involved in essential pathways including lipogenesis, heme-biosynthesis, redox homeostasis, and glycolysis. The lead targets were validated in a range of cell lines and additional assays performed to investigate the effect upon cell cycle and cell death. UROS, the fourth step of heme synthesis, was further investigated and depletion of this enzyme significantly inhibited prostate cancer proliferation and migration, promoted cell cycle arrest and induced cell death. Further, inhibition of heme synthesis using the inhibitor succinylacetone was found to significantly induce caspase-independent cell death and to sensitise cells to ROS. Importantly, the inhibitory activity of succinylacetone in combination with ROS showed specificity for cancer cell lines. Targeting heme synthesis therefore represents a novel targeted treatment option for prostate cancer and further work is needed to develop this into a therapeutic strategy.