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The prevalence of chronic wounds such as diabetic foot ulcers (DFUs) is growing at an epidemic rate in the United Kingdom, with a high cost burden for the National Health Service and are prone to infections. Mixed bacterial-fungal infections are more complicated than single microbe infections with enhanced severity of DFUs. Moreover, recently developed medicated dressings target only either bacterial or fungal infection. This study therefore aims to develop advanced bioactive calcium alginate-based dressings that will deliver therapeutically relevant doses of broad-spectrum antibiotic and antifungal drugs to target mixed infections in DFUs. The therapeutic calcium alginate (CA) based dressings were prepared as films and wafers by solvent casting and freeze-drying technique respectively, with ciprofloxacin (CIP) and fluconazole (FLU) as model antibacterial and antifungal drugs respectively. The dressings were physically characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and texture analysis. Further functional characterization for exudate handling properties included porosity, water absorption (Aw), equilibrium water content (EWC), swelling index (Is), water vapour transmission rate (WVTR), evaporative water loss (EWL) and moisture content, drug dissolution by HPLC, in vitro antimicrobial and cell viability (biocompatibility) studies. Further, the exudate handling, antimicrobial and cell viability properties of the dressings were compared with commercial silver and CA based Algisite Ag® and hydrogel Actiformcool®. The drug loaded films were transparent, flexible, uniform, durable and elastic while drug loaded wafers were soft, uniform texture and thickness, and pliable in nature. SEM investigation showed differences in surface morphology between films (continuous sheets) and wafers (porous matrix). XRD confirmed the presence of calcium carbonate in the dressings and FTIR showed that the addition of drug caused the shift of peaks towards a higher wavenumber due to hydrogen bonding formation. Wafers showed better wound dressing properties than films and the commercial dressings for swelling, EWC (91 ̶ 95%), Aw (1085 ̶ 2377%) and moisture content (9 ̶ 17%) due to high porosity. The dressings showed initial fast release followed by sustained drug release which completely eradicated all causative bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and reduced fungal load (Candida albicans) 10 fold within 24 h. Moreover, the medicated dressings were found to be highly biocompatible (> 70% cell viability over 72 h) with human primary adult keratinocytes and showed maximum wound closure (in vitro scratch assay) within 7 days. CA wafers and films appeared to be potential delivery systems for delivering antimicrobial agents to mixed infected DFUs.

Pathogenic bacteria are responsible for around two million deaths and many reported hospitalization per year worldwide. Streptococcus pyogenes is a Gram +ve bacteria responsible for invasive and non-invasive infections. Traditional molecular and immunological detection systems are usually time consuming, require well equipped laboratory facilities and skilled personnel. Biosensors are ideal for the early detection of microorganisms because of their on-site, sensitive and rapid detection capability and electrochemical impedance offers a highly sensitive signal following target analyte interaction. In this study, immunosensors were fabricated on commercial screen printed gold electrodes to detect S. pyogenes. Electrochemical impedance spectroscopy (EIS) was carried out as the primary technique with other supporting methods including cyclic voltammetry (CV), on-sensor chemiluminescence characterization (termed ‘Midland blotting’) and fluorescence microscopy to successfully detect bacteria. With unstable impedance using conducting polymer and inconsistent signal using self-assembled monolayer (SAM), a non-conducting polymer, polytyramine was selected and optimized. Polyclonal antibodies against heat killed intact S. pyogenes was immobilized using two methods. First, biotinylated antibodies were immobilized via biotin-NeutrAvidin; second, reduced half antibody fragments were coupled using a bifunctional linker. The latter produced a higher signal due to the oriented half antibodies and close distance from the sensor surface. The surface density of antibodies was found to be critically important to get optimum signal. S. pyogenes cell surface proteins M and H, contributed towards the non-specific signal and this was reduced using protein A/ G as specific blocking agents. In addition, an attempt was made to polymerize novel phenolic copolymers in order to minimize non relevant protein interference from biological samples. The optimized full antibody based sensors detected S. pyogenes both in buffer to 104 cells/ml and in spiked 50% human saliva in PBS (v/v) with high specificity and selectivity.

The wrong way driving (WWD) crash differs significantly from all other crashes in terms of severity. Despite much valuable research on WWD crash severity, there is no robust study on developing a WWD crash severity distribution table that could be used for crash predictive methods by the Highway Safety Manual (HSM). HSM has severity distribution tables for all crashes that are used in quantitative safety analyses. Using the same tables for quantitative WWD crash analysis may produce inaccurate results since WWD crash severity is significantly different. Current severity distribution tables in the HSM have been prepared using Highway Safety Information System (HSIS) crash database. Unfortunately, a structured guideline that could extract true WWD crashes from the HSIS database is still absent. Therefore, the current study attempts to extract true WWD crash from HSIS crash database. And using the extracted WWD crash database, the study proposes different WWD crash severity distribution tables for freeway facilities. Finally, the study shows practical applications of extracted WWD crash database and proposed WWD crash severity distribution tables. These applications are expected to help future researchers of WWD crashes, safety analysts and transportation policymakers.

Pericytes represent a population of potential mesenchymal stem cells (MSC) that reside within a perivascular niche until they are required in normal homeostasis and the response to injury. Their mesenchymal capacities for multipotent differentiation, immune modulation and release of trophic factors hold great promise for regenerative therapies. Pathological expression of these potentials has been described in disease states, while acute or chronic inflammation following injury can lead to the production of signalling molecules that ultimately drive these progenitors to a fibrotic fate. The aim of this work was to explore how fate decisions of pericytes are regulated by their niche (in the setting of osteogenesis), and in the response to acute and chronic injury (in the setting of fibrosis). It was hypothesized that interactions between pericytes and endothelial cells (EC) within their perivascular niche are responsible for regulating mesenchymal differentiation. The osteogenic, adipogenic and chondrogenic potential of pericytes following isolation from multiple human organs was confirmed. The interactions between pericytes and EC in 2D and 3D coculture and the production of basement membrane proteins in these settings were confirmed. The osteogenic differentiation of pericytes was accelerated by EC but no influence of EC on the adipogenic and chondrogenic differentiation of pericytes was detected. Furthermore, data indicated that the influence on pericyte osteogenic potential by EC may occur through wnt signaling. The activation of TGFβ (transforming growth factor beta) through αv integrins has been suggested as central mediator of fibrosis in multiple organs. We hypothesized that selective αv integrin deletions in PDGFRβ (platelet derived growth factor receptor beta) expressing pericytes identifies a targetable pathway regulating fibrosis in skeletal muscle. We report that PDGFRβ-Cre inactivates genes in murine skeletal muscle pericytes with high efficiency. Deletion of the αv integrin subunit in pericytes protected mice from chemical injury induced skeletal muscle fibrosis. Pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated muscle fibrosis, even when administered after fibrosis was established.

The endothelium plays a pivotal role in the maintenance of vascular homeostasis and its dysregulation promotes vascular complications. This thesis proposes that heme oxygenase-1 (HO-1), an anti-inflammatory enzyme with antioxidant properties, is endothelial protective factor that prevents endothelial injury induced by cisplatin or activated neutrophils. Specifically, this thesis aimed to test (i) that overexpression of HO-1 prevents cisplatin-induced endothelial injury and suppresses caspase activity; (ii) whether neutrophil-endothelial cell activation resulted in the release of soluble Flt-1 (sFlt-1) and soluble endoglin (sEng), the two anti-angiogenic factors known to induce the clinical signs of preeclampsia; (iii) whether HO-1 prevented activated neutrophils from stimulating the release of these factors from the endothelium; (iv) the relative contribution and the co-dependency of neutrophil activation and anti-angiogenic growth factors in preeclampsia where systemic endothelial dysfunction is known to occur. This thesis shows that cisplatin inhibited human umbilical vein endothelial cells (HUVEC) metabolism as measured by MTT assay and resulted in the release of placenta growth factor (PlGF). Immunoblotting confirmed that cisplatin increased cleaved caspase-3 expression in HUVEC. These effects of cisplatin were attenuated in HUVEC infected with adenovirus encoding HO-1 and the effects were exacerbated when HO-1 was silenced by siRNA. Furthermore, cisplatin stimulated PlGF release was suppressed by the overexpression of HO-1. In addition, HO-1 overexpression inhibited angiogenesis as determined by vascular endothelial growth factor-induced capillary tube formation on Matrigel coated plates. Thus these studies indicate that agents which upregulate HO-1 could increase the effectiveness and tolerability to cisplatin in cancer treatment. Although neutrophils are early contributors to endothelial cell activation, no studies have determined their contribution to the release of sFlt-1 and sEng. We therefore investigated the effect of activated neutrophils on the release of sFlt-1 and sEng in endothelial/neutrophil co-cultures and in the circulation of women with normal pregnancy and preeclampsia. LPS-mediated neutrophil activation stimulated the release of sEng but not sFlt-1 from endothelial cells in culture. In the absence of neutrophils, overexpression of HO-1 in HUVEC downregulated the release of sEng. In contrast, HO-1 overexpression failed to inhibit the release of sEng in the presence of activated neutrophils. The release of sEng by activated neutrophils-endothelial cell cocultures appears to be mediated by metalloproteinases (MMP) as the broad-spectrum MMP inhibitor (GM6001) attenuated sEng release. Clinical studies demonstrated that sEng, pro-inflammatory interleukin-6 (IL-6) and the soluble markers of neutrophil activation (α-defensins and calprotectin) were all elevated in women with preeclampsia. We identified a direct correlation between neutrophil activation and IL-6 release. However, no correlation could be established between these factors and sEng release in preeclampsia. Hence, these results provide compelling clinical evidence to show that the increase in neutrophil activation and IL-6 release during preeclampsia are unlikely to significantly contribute to the clinical signs of preeclampsia as they fail to correlate directly with the anti-angiogenic factors, which form the final common pathway to the clinical signs of preeclampsia and systemic endothelial dysfunction.