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Nature has evolved over millions of years to develop advanced biological machines which cooperatively perform a myriad of complex operations in 'one pot' with unparalleled efficiency. Using chemical fuels such as adenosine triphosphate to power endergonic reactions, Nature can selectively control reagents to achieve directional motion and even pumping of key intermediates along metabolic pathways. Being able to mimic this level of molecular precision would prove to be an extremely powerful technique across multiple disciplines of science. Whether within construction materials, surface chemistry or advanced drug delivery, properties on the nanoscale have huge implications on the macroscale. Herein, a series of artificial molecular machines are described which utilise stimuli to afford directional motion on the molecular scale to perform a range of desirable applications.

Current approach for ocular wound healing involves bandages combined with repeated administration of therapeutic eye drops. There is a need to develop ocular medicated bandages as a convenient and cost-effective strategy for wound healing. This project aimed to develop a controlled release biocompatible bandage consisting of 10-50 µm drug-incorporated poly (lactic-co-glycolic acid) (PLGA) microparticles (MP) and eggshell membrane (ESM) for ocular wound healing application. The eggshell membrane is a natural material with unique physical and biological properties. No studies have demonstrated nor reported its potential as a novel ocular wound dressing yet. ESM samples were isolated from the eggshell by either manual peeling (ESMstrip) or, via extraction, using in-house developed acetic acid or ethylenediaminetetraacetic acid, EDTA, protocols Energy-dispersive X-ray spectroscopy (EDS) confirmed that the ESM samples contained carbon, nitrogen, oxygen, and sulphur, with no traces of calcium residues from the extraction process. The extraction method (acetic acid and EDTA) did not alter the chemical bonding structures of the ESM, and the chemical composition of the fibrous proteins of the ESM was further clarified using Fourier transform infrared (FTIR) spectroscopy. The ESM has a three-layer composite structure, according to scanning electron microscopy (SEM) analyses: an inner layer that is continuous, dense, and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane), and an outer layer (outer shell membrane) with larger fibres. Furthermore, optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical/physical profiling of the ESM samples were performed and showed suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. A sub-study presented a design of experiment technique (DOE) known as Taguchi Design for the generation of a formulation model for optimizing the various factors shown to affect MP size when fabricated using the solvent emulsion technique and to produce the most appropriate MP for topical drug delivery. The first optimization step using the L12 design showed that all parameters significantly influenced the particle size of the fabricated MP with exception of the concentration of PVA in the hardening bath. In contrast, the L18 design results showed that the molecular weight of PLGA does not significantly affect the particle size. The optimized formulation was identified to be run 16 from the L18 OA design. The mean particle size was found to be 23.0 µm. that falls under the required range that is 10 - 50 µm. The results from this study showed that the application of Taguchi design can be used to predict and determine the best combination of process parameters that can provide the optimal response condition- in this case, to identify the most appropriate microparticle size for topical application. Additional studies were conducted with the formulation model to fabricate BSA-loaded PLGA-MP as well as their characterization using microscopy, size distribution and drug release profile. The MP were successfully attached onto the ESM using a chemical method (NaOH). The presence of the loaded MP did not compromise the transparency of the bandage. The FTIR spectra reveal the crosslink of the ESMs absorption peak with the absorption peak of the MP. The 2 in 1 bandage consisting of MP and ESM demonstrated high biocompatibility, durability, flexibility, fluid absorption capacity, thermal stability and a sustained drug release profile. These findings show that the ESM could be used in a variety of regenerative medical and/or biotechnological applications, such as ocular and skin wound dressings.

Poorly soluble drugs pose a major challenge in the field of drug delivery, as they are considered difficult to deliver. Many techniques can be possibly used to aid in improving the solubility profiles of those compound and improve their delivery. Those techniques has shown to be effectively used in the preparation of orally-administered dosage forms. Nonetheless, they can also be useful in delivering those compounds via other routes of administration as well. Transdermal drug delivery involves the application of a drug onto the skin to exert systematic effects. This administration route had drawn the attention of scientists over the last few decades, as the skin is considered to be the largest and most accessible organ in the human body. However, relatively few drugs possess the required characteristics to penetrate the skin passively. Microarray patches (MAPs) are novel transdermal drug delivery devices, capable of increasing the permeability of the skin by creating micro pores within the skin surface, thus, reduce its barrier function and enhance the permeation of molecules. Furthermore, long-acting dosage forms, capable of delivering therapeutic doses of drugs over prolonged periods of times, can enhance the compliance of patients to their treatment regimens, and thereby improve the quality of their lives. This thesis had focused on the development of polymeric MAPs systems, using solubility enhancing techniques, to facilitate the depot delivery of the hydrophobic atorvastatin (ATR) to obtain a long-acting effect. For this purpose, hydrogel-forming MAPs (HF-MAPs) were initially explored. Four Gantrez® S-97-based HF-MAPs formulations were initially prepared and characterised in terms of their mechanical properties, swelling and ATR diffusion across their swollen films. Afterwards, a solid dispersion (SD) technique was used to formulate ATR-reservoirs using PEG 200 and PEG 6,000 as a matrix. A drug loading of ~ 15 mg was obtained in each SD reservoir. Furthermore, dissolving MAPs (D-MAPs) were also investigated, where either ATR microparticles (MPs) or nanocrystals (NCs) were formulated into D-MAPs, in combination with the biocompatible water-soluble excipients: poly(vinyl) pyrrolidone (PVP) and poly(vinyl) alcohol (PVA). In this part, particle size reduction was explored as a solubility enhancing technique. MP-loaded MAPs had a drug content of ~ 5 mg, whereas NC-loaded MAPs contained ~ 2.5 mg of ATR. Both HF-MAPs combined with ATR PEG-based SD reservoirs system and ATR-containing D-MAPs where characterised in terms of drug content, thermal behaviour, insertion efficiency, mechanical properties, and in vitro drug release and recovery studies. Subsequently, ex vivo permeation, and skin deposition studies of the candidates of all formulations were conducted, from which the lead HF-MAPs and ATR SD reservoir formulation along with the lead D-MAPs formulation were brought forward for further in vivo pharmacokinetics investigation using Sprague Dawley rats. The result had shown, for the first time, the long-acting delivery of ATR via polymeric MAPs system (HF-MAPs and D-MAPs) over two weeks, where therapeutically-relevant concentrations of ATR (> 20 ng/mL) were obtained throughout the study from both MAP cohorts. It had also verified for the first time, the versality of HF-MAPs in the depot-delivery of ATR, from which therapeutically-relevant plasma concentrations were achieved for over 14 days. When compared to the oral group, the use of MAPs improved the overall pharmacokinetics profile of ATR in plasma, where significantly higher AUC values resulting in ~10 folds higher systemic exposure levels were obtained. The findings of this thesis provide a promising alternative for the oral administration of ATR, as the long-acting features of this method can ultimately be highly effective in enhancing the patients compliance and improving the quality of their lives. Looking towards large-scale production, this work provides evidence of a suitable formulation strategy for poorly soluble drugs to be delivered using polymeric MAPs. Nonetheless, pharmacodynamic assessment of the efficacy of ATR in hyperlipidaemia animal models must be initially determined, as well as considering industrial regulatory standards.

Background and Aim: Orally disintegrating tablets (ODTs) combined with multiparticulate formulations of modified-release (MR) properties are considered more efficient medicinal options than traditional immediate-release formulations. ODTs & MR multiparticulates provide ease of medicinal use for dysphagic patients allowing a modified drug release to reduce the undesirable side effects, and the frequent daily dose would be eliminated, thus enhancing patient compliance. Nevertheless, the compaction of multiparticulates is challenging, causing polymeric damage leading to a loss in integrity and a rapid drug release. This project aims to prepare ODTs-MR of indomethacin (IND) and propranolol hydrochloride (PRH) by enhancing mechanical properties and the parameters of ODTs and multiparticulates production. Methods: Two developed and validated HPLC methods (according to ICH guidelines) were utilized to detect and quantify indomethacin (IND) and propranolol hydrochloride (PRH) release from the prepared formulations. The direct compression method was used to prepare ODTs, extrusion spheronization and spray drying to prepare the multiparticulates. The mechanical properties of ODTs and multiparticulates were assessed using texture analysis and hardness testing. Through the project, all formulations were examined using disintegration time, dissolution studies (pH 1.2 and 6.8), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and stereomicroscope. Results and discussion: IND loaded microparticles of Eudragit L100 were successfully delayed IND release in pH 1.2 buffer using acetone as a solvent. The low value of YM, using the appropriate solvent type and setting feed concentration at 5% w/v, was appropriate for modifying IND release. Similarly, Eudragit L100 was used to prepare IND loaded pellets. Lactose and mannitol (63, 125 and 500 µm) ODTs comprised of IND loaded pellets disintegrated in less than 30 seconds with acceptable mechanical properties of less than 1.5 MPa and an adequate elastic profile. Also, ODTs-pellets from lactose and mannitol delayed IND release in acidic media to less than 1.07% at 120 minutes while released more than 93% at 120 minutes in phosphate buffer (pH 6.8). Furthermore, the tensile strength of IND pellets of Eudragit L100 (45, 63 and 90 µm) was directly proportional to YM (p<0.05), offering enough support to maintain their integrity under compression. In addition, the in vitro release study showed a delayed release of IND in the acidic media (pH 1.2) and an immediate release in the buffer media (pH 6.8). On the other hand, the matrix system was unapplicable to delay PRH release from Eudragit RS based pellets despite changing the ratio and type of plasticisers. However, PRH pellets of reservoir system using Eudragit RS, Eudragit RL and combination of Eudragit RS: RL (1:1) w/w showed that the mechanical properties of the coated pellets differ significantly from the uncoated batch (p<0.05). Besides, ODTs reservoir-pellets were able to modify PRH release to ≤ 53.9% in acidic media and ≤ 47.2 % in phosphate buffer for 30 minutes, while the uncoated pellets released > 75% of PRH. Alongside, EDEM® successfully simulated the compaction process and showed that increasing compression force at the beginning of compression strengthens the interparticulate bonds at the surfaces resulting from the low surface roughness. Conclusion: Matrix system to sustain IND release was achieved using Eudragit L100 microparticles and pellets embedded in the ODTs. The lowest percentage of propranolol release was attained from the reservoir system of Eudragit RS and RL pellets embedded in the ODTs. All formulations of ODTs disintegrated in less than 30 seconds. A balance between Young's modulus and tensile strength is essential to enhance the integrity of the multiparticulates and, eventually, drug release.

Patients with sickle cell disease (SCD) receive regular blood transfusions, which can lead to alloimmunisation due to exposure to different red blood cell (RBC) antigens. The spleen is frequently damaged in these patients, resulting in higher numbers of red cell-derived particles (RCDP) and autophagic vesicles (AV). Most RCDP, produced through membrane budding, are right-side out and expose the external domains of RBC proteins, whereas AV are inside-out, exposing cytoplasmic domains. Since SCD patients have higher numbers of RCDP and AV, we aimed to characterise these particles and compare with healthy donor plasma and with storage vesicles (SV) from outdated blood. We also investigated the use of these particles to induce tolerance to RBC antigens, in a murine model. RCDP and AV from SCD and healthy plasma were isolated using immunomagnetic separation. Results using imaging flow cytometry (IS) showed that RBC markers like glycophorins A and C, band 3, glucose transporter1 and phosphatidylserine, were detected in all particles, confirming they originated from RBC. RCDP were more prevalent and generally larger than AV but both particle types were more abundant in SCD than healthy plasma. IS was more sensitive for small particle detection than flow cytometry. The latter detected large ghost membranes. All particle types were detected by transmission electron microscopy (TEM) and immunogold staining against extracellular or cytoplasmic GPA domains further confirmed their RBC origin. TEM, IS and dynamic light scattering showed that RCDP were larger than AV. A model system was developed to investigate tolerance induction in vivo and indicated that both SV and ghosts could be used to prime host animals. In conclusion, small RCDP and AV from SCD and healthy plasma have been characterised for first time, using IS. It may be possible to use particles from blood cells induce tolerance to RBC antigens and thereby reduce alloimmunisation.

The applications of magnetism are far reaching, and much work has been done to make use of magnetic properties to develop viable applications. Two key areas are solid state memory, for example MRAM and hard discs, and the use of superparamagnetic nanoparticles in biological applications by using mechanical actuation. However, little has been done to bridge the gap between these two areas and bring to reality the possibility of carrying data in a liquid of magnetic particles. Ferrofluids to date are mostly simple iron oxide particles suspended in liquid, whereas this project uses micron lithography on thin film films of magnetic material which can be lifted off into solution to create an artificial ferrofluid of advanced materials. We hope to develop such a fluid with the same functionalisation which can be achieved through the thin film structures on a solid substrate. The particles can be redeposited onto a substrate to 'tag' it. By controlling the magnetic properties of batches of particles, we can detect the presence or absence of a given particle type hence providing a yes/no bit. In this thesis we first use the shape anisotropy of rectangular particles patterned from ultrathin films of Permalloy to control their magnetic properties. The extended shape introduces in-plane uniaxial anisotropy, with hysteresis along the easy axis. The dimensions of the rectangle determine the demagnetizing field so alter the coercivity. We can detect the presence of a particular particle shape by whether or not there is a switch measured at its specific coercivity. We characterize the particles and find that the coercivity and the ferromagnetic resonance peaks are specific to the particle dimensions. We also demonstrate that the particles can be lifted off into solution, redeposited under an applied field and detected in their dispersed form. However, the number of achievable channels is very low so we move on to an alternative system. We fabricate discs from magnetic multilayers consisting of ultrathin CoFeB films with interfacial perpendicular magnetic anisotropy which are antiferromagnetically interlayer exchange coupled via the Rudermann-Kittel-Kasuya-Yoshida interaction. They form synthetic antiferromagnets (SAFs) with uniaxial anisotropy along the surface normal. The coupling strength can be tuned by inserting ultrathin Pt layers between the CoFeB and the central Ru layer, which act to attenuate the exchange coupling. We characterize a range of multilayers each with specific coupling strengths and therefore different switching fields, which will be the basis of the channels of the tag. These are then patterned and lifted off into solution, then measured when redeposited to confirm the retention of their magnetic properties. We also investigate the time-dependence of the switching fields of individual CoFeB thin films and of the SAF bilayer films. In conclusion, we create a multi-channel tag from SAF discs with tuneable switching fields and demonstrate that the properties of the continuous film can be retained when patterned into discs which are lifted off into solution and redeposited onto a substrate.

37 million people are currently living with HIV worldwide. With the development of Antiretroviral Therapy (ART) AIDS mortality has reduced, with cardiovascular disease (CVD) now the leading cause of HIV deaths. HIV associated CVD has been linked to chronic immune activation through the association of inflammatory biomarkers, clinical events and asymptomatic atherosclerosis; even from a young age. Microparticles (MPs), markers of cellular activation and injury, are also elevated in adults with HIV, and demonstrate the capacity to contribute to inflammation, endothelial dysfunction and coagulation. Monocytes play a key role in atherosclerosis initiation and display an activated, pro-inflammatory phenotype in paediatric HIV. Monocytic MPs (MMPS) are elevated in adults with HIV, thus the aim of this thesis was to quantify MMPS in HIV infected children and investigate their effects on monocytic function. A novel negative isolation method was first optimised to enable the study of human monocyte function 'ex vivo'. This isolation procedure allowed the negative enrichment of all three circulating monocyte subsets (Classical, Non-classical and Intermediate), with high purity and minimal activation. Extracted monocytes displayed minimal alterations in marker expression while retaining their phagocytic, migratory and cytokine secretion function 'ex vivo'. Using a monocytic cell line (THP-1), MP release under different stimulatory and apoptotic conditions was investigated. Under stimulatory conditions with cytokines and a calcium ionophore (A23187), MPs were released in a concentration-dependent manner, with endotoxin stimulation resulting in the highest quantity. Furthermore, MPs displayed a similar trend in surface marker expression in comparison to their parent cell. Next, we investigated the effects of THP-1 derived MMPS on human monocyte function employing the isolation method established. MMPS evoked the release of pro-inflammatory cytokines in addition to enhancing CD11b expression, adherence and transendothelial migration. This work demonstrates that MMPS activates monocytes and enhances endothelial migration, presenting a mechanism that contributes to atherosclerosis pathogenesis. Finally, MMPS and MPs from an endothelial, platelet and T cell origin were enumerated in children with HIV infection pre/post ART initiation and healthy sex-age-matched controls. Plasma samples were collected during the CHAPAS-3 CV sub-study, from which we analysed 16 children receiving ART (≥2 years), 11 treatment-naïve children and 15 controls. Circulating MPs from an endothelial, platelet and T cell origin found in children with HIV infection normalised to levels found in healthy controls, following ART initiation and experience. MMPS were elevated within the treatment-naïve cohort and remained elevated despite treatment intervention; this elevation was also observed in the treatment-experienced cohort. The persistence of elevated MMPS indicates consistent monocyte activation despite successful viral suppression, supporting the activated phenotype reported in literature. This data combined with the functional implications of MMPS on monocytic function described in this thesis elucidates a mechanism that contributes to elevated vascular inflammation reported within this population.

End-stage renal disease patients exhibit markedly elevated cardiovascular risk when compared to the general population, as well as chronic systemic inflammation as a result of aberrant immune system activation. Circulating microparticles are also elevated, driving both inflammation and thrombosis and further increasing cardiovascular risk. Regular, moderate intensity aerobic exercise may be a therapeutic strategy for the impaired immune function and pro-inflammatory environment seen in these patients, however previous studies investigating this effect have been methodologically limited. The studies included in this thesis aimed to characterise the impact of acute and regular aerobic exercise on immune function, markers of inflammation and microparticles in end-stage renal disease patients. It is hypothesised that aerobic exercise will have an anti-inflammatory impact on the immune system and cytokine environment and reduce circulating microparticle thrombotic potential.

The first part of this project was inspired by the fascinating internal porosity of Polypore fungi, characterised by channel-like and micro-fibrous porosities, which create a highly efficient directional release system for their basidiospores. An initial analysis of their natural porous structures revealed the effect of their morphology on the sorption of liquids; sections of the channel-like regions of Ganoderma applanatum fungi were first dip-coated with octadecyltrichlorosilane to achieve superhydrophobicity (water contact angle higher than 150°) and selective sorption of oils and organic solvents. The treated fungi showed oil sorption capacity between 1.8 (rapeseed oil) and 3.1 g/g (chloroform), and the combination of channel-like porosity and hydrophobic properties suggested its use as naturally derived filtration element in micro fluidic networks, showing the possibility of efficiently separating water from chloroform. The second part of this research work replicated the Polypore fungi porosity through the fabrication of polymeric membranes by means of immersion precipitation technique, a specific variant of phase inversion methodologies, thus biomimicking the dual porosity of Polypore fungi. Poly(vinylidene fluoride) (PVDF) has been widely adopted to produce porous membranes due to its membrane forming capability and outstanding mechanical, chemical and thermal properties. N,N-dimethylformamide (DMF) (solvent) and water (coagulant) were then chosen as part of the PVDF membrane forming system to achieve the formation of a net separation between finger-like and sponge-like porosity regions within the membrane thickness. By precisely varying the coagulation bath temperature, the extension of the finger-like region was controlled; the use of 90 °C as temperature for the coagulant led to a predominance of sponge-like morphologies within the PVDF membranes, where only ≈2.5 % of the membrane thickness at the coagulation bath/casting solution interface was characterised by macrovoids; the macrovoids disappearance at high temperatures was addressed to the rapid polymer precipitation and skin layer formation at the early stage of the casting solution phase separation, which prevented the fast solvent/coagulant mass exchange within the inner casting solution layers. On the other hand, lowering the coagulation bath temperature led to a significant increase of the macrovoids length, which were present in more than 50% of the membrane thickness when the temperature was decreased below 20 °C. Also, the production of PVDF membranes at low coagulant temperatures affected the shape of the macrovoids, and finger-like porosity was obtained; this effect was addressed to the early gelation of the polymer-rich phase during the occurrence of phase separation, which generated a contraction force around the macrovoids which turned into narrow and long finger-like pores. Finally, the resulting membrane morphologies, which mimicked the Polypore fungi porosity, showed directional sorption and release of therapeutic essential oils to be applied as drug delivery systems. Novel research from the past years has shown the possibility of combining phase inversion techniques with more recent scaffolds production technologies, such as electrospinning, to achieve superior membrane properties. The last part of this work developed an innovative approach to improve the mechanical properties of polycaprolactone (PCL) electrospun membranes by first dip-coating them into PVDF casting solution and then inducing phase separation through water vapor. The slow phase separation of PVDF caused the solid-liquid demixing to occur and lead to the formation of PVDF spheres with diameters ranging from 4.8 to 10.3 m; the electrospun polycaprolactone nanofibers acted as nucleation sites, and the PVDF spherulites grew onto the porous surface of the fibers and physically bonded multiple fibers together. The fabricated composite scaffolds showed increased mechanical properties if compared to the polycaprolactone mats alone; the ultimate tensile strength, Young's modulus and elongation at break increased from 1.2 MPa, 62.9 kPa, 634.1 % for PCL electrospun mats to 2.1 MPa, 764.5 % and 136.7 kPa for the fabricated PCL-PVDF composite scaffolds, respectively. Finally, cytotoxicity studies using neuroblastoma cells showed the biocompatibility of the fabricated composite scaffolds and suggested their potential to be applied in neural tissue engineering.

Colloids, broadly defined as particles small enough to not sediment under gravitational force while dispersed in a continuous medium, are of great scientific and industrial importance. They are present in foods, cosmetics, paints and other materials, often in a jammed or a gelled state. This dissertation explores the self-assembly of hard and soft colloidal particles into mesoscopic structures through modification and control of their surface potentials. Due to its selective binding properties and thermal reversibility, deoxyribonucleic acid (DNA) was used to coat spherical polymeric colloids and subsequently have them assemble into high density colloidal gels. The formation and morphology of one- and two-component colloidal gels was investigated; the use of in-house synthesised fluorinated latex colloids (n = 1.36) allowed for refractive indexmatching of such systems in aqueous solutions, greatly aiding the imaging process deep inside dense gels. Additionally, the diffusion of tracer particles in the confinement of the gel structure was investigated. Furthermore, using the same coating technique, oil droplets (ODs) were functionalised with DNA: various sizes and configurations of DNAcoated ODs were investigated with the goal of creating functional oil-in-water emulsions for possible industrial and biotechnological applications. Taking a different assembly approach, 200 nm diameter fluorinated latex particles without any DNA coating were assembled into photonic crystals which, rather than showing iridescence, displayed strong single colour reflectance along with high transmittance, with the reflection colour varying with interparticle distance. Jumping back to colloidal assembly via DNA, rod-sphere structures made from DNA-coated gold nanoparticles and long DNA-coated virions were explored, demonstrating how anisotropic building blocks could create composite structures with increased porosity. Finally, an external magnetic field was shown to aid the assembly of superparamagnetic DNA-coated colloids into long linear constructs, while an additional coating of colloids was shown to stabilise the constructs after the magnetic field had been switched off.

Progression and regression of renal fibrosis is observed in patients with diabetic nephropathy (DN). The underlying pathways, especially those that promote regression of fibrosis, remain poorly understood in part due to the fact that most rodent DN models only mirror the early features of human DN. Another obstacle for optimizing treatment strategies is that albuminuria, the current gold standard biomarker of renal damage in DN, often lacks sensitivity and specificity for identification of those patients with diabetes who are at risk of a rapid decline in renal function. A novel DN model, in which diabetes was induced with streptozotocin in Cyp1a1mRen2 rats and hypertension was generated by inducing renin transgene expression with dietary indole-3-carbinol (I-3-C), mimicked many of the key biochemical, pathological and transcriptomic changes observed in the kidney of patients with DN. Recently, the model was extended to include a ‘reversal phase’ in which glycaemia was tightly controlled and blood pressure normalized for eight weeks after an ‘injury phase’ of 28 weeks. The present study aims to employ this novel rodent model to examine pathways activated in the kidney during and following reversal of hyperglycaemia and hypertension and to identify new biomarkers that might complement albuminuria in assessing risk of renal deterioration in patients with diabetes. Methods Tissue and urinary specimen from the Cyp1a1mRen 2 model of DN were analysed by realtime-PCR, Western-Blot, ELISA and staining techniques including immunohistochemistry, immunofluorescence and zymography. To establish in-situ zymography a model of ureteric obstruction was used. Urinary peptidomic analysis as well as measurement of urinary exosomes and microparticles was performed in the model and in patients with DN utilizing liquid chromatography/tandem mass-spectrometry, nanoparticle tracking analysis (NTA) or flow cytometry. Results Tight control of blood glucose and blood pressure during an 8 week ‘reversal phase’ did not significantly reverse the degree of renal fibrosis accrued during a 28wk ‘injury phase’. However, it did result in a reduction in expression of genes encoding myofibroblast markers and extracellular matrix (ECM) proteins. Genes that were up-regulated during both injury and reversal phases were implicated in adaptive immunity, phagocytosis, lysosomal processing and degradative metalloproteinases (MMPs). Paradoxically MMP activity was massively reduced during both injury and reversal phases. This may be due to an elevated level of tissue inhibitor of metalloproteinase-1 (TIMP-1) protein in both phases. After separating TIMP1 from MMP in renal tissue homogenates from animals of both the injury and reversal phases using gel electrophoresis, MMP activity was restored above that of controls. For biomarker discovery peptidomic analysis was performed on urine from rats at baseline and during the injury and reversal phases of the Cyp1a1mRen2 model of DN and from patients with moderately advanced DN and from normal controls. The use of two different search and analyse tools (Maxquant, Progenesis QI) resulted in the discovery of significantly altered peptides in the urine in rodent and human DN. Further studies focused on peptides derived from those proteins for which the corresponding gene was similarly regulated in the DN model and in human DN. Urinary epidermal growth factor (uEGF) matched these criteria as the reduction of excretion during the injury phase in the DN model was paralleled by reduced EGF protein expression in renal tissue. Key biomarker candidates identified in the first two chapters were measured in urinary specimens of patients from the Edinburgh Type 2 Diabetes study (ET2DS) to test translational utility. MMP7 and other candidates, such as osteopontin or vascular endothelial growth factor (VEGF) were not of value in predicting renal outcomes. Reduced uEGF was significantly associated with increased mortality rate. In a subgroup of 642 study participants who were normoalbuminuric and had a preserved renal function at baseline, a lower uEGF to creatinine ratio was a risk factor for either developing an estimated glomerular filtration rate less than 60 ml/min per 1.73m2, rapid (over 5% per annum) decline in renal function or the combination of both. The latter remained significant after correction for other covariates. Addition of uEGF resulted in a marginal improvement in a model derived from traditional risk factors for predicting rapid decline and the composite end-point. Urinary microparticle (20nm-1000nm) analysis was established in the rodent DN model and translated to patients with DN. Total urinary exosomes (20nm-100nm) or exosomes derived from specific renal cell types including podocytes and tubular cells, increased during the injury phase in the Cyp1a1mRen2 model followed by a decrease after reversal phase. In a pilot study comprising participants with advanced chronic kidney disease, the urinary exosome concentration correlated with renal function. In the ET2DS an increased exosome concentration at baseline indicated a higher risk for renal deterioration during four years follow-up even after correction for baseline eGFR. Urinary microvesicles (100nm-1000nm) concentration increased during the injury phase in the DN model though correlation with renal function in humans was only significant if kidney-specific marker (podocalyxin) positive microvesicles were measured. Conclusion Normalisation of hyperglycaemia and hypertension in the DN model allows the study of genetic and protein regulation during the injury and reversal phases. ECM-production but not ECM-degradation genes are down-regulated during the reversal phase. The lack of reduction in ECM during the reversal phase might be caused by persistently reduced MMP activity due to the presence of TIMP-1. Targeting TIMP might be a treatment strategy to promote reduction of renal fibrosis. For the first time, the analysis of urinary peptidomics was integrated with previous transcriptomic findings in the Cyp1a1mRen2 model and patients with DN for biomarker discovery. The approach was validated using different analysis tools and successfully identified candidate markers which were increased or reduced in DN. Candidates included uEGF, which identified patients with DN who were at risk of a rapid decline of renal function. Though the marker requires further confirmation in other cohorts, it might be especially useful for patients with type 2 diabetes, in whom renal decline is often uncoupled from the development of albuminuria. Finally, the DN model helped to develop the methodology of microparticle analysis. For the first time a potential prognostic value of urinary exosome analysis in patients with diabetes has been demonstrated. Future work will include further optimisation of the methodologies, including labelling of microparticles with multiple antibodies and increasing study participant numbers.

Cell-derived microvesicles (MVs) are naturally released into the human circulation and an increase in the concentration of certain MV populations have been observed after exercise. However, the MV appearance dynamics, the exercise-related stimuli that induce their formation and physiological relevance are poorly understood. Hence, the overall objectives of this thesis were to: 1) characterise the circulating platelet (PMV) and endothelial-derived MVs (EMVs) responses during exercise and recovery, as well as their arteriovenous dynamics, 2) investigate the potential role of haemodynamic forces on MVs formation in vivo by vascular shear stress manipulations, and 3) explore the putative proliferative, chemotactic and angiogenic potential of exercise-derived MVs upon human vascular endothelial cells in vitro. Chapter 5 of this thesis describes the time-course of MV appearance in response to prolonged cycling, and demonstrates that intravascular [PMV] increases during and after exercise performed in the heavy intensity domain, whereas [EMV] remains unaltered. Moreover, [PMV] during exercise was related to estimates of vascular shear stress and plasma noradrenaline levels. Results from chapter 6 revealed that PMVs increased in the arterial circulation during passive heat stress, and in the arterial as well as venous circulation during short duration very heavy exercise engaging either a large or small muscle mass. The increases in [PMV] were not directly linked to local changes in vascular shear stress through heat stress and exercise, indicating a systemic PMV response. Finally, chapter 7 revealed that exercise-derived MVs supported endothelial proliferation and migration, while displaying pro-angiogenic potential in vitro. In conclusion, results of this thesis provide original information about MV dynamics, by demonstrating that PMV increase systemically in the circulation not only after but during exercise involving a small and large muscle mass. This MV response seems to be modulated by exercise intensity, and is only partially linked to levels of vascular shear stress. Moreover, circulating MVs produced during exercise present stimulatory angiogenic and mitogenic effects upon endothelial cells in vitro, suggesting a novel potential link between vascular adaptation and exercise training.

Microparticles (MP) are submicron structures produced by all cells upon activation or apoptosis that act as a non-soluble means of communication between cells. They ferry proteins, bioactive lipids, RNA and receptors, as well as ridding cells of redundant organelles and toxins. They have been recently investigated for their pathophysiological role and as potential biomarkers/effectors in many diseases. In severe sepsis, studies of MP so far have produced inconsistent and even conflicting results. In this project, it was demonstrated that cell derived MP subsets vary according to the cause of severe sepsis (community acquired pneumonia (CAP) or faecal peritonitis (FP)), where CAP patients had higher levels of circulating MP. Surprisingly, FP patients MP levels were comparable to healthy volunteers. Further stratification of MP subsets according to their expression of the protein alpha-2-macroglobulin (A2M) has yielded better differentiation between the two diseases. The A2M expressing MP were significantly higher in survivors of community acquired pneumonia sepsis, but there was no similar association in patient with FP. Granulocyte macrophage colony-stimulating factor (GM-CSF) and interferon γ (IFN-γ) are being studied as possible adjuvant therapies in sepsis. They seem to reverse the immune-paresis that ensues after the initial insult. MP produced from whole blood stimulated with GM-CSF and IFN-γ were studied in this project. Both GM-CSF and IFN-γ increased MP expressing A2M over control. These MP elicited a pro-inflammatory phenotype when incubated with neutrophils or endothelial cells which may contribute to the potential benefits of GM-CSF and IFN-γ in severe sepsis.

Mutagenesis screening in mice is an effective means of identifying essential genes in cardiovascular development. The l11Jus8 (L8) mutant mouse line was originally isolated from a region-specific N-ethyl-N-nitrosourea (ENU) chemical mutagenesis screen and exhibited an autosomal recessive mid-gestational embryonic lethal phenotype characterised by haemorrhage in the thoracic cavity, blood pooling in the heart, right ventricular dysmorphology and yolk sac vascular degeneration. Prior work mapped the L8 mutation to a ~2.77Mb region on mouse chromosome 11. The aim of this study was to further characterise the L8 mutant phenotype and identify the L8 causative mutation. Phenotypic characterisation conducted here confirmed mid-gestational lethality, haemorrhage and yolk sac vascular degeneration in L8 mutants. Histological analysis of L8 mutants demonstrated presence of fragmented cell nuclei and loss of myocardial integrity in embryonic atrial myocardium. Areas of fragmented cell nuclei did not exhibit positive staining for apoptosis. Furthermore, L8 mutants did not appear to experience typical cardiac defects in aspects including myocardial or smooth muscle differentiation, cell proliferation, ECM production, myocardial hypoplasia/hyperplasia, basement membrane components or observable aberrations in cardiac conduction. L8 mutants exhibited atypical cardiac defects including sudden cessation of heartbeat with morphological indicators of necrosis such as swelling of mitochondria and release of microparticles both from atrial myocardial cells. The L8 mutant appears to represent a novel combination of cardiac defects or novel defects with secondary cardiac phenotypes. Sequencing of the coding exons and splice junctions of 22 candidate genes within the ~2.77Mb L8 locus did not identify the causative mutation. The L8 locus was therefore further refined to a ~1.16Mb region including 20 genes. Sanger sequencing of 10 of these genes plus targeted sequence capture and SOLiD sequencing of the region did not identify a potential L8 mutation. Given the refinement of the candidate locus and advances in sequencing technology and analysis, further sequencing will likely identify the L8 mutation and confirm the cause of the embryonic lethal phenotype.

One approach that has demonstrated success in the field of molecular imaging utilizes microparticles of iron oxide (MPIO) conjugated to specific antibodies and/or peptides to provide contrast effects on MRI in relation to the molecular expression of a specified target. The experimental aims of this thesis were 1) to investigate the ability of VCAM-1 and P-selectin targeted MPIO to detect the expression of VCAM-1 and P-selectin on the activated endothelium in-vitro and in-vivo in mouse models of renal and cerebral ischemia reperfusion injury, and 2) develop a novel contrast agent for imaging αvβ3-integrin expression in angiogenesis using RGD peptide conjugated MPIO (RGD-MPIO) in-vitro. MPIO (1.0 µm) were conjugated to monoclonal antibodies against VCAM-1 (VCAM-MPIO) or P-selectin (PSEL-MPIO). In vitro, MPIO bound in a dose-dependent manner to tumor necrosis factor (TNF)-alpha stimulated sEND-1 endothelial cells when conjugated to VCAM-1 (R² = 0.88, P<0.01) and P-selectin antibodies (R² = 0.93, P<0.01), reflecting molecular VCAM-1 and P-selectin mRNA and protein expression. Mice subjected to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO and PSEL-MPIO (4.5 mg iron/kg body weight). In ischemic kidneys, MR related contrast effects of VCAM-MPIO were 4-fold higher than unclamped kidneys (P<0.01) and 1.5-fold higher than clamped kidneys of PSEL-MPIO injected mice (P<0.05). VCAM-MPIO binding was less evident in IRI kidneys pre-treated with VCAM-1 antibody (P<0.001). VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R² = 0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. In mice subjected to cerebral ischemia, contrast volume was 11-fold greater in animals injected with VCAM-MPIO versus control IgG-MPIO (P<0.05). Finally, S-nitroso-N-acetylpenicillamine (SNAP) stimulated HUVEC-C cells, which express αvβ3-integrin, showed 44-fold greater RGD-MPIO binding than unstimulated cells (P<0.001) and 4-fold greater RGD-MPIO binding than SNAP stimulated cells blocked with soluble RGD peptide (P<0.001) in-vitro. This thesis demonstrated that targeted MPIO exhibited contrast effects that defined and quantified the molecular expression of specific targets through the use of high-resolution MRI in in-vitro and in-vivo models of vascular inflammation.