Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

Computer aided design and analysis of cold formed sections

The work reported in this thesis is concerned with the improvement and expansion of the assistance given to the designer by the computer in the design of cold formed sections. The main contributions have been in four areas, which have consequently led to the fifth, the development of a methodology to optimise designs. This methodology can be considered an `Expert Design System' for cold formed sections. A different method of determining section properties of profiles was introduced, using the properties of line and circular elements. Graphics were introduced to show the outline of the profile on screen. The analysis of beam loading has been expanded to beam loading conditions where the number of supports, point loads, and uniform distributive loads can be specified by the designer. The profile can then be checked for suitability for the specified type of loading. Artificial Intelligence concepts have been introduced to give the designer decision support from the computer, in combination with the computer aided design facilities. The more complex decision support was adopted through the use of production rules. All the support was based on the British standards. A method has been introduced, by which the appropriate use of stiffeners can be determined and consequently designed by the designer. Finally, the methodology by which the designer is given assistance from the computer, without constraining the designer, was developed. This methodology gives advice to the designer on possible methods of improving the design, but allows the designer to reject that option, and analyse the profile accordingly. The methodology enables optimisation to be achieved by the designer, designing variety of profiles for a particular loading, and determining which one is best suited.

Advanced fibre gratings fabricated in standard and infrared glass fibres by ultraviolet and near-infrared-femtosecond lasers

In this thesis, I describe studies on fabrication, spectral characteristics and applications of tilted fibre gratings (TFGs) with small, large and 45° tilted structures and novel developments in fabrication of fibre Bragg gratings (FBGs) and long period gratings (LPGs) in normal silica and mid-infrared (mid-IR) glass fibres using near-IR femtosecond laser. One of the major contributions presented in this thesis is the systematic investigation of structures, inscription methods and spectral, polarisation dependent loss (PDL) and thermal characteristics of TFGs with small (<45°), large (>45°) and 45° tilted structures. I have experimentally characterised TFGs, obtaining relationships between the radiation angle, central wavelength of the radiation profile, Bragg resonance and the tilt angle, which are consistent with theoretical simulation based on the mode-coupling theory. Furthermore, thermal responses have been measured for these three types of TFGs, showing the transmission spectra of large and 45° TFGs are insensitive to the temperature change, unlike the normal and small angle tilted FBGs. Based on the distinctive optical properties, TFGs have been developed into interrogation system and sensors, which form the other significant contributions of the work presented in this thesis. The 10°-TFG based 800nm WDM interrogation system can function not just as an in-fibre spectrum analyser but also possess refractive index sensing capability. By utilising the unique polarisation properties, the 81 °-TFG based sensors are capable of sensing the transverse loading and twisting with sensitivities of 2.04pW/(kg/m) and 145.90pW/rad, repectively. The final but the most important contribution from the research work presented in this thesis is the development of novel grating inscription techniques using near-IR femtosecond laser. A number of LPGs and FBGs were successfully fabricated in normal silica and mid-IR glass fibres using point-by-point and phase-mask techniques. LPGs and 1st and 2nd order FBGs have been fabricated in these mid-IR glass fibres showing resonances covering the wavelength range from 1200 to 1700nm with the strengths up to 13dB. In addition, the thermal and strain sensitivities of these gratings have been systematically investigated. All the results from these initial but systematic works will provide useful function characteristics information for future fibre grating based devices and applications in mid-IR range.

In situ mineralization by the release of calcium and phosphate ions from nanogels

Biomimetic hydroxyapatite was synthesized by the controlled release of calcium and phosphate ions from poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAAm-co-AA)) nanogels. Mixing nanogels containing calcium chloride (CaCl2 ·2H2O) and nanogels containing sodium hydrogen phosphate (Na2HPO4·2H2O) in simulated body fluid (SBF) at physiological conditions of 37 °C and pH 7.4, biomimetic hydroxyapatite was obtained. By studying separately the loading and controlled release of the salts from the nanogels, adequate conditions were chosen to synthesize the hydroxyapatite: Calcium loaded (Ca-loaded) nanogels (1000 mg/ml; 400:3) and inorganic phosphate loaded (Pi-loaded) nanogels (90 mg/ml; 12:1) in a ratio of 2:1 were placed in SBF solution. The obtained powders characterization showed that a low crystalline and substituted hydroxyapatite similar to bone apatite was formed. Such a strategy could be used in medical and dental procedures to induce rapid inorganic mineral formation from a nanogel-containing biomaterial. © 2012 American Scientific Publishers. All rights reserved.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives - Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6'-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn-glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B–ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods - Liposomal were prepared at a 5?:?1 DDA–TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings - As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion - These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system.

Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants

Objectives Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) combined with trehalose 6,6′-dibehenate (TDB) elicit strong cell-mediated and antibody immune responses; DDA facilitates antigen adsorption and presentation while TDB potentiates the immune response. To further investigate the role of DDA, DDA was replaced with the neutral lipid of distearoyl-sn- glycero-3-phosphocholine (DSPC) over a series of concentrations and these systems investigated as adjuvants for the delivery of Ag85B-ESAT-6-Rv2660c, a multistage tuberculosis vaccine. Methods Liposomal were prepared at a 5: 1 DDA-TDB weight ratio and DDA content incrementally replaced with DSPC. The physicochemical characteristics were assessed (vesicle size, zeta potential and antigen loading), and the ability of these systems to act as adjuvants was considered. Key findings As DDA was replaced with DSPC within the liposomal formulation, the cationic nature of the vesicles decreases as does electrostatically binding of the anionic H56 antigen (Hybrid56; Ag85B-ESAT6-Rv2660c); however, only when DDA was completed replaced with DSPC did vesicle size increase significantly. T-helper 1 (Th1)-type cell-mediated immune responses reduced. This reduction in responses was attributed to the replacement of DDA with DSPC rather than the reduction in DDA dose concentration within the formulation. Conclusion These results suggest Th1 responses can be controlled by tailoring the DDA/DSPC ratio within the liposomal adjuvant system. © 2013 Royal Pharmaceutical Society.

Effects of ball-milling on PLGA polymer and its implication on lansoprazole-loaded nanoparticles

PLGA is a biodegradable polymer utilised widely in pharmaceutical research for the encapsulation of a wide range of drugs as nano particulate systems. This study investigates the impact of rotary ball milling on the physical properties of PLGA and its influence on nanoparticle formation prepared using the solvent displacement technique. By applying mechanical stress to the polymer and altering its physical appearance and molecular weight, the loading of lansoprazole within the nanoparticles was increased to 96%, with a reduction in particle size. The results indicate that rotary ball milling significantly reduces particle size, increases lansoprazole loading and improves the release profile for lansoprazole loaded PLGA nanoparticles.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.

Dynamics of ecosystem metabolism and flocculent detritus transport in estuarine Taylor River

Estuaries and estuarine wetlands are ecologically and societally important systems, exhibiting high rates of primary production that fuel offshore secondary production. Hydrological processes play a central role in shaping estuarine ecosystem structure and function by controlling nutrient loading and the relative contributions of marine and terrestrial influences on the estuary. The Comprehensive Everglades Restoration Plan includes plans to restore freshwater delivery to Taylor Slough, a shallow drainage basin in the southern Everglades, ultimately resulting in increased freshwater flow to the downstream Taylor River estuary. The existing seasonal and inter-annual variability of water flow and source in Taylor River affords the opportunity to investigate relationships between ecosystem function and hydrologic forcing. Estimates of aquatic ecosystem metabolism, derived from free-water, diel changes in dissolved oxygen, were combined with assessments of wetland flocculent detritus quality and transport within the context of seasonal changes in Everglades hydrology. Variation in ecosystem gross primary production and respiration were linked to seasonal changes in estuarine water quality using multiple autoregression models. Furthermore, Taylor River was observed to be net heterotrophic, indicating that an allochthonous source of carbon maintained ecosystem respiration in excess of autochthonous primary production. Wetland-derived detritus appears to be an important vector of energy and nutrients across the Everglades landscape; and in Taylor River, is seasonally flushed into ponded segments of the river where it is then respired. Lastly, seasonal water delivery appears to govern feedbacks regulating water column phosphorus availability in the Taylor River estuary.

Phosphorus budgets in Everglades wetland ecosystems: the effects of hydrology and nutrient enrichment

The Florida Everglades is a naturally oligotrophic hydroscape that has experienced large changes in ecosystem structure and function as the result of increased anthropogenic phosphorus (P) loading and hydrologic changes. We present whole-ecosystem models of P cycling for Everglades wetlands with differing hydrology and P enrichment with the goal of synthesizing existing information into ecosystem P budgets. Budgets were developed for deeper water oligotrophic wet prairie/slough (‘Slough’), shallower water oligotrophic Cladium jamaicense (‘Cladium’), partially enriched C. jamaicense/Typha spp. mixture (‘Cladium/Typha’), and enriched Typha spp. (‘Typha’) marshes. The majority of ecosystem P was stored in the soil in all four ecosystem types, with the flocculent detrital organic matter (floc) layer at the bottom of the water column storing the next largest proportion of ecosystem P pools. However, most P cycling involved ecosystem components in the water column (periphyton, floc, and consumers) in deeper water, oligotrophic Slough marsh. Fluxes of P associated with macrophytes were more important in the shallower water, oligotrophic Cladium marsh. The two oligotrophic ecosystem types had similar total ecosystem P stocks and cycling rates, and low rates of P cycling associated with soils. Phosphorus flux rates cannot be estimated for ecosystem components residing in the water column in Cladium/Typha or Typha marshes due to insufficient data. Enrichment caused a large increase in the importance of macrophytes to P cycling in Everglades wetlands. The flux of P from soil to the water column, via roots to live aboveground tissues to macrophyte detritus, increased from 0.03 and 0.2 g P m−2 yr−1 in oligotrophic Slough and Cladium marsh, respectively, to 1.1 g P m−2 yr−1 in partially enriched Cladium/Typha, and 1.6 g P m−2 yr−1 in enriched Typha marsh. This macrophyte translocation P flux represents a large source of internal eutrophication to surface waters in P-enriched areas of the Everglades.

Neuromuscular Changes in Older Adults during the Lateral Step Task

Older adults may have trouble when performing activities of daily living due to decrease in physical strength and degradation of neuromotor and musculoskeletal function. Motor activation patterns during Lateral Step Down and Step Up from 4-inch and 8-inch step heights was assessed in younger (n=8, 24.4 years) and older adults (n=8, 58.9 years) using joint angle kinematics and electromyography of lower extremity muscles. Ground reaction forces were used to ascertain the loading, stabilization and unloading phases of the tasks. Older adults had an altered muscle activation sequence and significantly longer muscle bursts during loading for the tibialis anterior, gastrocnemius, vastus medialis, bicep femoris, gluteus medius and gluteus maximus muscles of the stationary leg. They also demonstrated a significantly larger swing time (579.1 ms vs. 444.8 ms) during the step down task for the moving leg. The novel data suggests presence of age-related differences in motor coordination during lateral stepping.

Isotopic data of atmospheric methane from EDML and Vostok ice cores over a full glacial cycle

The response of natural CH4 sources to climate changes will be an important factor to consider as concentrations of this potent greenhouse gas continue to increase. Polar ice cores provide the means to assess this sensitivity in the past and have shown a close connection between CH4 levels and northern hemisphere temperature variability over the last glacial cycle. However, the contribution of the various CH4 sources and sinks to these changes is still a matter of debate. Contemporaneous stable CH4 isotope records in ice cores provide additional boundary conditions for assessing changes in the CH4 sources and sinks. Here we present new ice core CH4 isotope data covering the last 160,000 years, showing a clear decoupling between CH4 loading and carbon isotopic variations over most of the record. We suggest that d13CH4 variations were not dominated by a change in the source mix but rather by climate- and CO2-related ecosystem control on the isotopic composition of the methane precursor material, especially in seasonally inundated wetlands in the tropics. In contrast, relatively stable d13CH4 intervals occurred during large CH4 loading changes concurrently with past climate changes implying that most CH4 sources (most notably tropical wetlands) responded simultaneously.

Physical Insights, Steady Aerodynamic Effects, and a Design Tool for Low-Pressure Turbine Flutter

The successful, efficient, and safe turbine design requires a thorough understanding of the underlying physical phenomena. This research investigates the physical understanding and parameters highly correlated to flutter, an aeroelastic instability prevalent among low pressure turbine (LPT) blades in both aircraft engines and power turbines. The modern way of determining whether a certain cascade of LPT blades is susceptible to flutter is through time-expensive computational fluid dynamics (CFD) codes. These codes converge to solution satisfying the Eulerian conservation equations subject to the boundary conditions of a nodal domain consisting fluid and solid wall particles. Most detailed CFD codes are accompanied by cryptic turbulence models, meticulous grid constructions, and elegant boundary condition enforcements all with one goal in mind: determine the sign (and therefore stability) of the aerodynamic damping. The main question being asked by the aeroelastician, ``is it positive or negative?'' This type of thought-process eventually gives rise to a black-box effect, leaving physical understanding behind. Therefore, the first part of this research aims to understand and reveal the physics behind LPT flutter in addition to several related topics including acoustic resonance effects. A percentage of this initial numerical investigation is completed using an influence coefficient approach to study the variation the work-per-cycle contributions of neighboring cascade blades to a reference airfoil. The second part of this research introduces new discoveries regarding the relationship between steady aerodynamic loading and negative aerodynamic damping. Using validated CFD codes as computational wind tunnels, a multitude of low-pressure turbine flutter parameters, such as reduced frequency, mode shape, and interblade phase angle, will be scrutinized across various airfoil geometries and steady operating conditions to reach new design guidelines regarding the influence of steady aerodynamic loading and LPT flutter. Many pressing topics influencing LPT flutter including shocks, their nonlinearity, and three-dimensionality are also addressed along the way. The work is concluded by introducing a useful preliminary design tool that can estimate within seconds the entire aerodynamic damping versus nodal diameter curve for a given three-dimensional cascade.

Inheritance of the CENP-A chromatin domain is spatially and temporally constrained at human centromeres.

BACKGROUND: Chromatin containing the histone variant CENP-A (CEN chromatin) exists as an essential domain at every centromere and heritably marks the location of kinetochore assembly. The size of the CEN chromatin domain on alpha satellite DNA in humans has been shown to vary according to underlying array size. However, the average amount of CENP-A reported at human centromeres is largely consistent, implying the genomic extent of CENP-A chromatin domains more likely reflects variations in the number of CENP-A subdomains and/or the density of CENP-A nucleosomes within individual subdomains. Defining the organizational and spatial properties of CEN chromatin would provide insight into centromere inheritance via CENP-A loading in G1 and the dynamics of its distribution between mother and daughter strands during replication. RESULTS: Using a multi-color protein strategy to detect distinct pools of CENP-A over several cell cycles, we show that nascent CENP-A is equally distributed to sister centromeres. CENP-A distribution is independent of previous or subsequent cell cycles in that centromeres showing disproportionately distributed CENP-A in one cycle can equally divide CENP-A nucleosomes in the next cycle. Furthermore, we show using extended chromatin fibers that maintenance of the CENP-A chromatin domain is achieved by a cycle-specific oscillating pattern of new CENP-A nucleosomes next to existing CENP-A nucleosomes over multiple cell cycles. Finally, we demonstrate that the size of the CENP-A domain does not change throughout the cell cycle and is spatially fixed to a similar location within a given alpha satellite DNA array. CONCLUSIONS: We demonstrate that most human chromosomes share similar patterns of CENP-A loading and distribution and that centromere inheritance is achieved through specific placement of new CENP-A near existing CENP-A as assembly occurs each cell cycle. The loading pattern fixes the location and size of the CENP-A domain on individual chromosomes. These results suggest that spatial and temporal dynamics of CENP-A are important for maintaining centromere identity and genome stability.

Development of nanostructured hydrogel for spatial and temporal controlled release of active compounds

L’utilisation de nanovecteurs pour la livraison contrôlée de principes actifs est un concept commun de nous jours. Les systèmes de livraison actuels présentent encore cependant des limites au niveau du taux de relargage des principes actifs ainsi que de la stabilité des transporteurs. Les systèmes composés à la fois de nanovecteurs (liposomes, microgels et nanogels) et d’hydrogels peuvent cependant permettre de résoudre ces problèmes. Dans cette étude, nous avons développé un système de livraison contrôlé se basant sur l’incorporation d’un nanovecteur dans une matrice hydrogel dans le but de combler les lacunes des systèmes se basant sur un vecteur uniquement. Une telle combinaison pourrait permettre un contrôle accru du relargage par stabilisation réciproque. Plus spécifiquement, nous avons développé un hydrogel structuré intégrant des liposomes, microgels et nanogels séparément chargés en principes actifs modèles potentiellement relargués de manière contrôlé. Ce contrôle a été obtenu par la modification de différents paramètres tels que la température ainsi que la composition et la concentration en nanovecteurs. Nous avons comparé la capacité de chargement et la cinétique de relargage de la sulforhodamine B et de la rhodamine 6G en utilisant des liposomes de DOPC et DPPC à différents ratios, des nanogels de chitosan/acide hyaluronique et des microgels de N-isopropylacrylamide (NIPAM) à différents ratios d’acide méthacrylique, incorporés dans un hydrogel modèle d’acrylamide. Les liposomes présentaient des capacités de chargement modérés avec un relargage prolongé sur plus de dix jours alors que les nanogels présentaient des capacités de chargement plus élevées mais une cinétique de relargage plus rapide avec un épuisement de la cargaison en deux jours. Comparativement, les microgels relarguaient complétement leur contenu en un jour. Malgré une cinétique de relargage plus rapide, les microgels ont démontré la possibilité de contrôler finement le chargement en principe actif. Ce contrôle peut être atteint par la modification des propriétés structurelles ou en changeant le milieu d’incubation, comme l’a montré la corrélation avec les isothermes de Langmuir. Chaque système développé a démontré un potentiel contrôle du taux de relargage, ce qui en fait des candidats pour des investigations futures.