Glaucoma is a group of optic nerve degenerative diseases, which leads to gradual and permanent vision loss. Recent developments in the field of gene therapy have proposed increasingly promising treatments for glaucoma, in the form of delivery of neuro-protective or neuro-regenerative genes to the retina. Despite these developments, there are concerns related to the biocompatibility and invasiveness of common gene delivery systems, since they are commonly mediated by viral gene carriers and invasive administration methods. Non-viral gene delivery systems offer a safe and increasingly efficient alternative to deliver therapeutic genes to the retina. An example of these systems is gemini-phospholipid nanoparticles (GL-NPs), which have been successfully used to deliver genes in similarly challenging anatomical settings, such as the skin. The objective of this thesis is to demonstrate the potential of GL-NPs, as candidate gene delivery vehicles for topically administered genes, targeted to the retina. The dicationic gemini surfactant, 12-7NH-12 was used, along with the helper lipids, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), to prepare various types of GL-NPs, and assess their transfection efficiency in the rat retinal ganglion cell line (RGC-5). The transfection efficiency was evaluated using flow cytometry, as a function of several physical and chemical parameters of GL-NPs. These include a range of charge ratios (5:1 to 15:1 ????), helper lipid composition (several DOPE: DPPC ratios), order of assembly (plasmid-gemini + lipid versus gemini-lipid + plasmid), and manufacturing method of helper lipid vesicles (thin film versus high pressure homogenization method). Size and zeta (??) potential characterization of GL-NPs was carried out in parallel, using dynamic light scattering, to relate the physical parameters of GL-NPs to their respective transfection efficiency. A comprehensive toxicological evaluation was undertaken to assess the extent of GL-NP???s toxicity in RGC-5 cells, using the resazurin-based PrestoblueTM cell toxicity assay. Optimized GL-NPs were used to induce expression of the brain derived neurotrophic factor (BDNF) in RGC-5 cells, and were assessed in terms of their capacity to induce neurite outgrowth. Quantification of neurite outgrowth was carried out by measuring average neurite length in RGC-5 cells, by confocal microscopic imaging of immunostained neurites. Furthermore, confocal microscopic studies were carried out to assess the extent of GL-NP???s corneal permeation in a 3-D human corneal epithelial (HCE) model. A parallel toxicological evaluation was completed to ensure GL-NP???s biocompatibility with the corneal epithelial cells. Finally, GL-NP biodistribution pattern and gene transfer capacity was assessed in a mouse model, following topical and intravitreal administration. The transfection efficiency in RGC-5 cells, which ranged between 2.1 ?? 0.3% and 14.5 ?? 1.4%, was highly dependent on GL-NP???s charge ratio, helper lipid composition, order of assembly, and manufacturing technique. GL-NPs at 10:1 ???? charge ratio, assembled with homogenized DOPE (25%)-DPPC (75%) helper lipid vesicles, in the plasmid-gemini + lipid order, mediated the highest transfection efficiency in RGC-5 cells. These GL-NPs had a size of 222.8 ?? 4.2 nm and a ?? potential of +33.5??2.9 mV. Optimized GL-NPs were highly biocompatible with both RGC-5 and HCE model cells, with viability values ranging between 94.8 ?? 6 % to 100 ?? 3.4 %. Assessment of corneal permeation showed that GL-NPs were able to bind to the corneal epithelial surface and achieve a moderate permeation depth (35-40 ??m), following topical application in the HCE model. Intravitreal injection of the non-viral GL-NPs in mice has successfully led to their localization within the nerve fiber layer (NFL) of the retina. Finally, GL-NPs were non-invasively delivered to several anterior chamber tissues, including the limbus, the iris and conjunctiva, following topical administration. GL-NPs offer several advantageous features critical to topical and intravitreal ocular administration of gene carriers, including in vitro corneal binding and effective biodistribution following in vivo topical and intravitreal administration, high biocompatibility, and a highly tunable transfection efficiency. The current data presents 12-7NH-12 GL-NPs as a promising candidate for ocular gene therapy applications.

This thesis describes the design and construction of a Brewster Angle Microscope (BAM) to investigate monolayer films at the air/water interface. A Schiff base coordination polymer, Cu(ll) 5,5'-methylenebis(N-hexadecyl- sallcylideneamine (poly(CuMBSH)), was initially investigated using the BAM equipment. This material is being developed as the active material in vapour sensing devices produced by the Langmuir Blodgett (LB) method. Transfer of the monomer (MBSH) to the substrate was found to be poor but poly(CuMBSH) had a good deposition ratio (>0.95). The film was polymerised at the air/water interface by injecting a solution of Cu(ll) ions into the subphase. BAM was able to record the polymerisation of MBSH to poly(CuMBSH) in real time. Qualitative image analysis indicates a reordering of the material at the interface and a decrease in film thickness. The technique of BAM clearly displays the change in structure between the monomer film and the polymer film and that the poly(CuMBSH) film is homogenous at the micron level. Poly(para-phenylenevinylenes) PPV derivatives are presently being examined as potential LED devices. One method used to produce such devices is the LB method. BAM was used to investigate ordering of the monolayer prior to deposition. Results by the Physics and Engineering department indicated that if subphase contained water that had been left to stand for several days film transfer was improved. A range of BAM experiments were conducted with varying subphases to determine the cause of this effect. A new technique of determining the surface excess concentration was developed in this work which is known as Brewster Reflectivity (BAR). The reflectivity of simple surfactants; sodium dodecylbenzenesulfonate, Getyltrimethylammonium bromide, tetradecyltrimethylammonium bromide and dodecyltrimethyl-ammonium bromide at concentrations above and below the critical micelle concentration (CMC) were correlated to surface excess. This is a new, simple, non-invasive method for probing the surface excess using intrinsic properties of the system.

Bibliography: leaves 209-254. / viii, 254, [39] leaves, [17] leaves of plates : ill. 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Attempts to construct an evolutionary overview of the regulation of surfactant secretion among the vertebrates. A detailed whole animal and in vitro study of the factors that control surfactant secretion and function in the central Australian agamid lizard Pogona vitticeps was undertaken. Type II pneumocytes were also isolated and cultured from Australian lungfish, North American bullfrogs and fat-tailed dunnarts. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1999

Pulmonary surfactant is a complex mixture of lipids and proteins that lowers surface tension, increases lung compliance, and prevents the adhesion of respiratory surfaces and pulmonary oedema. Pressure can have an enormous impact on respiratory function, by mechanically compressing tissues, increasing gas tension resulting in increased gas absorption and by increasing dissolved gas tensions during diving, resulting in the formation of bubbles in the blood and tissues. The lungs of diving mammals have a huge range of morphological adaptations to enable them to endure the extremely high pressures associated with deep diving. Here, I hypothesise that surfactant will also be modified, to complement the morphological changes and enable more efficient lung function during diving. Molecular adaptations to diving were examined in surfactant protein C (SP-C) using phylogenetic analyses. The composition and function of pulmonary surfactant from several species of diving mammals was examined using biochemical assays, mass spectrometry and captive bubble surfactometry. The development of surfactant in one species of diving mammal (California sea lion), and the control of surfactant secretion using chemical and mechanical stimuli were also determined. Diving mammals showed modifications to SP-C, which are likely to lead to stronger binding to the monolayer, thereby increasing its fluidity. Phospholipid molecular species concentrations were altered to increase the concentration of more fluid species. There was also an increase in the percentage of alkyl molecular species, which may increase the stability of the monolayer during compression and facilitate rapid respreading. Levels of SP-B were much lower in the diving species, and cholesterol was inversely proportional to the maximum dive depth of the three species. Surface activity of surfactant from diving mammals was very poor compared to surfactant from terrestrial mammals. The newborn California sea lion surfactant was similar to terrestrial mammal surfactant, suggesting that these animals develop the diving-type of surfactant after they first enter the water. The isolated cells of California sea lions also showed a similar response to neuro-hormonal stimulation as terrestrial mammals, but were insensitive to pressure. These findings showed diving mammal surfactant to have a primarily anti-adhesive function that develops after the first entry into the water, with a surfactant monolayer, which would be better suited to repeated collapse and respreading. / Thesis (Ph.D.)--School of Earth and Environmental Sciences, 2005.

Thesis (M.Sc.)--University of Adelaide, Department of Environmental Biology, 2001. / Includes bibliographical references.

Zugl.: Berlin, Freie Universiẗat, Diss., 2008.

Thesis supervisor: Madeline A. Shea. Includes bibliographical references (p. 195-206).

Bibliography: leaves 209-254. Attempts to construct an evolutionary overview of the regulation of surfactant secretion among the vertebrates. A detailed whole animal and in vitro study of the factors that control surfactant secretion and function in the central Australian agamid lizard Pogona vitticeps was undertaken. Type II pneumocytes were also isolated and cultured from Australian lungfish, North American bullfrogs and fat-tailed dunnarts.

In this diploma thesis has been studied system of hydrogels based on interaction between polyelectrolyte and surfactants, especially their solubilizing properties. As a surfactant was used ionic cationic cetyltrimethylammonium bromide (CTAB). As a polyelectrolyte was chosen native anionic hyaluronan with various molecular weight - 0,3; 0,9; 1,6 MDa and with different concentration - 2%, 4%, 6% wt. Solubilization experiments were realized with hydrophobic dye sudan red G. The whole system was prepared in the simpliest model system of physiological solution 0,15 M NaCl and in water. At the prepared gels was investigated their stability, influence of the environment, molecular weight and concentration on solubilization. Gels were prepared in rate hyaluronan-surfactant 1:1. The purpose of this diploma thesis was to find and optimize the appropriate procedure for determining solubilization capacity by using UV-VIS spectrophotometry.

Neste trabalho estudou-se a formação de complexos entre polieletrólitos e surfatantes de cargas opostas e uma nova rota para sintetizar partículas poliméricas estáveis, na qual a polimerização ocorre dentro dos complexos. O comportamento de misturas formadas pelo poliânion carboximetilcelulose (CMC) e o surfatante catiônico brometo de cetiltrimetilamônio (CTAB) e pelo policátion quitosana (CH) e o surfatante aniônico dodecilsulfato de sódio (SDS) foi investigado na interface líquido-ar por medidas de tensão superficial e na solução através de medidas de turbidez e condutividade. Foi avaliado o efeito da massa molar viscosimétrica média e da densidade de carga dos polieletrólitos na formação dos complexos. A síntese de poliestireno e poli(metacrilato de metila) em emulsão foi realizada na concentração de agregação crítica (cac) do sistema CMC/CTAB. As partículas híbridas foram caracterizadas por medidas de potencial zeta, espalhamento de luz e microscopia eletrôncia de varredura. Todas as dispersões foram estáveis na força iônica de 2,0 mol L-1 de NaCl durante quatro dias. A estabilidade coloidal foi atribuída à presença de uma camada de hidratação formada pela CMC em volta das partículas. Esta nova rota sintética tem a vantagem de produzir partículas de látex estabilizadas com uma quantidade reduzida de surfatante. / This work reports the complex formation between polyelectrolytes and opposite charged surfactants, and a new procedure to synthesize stable polymeric particles, where the polymerization takes place inside the complex. The behavior of mixtures of carboxymethylcellulose (CMC) and the cationic surfactant cetyltrimethylammonium bromide (CTAB), and chitosan (CH) and the anionic surfactant sodium dodecilsulfate (SDS) was investigated in the diluted range at the liquid-air interface by means of surface tension, and in the bulk solution by turidity and conductivity measurements. The effect of the molecular weight and charge density of the polyelectrolytes on the complexation with oppositely charged surfactants was studied. The synthesis of polystyrene or poly(methyl methacrylate) was carried out by emulsion polymerization in the critical aggregation concentration (cac) of the CMC/CTAB system. The hybrid particles were characterized by zeta potential, and light scattering measurements, and scanning electron microscopy. All dispersions were stable in the ionic strength of 2.0 mol/L NaCl at least during four days. The colloidal stability was attributed to the presence of a hydrated CMC layer around the particles. The present procedure brings the advantage of synthesizing and stabilizing particles with funciotnal groups on the surface in a one-step method using very small amounts of surfactant, a friendly condition for the environment.