No description available.

The presence of cholesterol is critical in defining a dynamic lateral structure in pulmonary surfactant membranes, including the segregation of fluid-ordered and fluid-disordered phases. However, an excess of cholesterol has been associated with impaired surface activity both in surfactant models and in surfactant from injured lungs. It has also been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films. In the present study, we have analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholesterol and on the ability of lipid/protein complexes containing surfactant proteins and cholesterol to form and re-spread interfacial films capable of producing very low surface tensions upon repetitive compression-expansion cycling. We have also analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

The objective of this thesis was to further develop a methodology for surface tension measurement called Axisymmetric Drop Shape Analysisn(ADSA) and to adapt it to studies of lung surfactants, i.e. the material that coats and facilitates the functioning of the lungs of all mammals. The key property of a functioning lung surfactant is its surface tension, which can reach extremely low values. Such values are difficult to measure; but a certain configuration of ADSA, using a constrained sessile drop (ADSA--CSD), is capable of performing such measurements. Clinically, lung surfactant films can be altered from both sides, i.e. from the airspace as well as from the bulk liquid phase that carries the film. Therefore, being able to access the interface from both sides is important. Here, ADSA--CSD was redesigned to be used as a micro film balance allowing access to the interface from both gas- and liquid-side. This allows deposition from the gas side as well as complete exchange of the bulk liquid phase. The new design was used to study lung surfactant inhibition and inhibition reversal. A dynamic compression-relaxation model (CRM) was developed to describe the mechanical properties of lung surfactant films by investigating the response of surface tension to changes in surface area. The model evaluates the quality of lung surfactant preparations -- beyond the minimum surface tension value -- and calculates the film properties, i.e. elasticity, adsorption and relaxation, independent of the compression protocol. The accuracy of the surface tension measurement can depend on drop size. A detailed analysis of drop shapes and accuracy of measured surface tension values was performed using a shape parameter concept. Based on this analysis, the design of ADSA--CSD was optimized to facilitate more accurate measurements. The validity analysis was further extended to the more conventional pendant drop setup (ADSA--PD). An overall upgrade of both hardware and software of ADSA--CSD, together with extensive numerical work, is described and applied to facilitate a more efficient operation. Finally, it is noted that the ADSA--CSD setup developed here can be used for a wide range of colloid and surface chemical applications.

The objective of this thesis was to further develop a methodology for surface tension measurement called Axisymmetric Drop Shape Analysisn(ADSA) and to adapt it to studies of lung surfactants, i.e. the material that coats and facilitates the functioning of the lungs of all mammals. The key property of a functioning lung surfactant is its surface tension, which can reach extremely low values. Such values are difficult to measure; but a certain configuration of ADSA, using a constrained sessile drop (ADSA--CSD), is capable of performing such measurements. Clinically, lung surfactant films can be altered from both sides, i.e. from the airspace as well as from the bulk liquid phase that carries the film. Therefore, being able to access the interface from both sides is important. Here, ADSA--CSD was redesigned to be used as a micro film balance allowing access to the interface from both gas- and liquid-side. This allows deposition from the gas side as well as complete exchange of the bulk liquid phase. The new design was used to study lung surfactant inhibition and inhibition reversal. A dynamic compression-relaxation model (CRM) was developed to describe the mechanical properties of lung surfactant films by investigating the response of surface tension to changes in surface area. The model evaluates the quality of lung surfactant preparations -- beyond the minimum surface tension value -- and calculates the film properties, i.e. elasticity, adsorption and relaxation, independent of the compression protocol. The accuracy of the surface tension measurement can depend on drop size. A detailed analysis of drop shapes and accuracy of measured surface tension values was performed using a shape parameter concept. Based on this analysis, the design of ADSA--CSD was optimized to facilitate more accurate measurements. The validity analysis was further extended to the more conventional pendant drop setup (ADSA--PD). An overall upgrade of both hardware and software of ADSA--CSD, together with extensive numerical work, is described and applied to facilitate a more efficient operation. Finally, it is noted that the ADSA--CSD setup developed here can be used for a wide range of colloid and surface chemical applications.

[pt] O surfactante pulmonar é um sistema lipo-proteico que atua na interface alveolar com vital importância para manter funcional a mecânica respiratória. Os comprometimentos na sua função estão associados a diversas infecções pulmonares. Os sistemas de administração de fármacos baseados em surfactantes pulmonares derivados de animais são complexos, dificultando a compreensão do papel individual das moléculas hóspedes nas suas interações com a membrana. Aqui apresentamos uma caracterização de um extrato surfactante de pulmão porcino de grau clínico misturado com os antibióticos Levofloxacina e Claritromicina, usando uma abordagem multi-técnica – em conjunto com a metodologia de monocamadas de Langmuir– consistindo de isotermas de pressão de superfície-area, microscopia de ângulo de Brewster (BAM), espectroscopia de reflexão-absorção do infravermelho com modulação da polarização (PM-IRRAS), reflectometria de nêutrons (NR), ensaios in vitro e simulações de dinâmica molecular. Avaliou-se o efeito de ambos os antibióticos na estrutura das monocamadas de surfactantes de origem porcino bem como em monocamadas de DPPC. Foi revelado que a estabilidade / integridade das monocamadas é preservada na presença de ambas as drogas. Os sistemas mistos de antibiótico / surfactante pulmonar aumentam a atividade antibacteriana contra bactérias Gram-positivas (Bacillus cereus) e Gram-negativas (Escherichia coli). Essas descobertas fornecem novas percepções sobre a otimização de sistemas eficientes de administração de medicamentos para o tratamento de condições patológicas no nível respiratório. / [en] The lipo-proteic surfactant system acting at the alveolar interface is of vital importance for keeping functional the respiratory mechanics. Its impairments are associated with several pulmonary infections. Drug delivery systems based on animal-derived lung surfactants are complex making it difficult to understand the individual role of guest molecules in membrane interactions. Here we present a characterization of a clinical-grade porcine lung surfactant extract mixed with the antibiotics Levofloxacin and Clarithromycin, using a multi-technique approach –in conjunction with the Langmuir-monolayer methodology– consisting of surface pressure-area isotherms, Brewster angle microscopy (BAM), polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), neutron reflectometry (NR), in vitro assays, and molecular dynamics simulations. The effect of both antibiotics in the structure of porcine lung surfactant monolayers as well as in DPPC monolayers was examined. It was revealed that the stability/integrity of the monolayers is preserved in the presence of both drugs. The mixed antibiotic/lung surfactant systems enhance the antibacterial activity against Gram-positive (Bacillus cereus) and Gram-negative (Escherichia coli) bacteria. These findings provide new insights into the optimization of efficient drug delivery systems for the treatment of pathological conditions at the respiratory level.

No description available.

The overall objective of this PhD thesis research is to understand and control phase separation in mixed perfluorinated fatty acid-phospholipid surfactant systems that have applications as pulmonary surfactant (PS) mixtures, with an ultimate view of controlling film composition, morphology and mechanical properties. In this context the interaction between perfluorooctadecanoic acid (C18F), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the major component of native PS extract, and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) has been explored in Langmuir monolayers and Langmuir–Blodgett (LB) films using a combination of atomic force microscopy (AFM), fluorescence microscopy (FM) and Brewster angle microscopy (BAM) measurements. Thermodynamic and morphological studies of binary and ternary mixed films made of C18F, DPPC and DPPG indicated that both the phospholipids and C18F were miscible over a wide range of compositions. The mixed phospholipid-C18F films contained multimolecular aggregates that were highly enriched in the phospholipids. Furthermore, it was found that the magnitude of the DPPC-C18F interaction could be modulated by altering the concentration of sodium ions in the underlying subphase. Using a highly simplified lung mimic fluid (pH 7.4, 150mM NaCl), DPPC and C18F became fully immiscible. Moreover, the performance characteristics of the mixed films demonstrated the usefulness of C18F as an additive for PS formulations. The effectiveness of a PS protein mimicking peptide was evaluated against DPPC to allow comparison with previous measurements of DPPC-C18F mixed system. The mixing thermodynamics of the peptide and DPPC in Langmuir monolayer implied a repulsive interaction between the film components. The hysteresis response of the mixed monolayer films indicated that the lipid-protein mixture improved the re-spreading of DPPC films. Moreover, molecular-level organization of the mixed films explored by both FM and BAM confirmed the formation of liquid-expanded DPPC domains in the presence of minute amount of the peptide. In order to obtain a thorough understanding of the effect of the deposition process and surfactant tail polarities on the interfacial behavior of perfluorocarbon-hydrocarbon mixed monolayer films, both BAM and AFM measurements of arachidic acid (C20) with perfluorotetradecanoic acid (C14F) and palmitic acid (C16) with C18F mixed monolayer were performed. These measurements revealed that film morphology was minimally perturbed upon its deposition onto solid substrates. Coarse grained molecular dynamics (MD) simulations of films comprised of DPPC molecules with tails of various polarities suggested that the phase separation between the monolayer components could be controlled by varying surfactant tail polarities.