Characterisation of the plasma membrane subproteome of bloodstream form Trypanosoma brucei

Proteome analysis by conventional approaches is biased against hydrophobic membrane proteins, many of which are also of low abundance. We have isolated plasma membrane sheets from bloodstream forms of Trypanosoma brucei by subcellular fractionation, and then applied a battery of complementary protein separation and identification techniques to identify a large number of proteins in this fraction. The results of these analyses have been combined to generate a subproteome for the pellicular plasma membrane of bloodstream forms of T. brucei as well as a separate subproteome for the pellicular cytoskeleton. In parallel, we have used in silico approaches to predict the relative abundance of proteins potentially expressed by bloodstream form trypanosomes, and to identify likely polytopic membrane proteins, providing quality control for the experimentally defined plasma membrane subproteome. We show that the application of multiple high-resolution proteomic techniques to an enriched organelle fraction is a valuable approach for the characterisation of relatively intractable membrane proteomes. We present here the most complete analysis of a protozoan plasma membrane proteome to date and show the presence of a large number of integral membrane proteins, including 11 nucleoside/nucleobase transporters, 15 ion pumps and channels and a large number of adenylate cyclases hitherto listed as putative proteins.

Membrane trafficking of aquaporin 1 is mediated by protein kinase C via microtubules and regulated by tonicity

It is well-known that the rapid flow of water into and out of cells is controlled by membrane proteins called aquaporins (AQPs). However, the mechanisms that allow cells to quickly respond to a changing osmotic environment are less well established. Using GFP-AQP fusion proteins expressed in HEK293 cells, we demonstrate the reversible manipulation of cellular trafficking of AQP1. AQP1 trafficking was mediated by the tonicity of the cell environment in a specific PKC- and microtubule-dependent manner. This suggests that the increased level of water transport following osmotic change may be due a phosphorylation-dependent increase in the level of AQP1 trafficking resulting in membrane localization.

Plasma membrane abundance of human aquaporin 5 is dynamically regulated by multiple pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.

Four methods for measuring the solubilities of gases and vapors in liquids and polymers

Peer reviewed

Four methods for measuring the solubilities of gases and vapors in liquids and polymers

Peer reviewed

Determination of gases in sediments

A method was developed for sampling and analysis of gases contained in interstitial water. Interstitial water was separated from sediments with a filter press-type sediment squeezer and transferred to a specially designed sampler-stripper. Gases contained in the interstitial water were stripped from solution and analyzed by gas chromatography. Argon, N2, and CH4 have been measured in sediments. The technique can be used to measure total CO2 and H2S with minor modifications.

Evaluation of the Genotoxicity of Chitosan Nanoparticles for Use in Food Packaging Films

The use of nanoparticles in food packaging has been proposed on the basis that it could improve protection of foods by, for example, reducing permeation of gases, minimizing odor loss, and increasing mechanical strength and thermal stability. Consequently, the impacts of such nanoparticles on organisms and on the environment need to be investigated to ensure their safe use. In an earlier study, Moura and others (2008a) described the effect of addition of chitosan (CS) and poly(methacrylic acid) (PMAA) nanoparticles on the mechanical properties, water vapor, and oxygen permeability of hydroxypropyl methylcellulose films used in food packaging. Here, the genotoxicity of different polymeric CS/PMAA nanoparticles (size 60, 82, and 111 nm) was evaluated at different concentration levels, using the Allium cepa chromosome damage test as well as cytogenetic tests employing human lymphocyte cultures. Test substrates were exposed to solutions containing nanoparticles at polymer mass concentrations of 1.8, 18, and 180 mg/L. Results showed no evidence of DNA damage caused by the nanoparticles (no significant numerical or structural changes were observed), however the 82 and 111 nm nanoparticles reduced mitotic index values at the highest concentration tested (180 mg/L), indicating that the nanoparticles were toxic to the cells used at this concentration. In the case of the 60 nm CS/PMAA nanoparticles, no significant changes in the mitotic index were observed at the concentration levels tested, indicating that these particles were not toxic. The techniques used show promising potential for application in tests of nanoparticle safety envisaging the future use of these materials in food packaging.

Lysossomal membrane stability of the brown mussel Perna perna (Linnaeus) (Mollusca, Bivalvia) exposed to the anionic surfactant Linear Alkylbenzene Sulphonate (LAS).

The effects of the Linear Alkylbenzene Sulphonate (LAS) were evaluated on the mussel Perna perna (Linnaeus, 1758), using a cellular level biomarker. The Neutral Red Retention Time (NRRT) assay was used to estimate effects at cellular levels. Significant effects were observed for the NRRT assay, even in low concentrations. The effects at cellular level were progressive, suggesting that the organisms are not capable to recover of such increasing effects. Additionally, the results show that the levels of LAS observed for Brazilian coastal waters may chronically affect the biota.

Influence of mini-Percoll techniques in sperm capacitation and plasma membrane integrity of ram frozen-thawed sperm.

2016

Modeling of non-isothermalvapor membrane separation with thermodynamic models and generalized mass transfer equations

A rigorous unit operation model is developed for vapor membrane separation. The new model is able to describe temperature, pressure, and concentration dependent permeation as wellreal fluid effects in vapor and gas separation with hydrocarbon selective rubbery polymeric membranes. The permeation through the membrane is described by a separate treatment of sorption and diffusion within the membrane. The chemical engineering thermodynamics is used to describe the equilibrium sorption of vapors and gases in rubbery membranes with equation of state models for polymeric systems. Also a new modification of the UNIFAC model is proposed for this purpose. Various thermodynamic models are extensively compared in order to verify the models' ability to predict and correlate experimental vapor-liquid equilibrium data. The penetrant transport through the selective layer of the membrane is described with the generalized Maxwell-Stefan equations, which are able to account for thebulk flux contribution as well as the diffusive coupling effect. A method is described to compute and correlate binary penetrant¿membrane diffusion coefficients from the experimental permeability coefficients at different temperatures and pressures. A fluid flow model for spiral-wound modules is derived from the conservation equation of mass, momentum, and energy. The conservation equations are presented in a discretized form by using the control volume approach. A combination of the permeation model and the fluid flow model yields the desired rigorous model for vapor membrane separation. The model is implemented into an inhouse process simulator and so vapor membrane separation may be evaluated as an integralpart of a process flowsheet.

Membrane design and characterisation by incorporation of ionic liquids

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Química, especialidade de Operações Unitárias e Fenómenos de Transferência, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia