Abstract The carbonic anhydrase (CA) gene family contains eleven active members, the basic physiological functions of which are linked to the interconversion of carbon dioxide and bicarbonate (CO2 + H2O ⇔ H+ + HCO3⇔ H2CO3-). They participate in a variety of physiological processes that involve pH regulation, CO2 and HCO3- transport and water and electrolyte balance, and some new functions have also been suggested recently. A novel tumour-associated antigen, MN, containing a CA-domain and named MN/CA IX, has been found to promote cell proliferation when transfected into NIH3T3 cells and has also been shown to be a potential biomarker for neoplasia in the uterine cervix. The present study examines the expression of MN/CA IX in the normal alimentary tract by immunohistochemistry and compares it with the expression of cytoplasmic CA I, CA II, apical plasma membrane associated CA IV and secretory CA VI. The distribution of mitochondrial CA V is examined by immunohistochemistry and Western blotting. The value of MN/CA IX as a potential biomarker of gastrointestinal tumours is assessed in a series of colorectal and hepatobiliary neoplasms. A positive immunoreaction for MN/CA IX was detected in the basolateral plasma membrane of the gastric, intestinal and biliary epithelium, but was confined to the proliferating cryptal enterocytes in the human gut, suggesting a role in cellular proliferation. In colorectal tumours, MN/CA IX immunoreaction was also located in the proliferative zone, indicating that it could be a useful marker of cellular proliferation. In the case of hepatobiliary tumours a positive signal was mainly associated with tumours of biliary epithelial parentage. These results demonstrate that MN/CA IX has a unique expression pattern in the alimentary tract relative to other CAs. Its localization and enzymatic properties suggest that it may have a dual function in the gastrointestinal epithelium. Through its CA activity it could participate in the regulation of carbon dioxide/bicarbonate homeostasis, while its localization to the basolateral surfaces of proliferating cryptal enterocytes suggests that it may serve as a ligand or receptor for one or more other proteins that regulate intercellular communication and/or cell proliferation. MN/CA IX may also serve as a new biomarker of gastrointestinal tumours.

Chemical and electrochemical methods for depositing platinum microparticles at the membrane-electrode interface of solid polymer fuel cell oxygen reduction electrodes have been developed and studied.

Considerable evidence has emerged in recent years to suggest that the cell plasma membrane handles univalent and divalent cations abnormally in patients with untreated essential hypertension. Many of the phenomena originally discovered in patients with the established disease have now been found to occur in the genetically hypertension-prone offspring of hypertensive patients when their blood pressure is normal. The studies described in this thesis were designed to investigate the mechanisms which might explain these disturbances of membrane function. The first experiments were performed to investigate whether a circulating blood-borne factor might be present in excess in hypertensive patients and their relatives and by exposing cells from subjects with normal blood pressure and no family history to serum from patients and their offspring, the object was to try and reproduce the findings in hypertension. These studies were negative. Because of many reports of an overactive sympathetic nervous system in hypertension leucocytes were exposed to noradrenaline and this was found to influence sodium transport in cells from control subjects suggesting that at least some of the phenomena described in hypertension might be related to autonomic dysfunction. An alternative hypothesis to explain these abnormalities is that there is a genetically predetermined disturbance of the physicochemical structure of the plasma membrane which alters its function. The abnormality might well lie within the lipid fraction of the cell membrane. Attempts to alter this were undertaken using changes in dietary fat intake. These lowered blood pressure slightly and indeed altered sodium influx. The final series of experiments involved detailed examination of one fraction of plasma membrane phospholipids which is highly metabolically active, namely the phosphoinositides, and indeed using red cells it was possible to demonstrate that these lipids are overactive in the early stages of hypertension. These findings suggest that the plasma membrane is structurally abnormal in hypertension, the abnormality may reside in the phospho- inositide lipids and may possibly be susceptible to dietary manipulation.

The goal of this thesis was to design, synthesize and test a chemical switch for control of membrane fusion. Control of the shape of the molecules that comprise a membrane should induce a phase change in the membrane. According to current views of membrane fusion, the phase change should also facilitate formation of fusion intermediates hence should provoke membrane fusion. The design thus focused on synthetic lipid targets that have controllable shape changes. Specifically the incorporation of the hemithioindigo (HT) photochemical switch into the fatty acid chains of phospholipids was deemed a solution to the design problem. The synthesis of four phosphatidylcholine (PC) analogues bearing two hemithioindigo moieties was accomplished. The successful synthesis starts from bromophenols. The bromide is extended to a nitrile via the Heck reaction with acrylonitrile. The thiophenol is converted to a thioindoxyl which is coupled with an aromatic aldehyde to produce the HT core. “Solventless” hydrolysis of the nitrile produces a carboxylic acid that can be coupled to a phosphoglycerol to give the target lipids. The synthetic process is both efficient and modular. All new compounds were characterized by NMR, MS and elemental analysis. The photochemistry of various HT derivatives was studied to confirm the expected photoisomerization in both homogenous solutions and vesicle bilayers. Although the UV-Vis spectra become rather insensitive to the presence of different isomers, there is evidence to confirm the Z-E switching in a range of organic solvents and in vesicles. Apparent bleaching of the HT-Iipid may indicate a photochemical dimerization reaction although isomerization would also be consistent with the data. Fusion was explored by manufacturing PS vesicles with varying concentrations and isomers of HT-lipid, and was monitored with the Terbium/Dipicolinic acid aqueous contents mixing assay (Tb/DPA assay). The sensitivity of this assay was lower than originally expected due to inner filter effects resulting in self-quenching the complex luminescence. The available data suggest that the synthetic HT-lipids disturb the membrane structure. Spontaneous fusion, apposition without metal cations, and contents leakage are some of the observations of the complexity of this system. HT-lipids in one population of vesicles are able to interact with a second population of vesicles, presumeably via membrane mixing. These results confirm that shape is a key factor in the integrity of membranes, and that second generation HT-lipids have the potential to control membrane fusion. / Graduate

Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...

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The Centrifugal Membrane and Density Separation (CMDS) process is a novel type of membrane process that exploits the action of a centrifuge to generate process pressure for reverse osmosis and nanofiltration. The centrifuge could potentially enhance flux and alleviate fouling of the membrane as a result of the hydrodynamic environment of the centrifuge. All experimental work has been conducted on a prototype model of the CMDS process. The apparatus allows a membrane module to be fixed in space at a specified orientation, with respect to the rotation. This orientation in space is denoted by the terms “pitch, roll and yaw” (p,r,y). Experiments have been done using brine feed solutions at various concentrations to determine if the CMDS process minimizes the effects of concentration polarization. An example of this was illustrated with a 54% flux enhancement relative to a conventional membrane process using a 35000 ppm NaCl feed solution. Colloidal feed solutions were also used to examine how the CMDS process enhances flux in a fouling environment. These feed solutions include 21 g/L silica and 300 mg/L humic acid, with typical relative flux enhancement factors (κ) found to be 0.59 and 0.14, respectively. The final group of experiments examined the use of 50 g/L whey feed solutions with nanofiltration membranes. Results obtained here indicate that the centrifugal action enhanced the flux with an absolute flux enhancement factor (κ′) of 17.5 L/m² hr. These experiments have shown that a given orientation (90,270,0) best enhances the flux of a membrane with respect to colloidal fouling, while showing that another orientation (90,180,0) best reduces the effects of concentration polarization. Scanning electron microscopy (SEM) and an energy dispersive x-ray (EDX) detector have helped to examine the nature of the fouling layers and determine how well the layers adhere to the surface of the membrane. It was determined that in some cases, the fouling layer adhered better to the surface of a membrane used in the CMDS process. However, as the fluxes were typically higher in the dynamic process, it leads to the conclusion that the fouling layer on the CMDS membranes is more permeable. From the experimental work it has been concluded that the forces at work in the CMDS process create sufficient secondary flow instabilities to reduce the effects of fouling and concentration polarization on the membrane surface. The significance of this process with respect to industrial applications is considered, and the process is deemed feasible for such applications. / Graduate

Forward osmosis (FO) is an osmotically driven process that uses a high concentration draw solution to pull water across a semipermeable membrane from a feed solution. Wastewater, seawater, or other contaminated water sources may be used as a feed solution. In FO, the final product is not clean water but a diluted draw solution. However, FO may be combined with another process, e.g. reverse osmosis (RO). The resulting hybrid process offers advantages compared to the RO process in, for example, seawater desalination. Thin-film composite (TFC) membranes have been used in pressurized processes such as RO due to their thick porous support layer and their ability to endure high hydrostatic pressures. However, the presence of a thick porous layer is detrimental for FO processes. It is responsible for the internal concentration polarization (ICP) inside the membrane, reducing the osmotic driving force and the overall water flux. The characterization of membranes in FO applications is key for understanding how different intrinsic parameters affect membrane performance. In this work, a previously developed methodology for characterizing TFC membranes was improved. Experimental data was obtained from a laboratory-scale FO system, and the experimental data was used to determine three intrinsic transport parameters, namely the water permeance, the salt permeance and the porous layer structural parameter. With this method, the characterization of TFC membranes can be achieved based exclusively on FO data. A sensitivity analysis has highlighted the impact of the intrinsic transport parameters on an FO membrane performance.

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