Browsing by Subject "Membrane"

Now showing 1 - 11 of 11

Cell free expression in emulsions and vesicles.
(2010-08) Monina, Nadezda
In this study we address the effects that membrane composition has on expression within two common forms of encapsulation: the single layered micelle and the bilayered vesicle. We then proceed to show the effects of oxygen on transcription machinery and the role the membrane plays in optimizing oxygen concentration for protein expression. In each case, the surfactant encapsulation is prepared in mineral oil and encloses an Escherichia coli cytoplasmic extract. Measurement of expression efficiency is conducted by both endpoint and kinetic measurement of eGFP (enhanced green fluorescent protein) concentrations via fluorescent microscopy. In the case of micelle encapsulation, results show that micelles of long block copolymer (LBCP) have the highest rate of expression as well as final concentration at 8 hours. Results also show a large discrepancy in eGFP expression between well oxygenated and less oxygenated environments. Similarly, a comparison between lipids suggests longer fatty acid tail length corresponds to more effective inhibition of oxygen diffusion leading to greater expression efficiency. Further expression tests in vesicles show membranes composed of phosphotidylcholine (PC) lipid mixed with LBCP have the most efficient eGFP as well as α hemolysin expression among vesicles of differing composition. With this information comes a newfound knowledge of the effects of synthetic membrane composition, oxygen concentration and the relationship between these two parameters on protein expression.
Communication in Membrane Repair
(2015-06) Mahling, Ryan W
The force generated by muscle cells places a high amount of stress on their plasma membranes creating lesions which must be effectively repaired in order for the cell to survive. Multiple proteins have been implicated in the membrane repair process, one of which is dysferlin, a seven C2 domain containing protein. Of the seven C2 domains within dysferlin, only the C2A domain exists in two isoforms and has been suggested to be the Ca2+ sensor within dysferlin. Mutations within dysferlin have been found to cause several types of muscular dystrophies including Limb-Girdle muscular dystrophy, Myoshi Myopathy and Distal Anterior Compartment Myopathy. In vivo studies have revealed that after membrane rupture, dysferlin interacts with multiple proteins including annexin A2. In order to gain a better understanding of how this system functions, this author used methods including differential scanning calorimetry (DSC) and spectroscopy (fluorescence and circular dichroism) to examine both isoforms of the C2A domain and annexin A2. All three proteins were found to be marginally stable suggestive of a system that is highly capable of information propagation. From this, a picture emerges where mutations within dysferlin could result in a dramatic shift in the conformational ensemble available to the protein, which would interfere with its ability to properly interact and communicate with the other members of the membrane repair machinery. This would result in the loss of the ability to properly repair the membrane.
Copolymer-based membrane stabilizers for Duchenne Muscular Dystrophy
(2016-04) Houang, Evelyne
The overarching objective of this work centers on a structure-function approach to investigate the mechanism of action of synthetic copolymer-based membrane stabilization in the context of Duchenne Muscular Dystrophy (DMD). The guiding theme is the investigation of mechanism of interaction of membrane stabilizing copolymers using cellular and whole animal physiology, chemical engineering, and supercomputational approaches. DMD is an X-linked recessive disease of marked striated muscle deterioration affecting 1 in 3500-5000 boys. DMD results from the lack of the cytoskeletal protein dystrophin, which is essential for maintaining the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle degeneration, along with clinically significant cardiomyopathy. There is no cure for DMD patients, or any effective treatment to halt, prevent or reverse DMD striated muscle deterioration. The primary pathophysiological defect in DMD is the marked susceptibility to contraction-induced membrane stress and the subsequent muscle damage and degeneration that occurs due to loss of muscle membrane barrier function. In this context, a unique therapeutic approach is the use of synthetic membrane stabilizers to prevent muscle damage by directly stabilizing the dystrophin-deficient muscle membrane. The triblock copolymer poloxamer 188 (P188) has numerous features that make it an attractive synthetic membrane stabilizer candidate for DMD treatment and has been demonstrated to target and stabilize damaged membranes in various pathophysiological contexts. The efficacy of P188 in protecting the dystrophic myocardium has been well established, but its effect on the dystrophic skeletal muscle has remained unclear. This work for the first time demonstrates that P188 stabilizes the dystrophic skeletal muscle membrane in vivo and protects it against the mechanical stress associated with lengthening contractions. This result validates P188 as a therapeutic strategy to directly target the hallmark of DMD: impaired membrane stability in all striated muscles. Very little is known on how P188 interacts with and stabilizes biological membranes. To fundamentally probe the mechanism of action of synthetic copolymers as membrane stabilizers, a structure-function approach was undertaken. The aim was to gain insight into the essential critical chemical parameters of copolymers in terms of membrane interacting properties. This work shows for the first time that copolymer mass, composition, architecture, and functional end group chemistries significantly define mechanism of action at the membrane. Based on these insights, an “anchor and chain” model is advanced whereby membrane interaction is critically dependent on end group hydrophobicity. Finally, leveraging the power of supercomputational approaches, Molecular Dynamics simulations were developed to further evaluate and understand copolymer-membrane interactions at atom level resolution. Using increases in surface tension applied to the lipid bilayer, an area-per- lipid dependence of adsorption vs. insertion was uncovered, supporting the hypothesis that copolymers insert into areas of decreased lipid density and then are “squeezed-out” once membrane integrity is restored. Collectively, these findings shed new light on block copolymer dynamic interaction with biological membranes.
Dispersible exfoliated zeolite nanosheets and their application in high performance zeolite membrane
(2013-10) Agrawal, Kumar Varoon
In the wake of the energy crisis, an efficient separation technology such as membrane is required to replace the energy intensive processes like distillation. High performance zeolite membrane can be fabricated by coating of a thin film of high-aspect-ratio zeolite nanosheets on a porous support. However, the synthesis of highly crystalline and morphologically intact zeolite nanosheets by the direct hydrothermal synthesis has been challenging. Successful reports on the synthesis of zeolite nanosheets by the exfoliation of their layered structure exist, but the synthesis routes provided in these reports often lead to significant damages to the structure and the morphology of nanosheets. This dissertation focuses on the development of a scalable method for the synthesis of zeolite nanosheets, while preserving their structure and the morphology. MWW and MFI nanosheets were prepared by polymer melt compounding of their layered precursors with polystyrene. Zeolite nanosheets were extracted out of the polymer matrix by solution processing of the zeolite-polymer nanocomposite. Exfoliated nanosheets and their coatings were then characterized by the scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), nuclear magnetic resonance (NMR), and X-ray diffraction (XRD). A compact, oriented, 300-nm thick zeolite film was fabricated on a symmetric alumina support by a one-step filter coating method. This nanosheet film demonstrated molecular sieving capabilities after a mild hydrothermal treatment. Density gradient centrifugation was used to purify the zeolite nanosheets from the polymer matrix, and the large unexfoliated particles, resulting in a two-fold increase in the yield of nanosheets in the final coating suspension. Sub-100 nm thick films of these nanosheets were made on a symmetric alumina supports. Nanosheet films with thickness ranging from 10 nm to 100 nm were prepared on an asymmetric silica supports. In-plane secondary growth of these films by the impregnation growth method led to b-oriented, 100-150 nm thick zeolite film that separated xylene isomers with separation factors of 100-800, while providing a high permeance of p-xylene (4 x 10-7 moles/m2-s-Pa).</
Exfoliated zeolite sheets and block copolymers as building blocks for composite membranes.
(2009-08) Maheshwari, Sudeep
Mixed matrix materials, comprising of zeolites incorporated in suitable matrix (polymeric or inorganic), are promising as future membrane materials with high permselectivity. However, they suffer from the drawback of low productivity due to increase in the membrane thickness by incorporation of micron-sized zeolites crystals as well as the low-permeability matrices employed currently. Nanocomposite membranes, consisting of thin zeolite sheets (~2 nm) embedded in an appropriate matrix, can provide a solution to this problem. This thesis addresses some of the material challenges to make such nanocomposite membranes. A high permeability polymer was synthesized by combining the glassy polystyrene (PS) with the rubbery polydimethylsiloxane (PDMS) in a block copolymer architecture. The mechanical toughness of the material was optimized to facilitate the fabrication of thin free standing films and its gas transport properties were evaluated. The PS-PDMS-PS triblock copolymers were successfully hydrogenated for the first time to obtain the PCHE-PDMS-PCHE triblock copolymers (PCHE stands for polycyclohexylethylene). The hydrogenation reaction proceeded without any polymer chain breaking and the resultant polymer showed some interesting, rather unexpected thermodynamic properties. These polymeric materials are potentially useful as the matrix of nanocomposite membranes. Highly crystalline zeolite sheets were obtained by exfoliation of zeolite lamellae. Preservation of crystal morphology and pore structure, which presents a major challenge during the exfoliation process, was successfully addressed in this work by judicious choice of operating conditions. Lamellae were exfoliated by surfactant intercalation and subsequently melt processing with polymers, resulting in polymer nanocomposites containing thin zeolite sheets (~2.5 nm) with well preserved pore structure. A method to obtain polymer-free exfoliated sheets was also developed to facilitate the fabrication of inorganic composite membranes. These zeolite sheets can be used as the selectivity-enhancement additive in composite membranes.
Mechanisms and models of dehydration and slow freezing damage to cell membranes
(2010-10) Ragoonanan, Vishard
Cell preservation is accomplished primarily by two methods: cryopreservation and dehydration, with the former being the standard technique used. In order to optimize and develop cell preservation protocols for cells that are difficult to preserve or whose end application is incompatible with current cell preservation protocls and to advance preservation by dehydration, a better understanding of the freeze- and dehydration-induced changes to the cell membrane is required. Despite a large body of literature on the topic, the mechanisms of damage to cells during slow freezing and dehydration are still ambiguous. The objective of this study is to investigate the mechanisms of damage to the cell membrane during slow freezing and dehydration and expand our outlook beyond the cell membrane to its underlying support, the cytoskeleton. In this study, we used several model systems to investigate slow freezing and dehydration. We used a liposome model to gather basic information on changes that can occur to a simple membrane system during freezing. This study revealed that eutectic formation was capable of dehydrating the membrane at low temperatures which may be contribute to alteration of the post-thaw membrane structure. We used a bacteria model to investigate the role of the phase transition and immediate versus slow osmotic stress on post-rehydration viability. This study revealed that going through a lyotropic membrane phase transition was detrimental to post-rehydration viability. This study also demonstrated that a rapidly applied osmotic stress was more detrimental to the structure/ organization of the membrane than gradual osmotic stress. We then subjected a model mammalian cell to both hyperosmotic stress and freeze-thaw and investigated both the membrane and cytoskeletal responses. Osmotic stress experiments suggested that alterations in membrane structure (i.e., surface defects and lipid dissolution) were directly dependent on the change in the chemical potential of water. These experiments also suggest that cell shrinkage and the resulting formation of membrane protrusions negatively affect viability upon return to isotonic conditions. It was found that membrane morphology in the dehydrated state and post-hyperosmotic viability was dependent on the stiffness of the cytoskeleton. Freeze/ thaw experiments suggested that ice-cell interaction decreases post-thaw viability. However, similar to osmotic stress experiments, cell shrinkage and cytoskeletal stiffness negatively impact post-thaw viability. We suggest the resulting membrane morphology due to cell shrinkage is also responsible for damage during freeze/ thaw. The various mechanisms discovered and the models proposed can be used in developing new protocols for cell preservation and for cell destruction (e.g. cryosurgery).
Membrane-Targeting Approaches for Enhanced Cell Destruction with Irreversible Electroporation
(2014-05) Jiang, Chunlan
Irreversible Electroporation (IRE) has gained increasing popularity in the cancer treatment field during the past decade due to many advantages over other focal therapies. Despite early success in pre-clinical and clinical IRE trials, in vivo studies have shown that IRE suffers from an inability to destroy large volumes of cancer tissue without repeating treatment and/or increasing the applied electrical dose to dangerous levels. There are approaches to expand the treatment volume by IRE with the addition of chemotherapeutic or cytotoxic agents. While these studies demonstrated improved cell killing, the focus was on enhancing the ability of chemotherapeutic drugs or cytotoxic agents to enter and kill the cancer cells rather than enhancing the efficacy of IRE itself. Therefore, the aim of this work is to investigate the ability to increase the destructive capability of IRE without relying on cytotoxic drugs. Specifically, mechanisms that directly modify membrane properties should reduce the voltage threshold for lethal permeabilization and therefore increase the efficacy of cell killing and therefore the volume treated after a given IRE level. Two methods to achieve these changes are proposed in this study: 1) addition of surfactant (e.g. Dimethyl sulfoxide, or DMSO) to directly interact with membrane lipids thereby changing membrane line tension and surface tension, and 2) use of pulse timing (i.e. introduction and persistence of defects in the membrane between pulses). Here then we began by Investigation of IRE enhancement in vitro to understand the impacts of our proposed mechanisms and their ideal working parameters. We found that the best enhancement effect was achieved with addition of 5% v/v DMSO, which resulted in a significant increase of 75% more cell destruction compared to baseline IRE. Similarly with pulse timing, when dividing the pulses into three trains with 30s delays in between, an enhancement of 67% more cell destruction was achieved compared to baseline IRE. Next we tested our IRE enhancement approaches in an in vivo dorsal skin fold chamber (DSFC) model of prostate cancer with optimal parameters selected from our in vitro experiments. The results reproducibly showed that more than 120% and 101% enhancement in the treatment volume were achieved by the addition of DMSO and pulse timing, respectively, with two independent injury assessment methods (histological and perfusion defect). Finally, we translated one of the enhancement approaches (pulse timing) to an in vivo hind limb model of prostate cancer and demonstrated that more than 33% additional tumor destruction and 2 weeks longer tumor growth delay could be achieved compared to baseline IRE treatment without relying on any cytotoxic drugs or agents. Because DMSO is commercially available and regularly used at low concentrations (<10% v/v) in clinic, this approach could easily be integrated into current IRE procedures to increase the treatment efficacy. In addition, introducing pulse timing delays in IRE also increases the destructive potential of IRE without the introduction of any foreign agents into the body. Further opportunities exist in improving the adjuvant delivery methods, optimizing the pulse timing delivery approach and understanding the fundamental mechanisms of IRE. Nevertheless, we suggest that the simple and safe nature of our proposed approaches compared with cytotoxic drugs may help to translate IRE into the clinic.
Modeling, film formation, and material synthesis for performance optimization of mixed matrix membranes.
(2009-01) Sheffel, Joshua Alexander
Mixed matrix membranes offer the hope of improving the performance of a separating membrane by dispersing a second phase within it. By combining the processability of a continuous phase (the matrix) with the separation characteristics of a dispersed phase (the flake), mixed matrix membranes aim to provide a step-change improvement in membrane performance without dramatically increasing the cost of membrane technology. In this dissertation, a numerical model for the performance of mixed matrix membranes is presented that accounts for effects such as competitive adsorption and concentration-dependent diffusivities. It is shown that these effects are vital for the modeling of a membrane containing zeolite flakes. This insight is then used to formulate a semi-empirical model for mixed matrix membrane performance that does not require extensive numerical calculations. Through a series of case studies on relevant gas and vapor separations, these models are applied to material and process design for mixed matrix membranes. Finally, experimental aspects of mixed matrix membrane formation are presented, including the synthesis of layered aluminophosphate molecular sieves and the fabrication of mesoporous silica/silicalite-1 zeolite films.
Nanoporous materials from ABAC tetrablock terpolymers
(2013-07) Jackson, Elizabeth
This dissertation describes efforts towards the preparation of tough nanoporous membranes from ABAC tetrablock terpolymers. This architecture was strategically chosen to combine an etchable C block, PLA, with a mechanically tough ABA triblock into one ABAC terpolymer. Multiple series of poly(styrene-b-isoprene-bstyrene-lactide) (PS-PI-PS-PLA) tetrablock terpolymers were synthesized. Morphological behavior was characterized for terpolymers containing both a 50:50 and 30:70 PS:PI ratio with between 0 and ~20% PLA by volume. Observed bulk morphologies include hexagonally packed cylinders (HEX), core(PLA)-shell(PS) cylinders (CSC), and a PLA sphere in cylinder morphology. Mechanical properties of PS-PEEP-PS-PLA tetrablocks were also investigated. All materials exhibited mechanical properties characteristic of tough thermoplastic elastomers. Composite membranes were prepared from a thin film of PS-PI-PS-PLA terpolymer and a macroporous polyethersulfone support. Described within are the efforts related to the fabrication and filtration performance of these nanoporous PSPI-PS composite membranes. As part of this process, solvent casting and annealing conditions were varied to investigate effects on tetrablock thin film morphology. Optimum conditions were determined to achieve PS-PI-PS-PLA films with perpendicular PLA cylinder orientation. These conditions included use of a mixed solvent system and the addition of a small amount of homopolymer PLA. Highly ordered films with vertically oriented nanopores were obtained.
The thermodynamic basis for the binding of lipids to annexin a5
(2009-12) Knutson, Kristofer James
Protein-membrane interactions are a vital mechanism of propagating signals both across the membrane and between cells. To control the magnitude and specificity of this type of cell signaling at the membrane, clustering of similar lipids and proteins has been observed in the cell via the formation of lipid microdomains. To address the thermodynamic basis of lipid induced signal propagation, we investigated how lipid microdomains form in response to annexin a5 binding to model membranes using Isothermal Titration Calorimetry (ITC). Annexins are known to bind to negatively charged (e.g., phosphatidylserine [PS]) membranes in a Ca2+-dependent manner. Based on Differential Scanning Calorimetry (DSC) results, we suggest that annexin functions to order lipid acyl chains upon binding and that the ordering of phospholipids can lead to the formation of microdomains. Using ITC, we have analyzed the membrane binding affinity of annexin for both gel and fluid state mixtures. Binding analysis of these isotherms shows that annexin binds fluid state mixtures with a significantly lower Kd than gel state (acyl chain ordered) lipids, which would be consistent with the hypothesis that binding of annexin a5 orders the acyl chains of the phospholipids. In addition, because the binding is entropically dominated but exhibits greater affinity for fluid compared to gel state lipids, we suggest that annexin binding is driven by the release of water molecules and ions as fluid lipids have more waters of hydration. Interestingly, the enthalpy associated with the binding process for both gel and fluid state lipid mixtures is small, indicative of a weak enthalpic association and suggestive of entropically mediated binding. We also present the binding of Eu3+ by a lanthanide binding complex (Tetra(N-(tert-butyl)-acetamide)-1,13-diamino-3,6,9-trioxadecane).
Zeolite MFI Membranes Towards Industrial Applications
(2020-11) Duan, Xuekui
Zeolite membranes have been the interest of research for decades due to their potentials in various separation applications including gas separation, water purification, pervaporation, etc. Among the zeolite materials studied, MFI zeolite (Silicalite-1 and ZSM-5) is one of the major subjects of research, mainly because of its suitability for the separation of hydrocarbons, such as n-butane from iso-butane and para-xylene from its isomers. Besides, all-silica Silicalite-1 and high-silica ZSM-5 have been explored for organic/water pervaporation as well by utilizing their high hydrophobicity. Despite years of research efforts on these applications, the industrialization of MFI membranes has not been achieved. One reason is that the cost associated with the fabrication of these membranes is too high to be commercially attractive. The high-cost, specially engineered silica membrane supports account for a major share of the total cost. Alternative supports such as polymeric supports and low-cost and commercially available alumina supports are possible substitutes to explore. Another problem is the lack of demonstration of high membrane separation performance at industrially relevant conditions (high temperature and high pressure). It is thus the goal of this thesis to address these problems and make progress towards the commercialization of MFI membranes. First, the recent advances of MFI zeolite membranes were reviewed. Then, the fabrication of high-performance MFI membranes using aqueous dispersions of open-pore, two-dimensional MFI zeolite nanosheets on low-cost polymeric substrates was demonstrated. Next, progress towards making MFI membranes on alumina supports has been made. Despite these efforts to use other supports, we failed to make high-performance membranes as comparable to the silica-supported ones. Besides these efforts, ultra-thin MFI membranes fabricated using dc-5 nanosheets as seeds were showed to have high xylene isomer separation performance at industrial conditions and high performance for H2/hydrocarbons separation and ammonia/H2/N2 separation. These works demonstrated the potential of high-performance MFI membranes for energy-efficient separation processes in industrial conditions.

University Digital Conservancy

Browse by Subject

Browsing by Subject "Membrane"