published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Our group previously reported that caveolin-1 (cav-1) was down-regulated by nitric oxide (NO) during cerebral ischemia and reperfusion (I/R). However, the role of cav-1 in regulating blood-brain barrier (BBB) permeability is unclear yet. This study aims to address whether the loss of cav-1 induced by NO production affects BBB permeability. Data showed that the expression of cav-1 in isolated cortical microvessels was down-regulated in ischemia-reperfused rat brains subjected to middle cerebral artery occlusion (MCAO). Treatment of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, reserved cav-1 expression, inhibited matrix metalloproteinases (MMPs) activity and reduced the BBB permeability. Moreover, cav-1 knockdown remarkably increased MMPs activity in the culture medium of brain microvascular endothelial cells. Cav-1 deficiency mice displayed higher MMPs activity and BBB permeability than that of the wild-type mice. Interestingly, the effects of L-NAME on MMPs activity and BBB permeability were partly reversed in cav-1 deficiency mice. These results suggest that cav-1 plays important roles in regulating MMPs activity and BBB permeability in cerebral I/R injury. After completing the mechanism study, I investigated the potential drug candidate that targets cav-1 for protecting BBB and neuronal damage during cerebral I/R. Results showed that calycosin, an isoflavones from Astragali Radix, up-regulated the expression of cav-1 and inhibited MMPs activity, and decreased the BBB permeability in the MCAO ischemia-reperfused rat brains. I further investigated the neuroprotective effects of isoflavones of Astragali Radix, with in vitro oxygen glucose deprivation (OGD) model and in vivo cerebral ischemia-reperfusion models. In addition to calycosin and formononetin, two major isoflavones in Astragali Radix, daidzein was also included because it is a metabolite of formononetin after absorption. Results showed that all three isoflavones decreased infarction volume and neurological scores in MCAO rats and dose-dependently attenuated neuronal death induced by L-glutamate treatment and oxygen-glucose deprivation plus reoxygenation (OGD/RO). The neuroprotective effects were inhibited by estrogen receptors (ER) antagonist ICI 182,780. Interestingly, combine treatment of isoflavones displayed synergistic effects in both OGD/RO and L-glutamate induced neuronal injury models, as well as in MCAO cerebral ischemia-reperfusion rat brains. Mechanistically, estrogen receptor antagonist partly reduced the synergism in these models. PI3K/Akt activation was synergistically induced by treatment of those isoflavones simultaneously. In summary, cav-1 could be a potential therapeutic target for protecting the BBB in the treatment of cerebral I/R injury. Major findings in this thesis include: 1) Cav-1 plays an important role in maintaining BBB integrity through inhibition of MMPs activity. NO induced MMPs activities and BBB leakage are partially mediated by the down-regulation of cav-1 during cerebral I/R injury. 2) Calycosin treatment reserved cav-1 expression and reduced BBB permeability. 3) Isoflavones synergistically protected neurons against I/R-induced neuronal insults both in vitro and in vivo. The works provide a valuable step towards the clarification of the physiological and pathophysiological functions of cav-1, and a new clue for developing isoflavones as agents targeting cav-1 for the prevention or treatment of ischemic stroke. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

The driving force behind research in alternative clean and renewable energy has been the desire to reduce emissions and dependence on fossil fuels. In the United States, ground vehicles account for 30% of total carbon emission, and significantly contribute to other harmful emissions. This issue causes environmental concerns and threat to human health. On the other hand, the demand on fossil fuel grows with the increasing energy consumption worldwide. Particularly in the United States of America, transportation absorbs 75% of this energy source. There is an urgent need to reduce the transportation dependence on fossil fuel for the purpose of national security. Polymer electrolyte membrane (PEM) fuel cells are strong potential candidates to replace the traditional combustion engines. Even though research effort has transferred the fuel cell technology into real‒world vehicle applications, there are still several challenges hindering the fuel cell technology commercialization, such as hydrogen supply infrastructure, cost of the fuel cell vehicles, on‒board hydrogen storage, public acceptance, and more importantly the performance, durability, and reliability of the PEM fuel cell vehicles themselves. One of the key factors that affect the fuel cell performance and life is the run‒time thermal and water management. The temperature directly affects the humidification of the fuel cell stack and plays a critical role in avoiding liquid water flooding as well as membrane dehydration which affect the performance and long term reliability. There are many models exists in the literature. However, there are still lacks of control‒oriented modeling techniques that describe the coupled heat and mass transfer dynamics, and experimental validation is rarely performed for these models. In order to establish an in‒depth understanding and enable control design to achieve optimal performance in real‒time, this research has explored modeling techniques to describe the coupled heat and mass transfer dynamics inside a PEM fuel cell. This dissertation is to report our findings on modeling the temperature dynamics of the gas and liquid flow in the porous media for the purpose of control development. The developed thermal model captures the temperature dynamics without using much computation power commonly found in CFD models. The model results agree very well with the experimental validation of a 1.5 kW fuel cell stack after calibrations. Relative gain array (RGA) was performed to investigate the coupling between inputs and outputs and to explore the possibility of using a single‒input single‒output (SISO) control scheme for this multi‒input multi‒output (MIMO) system. The RGA analyses showed that SISO control design would be effective for controlling the fuel cell stack alone. Adding auxiliary components to the fuel cell stack, such as compressor to supply the pressurized air, requires a MIMO control framework. The developed model of describing water transport in porous media improves the modeling accuracy by adding catalyst layers and utilizing an empirically derived capillary pressure model. Comparing with other control‒oriented models in the literature, the developed model improves accuracy and provides more insights of the liquid water transport during transient response. / text

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Sustainable algae biofuel production is rising in demand, and the need to establish an efficient and proper algae harvesting method is extremely essential. Membrane filtration technology seems to be the most promising as a solid-liquid separation process. However, fouling seems to be the major problem for membranes. There is limited research on how to solve the problem of fouling, and cake buildup inside the membranes. A novel membrane design is required to solve the problem of fouling and cake buildup inside the membranes. The objective of this research is to construct a novel two way membrane design for algae biomass harvesting and water recycling. The methods used include culturing algae species, filtering them through the membrane module, and sample analysis for determining the water quality. The results show that the present filtration model had no fouling, or cake buildup as opposed to the previous filtration model. The present model permeate has a very low optical density of 0.007 absorbance at 750 nanometers. This result shows that permeate is completely devoid of algae.

Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa. Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE. Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å. / Chemistry and Chemical Biology

NMR crystallography is a new and developing area. Unlike solution state NMR, solid state NMR has the potential for structural studies of large, motionally restricted biological macromolecules, such as proteins in crystals which may, or may not, diffract. However, finding the best and the most useful sample form and geometry is still a major obstacle to rapid progress. Little has been reported about protein sample preparation for any class of protein for NMR crystallography, mainly since the availability of NMR labelled proteins is still not routine, especially for eukaryotic membrane proteins. The amino acid L-glutamate is the major excitatory neurotransmitter in the brain. Details of glutamate binding to any of its main brain or sensory receptors are not well resolved at the atomic level. In an effort to resolve the glutamate binding mechanism by solid state NMR methods, full-length taste and brain mGluR4 were expressed in E. coli, but proved to be toxic for the cells. The ligand-binding domains (LBD) of mGluR4, with various fusions for the periplasmic expression and with various fusions for expression in the cytoplasm therefore, were used. Solubilisation and then purification of the LBD from inclusion bodies is still under way, no crystals of mGluR4 for NMR were, therefore, grown. Initial NMR spectra of labelled 13C, 15N and 17O glutamate have been recorded to verify sensitivity requirements. Using homology modelling, a model for the truncated version of the ligand binding domain of mGluR4 has been constructed as a basis for designing solid state NMR experiments to probe the ligand binding site in the receptor. Bacteriorhodopsin is a large membrane protein and a model for G-protein coupled receptors (GPCRs). Spectra of bacteriorhodopsin produced in H. salinarium in purple membrane are reported here and compared to spectra of the protein crystallised from bicelles. Optimal conditions for producing spectra suitable for spectral assignment are reported as an initial step towards spectral resolution. Three differently labelled samples of bacteriorhodopsin were prepared to test the applicability of the various assignment strategies and the effects of deuteration on quality of solid state NMR spectra of a large, crystalline membrane protein.