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The outer membrane palmitoyltransferase PagP possesses regioselectivity for the palmitoylation of the (R)-3-hydroxymyristate chain at position 2 on the proximal glucosamine unit of lipid A. The residues Arg45 and Arg49 in the L1 loop appear to poise their guanidinium groups so as to interact with the proximal and distal phosphate groups at positions 1 and 4’ of lipid A, respectively. Both single and double substitution of these arginine residues with serine has no effect on the folding, stability, phospholipase and palmitoyltransferase activities. Additionally, the arginine to serine substitutions display wild-type regioselectivity and specific activity in the palmitoylation of the biosynthetic precursor lipid IVA as indicated by collision induced fragmentation MS/MS. In vivo, lipid A analysis in a msbB/pagP deletion strain shows no difference in acylation pattern as compared to the wild-type. These results establish both in vitro and in vivo that the arginine to serine substitutions have no effect on lipid A regioselectivity. / Thesis / Bachelor of Science (BSc)

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Abstract Heavy metals are used in many industrial processes and their cations are either valuable or environmentally harmful to discharge in wastewater. Thus, it is necessary to separate heavy metals in wastewater treatment. Amongst several technical methods of separation, use of permeable membranes is an important one. For separation processes, membranes can be selective towards a target heavy metal cation either against mono-valent common cations or against other similar heavy metal cations. Synthesis of selective permeable membranes for separation purposes is an area of continuous research to meet specific needs in different applications. One of the common applications of selective separation by a permeable membrane is cation/anion separation processes using cation exchange and anion exchange membranes. Another application of selective permeable membranes is the separation of mono-valent cations from other higher valence cations. Some researchers have focused on specific selective separation of heavy metal cation from other heavy metal cations having the same valent charge. Some use chelating particulates dispersed in a neutral polymeric membrane matrix and others applied a thin chelating film on the surface of a commercial cation exchange membrane. In this work, the synthesis of novel permeable selective membranes and their use for selective separation between two di-valent heavy metal cations is presented. Three different sets of membranes were prepared in non-imprinted and imprinted forms. The ion imprinted membranes form is prepared by pre-reacting the target metal ion with the selective chelating monomer before applying in situ polymerization step, and in the non-imprinted membranes form this step is not considered. Their morphological and chemical structures were determined and their separation performances were investigated using a diffusion dialysis technique. The first membrane (non-imprinted polyvinylidene fluoride-divinylbenzyl-triethylenetetramine (PVDF/diVB-TETA) and Cu-imprinted PVDF/diVB-TETA-Cu forms) was prepared by in situ polymerization of the chelating monomer divinylbenzyl-triethylenetetramine diVB-TETA (or diVB-TETA-Cu) within a PVDF substrate, using a divinylbenzene cross-linker. Fourier transform infrared FT-IR spectroscopy showed the successful in situ polymerization of the chelating monomer within the PVDF texture. The permeation study showed that the ion-imprinted membrane has a Cu2+ selectivity factor of 3.78, while the non-imprinted membrane has a Cu2+ selectivity factor of 1.65. In addition the Cu2+ permeation flux in the imprinted membrane is 3.9 time that in the non-imprinted membrane For the second membrane, the synthesis is similar to the first membrane for both non-imprinted and imprinted forms (polyvinylidene fluoride-divinylbenzyl-triethylenetetramine-N,N'-methylenebis(acrylamide) (PVDF/diVB-TETA-N) and PVDF/diVB-TETA-N-Cu respectively), except that the used cross-linker was N,N'-methylenebis(acrylamide). In addition, sodium 4-vinylbenzyl sulfonate was added in selected percentages, (5-15% mol), to enhance the permeation flux. FT-IR spectroscopy analysis of the prepared membranes confirmed the chemical structure of diVB-TETA-N and sulfonate group into PVDF. Permeation and selective separation studies for the prepared membranes showed that the ion-imprinted membrane has a higher selectivity for copper permeation over the non-imprinted membrane. However imprinted membrane showed a lower flux for the permeated cations than that of the non-imprinted membranes The addition of the sulfonate groups to the prepared membranes enhanced the flux of the permeated cations, but the copper selective permeation decreased for both types (non-imprinted and ion-imprinted). Moreover, the ion-imprinted membrane PVDF/diVB-TETA-N-Cu showed a lower flux for the permeated cations than that of the non-imprinted membranes PVDF/diVB-TETA-N. Selective separation factors decreased to unity when the content of the sulfonate groups increased to 15% mol. Ion imprinted membrane prepared with 10% of sulfonate group showed optimum copper selectivity factor (α = 30304) and permeation flux for copper (0.4949 μmol cm‒2 h‒1) The third membrane (non-imprinted Selemion TM cation exchange membrane-divinylbenzyl-triethylenetetramine (CMV-S/diVB-TETA) and ion-imprinted CMV-S/diVb-TETA-Cu forms) was prepared by in situ polymerization of the chelating monomer, diVB-TETA (or diVB-TETA-Cu), on the surface of the commercial cation exchange membrane, Selemion, using divinylbenzene as cross-linker. FT-IR spectroscopy confirmed the chemical structure of the chelating polymer on the CMV-S membrane surface. Permeation study showed that ion-imprinted CMV-S/diVB-TETA-Cu membrane reached high separation factor (α = 17), yet the flux is low (0.0391 μmol cm‒2 h‒1). Non-imprinted CMV-S/diVB-TETA membrane of thickness (0.115±0.005 mm) using cross-linker (10% DVB) showed reasonable copper selectivity factor (α = 2.723) and permeation flux (0.433 μmol cm‒2 h‒1) / Thesis / Doctor of Philosophy (PhD)

Diacylglycerol kinases (DGK’s) tightly regulate the intracellular levels of diacylglycerol (DAG) and phosphatidic acid (PA). DAG is an important intermediate in lipid biosynthetic pathways and acts as a lipid second messenger in a number of signaling pathways. Similarly, since PA serves as a potent signaling lipid and is a precursor for lipid biosynthesis, intracellular PA levels must be tightly regulated. There are ten isoforms of DGK in mammals, but we have decided to focus solely on the epsilon form (DGKε) in this work. DGKε is the only isoform that shows specificity for the acyl chains of its DAG substrate; as a consequence, it contributes to the dramatic enrichment of cellular lipids with sn-1 stearoyl and sn-2 arachidonoyl. The most notable example is the highly enriched bioactive lipid 1-stearoyl-2-arachidonoyl phosphatidylinositol. We have purified active human DGKε to near homogeneity and thoroughly characterized its stability as well as examined its secondary structure with CD. We also purified a truncated form (DGKε Δ40) that shows increased stability compared to the full-length protein. Our purified fractions are well suited for a wide range of exciting applications and studies. We have begun incorporating DGKε into liposomes in order to develop a liposome-based assay, which would be a dramatic improvement over the presently used micelle-based assay. This purification also allows for high throughput screens of chemical compounds to test for a specific inhibitor. These studies will reveal valuable information about the structural and functional properties of DGKε and will aid in the development of therapies for DGKε-related diseases. / Thesis / Master of Science (MSc)

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The outer membrane (OM) is a formidable barrier that has made antibiotic drug discovery in Gram-negatives exceedingly difficult. Many antibiotics which are effective against Gram-positive bacteria cannot permeate the Gram-negative OM to reach their intracellular targets. Thus, it is important to explore unconventional approaches to overcome the intrinsic resistance conferred by the OM. Herein, we used both genetic and chemical means to compromise OM integrity to potentiate the activity of large-scaffold antibiotics against Escherichia coli. First, we mapped the genetic interaction network of OM biosynthetic genes using synthetic genetic arrays (SGAs) to reveal permeability determinants of the E. coli OM. This led to the creation of a publicly accessible dataset of ~155,400 double deletion strains with growth data in the presence of the large-scaffold antibiotics rifampicin and vancomycin. Investigations of a subset of synthetic sick interactions revealed connectivity in the context of permeability between lipopolysaccharide (LPS) inner core biosynthetic genes and an enigmatic gene involved in enterobacterial common antigen (ECA) regulation. Second, we leveraged a chemical screening platform based on the observation that disruption of the E. coli OM leads to antagonism of vancomycin activity at cold temperatures to uncover molecules that potentiate Gram-positive-targeting antibiotics at 37 ºC. Two of these compounds, liproxstatin-1 and MAC-0568743, were characterized to bind to LPS and disrupt OM integrity specifically without impacting the inner membrane (IM). Third, we performed genetic and chemical screening to unearth targets capable of potentiating the activity of Gram-positive-targeting antibiotics against E. coli. This validated the OM as a valuable target for antibiotic adjuvants and led to the discovery of two membrane active compounds and an inhibitor of lipid A biosynthesis. Overall, this thesis emphasizes the importance of elucidating biological factors contributing to OM permeability and the attractiveness of the OM as a target for antibiotic potentiators. / Thesis / Doctor of Philosophy (PhD)

Climate change, industrial and agricultural activities, and population growth exacerbate global water stress. A variety of advanced technologies have been studied to alleviate water scarcity and water pollution. Membrane technology owing to its low footprints and ease of operation, has drawn intensive attention for water purification and wastewater treatment. Further, integrating membrane technology, electrochemistry and catalysis can improve separation and selectivity of the filtration process. This work aims to fabricate high-performance active and reactive ultrafiltration membranes involving electrically conductive membranes, catalytic membranes and electrocatalytic membranes. Their use in water treatment inspires the development of advanced functionalized membranes and further accelerates the transition to industrial applications. / Thesis / Doctor of Engineering (DEng)

Listeria monocytogenes is an enteric pathogen that can replicate within bile, yet this capability differs between strains. This project analyzed whether the pathogenic potential of the strain affects the ability to resist bile. We tested this hypothesis by examining the effect of bile on the morphology of a virulent strain (EGD-e) and an avirulent strain (HCC23) under aerobic and anaerobic conditions. Our data showed that exposure to bile greatly impacted the growth of HCC23. Additionally, scanning electron microscopy and transmission electron microscopy analyses indicated that bile affects the cell envelope of EGD-e and HCC23 differently. Our results suggest that differences exist in the ability of EGD-e and HCC23 to survive and replicate in the presence of bile. We propose that the virulence capability of L. monocytogenes directly correlates to its ability to resist the detergent properties of bile.

No description available.