Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

This thesis explores structural and electronic properties of layered materials at the nanometre scale. Room temperature and low temperature ultrahigh vacuum scanning probe microscopy (scanning tunneling microscopy, scanning tunneling spectroscopy, atomic force microscopy) is used as the primary characterization method. The main findings in this thesis are: (a) observations of the atomic lattice and imaging local lattice defects of semiconducting ReS2 by scanning tunneling microscopy, (b) measurement of the electronic band gap of ReS2 by scanning tunneling spectroscopy, and (c) scanning tunneling microscopy study of 1T-TaS2 lattice and chemically functionalizing its defects with magnetic molecules.

The reactions of atomic hydrogen with methanethiol and that of atomic hydrogen with carbon disulfide were studied experimentally using flash-photolysis resonance-fluorescence techniques. Rate constants were determined over a range of temperatures and pressures, and through analysis and comparison to theoretical work details of the reactions were ascertained.

Proteins are substances with great usage. For industrial usage, proteins are often taken from their natural enviroment. In foreign environment, it proteins can unfold and their function can be compromised. This is the reason for stabilization of proteins and one of ways to stabilization is using disulphide bonds. This work describes basic terms related to protein stabilization - proteins, their structure and interactions within them, basic terms from thermodynamics. Problem of protein stability is discussed and the factors which stabilize or destabilize protein are enumerated with the emphasis on disulphide bonds. Existing approaches to disulphide bonds design, dataset for testing own tool are described. Implementation of the tool using geometrical properties of the bonds and fl exibility of places in protein is described. The tool was tested on proteins with native disulfide bonds and compared to existing tools, also metrics FRO (fractional rank order) was used. Native disulfide bond was found in 64 % of cases, in 60 % of cases this native disulfi de bond was in the first quarter of ordered found disulfi de bonds.

abstract: A highly uniform and repeatable method for synthesizing the single-layer transition metal dichalcogenides (TMDs) molybdenum disulfide, MoS2, and tungsten disulfide, WS2, was developed. This method employed chemical vapor deposition (CVD) of precursors in a custom built cold-wall reaction chamber designed to allow independent control over the growth parameters. Iterations of this reaction chamber were employed to overcome limitations to the growth method. First, molybdenum trioxide, MoO3, and S were co-evaporated from alumina coated W baskets to grow MoS2 on SiO2/Si substrates. Using this method, films were found to have repeatable coverage, but unrepeatable morphology. Second, the reaction chamber was modified to include a pair of custom bubbler delivery systems to transport diethyl sulfide (DES) and molybdenum hexacarbonyl (MHC) to the substrate as a S and Mo precursors. Third, tungsten hexacarbonyl (WHC) replaced MHC as a transition metal precursor for the synthesis of WS2 on Al2O3, substrates. This method proved repeatable in both coverage and morphology allowing the investigation of the effect of varying the flow of Ar, varying the substrate temperature and varying the flux of DES to the sample. Increasing each of these parameters was found to decrease the nucleation density on the sample and, with the exception of the Ar flow, induce multi-layer feature growth. This combination of precursors was also used to investigate the reported improvement in feature morphology when NaCl is placed upstream of the substrate. This was found to have no effect on experiments in the configurations used. A final effort was made to adequately increase the feature size by switching from DES to hydrogen sulfide, H2S, as a source of S. Using H2S and WHC to grow WS2 films on Al2O3, it was found that increasing the substrate temperature and increasing the H2S flow both decrease nucleation density. Increasing the H2S flow induced bi-layer growth. Ripening of synthesized WS2 crystals was demonstrated to occur when the sample was annealed, post-growth, in an Ar, H2, and H2S flow. Finally, it was verified that the final H2S and WHC growth method yielded repeatability and uniformity matching, or improving upon, the other methods and precursors investigated. / Dissertation/Thesis / Doctoral Dissertation Physics 2019

No description available.

Adiponectin, a hormone that homo-oligomerizes into trimer, hexamer, or higher molecular weight (HMW) species, is involved in maintaining insulin sensitivity in muscle and liver. Interestingly, its functions appear to be oligomer-specific. Recent data suggest that HMW levels are decreased in obesity and insulin resistance, although, the cause for this decrease is not known. Impaired assembly to the octadecamer represents one possible reason for decreased HMW adiponectin in insulin resistance and type 2 diabetes, but mechanisms by which HMW adiponectin assembles are unknown. This dissertation discusses the progress that we have made regarding formation of HMW adiponectin in vitro.I found that disulfide bonds are important in the assembly process to octadecameric adiponectin, but are not required for stability of the octadecamer itself. We showed that hydrogen peroxide accelerated oligomerization to the octadecamer through formation of disulfide bonds, while alkylation of the cysteines led to inhibition of both oligomerization and disulfide bond formation. Using comparative native/denaturing polyacrylamide gel electrophoresis (PAGE), dynamic light scattering, and tandem mass spectrometry, we demonstrated that octadecamer is stable in the absence of disulfide bonds by using multiple biochemical and biophysical assays. In addition, oxidized adiponectin oligomerizes to octadecamer far slower than reduced adiponectin. To further evaluate the role of disulfide bonds in the formation to octadecamer, we analyzed the role of reduction potential on adiponectin oligomerization. We observed that under immediate oxidizing conditions, hexamers and trimers form. Oxidized hexamer can form HMW adiponectin through disulfide bond rearrangement using beta-mercaptoethanol (βME) or increasing the total concentration of glutathione under oxidizing conditions. To further understand the role of disulfide bonds, we showed that zinc increased the oligomerization to octadecamer. This effect was associated with decreased initial disulfide bonding during the assembly to the octadecamer. In summary, these data suggest the rate of disulfide bond formation and the ability to undergo disulfide bond isomerization are important in the oligomerization process of HMW adiponectin.

The interaction between carbon dioxide (CO2) and N-compounds such as 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) has been extensively studied in the Jessop Lab. Carbon disulfide (CS2) is the sulfur congener of CO2, although it exhibits different reactivity with N-containing compounds. This thesis presents the search for zwitterionic CO2-switchable polarity solvents, new and general reactivity of CS2 with amidines and guanidines, and attempts at using CO2 as a carbonyl source in the synthesis of nitrogen containing compounds. In the second chapter, the reactions of CO2 with various diamines are described. Spectroscopic methods and X-ray crystallography determined the structure of a solid zwitterionic carbamate salt of CO2 and N,N’-dimethyl-1,3-propanediamine. The polarity of the liquid zwitterionic carbamate salt formed with N,N,N’-trimethyl-1,3-propanediamine was measured using UV-Vis and the solvatochromic dye Nile red; its polarity was comparable to previous switchable polarity systems. The CO2 gravimetric uptake of the liquid zwitterionic carbamate salt was 28%, far greater than other solvents for the capture and release of CO2. In the third chapter, it was found that a variety of products can be accessed depending on the structure of the N-base (cyclic or acyclic) upon reaction of the base with CS2 at room temperature. The reaction of CS2 with cyclic amidines produced a cyclic trithioanhydride structure, forming a new C-C bond at a sp2-carbon beta to the imino nitrogen centre. When an amidine was acyclic it led to cleavage and formation of isothiocyanates in near quantitative yields. When a N-base had a N-H bond, CS2 can insert, forming a dimer in the presence of dichloromethane. In the fourth chapter, preliminary investigations are ascribed for synthesis of α-amino acids, amides, and ureas. Carboxylation of ketimines was detected, although the formed carboxylates from a variety of ketimines readily decomposed. Isomerization products of two ketimines were generated with DBU and CO2. Lewis acid catalysts were implemented towards the amidation of benzoylacetic acid and synthesis of ureas. Amidation of benzoylacetic acid did not occur in the presence of Lewis acid catalysts and CO2. Formation of a cyclic tetraalkylurea was afforded in low yields by the use of a diamine, CO2 and Lewis acid catalysts. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-12-01 19:16:55.257

Iron is the most abundant transition metal found in living systems and plays a crucial role in DNA biosynthesis. To accommodate higher replication rates, cancer cells require higher amounts of iron compared to non-neoplastic counterparts. This higher demand for iron renders cancer cells susceptible to iron deprivation, and exposure to iron chelators leads to growth arrest and cell death. Iron chelation strategies employing a wide variety of iron-binding scaffolds are currently under investigation for use in cancer treatment. Although these chelation approaches are effective against several cancer cell types, their use is limited due to toxicity ascribed to indiscriminate metal sequestration and induction of oxidative stress. Prochelation strategies in which the chelating unit remains inactive until triggered by a disease-specific event are expected to increase the specificity of chelation-based therapeutics. Chapter 1 provides an overview of chelation and prochelation based therapies as well as disulfide-based approaches in the design of prodrugs. In Chapter 2, the reduction activation mechanism of disulfide-masked thiosemicarbazone prochelators is described. Whereas disulfide-masked prochelators do not bind iron, reduction of the disulfide bond upon cellular uptake produces active chelators that readily bind intracellular iron. These systems are not active extracellularly; rather, they target the intracellular labile iron pool. We found that the antiproliferative activity of these disulfide-masked prochelators is dependent on the intracellular redox environment, with enhanced toxicity in more reducing conditions. The iron complexes resulting from exposure of cultured cells to the chelation systems were detected intracellularly by electron paramagnetic resonance in intact frozen cells. The compounds in our first series do not engage in intracellular redox chemistry and do not cause oxidative stress. In Chapter 3, the synthesis and characterization of a larger series of disulfide-masked prochelators featuring several classes of tridentate ligands is described. We investigated the iron-binding efficacy of the corresponding chelators, their ability to induce oxidative stress and their cell-cycle effects. We found that these prochelator systems, regardless of the identity of the donor set of atoms, do not result in the intracellular generation of oxidative stress. We also found that treatment of cultured cancer cells with prochelators results in cell-cycle arrest at G1/0 in non-synchronized cells and G2/M in G2-synchronized cells. In addition, we found that all classes of prochelators exhibit antiproliferative effects likely through induction of apoptosis. In Chapter 4, the syntheses and biological evaluations of disulfide-masked prochelators that feature carbohydrate targeting units are described. The sugar conjugates present increased aqueous solubility, compete as effectively as D-glucose for transporter-mediated cellular uptake, and are 6 to 11-fold more selective towards colorectal cancer compared to an aglycone that does not contain a targeting unit. The design of more potent prochelator systems, as well as the design of systems with improved selectivity and aqueous solubility are discussed in Chapter 5.