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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Design and Synthesis of Small-Molecule Protein-Protein Interaction Antagonists

Han, Xu January 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.
32

Investigating spatial distribution and dynamics of membrane proteins in polymer-tethered lipid bilayer systems using single molecule-sensitive imaging techniques

Ge, Yifan 12 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.
33

Biochemical And Genetic Studies of Quebec Platelet Disorder

Diamandis, Maria 05 1900 (has links)
<p> Inherited bleeding disorders can be caused by mutations affecting platelet, coagulation, or fibrinolytic proteins. Quebec platelet disorder (QPD) is a rare, autosomal dominant disorder associated with increased expression of the fibrinolytic enzyme urokinase plasminogen activator (uPA) in platelets. Individuals with QPD experience delayed-onset bleeding after hemostatic challenges that is attenuated with fibrinolytic inhibitor therapy. The aims of this thesis were to: 1) determine if increased platelet uPA contributes to QPD clot lysis in vitro; 2) investigate whether QPD individuals have increased urinary uPA, as some individuals experience hematuria; and 3) map the genetic locus of QPD, and look for the putative mutation. Studies of clot lysis indicated that QPD platelets induce a gain-of-function defect in fibrinolysis when platelets are incorporated into clots. This suggests that accelerated fibrinolysis may contribute to QPD bleeding. Studies of urinary uPA in QPD showed that uPA is not increased, indicating that hematuria in QPD is likely a consequence of increased platelet uPA. This finding also suggests that uPA overexpression in QPD may be megakaryocyte-specific. Linkage studies showed that QPD is strongly linked to a 2 megabase region on chromosome 10 that harbors the uPA gene, PLA U. No mutations in PLA U or its regulatory regions were identified; however, a common haplotype for a 32.5 kilobase region around PLA U, including inheritance of a rare, linked polymorphism, suggests this is the most likely locus for QPD. mRNA studies in QPD platelets showed that QPD selectively increases expression of the linked PLAU allele, without similar increases in megakaryocyte progenitors or in saliva. These findings implicate a cis-mutation near PLA U as the cause of QPD. This thesis provides novel insights on the fibrinolytic abnormality in QPD blood, and on the QPD genetic locus. which will be important for identifying the precise mutation that converts normally prohemostatic platelets to profibrinolytic cells. </p> / Thesis / Doctor of Philosophy (PhD)
34

Receptor mediated catabolism of plasminogen activators

Grimsley, Philip George, Medical Sciences, Faculty of Medicine, UNSW January 2009 (has links)
Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.

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