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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.
91

Engineering yeast G protein-coupled receptors for biosensor development

Matragrano, Joseph Antonio January 2020 (has links)
The ability to sense and respond to environmental stimuli is essential for the survival of all living things. As a result, nature has evolved an uncountable number of ways to detect environmental signals. At the cellular level, G protein-coupled receptors (GPCRs) are used by eukaryotes, including fungi and humans, to convert extracellular molecular binding events into intracellular responses. Recently, synthetic biologists have shown that biological sensing systems can be repurposed to suit human needs, developing tools such as diagnostic devices and drug screening platforms. In this thesis, I present work exploring the potential of fungal GPCRs to be used as sensing elements in yeast-based biosensors. Chapter 1 gives background information related to synthetic biology, biosensors, and yeast signaling pathways. Chapter 2 describes the development of the baker's yeast Saccharomyces cerevisiae into a diagnostic device for detection of fungal pathogens, using fungal GPCRs. In Chapter 3 I demonstrate that the substrate specificity of fungal GPCRs can be altered using directed evolution. Chapter 4 describes experiments further probing the native binding abilities of fungal GPCRs, specifically examining protein ligands. Finally, in Chapter 5 we move beyond fungal GPCRs and engineer yeast to detect other stimuli, in the context of an engineered living material.
92

Design of novel αvβ3 ligands as probes for imaging of tumour angiogenesis and site-directed delivery of cytotoxic drugs

Piras, Monica January 2014 (has links)
The dependence of tumour growth and metastasis on blood vessels makes tumour angiogenesis a rational target for therapy. Imaging of αvβ3 expression could potentially be used as a biomarker and an early indicator of efficacy of antiangiogenic treatments at a molecular level. Research efforts have mainly focused on the development of RGD-based radiolabelled αvβ3 inhibitors suitable for PET and SPECT imaging modalities that, owing to their high sensitivity, represent the most powerful tool for monitoring in vivo tumour angiogenesis. The aim of this multidisciplinary project was the design, synthesis and biological evaluation of novel αvβ3 ligands as molecular imaging probes. Three classes of integrin antagonists were designed: 1) triazole-based RGD mimetics that can be isotopically-labelled with tritium, fluorine and iodine radioisotopes by means of highly practical procedures, 2) RGD peptidomimetics incorporating the metabolically stable 2,2,2-trifluoroethylamine function as a peptide bond bioisostere and 3) RGD cyclopeptides conjugated with FDR, a novel prosthetic group allowing glycosylation and 18F-fluorination of aminooxy-functionalised molecules in one synthetic step. RGD-based strategies have also been used for selective tumour delivery of chemotherapeutic agents. A number of cytotoxic drugs have been conjugated to RGD peptides, providing experimental evidence that αvβ3 targeted chemotherapy strategies could be used as a powerful tool to reduce the toxicity and augment the therapeutic window of existing cytotoxic agents. In this work, we described the rational design of a novel targeted cytotoxic conjugate containing a triazole-based RGD peptidomimetic as tumour-homing motif of the potent antimitotic agent, paclitaxel. Preliminary in vitro studies were performed to assess the therapeutic potential of this targeted cytotoxic construct.
93

Identifying ligands of the C-terminal domain of cardiac expressed connexin 40 and assessing its involvement in cardiac conduction disease

Keyser, Rowena J. 12 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2007. / Connexins (Cx) are major proteins of gap junctions, dynamic pores mediating the relay of ions and metabolites between cells. Cxs 40, 43 and 45 are the predominant cardiac isoforms and their distinct distribution raises questions about their functional differences. Their cytoplasmic (C)-terminal domains are involved in protein-protein interactions. Furthermore, mutations in the myotonic dystrophy protein kinase (DMPK)-causative gene are associated with disruptions in cardiac conduction similar to that described for Cx knock-out mice. DMPK is a Cx43 ligand, raising the possibility that defects in Cx40 ligands may be involved in the development of cardiac conduction disturbances. We hypothesised that delineation of the protein ligands of the C-termini of Cx40 and of Cx45 (parallel study conducted by N Nxumalo) would help elucidate their functional roles. Yeast-two-hybrid methodology was used to identify putative Cx40 ligands. Primers were designed to amplify the C-terminus-encoding domain of the human Cx40 gene (Cx40), the DNA product was cloned into the pGBKT7 vector which was used to screen a cardiac cDNA library in Saccharomyces cerevisiae. Successive selection stages reduced the number of putative Cx40 ligand-containing colonies (preys) from 324 to 33. The DNA sequences of the 33 ligands were subjected to BLAST-searches and internet database literature searches to assign identity and function and to exclude false positive ligands based on subcellular location and function. Eleven plausible ligands were identified: cysteine-rich protein 2 (CRP2), beta-actin (ACTB), creatine kinase, muscle type (CKM), myosin, heavy polypeptide 7 (MYH7), mucolipin1 (MCOLN1), voltage-dependent anion channel 2 (VDAC2), aldehyde dehydrogenase 2 (ALDH2), DEAH box polypeptide 30 (DHX30), NADH dehydrogenase, 6, (NDUFA6), prosaposin (PSAP) and filamin A (FLNA). Cxs 40 and 45 showed differences in the classes of proteins with which they interacted; the majority of putative Cx40 interactors were cytoplasmic proteins, while Cx45 interactors were mitochondrial proteins. These results suggest that Cxs 40 and 45 are not only functionally different, but may also have different cellular distributions. Further analyses of these protein interactions will shed light on the independent roles of Cxs 40 and 45.
94

An investigation of the role of phosphorylation at Ser211 of the glucocorticoid receptor in ligand-specific transcriptional regulation

Stubsrud, Elisabeth 12 1900 (has links)
Thesis (MSc (Biochemistry))--University of Stellenbosch, 2005. / Endogenous glucocorticoids (GCs) modulate many physiological functions in the human body and synthetic GCs are the most effective therapy in the treatment of inflammation, autoimmune and endocrine disorders. However, the long-term usage of synthetic GCs is associated with severe side-effects. GCs mediate their effects through the ligand-dependent transcription factor, the glucocorticoid receptor (GR), either by causing an increase (transactivation) or a decrease (transrepression) in gene transcription. The bioactivity of a ligand in GR-mediated transcriptional regulation is established by a transcriptional doseresponse curve, where the potency (EC50 value) and the efficacy (maximal response) of the ligand are determined. A central question is how different GR ligands elicit their differential physiological responses for the same gene in the same cell. The main aim of this thesis is to investigate if the phosphorylation of GR at serine 211 (Ser211) correlates with the potency and/or efficacy of a particular ligand in transactivation and transrepression of gene expression.
95

Application of affinity mass sensor based on boronic acid derivatives.

January 2001 (has links)
Chow Ka-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 52-55). / Abstracts in English and Chinese. / Chapter 1 --- Introduction / Chapter 1.1 --- Chemical sensors --- p.1 / Chapter 1.2 --- Quartz crystal microbalance --- p.4 / Chapter 1.3 --- Concept of affinity mass sensor --- p.8 / Chapter 1.4 --- Film immobilization technologies --- p.9 / Chapter 1.5 --- Research outlines --- p.13 / Chapter 2 --- Experimental / Chapter 2.1 --- Sensor fabrication --- p.14 / Chapter 2.2 --- Flow-through cell --- p.16 / Chapter 2.3 --- Analysis procedures --- p.19 / Chapter 2.4 --- Response curve --- p.19 / Chapter 2.5 --- Experimental setup --- p.21 / Chapter 3 --- Detection of ascorbic acid by affinity mass sensor based on 3-aminophenylboronic acid / Chapter 3.1 --- Conventional analytical methods --- p.23 / Chapter 3.2 --- Research method - affinity mass sensor based on APBA --- p.24 / Chapter 3.3 --- To locate the binding site in ascorbic acid --- p.25 / Chapter 3.3.1 --- Steric energy calculated by molecular modeling --- p.26 / Chapter 3.4 --- Optimization of experimental variables --- p.29 / Chapter 3.4.1 --- Effect of pH --- p.29 / Chapter 3.4.2 --- Effect of sample volume --- p.30 / Chapter 3.4.3 --- Effect of flow velocity --- p.30 / Chapter 3.5 --- Calibration and Reproducibility --- p.32 / Chapter 3.6 --- Kinetic analysis --- p.33 / Chapter 3.7 --- Stability of sensor --- p.37 / Chapter 3.8 --- Interference studies --- p.37 / Chapter 3.9 --- Determination of ascorbic acid in real samples --- p.39 / Chapter 3.9.1 --- Results and Discussion --- p.39 / Chapter 3.10 --- Comparison with conventional ascorbic acid sensors --- p.42 / Chapter 3.11 --- Summary --- p.42 / Chapter 4 --- Boronic acid derivatives for the detection of sugars / Chapter 4.1 --- Scope of this work --- p.43 / Chapter 4.2 --- Results and Discussion --- p.44 / Chapter 4.3 --- Summary --- p.49 / Conclusion --- p.50 / References --- p.52 / List for tables --- p.56 / List for figures --- p.57 / Appendix I --- p.59 / Appendix II --- p.61
96

Fluorescent GFP chromophores as potential ligands for various nuclear receptors

Duraj-Thatte, Anna 18 May 2012 (has links)
Nuclear receptors are ligand activated transcription factors, where upon binding with small molecule ligands, these proteins are involved in the regulation of gene expression. To date there are approximately 48 human nuclear receptors known, involved in multiple biological and cellular processes, ranging from differentiation to maintenance of homeostasis. Due to their critical role in transcriptional regulation, these receptors are implicated in several diseases. Currently, 13% of prescribed drugs in the market are NR ligands for diseases such as cancer, diabetes and osteoporosis. In addition to drug discovery, the mechanism of function, mobility and trafficking of these receptors is poorly understood. Gaining insight into the relationship between the function and /or dysfunction of these receptors and their mobility will aid in a better understanding of the role of these receptors. The green fluorescent protein (GFP) has revolutionized molecular biology by providing the ability to monitor protein function and structure via fluorescence. The fluorescence contribution from this biological marker is the chromophore, formed from the polypeptide backbone of three amino acid residues, buried inside 11-stranded â-barrel protein. Synthesis of GFP derivatives of is based on the structure of the arylmethyleneimidazolidinone (AMI), creating a molecule that is only weakly fluorescent. Characterizing these AMI derivatives for other proteins can provide a powerful visualization tool for analysis of protein function and structure. This development could provide a very powerful method for protein analysis in vitro and in vivo. Development of such fluorescent ligands will prove beneficial for the nuclear receptors. In this work, libraries of AMIs derviatives were synthesized by manipulating various R groups around the core structure, and tested for their ability to serve as nuclear receptor ligands with the ability to fluoresce upon binding. The fluorogens are developed for steroidal and non-steroidal receptors, two general classes of nuclear receptors. Specific AMIs were designed and developed for steroid receptor estrogen receptor á (ERá). These ligands are showed to activate the receptor with an EC50 of value 3 ìM and the 10-fold activation with AMI 1 and AMI 2 in comparison to the 21-fold activation observed with natural ERá ligand, 17â-estradiol. These novel ligands were not able to display the fluorescence upon binding the receptor. However, fluorescence localized in nucleus was observed in case of another AMI derivative, AMI 10, which does not activate the receptor. Such ligands open new avenues for developing fluorescent probes for ERá that do not involve fluorescent conjugates attached to a known ERá ligand core. AMIs were also characterized for non-steroidal receptors,specifically the pregnane x receptor (PXR) and retinoic acid receptor á (RARá). To date, fluorogens which turn fluorescence upon binding and activate the receptor have not been developed for these receptors. With respect to PXR, several AMI derivatives were discovered to bind and activate this receptor with a fold-activation better than the known agonist, rifampicin. The best characterized AMI derivative, AMI 4, activates the receptor with an EC50 of value 6.3 ìM and the 154-fold activation in comparison to the 90-fold activation and an EC50 value of 1.3 ìM seen with rifamipicin. This ligand is not only able to activate PXR but also displays fluorescence upon binding to the receptor. The fluroscence pattern was observed around the nucleus. Besides AMI 4, 16 other AMI derivatives are identified that activate PXR with different activation profiles. Thus, a novel class of PXR ligands with fluorescence ability has been developed. The AMI derivatives able to bind and activate RAR, also displayed activation profiles that were comparable to the wild-type ligand, all trans retinoic acid. These ligands activated the receptor with an EC50 value of 220 nM with AMI 109 in comparison to an EC50 value of 0.8 nM with the natural ligand for RARá. When these ligands were tested for fluorescence in yeast, the yeast were able to fluoresce only in the presence of the receptor and the AMI derivative, indicating that these agonists also have the ability to fluoresce.
97

Engineering Protein Molecular Switches To Regulate Gene Expression with Small Molecules

Rohatgi, Priyanka 29 November 2006 (has links)
Small molecule dependent molecular switches that control gene expression are important tool in understanding biological cellular processes and for regulating gene therapy. Nuclear receptors are ligand activated transcription factors that have been engineered to selectively respond to synthetic ligands and used as regulators of gene expression. In this work the retinoid X receptor (RXR), has been used to develop an inducible molecular switch with a near drug like compound LG335. Three RXR variants (Q275C; I310M; F313I), (I268A; I310A; F313A; L436F), (I268V; A272V; I310M; F313S; L436M) were created via site-directed mutagenesis and a structure based approach, such that they preferentially bind to the synthetic ligand LG335 and not its natural ligand, 9-cis retinoic acid. These variants show reverse ligand specificity as designed and have an EC50 for LG335 of 80 nM, 30 nM, 180 nM, respectively. The ligand binding domains of the RXR variants were fused to a yeast transcription factor Gal4 DNA binding domain. This modified chimeric fusion protein showed reverse response element specificity as designed and recognized the Gal4 response element instead of the RXR response element. The modified RXR protein did not heterodimerize with wild type RXR or with other nuclear receptor such as retinoic acid receptor. These RXR-based molecular switches were tested in retroviral vectors using firefly luciferase and green fluorescence protein and they maintain their inducible behavior with LG335. These experiments demonstrate the orthogonality of RXR variants and their possible use in regulating gene therapy.
98

Surface modification of nanoparticles for polymer/ceramic nanocomposites and their applications

Kim, Philseok 17 November 2008 (has links)
Polymer/ceramic nanocomposites benefit by combining high permittivities (r) of metal oxide nanoparticles with high dielectric strength and excellent solution-processability of polymeric hosts. Simple mixing of nanoparticles and polymer generally results in poor quality materials due mainly to the agglomeration of nanoparticles and poor miscibility of nanoparticles in host materials. Surface modification of metal oxide nanoparticles with phosphonic acid-based ligands was found to afford a robust surface modification and improve the processablity and the quality of nanocomposites. The use of phosphonic-acid modified barium titanate (BaTiO₃) nanoparticles in dielectric nanocomposites dramatically improved the stability of the nanoparticle dispersion and the quality of the nanocomposites. Surface modification of BaTiO₃ nanoparticles allowed high quality nanocomposite thin films in ferroelectric polymer hosts such as poly(vinylidene fluoride-co-hexafluoropropylene) with large volume fractions (up to 50 vol. %), which exhibited a remarkable combination of a high permittvity (35 at 1 kHz) and a high breakdown strength (210 V/µm) leading to a maximum energy storage density of 6.1 J/cm³. The effect of nanoparticle volume fractions on the dielectric properties of this nanocomposite system was investigated and compared with theoretical models. At high volume fraction of nanoparticles, the porosity of the nanocomposites was found to have important role in the dielectric performance. A combined effective medium theory and finite difference simulation was used to model the dielectric properties of high volume fraction dielectric nanocomposites with porosity. These results provide a guideline to optimize the volume fractions of nanoparticles for maximum energy density. Nanocomposites based on phosphonic acid-modified BaTiO₃ nanoparticles can also be used as printable high permittivity dielectrics in organic electronics. High volume fractions (up to 37 vol. %) of phosphonic acid-modified BaTiO₃ nanoparticles dispersed in cross-linked poly(4-vinylphenol) allowed solution-processable high permittivity thin films with a large capacitance density (~50 nF/cm²) and a low leakage current (10 8 A/cm²) suitable as a gate insulator in organic field-effect transistors (OFETs). Pentacene-based OFETs using these nanocomposites showed a low threshold voltage (< -2.0 V) and a large on/off current ratio (Ion/off 104 ~ 106) due to the high capacitance density and low leakage current of the gate insulator.
99

Facilitating multi-electron reactivity at low-coordinate cobalt complexes using redox-active ligands

Smith, Aubrey L. 23 August 2011 (has links)
In this study, we describe a detailed investigation of cobalt complexes containing redox-active ligands. We have prepared an electronic series of the complex in three oxidation states: [CoIII(ap)2]-, CoIII(isq)(ap), and [CoIII(CH3CN)(isq)2]+. Characterization shows that the metal center remains cobalt(III) through the redox changes and indicates that the oxidation state changes occur with gain or loss of electrons from the ligand set. While CoIII(isq)(ap) reacts with halide radicals to form a new cobalt-halide bond in a single electron reaction, [CoIII(ap)2]- appears to be prone to multi-electron reactivity in reactions with sources of "Cl+". Both reactions occur with electrons derived from the ligand set. Mechanistic studies suggest a single, two electron step is responsible for the bond-formation. Similarly, [CoIII(ap)2]- reacts with alkyl halides to pseudo-oxidatively add the alkyl at the cobalt center. The product of the reaction can be isolated and fully characterized and was found to be best assigned as CoIII(alkyl)(isq)2. This assignment indicates that the reaction occurs, again, with the new bond formed with two electrons formally derived from the ligand set and with no change in oxidation state at the metal center. Mechanistic investigations of the pseudo-oxidative addition suggest the reaction is SN2-like. The reaction occurs with a wide scope of alkyl halides, including those containing beta-hydrogens. The cross-coupling reaction of CoIII(alkyl)(isq)2 with RZnX forms a new carbon-carbon bond. Similarly, the two electron oxidized complex [CoIII(CH3CN)(isq)2]+ reacts with organozinc reagents to couple two carbon nucleophiles and form a new carbon-carbon bond. Both reactions are successful with both sp2 and sp3 carbons. When followed substoichiometrically, the homocoupling reaction can be observed to form CoIII(alkyl)(isq)2. This indicates that the homocoupling and cross-coupling reactions proceed by the same mechanism. However, both reactions have low yields. The yield of the reactions are decreased by steric bulk of the alkyl or aryl fragments or around the metal center created by substituents on the ligand. Also, while the steric congestion disfavors the addition of the first alkyl fragment, the addition of the second alkyl fragment and subsequent rapid elimination of the coupling product is almost completely inhibited. This result also implies that the coupling of the two alkyl fragments is entirely inner-sphere requiring installation of both for coupling. In a complementary study, use of bidentate or tridentate stabilizing ligands in combination with one redox-active catechol-derived or amidophenol-derived ligand was investigated. With the synthesis of (triphos)CoII(cat) and the one electron oxidized [(triphos)CoII(sq)]+, it is evident that the oxidation occurs at the ligand and not the metal. Reaction of (triphos)CoII(cat) with a Cl+ reagent generated a new material which we tentatively describe as (triphos)CoIII(Cl)(sq). This implies that the two electrons used to create the new cobalt-halide bond are derived from both the ligand and the metal, one from each. We believe the complex is unreactive with organic halides due to the steric bulk surrounding the metal center. Similar cobalt complexes containing tridentate or bidentate phosphine ligands or a tridentate pyrazol ligand in combination with a catechol-derived or amidophenol-derived ligand resulted in unsuccessful synthesis or unstable complexes. Throughout the course of both of these studies, steric crowding at the metal center is a problem disfavoring the facilitated reactivity. We have however shown that the amidophenol ligands have favorable molecular orbital overlap with the cobalt to act as an electron reservoir and facilitate reactivity at the metal center. We have also shown that this combination can create a proclivity to facilitate multi-electron reactions at the metal that is naturally prone to radical reactions.
100

Characterization of the ligand-binding specificity and transcriptional properties of estrogen receptor homodimeric/heterodimeric complexes

Yuan, Xiaohui, January 2001 (has links)
Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 228-272). Also available on the Internet.

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