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1
Ambrogi, Martina. "Synthesis, characterization and conformational studies of arylbenzylmaleimides." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amslaurea.unibo.it/4251/.
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From the discoveries of Pasteur, stereochemistry has played an increasingly important role in the chemical sciences. In particular conformational study of molecules with axial chirality is object of intense research. Through Dynamic-NMR analysis and simulation of the spectra, the energy rotational barriers value of conformers are obtained. When this barrier is high sufficiently, atropisomeric stable compounds can be reached. They can be separated and used in stereo-synthesis and in packing processes.
3,4-bis-aryl maleimides, in which the aromatic groups are sufficiently bulky, generate atropisomeric stable configurations, that can be isolated at room temperature. The assignment of absolute configurations is performed through ECD analysis and comparison with computational calculations. The biological activities of maleimide derivatives widen the field of atropisomers application also in biological systems.
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Silenzi, Ilaria. "Synthesis, characterization and conformational studies of bis-phenothiazine-aryl-boranes." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2019. http://amslaurea.unibo.it/19201/.
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This thesis project presents a work based on the study of a particular class of amino-boranes, called bis-phenothiazine-aryl-boranes. The peculiarity of these compounds is the N-B-N chemical moiety and their complex conformational behaviour, due to the combination of steric hindrance and conjugation of the B-N bond. Our work is focused on two main products with different symmetry: bis-phenothiazine-2-methylnaphthyl-borane (2b) and bis-phenothiazine-anthracenyl-borane (2c). We firstly focused our attention on an effective way of synthesis, by optimizing both reaction conditions and purification. The products and co-products of interest were then characterized with NMR, mass spectroscopy and X-Ray diffraction on single crystals. The products were eventually analysed through conformational studies, by experimental techniques, such as dynamic NMR and EXSY, and by a theorical approach with DFT calculations.
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Zych, Andrew John. "Conformational characterization of abiotic secondary structure based on aromatic stacking /." Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3008484.
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Foschi, Simone. "Synthesis, Characterization and Conformational Studies of Modified Carbazole-bis-aryl-boranes." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amslaurea.unibo.it/20686/.
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During this project we have synthetized different compounds belonging to the class of amino-boranes for the study of bis-aryl-B=N system. We have decided to keep unchanged the aryl components and change only the amine to observe the effect of that on the B=N bond. The used amines are modified carbazoles with functional groups chosen to amplify or disempower the steric and the conjugation effect. We have found that the evaluation of steric barrier was possible studying the gearing aryls rotation around the C-B bonds, while the conjugation barrier is instead given by the energy needed to break the formal double bond B=N and allow the amine rotation. The work started with a proposed synthesis, improved for every reaction, then the products are characterized by NMR, fluorometric spectroscopy, mass spectrometry and X-Ray diffraction on single crystal. The following study on rotational energy barrier was possible theoretically through DFT calculation and experimentally with techniques like Dynamic NMR and EXSY. The fluorometric analysis was done for the study of the solvatochromic propriety of the products.
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Croasdale, Rebecca Alice. "Characterization of the conformational dynamics of the Nek2 leucine zipper domain." Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/27799.
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Nek2 is a cell cycle regulated protein kinase. Its expression and activity peak in S and G2 phase before the protein is targeted for degradation in mitosis. Nek2 activity promotes centrosome separation and bipolar spindle formation, processes that are essential for maintaining the fidelity of chromosome segregation and cell division. Importantly, Nek2 inhibition results in increased apoptosis and senescence in cancer cell lines and tumour xenografts making Nek2 an attractive target for chemotherapeutic intervention. The α-helix is the most common secondary structure in proteins, with 2-3% of all proteins adopting a coiled-coil structure. The coiled-coil motif mediates a diverse range of functions including DNA transcription, intracellular protein shuttling, membrane signalling, and coordination of the cell cycle. Nek2 contains two coiled-coil motifs in its C-terminal non-catalytic domain, the first of which forms a leucine zipper structure, whilst the second has recently been classified as a SARAH domain. The leucine zipper spans residues 304-340 and is responsible for Nek2 dimerization, autophosphorylation and activation. The Nek2 leucine zipper contains an unusual pattern of charged residues in the dimerization interface. Through studies on isolated fragments, we found that the Nek2 leucine zipper exists primarily as a dimer in solution, albeit with concentration dependent higher order oligomerization. However, the positioning of charged residues enabled the helices to undergo a register shift between two alternative heptad conformations on a timescale of 17s-1. This represents slow-intermediate exchange on the NMR timescale, greatly increasing the transverse and longitudinal relaxation rates leading to enhanced loss of signal intensity. As this precluded structure determination through NMR studies on wild-type fragments, a K309C mutant was generated that ‘locked’ the leucine zipper into one conformation. The significantly reduced relaxation rates confirming the presence of conformational dynamics and making structural determination possible in the future.
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Ieronymaki, Matthaia. "Immunological and Conformational characterization of synthetic peptide probes for autoimmune diseases." Thesis, Cergy-Pontoise, 2016. http://www.theses.fr/2016CERG0831/document.
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Les maladies auto-immunes sont des maladies chroniques et hétérogènes caractérisées par des réactions du système immunitaire acquis contre les propres tissus sains de l'organisme. Ces maladies affectent presque 5% de la population mondiale et en particulier les jeunes adultes. La complexité de leur spectre est énorme et même si leur étiologie est encore incertaine, il a été démontré que des facteurs génétiques et environnementaux sont impliqués dans le déclenchement du mécanisme pathologique. Cependant, il est nécessaire d'utiliser des outils diagnostiques et / ou pronostiques fiables pour le diagnostic précoce avant que des dommages cellulaires irréversibles ne se produisent et pour surveiller la progression de la maladie.De nombreuses études ont mis en évidence la présence de différents auto-anticorps dans le sérum de patients atteints de maladies auto-immunes. Les auto-anticorps qui sont spécifiques d’une maladie peuvent être utilisés en tant que biomarqueurs (BM) pour son diagnostic alors que les auto-anticorps qui diffèrent en fonction de l'état de la maladie peuvent être utilisés dans le suivi des patients. En fait, dans le cas de l'auto-immunité, un BM facilement détectable et fiable peut être représenté par le titre d'un auto-anticorps spécifique.Dans ce contexte, nous nous intéressons à deux maladies différentes, la sclérose en plaques (SEP) et la gammapathie monoclonale, en utilisant l'approche chimique inverse via le criblage de librairies de peptides par des sérums de patients.En particulier, l'importance des anticorps anti-myéline, et surtout, des anticorps anti-MOG (myéline oligodendrocyte glycoprotéine) est toujours l’objet de débats, soulignant la question très controversée d'un rôle pathogène putatif d'anticorps anti-MOG dans la SEP. Dans cette thèse, nous avons étudié le rôle de MOG comme auto-Ag putatif dans la SEP en utilisant le modèle expérimental d’encéphalomyélite auto-immune (EAE). Ainsi, afin d'évaluer la présence d'un mécanisme d’« epitope spreading » des cellules B, à savoir l'apparition d'une réponse dirigée vers des épitopes distincts de l'agent pathogène induisant la réponse immunitaire, nous avons synthétisé et testé en tant que sondes antigéniques cinq peptides synthétiques qui couvrent la séquence 1-117 de MOG.La seconde étude a porté sur la sélection d'un peptide mimant l'épitope minimal reconnu par l'anticorps monoclonal commercial anti- natural killer cell-1 humain (anti-HNK-1) en utilisant la résonance plasmonique de surface (SPR). L’épitope HNK-1 est considéré comme le déterminant antigénique de la glycoprotéine associée à la myéline (MAG), un composant quantitativement mineur des gaines de myéline. On observe que les patients atteints de troubles neurologiques auto-immuns, tels que la gammapathie monoclonale à IgM et la polyneuropathie démyélinisante, développent souvent des anticorps anti-MAG ciblant spécifiquement l’épitope HNK-1. Par conséquent, l'identification et la caractérisation de ces anticorps est pertinente. Le peptide choisi suite à notre étude pourrait ensuite être utilisé chez des patients atteints de troubles neurologiques pour le développement d'un outil de diagnostic fiable ou de surveillance de l'activité de la maladie par l'identification d'anticorps anti-HNK-1 dans le sérum des patientsAutoimmune diseases (ADs) refer to chronic and heterogeneous diseases with acquired immune system’s reactions against the body’s own healthy tissues. ADs affect more than 5% of the population worldwide and especially young adults. The complexity of their spectrum is enormous and even if their etiology is still unclear, it was demonstrated that both genetic and environmental factors are involved in triggering the pathological mechanism. Hence, a reliable diagnostic and/or prognostic tool for an early diagnosis of ADs before irreversible cellular damage occurs and for monitoring their progression is demanded.Numerous studies have revealed the presence of different autoantibodies (auto-Abs) in sera of patients suffering from ADs. Autoantibodies that are specific for a disease can be used as biomarkers (BMs) for its diagnosis while autoantibodies that differ depending on the disease state can be used in the follow up of the patients. Actually, in the case of autoimmunity, an easily detectable and reliable BM may be represented by the titer of a specific auto-Ab.In this context, we aimed to identify target(s) of the response for two different ADs, multiple sclerosis (MS) and monoclonal gammopathy, using the chemical reverse approach, which involves the screening of focused antigen (Ag) libraries with patients’ serum.In particular, the significance of anti-myelin antibodies, and especially, anti- Myelin Oligodendrocyte Glycoprotein (anti-MOG) antibodies is still matter of debate, underscoring the highly controversial issue of a putative pathogenetic role of anti-MOG antibodies in MS. In this thesis we investigated the role of MOG as putative auto-Ag in MS using the experimental autoimmune encephalomyelitis (EAE) model. Moreover, in order to assess the presence of a B-cell epitope spreading mechanism, i.e. the occurrence of a response directed toward epitopes distinct from the disease-inducing agent, we synthesized and tested as antigenic probes also five synthetic peptides covering the 1-117 sequence of MOG.The second issue focused on the selection of a peptide mimicking the minimal epitope recognized by the commercial available monoclonal antibody anti-human natural killer cell-1 (anti-HNK-1) using Surface Plasmon Resonance (SPR) technique. HNK-1 epitope, is considered as the antigenic determinant of myelin-associated glycoprotein (MAG), a quantitatively minor component of myelin sheaths. It is observed that patients affected by autoimmune neurological disorders, such as IgM monoclonal gammopathy and demyelinating polyneuropathy, often develop anti-MAG antibodies specifically targeting the HNK-1 epitope. Accordingly, identification and characterisation of these antibodies is relevant. The selected peptide could be subsequently used in earlier stage patients for the development of a novel and reliable diagnostic tool for anti-HNK-1 antibody identification in sera of patients affected by autoimmune neurological disorders monitoring disease activity
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Fisher, Charles. "Statistical Characterization of Protein Ensembles." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10223.
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Conformational ensembles are models of proteins that capture variations in conformation that result from thermal fluctuations. Ensemble based models are important tools for studying Intrinsically Disordered Proteins (IDPs), which adopt a heterogeneous set of conformations in solution. In order to construct an ensemble that provides an accurate model for a protein, one must identify a set of conformations, and their relative stabilities, that agree with experimental data. Inferring the characteristics of an ensemble for an IDP is a problem plagued by degeneracy; that is, one can typically construct many different ensembles that agree with any given set of experimental measurements. In light of this problem, this thesis will introduce three tools for characterizing ensembles: (1) an algorithm for modeling ensembles that provides estimates for the uncertainty in the resulting model, (2) a fast algorithm for constructing ensembles for large or complex IDPs and (3) a measure of the degree of disorder in an ensemble. Our hypothesis is that a protein can be accurately modeled as an ensemble only when the degeneracy of the model is appropriately accounted for. We demonstrate these methods by constructing ensembles for K18 tau protein, \(\alpha\)-synuclein and amyloid beta - IDPs that are implicated in the pathogenesis of Alzheimer's and Parkinson's diseases.
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Niyogi, Sandip. "Synthesis and characterization of molecules to study the conformational barriers of fluorocarbon chains." Thesis, University of North Texas, 2000. https://digital.library.unt.edu/ark:/67531/metadc2511/.
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Fluorocarbons are known to be stiffer than their hydrocarbon analogues, a property that underlines the extensive industrial application of fluorocarbon materials. Although there has been previous studies on the rotational barrier of molecules having fluorocarbon centers, a detailed systematic study is necessary to quantify flurocarbon stiffness. The molecules, Pyrene-(CF2)n-Pyrene, Pyrene-(CF2)n-F, Pyrene-(CH2)n-Pyrene and Pyrene-(CH2)n-H were therefore synthesized to enable the determination of the barrier to rotation of the carbon backbone in fluorocarbons. Conformational studies will be completed with steady-state and time-dependent emission spectroscopy.
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Cherwa, Jr James Edward. "Characterization of Scaffolding Proteins Altered in the Ability to Perform a Critical Conformational Switch." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/195476.
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Throughout recent history scientists have struggled to elucidate the biochemical and biophysical mechanisms that guide the assembly of macromolecular structures. The early models of "sub-assembly" or "self assembly" attempted to explain how individual components could interact in a precisely regulated manner to form higher-ordered complex biological structures. Subsequent studies, using viral systems as assembly models, demonstrated how protein-protein and protein-nucleic acid interactions assist in lowering the thermodynamic barriers that typically disfavor assembly.Due to their simplicity, viruses provide an ideal system to investigate the biophysical mechanisms that drive the assembly of complex biological structures. Proper virion assembly requires numerous macromolecular interactions that proceed along an ordered morphogenetic pathway. While structural proteins are incorporated into the final product, morphogenesis is equally dependent upon scaffolding proteins, which are not included in the mature virion. Since the identification of scaffolding proteins in the bacteriophage P22, homologues have been discovered in many systems. Scaffolding proteins play multiple roles during morphogenesis by inducing protein conformational switches and lowering the thermodynamic barriers to promote virion assembly, while ensuring the efficiency and fidelity of the final product.
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Braggin, Greg A. "Effect of Surfactant Architecture on Conformational Transitions of Conjugated Polyelectrolytes." DigitalCommons@CalPoly, 2015. https://digitalcommons.calpoly.edu/theses/1411.
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Water soluble conjugated polyelectrolytes (CPEs), which fall under the category of conductive polymers, possess numerous advantages over other conductive materials for the fabrication of electronic devices. Namely, the processing of water soluble conjugated polyelectrolytes into thin film electronic devices is much less costly as compared to the processing of inorganic materials. Moreover, the handling of conjugated polyelectrolytes can be performed in a much more environmentally friendly manner than in the processing of other conjugated polymers because conjugated polyelectrolytes are water soluble, whereas other polymers will only dissolve in toxic organic solvents. The processing of electronic devices containing inorganic constituents such as copper indium gallium selenide (CIGS), is much more expensive and poses much greater environmental risks because toxic metals may be released into landfills or waterways upon cell disposal.75 Because conjugated polyelectrolytes enjoy an assortment of advantages over other materials for the manufacturing of thin film electronic devices, there is globally vested interest in the researching of their properties. Despite the fact that CPEs can serve as efficient electron transport mediums, devices such as organic solar cells cannot realize their highest efficiencies unless the morphology of CPEs is precisely controlled. Charged surfactants can electrostatically and ionically interact with CPEs, and when introduced in specific concentrations, molar ratios, and temperature ranges, will aid in a ‘coil to rod’ transition of the CPE, wherein polymer chains undergo intramolecular transitions to obtain rigid-rod morphologies. The kinetics and thermodynamics of the ‘coil to rod’ transition are heavily dependent upon the type(s) of charged surfactant complexed with the CPE (i.e. on the surfactant architecture). By performing UV/Vis Spectroscopy and Fluorometry on dilute polymer/surfactant solutions, Polarized Optical Microscopy (POM) and Small Angle X-Ray Scattering (SAXS) on high concentration polymer/surfactant solutions, and Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) on solid-state polymer/surfactant samples, the role of various surfactant architectures on the kinetics and thermodynamics of the ‘coil to rod’ transition was studied. The liquid crystalline physical properties and the extent of solid state crystallinity were also investigated. Through an analysis of the data obtained from these various techniques, it was found that the ‘coil to rod’ transition is progressively favored when the alkyl chain length of a single tailed surfactant is sequentially increased, and that as the concentration of double-tailed surfactant increases, the ‘coil to rod’ transition is negated.
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Zhang, Xiaofan. "Synthesis, Characterization and Catalytic Studies of Chiral Gold Acyclic Diaminocarbene Complexes." Thesis, University of North Texas, 2016. https://digital.library.unt.edu/ark:/67531/metadc862827/.
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Chiral gold complexes have been applied in homogeneous catalytic reactions since 1986, in some cases with high enantioselectivity. Acyclic diaminocarbene (ADC) ligands are acyclic analogues of N-heterocyclic carbenes (NHCs) that have larger N-CCarbene-N angles and stronger donating ability. ADCs have been developed as alternatives to phosphine and NHC ligands in homogeneous gold catalysis. In 2012, a new series of chiral gold(I) ADCs were first developed by Slaughter's group and were shown to give remarkable enantioselectivities in some reactions. Because of the hindered rotation of the N-CCarbene bonds of ADC, chiral ADC substituents can easily get close to the metal center in some conformations, although two rotameric structures are formed if the chiral amine is nonsymmetric. The selective of specific ADC conformations was the initial focus of this study. Formational selectivity of one diastereomer of an ADC ligand during synthesis was examines by measuring the relative rates of diastereomer formation in a 1H NMR kinetic study. The potential for converting multiple conformational isomers of ADCs into a single conformation, or at least a simpler mixture, was examined. This study used the analogy that anti- isomer has electronic and structural similarity with urea/thiourea, raising the possibility that 1,8-naphthyridine can be used to favor certain conformations through a self-assembled hydrogen-bonding complex. Gold(I) is a soft carbophilic Lewis acid able to active C-C π bonds to nucleophilic attack, and ADC-gold complexes are potentially useful in this regard. Therefore, biaryl gold(I) ADC complexes were examine with silver salt additives in catalytic 1,6-enyne cyclization reaction. A detailed study found that the counteranion affects the regioselectivities of these reactions more than substituents on the ancillary ADC ligands.
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Lee, Alexis J. "Theoretical Approaches to the Characterization of Water, Aqueous Interfaces, and Improved Sampling of Protein Conformational Changes." ScholarWorks@UNO, 2012. http://scholarworks.uno.edu/td/1511.
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Methods to advance the understanding of water and other aqueous systems are devel- oped. This work falls into three areas: The creation of better interaction potentials for water, improved methods for sampling configurational space, and the applications of these methods to understand systems of interest. Charge transfer has been shown by ab initio methods to be important in the water–water and water–ion interactions. A model for treating charge transfer in liquid water and aqueous systems is presented in this manuscript. The model is called Discrete Charge Transfer (DCT) and is based on the commonly-used TIP4P/2005 model, which represents the charge distribution of water molecules with three charge sites. Such models have been very successful in reproducing many of the physical properties of water. Charge transfer is introduced by transferring a small amount of charge, -0.02e, from the hydrogen bond acceptor to the hydrogen bond donor, as has been indicated by electronic structure calculations. We have parameterized both polarizable and non-polarizable potentials, optimized to include charge transfer. Methods to surmount the obstacles incurred by the introduction of charge transfer, which involve the amount of charge transfer at large distances and implementation into Molecular Dynamics simulation, is presented, along with our results assessing the importance of charge transfer in liquid water and aqueous systems. Also presented is a method for improving eciency of a sampling technique, Replica Exchange, by reducing the number of replicas. The improved method is called Replica Exchange with Driven Scaling (REDS2).
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Pondaven, Simon Pierre. "Conformational Flexibility and Amyloid Core Characterization of Human Immunoglobulin Light Chain Domains by Multidimensional NMR Spectroscopy." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354113457.
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Dollery, Stephen. "Identification and characterization of low pH-triggered conformational changes in the herpes simplex virus glycoprotein B." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/176.
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Herpesviruses can enter host cells by pH-dependent endocytic pathways in a cell-specific manner. The role of pH in herpesvirus endocytosis is unclear. Herpes simplex virus (HSV) is a paradigm for virus membrane fusion via a complex of glycoproteins. HSV glycoproteins B, D and the heterodimer H-L are necessary and sufficient for membrane fusion. This work analyzes the structure and function of HSV glycoproteins B, D, and H-L at neutral pH, and at the physiological low-pH encountered during endocytic entry. It is demonstrated that mildly acidic low pH triggers specific conformational changes in HSV gB at a pH of 5.7 to 6.0. The antigenic structure of gB functional region I that is critical for fusion is specifically altered by mildly acidic pH both in vitro and during entry into host cells. Point mutations within gB functional region 1 that block membrane fusion still allow conformational changes in region 1. This suggests that specific hydrophobic residues are essential for fusion domain insertion into the host cell membrane but not conformational change. The detected conformational changes were reversible, similar to other class III fusion glycoproteins. Exposure to mildly acidic pH directly triggered the fusion function of HSV glycoproteins and caused gB, but not other glycoproteins, to become more hydrophobic. The oligomeric conformation of gB is altered at a similar pH range. In addition, several approaches were used to monitor gB throughout glycoprotein synthesis and maturation. It is shown that gB may cotranslationally fold and oligomerize as it is synthesized on the ribosome. As gB matures it then alters conformation and/or binding partner to form antigenically distinct populations of gB within the cell and virion. I conclude that intracellular low pH induces changes in gB conformation that, together with additional triggers such as receptor-binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.
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Raspadori, Andrea. "Characterization of the conformational space of the murine prion protein using single-molecule force spectroscopy techniques." Doctoral thesis, SISSA, 2014. http://hdl.handle.net/20.500.11767/4124.
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The conversion of the cellular prion protein (PrPC) to its infectious counterpart (PrPSc) is the initial step of prion diseases. These neurodegenerative disorders are characterized by different incubation times, sympthoms and disease phenotypes. Structural heterogenity of PrP aggregates is responsible for this biological diversity. Understanding the structural rearrangements of PrP at the monomeric and oligomeric level is essential to gain insights into its aggregation processes. However traditional “in-bulk” techniques can only provide ensemble-averaged information for monomer and oligomer structures. We applied single-molecule force spectroscopy to characterize the heterogeneous structural ensemble of the murine PrP at the monomeric and at the oligomeric level. By stretching chimeric protein construct carrying one MoPrP molecule we found that the protein folds with a two state mechanism. Less frequently the protein can adopt more extended conformations that encompass also the N-terminal domain. These structures might be involved in subsequent aggregation processes. We also developed an assay to characterize the oligomerization processes using multiple PrP constructs. By analyzing the extension of these constructs under tension we characterized the structure between different PrP moieties, under different conditions. We found that reciprocal PrP orientation affects the length and mechanical resistance of these structures but their events frequency. Comparing the structures observed from monomers, dimers, trimers and tetramers we found that their frequency of events and their average length increased by increasing the number of PrP moieties. Remarkably, decreasing pH to more acidic values resulted in a higher frequency of events that involved structures between PrP moieties only in multimeric constructs. Instead, increasing the ionic strength significantly diminished their frequency, indicating how solution conditions can strongly alter the conformational transitions. These results provide a new scenario on PrP misfolding and aggregation processes, characterizing their early aggregation steps under different reaction conditions.
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Symeonidou, Evgenia. "Synthesis, characterization and DFT study of new azaborinine compounds." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amslaurea.unibo.it/21700/.
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In the framework of this thesis, a wide-ranging study of azaborinine derivatives was conducted, with a particular interest towards 9,10-B,N-phenanthrenes, holding an isosteric B-N unit in the place of a C=C bond and incorporating a B-C chiral axis. For this purpose, a preliminary theoretical study for four compounds of this class was carried out: conformational analysis and rotational energy barriers, UV-Vis absorption and fluorescence emission as well as theoretical ECD spectra of the atropisomeric structures were calculated by means of DFT and TD-DFT. An experimental attempt to synthesize and characterize another derivative of this class followed. The main synthetic concept evolved around the obtention of anti-aromatic 9-borafluorene precursors by boron-tin exchange from the reaction of a 9-stannafluorene derivative with boron reagents, followed by an aromaticity-driven ring-opening reaction of these borafluorene precursors with an organic azide. Several approaches based on similar previous works were reproduced to the closest possible version, as no absolutely inert conditions could be achieved, but the expected products were not formed; in one case, an open structure was obtained instead, for the formation of which a mechanism was proposed.
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Posgai, Monica Therese. "Energetic and dynamic characterization of the IgA1:FcαRI interaction reveals long-range conformational changes in IgA1 upon receptor binding." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1354043317.
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Zhao, Shen. "Design and in vitro characterization of lipids with a pH-sensitive conformational switch and their liposomes for anticancer drug delivery." Scholarly Commons, 2018. https://scholarlycommons.pacific.edu/uop_etds/3574.
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The traditional anticancer drugs are distributed in vivo through systemic blood circulation with a very small portion reaching the tumor site. Targeted drug delivery systems are developed in efforts to concentrate the drug molecules in the tissue of interest while reducing the drug distribution to healthy tissues to reduce the side effects. Liposomes are colloidal systems composed of amphiphilic molecules that assemble into vesicle structures in aqueous media. They are common carriers for targeted drug delivery with the advantages of low toxicity, low immunogenicity and the ability of encapsulating both lipophilic and hydrophilic drugs.
Prior research indicated the advantages of triggered release in drug delivery systems. As a specific example, a series of trans-2-aminocyclohexanol based lipids (flipids) have been reported to illustrate a promising strategy to render pH-triggered drug delivery systems: pH-triggered conformational switch. Based on the foregoing, we hypothesize that incorporation of lipids with a pH-sensitive conformational switch and a long-saturated lipid tail can improve the anticancer activities of stealth liposomes. In this study, six new flipids with C-16 saturated hydrocarbon tails were designed. Such lipids were synthesized with high yields by introducing a catalyst (Copper (II) tetrafluoroborate) at a key step of the synthetic scheme.
pH-sensitive liposomes (fliposomes) composed of flipids were prepared and loaded with the anticancer drug doxorubicin with high encapsulation efficiency. The physicochemical properties of doxorubicin-loaded fliposomes were characterized and their pH-dependent leakage were investigated. The results showed that among all groups fliposomes containing the C-16 trans-2-morpholylcyclohexanol-based flipid (Mor-C16) exhibited the largest increase of release as the pH dropped form pH 7.4 to 6.0, indicating its good potential of serving as a component in pH-triggered drug delivery systems.
Three-dimensional multicellular spheroids (3D MCS) are self-assembled microscale tissue analogs in vitro. They better mimic the native and complex tumor microenvironment than the conventional two-dimensional cell culture systems. In this dissertation study, 3D MCS of six different human cancer cells were successfully cultured and their growing conditions were optimized to obtain 3D MCS of tight structure and reproducible size. The constructed 3D MCS carried heterogeneously distributed live and apoptotic cells as well as acidic inside pH based on confocal microscopic imaging studies.
The penetration of doxorubicin-loaded Mor-C16 fliposomes into 3D MCS was imaged by confocal microscopy in comparison to doxorubicin-loaded non pH-sensitive liposomes and free doxorubicin. The anticancer activities of doxorubicin-loaded Mor-C16 fliposomes against 3D MCS of three different cell lines was also evaluated by cell viability. Both the fliposome and the non pH-sensitive liposome formulations more efficiently penetrated into two of the three types of 3D MCS compared to free doxorubicin after 4h drug exposure. However, doxorubicin-loaded Mor-C16 fliposome imposed higher cytotoxicity to all three types of 3D MCS compared to doxorubicin-loaded non pH-sensitive liposome over 72 h drug exposure. Taken together, we propose that fliposomes achieved superior activity against 3D MCS by efficient penetration into 3D MCS, followed by enhanced release of the anticancer drug doxorubicin.
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TESTA, LORENZO. "Conformational transitions of the intrinsically disordered protein sic1 from the yeast saccharomyces cerevisiae. Towards structural and functional characterization of the whibitory complex with CDK1-CLB5." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/43673.
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Many naturally occurring proteins have been shown to lack rigid three-dimensional structure, existing instead as dynamic ensembles of inter-converting conformations. Many of these proteins acquire an ordered structure only upon binding to specific intracellular partners. In isolation, these proteins exhibit a highly dynamic structure that is resembling more the denatured rather than native state of “normal” globular proteins. Intrinsically disordered proteins (IDPs) have attracted a great deal of interest since it became clear that their lack of structural specificity is of physiological importance. Indeed, structural disorder characterizes a broad class of regulatory proteins, which all share the feature to bind multiple interactors. Intrinsic disorder is a common property of many protein types especially among the proteins involved in signal transduction and transcription regulation. The dynamic properties of IDPs are considered to be instrumental to adaptation to different interaction surfaces and to favor rapid formation and dissociation of the complexes, as required for efficient intracellular regulatory networks.
In the yeast Saccharomyces cerevisiae, Sic1 is a central regulatory protein of the cell cycle, acting as inhibitor of the cyclin-dependent protein kinases by forming ternary complexes with kinases and their cognate cyclins. A well established role of Sic1 is to control the timing of entrance in S phase during the yeast cell cycle by inhibiting the complex Cdk1-Clb5/6, whose activity is required for the G1-to-S transition. The inhibition is released upon ubiquitin-dependent Sic1 degradation, which is, in turn, triggered by Sic1 phosphorylation at multiple sites in its N-terminal region. Previous work has shown that Sic1 is disordered in its whole length, with some intrinsic propensity to form ordered helical structure. The Sic1 functional kinase-inhibitor domain (KID) corresponds to the C-terminal 70, out of 284, amino acids forming the native protein. This Sic1 C-terminal region is structurally and functionally related to the mammalian p21 and p27 KIDs that are located, instead, at the protein N-terminus. A detailed structural characterization of the mammalian proteins have been achieved, both on their unbound states and on the complexes with their partners.
Structural characterization of proteins in disordered conformation is technically difficult, but it is important to better understand folding transitions to ordered states. In this work, an in-depth description of Sic1 in the absence of interactors, both in terms of secondary and tertiary structure, is presented. A multiparametric analysis, which employed a set of complementary methods sensitive to distinct structural features, has been used to guarantee the description of such a highly dynamic and heterogeneous molecular ensemble.
First of all, a novel tool for extracting structural information from electrospray-ionization mass spectrometry (ESI-MS) data has been developed. It has been shown that the extent of protein ionization under nondenaturing conditions correlates with the solvent-accessible surface area (SASA), for either folded or unfolded proteins. Therefore, the technique has been employed to estimate the SASA of Sic1.
Fragments corresponding to the N- and C-terminal moieties of Sic1 have been produced, and circular dichroism (CD) data showed that the little content in secondary structure is distributed quite uniformly throughout the chain length, although the C-terminus is slightly more ordered than the N-terminus. Consistent with such evidence, conformational analysis by ESI-MS suggested that the Sic1 C-terminal domain is more structured than its complementary N-terminal domain. Thus, altogether, functional and structural features pointed to a modular organization of this protein, despite its disordered nature.
A more detailed description of the Sic1 KID has been achieved by integrating an array of biophysical data with all-atom molecular dynamics (MD) simulations. Highly dynamic helical elements are detected by Fourier-transformed infrared (FT-IR) spectroscopy, while protein tertiary structure has been probed by nondenaturing gel filtration, ESI-MS, and electrospray-ionization ion-mobility mass-spectrometry (ESI-IM-MS). The molecular ensemble of the isolated KID fragment has been found to interconvert between collapsed states of different compactness, with a small fraction of the population found in a highly compact state. MD simulations results has suggested a predominant role of electrostatic interactions in promoting the compaction in the Sic1 inhibitory domain. Moreover, comparison to the full-length protein hinted to a critical role of chain length in determining the overall compaction of Sic1.
Since no structural data are available for the Sic1-Cdk1-Clb5/6 ternary complex, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains unclear. In this work, the results about the heterologous expression and purification of Cdk1 and Clb5 are also presented. Cdk1 has been overexpressed as a histidine-tagged protein in Escherichia coli cells and CD analysis has revealed a similar fold to mammalian homologues. The full-length Clb5, on the contrary, underwent proteolytic degradation when expressed in E. coli cells, probably because of the high degree of disorder in the N-terminal region of the amino-acids sequence. The deletion of the first 156 residues has allowed the expression of the protein as inclusion bodies (IBs). A purification procedure has been then developed, so that IBs have been solubilized and refolded by on-column removal of denaturing conditions. The results concerning the expression and the characterization of Cdk1 and Clb5 represent a starting points for the production of a functional Cdk1-Clb5 complex and so, ultimately, for the complete description of Sic1 binding mechanism.
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Malloni, Wilhelm Massimiliano [Verfasser], and Hans Robert [Akademischer Betreuer] Kalbitzer. "AUREMOL-QTA, a program package for NMR based automated recognition and characterization of local and global conformational changes in proteins induced by ligand binding as external perturbation / Wilhelm Massimiliano Malloni. Betreuer: Hans Robert Kalbitzer." Regensburg : Universitätsbibliothek Regensburg, 2011. http://d-nb.info/1030178836/34.
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Rauch, Sebastian, Klaus-Jochen Eichhorn, Ulrich Oertel, Manfred Stamm, Dirk Kuckling, and Petra Uhlmann. "Temperature responsive polymer brushes with clicked rhodamine B: synthesis, characterization and swelling dynamics studied by spectroscopic ellipsometry." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-139314.
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Here, we report on a new temperature responsive polymer brush system with a terminal “click” functionality. Bifunctionalized poly(N-isopropylacrylamide) (PNiPAAm) with distinct functional end groups was synthesized by atom transfer radical polymerization (ATRP) and grafted to a modified silicon substrate. The presence of the active terminal alkyne functionality is validated using an azide-modified rhodamine B (N3-RhB) via copper(I) catalyzed alkyne–azide cycloaddition (CuAAC). The optical properties and swelling dynamics of an N3-RhB modified PNiPAAm brush are analyzed in dry state and in situ by VIS-spectroscopic ellipsometry (SE). The best-fit results are obtained using a Gaussian oscillator model and are confirmed by UV/VIS-spectroscopy. We observed evidence of interactions between the aromatic residues of the dye and the PNiPAAm amide groups, which significantly affect the swelling behavior of the modified polymer brushDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Rauch, Sebastian, Klaus-Jochen Eichhorn, Ulrich Oertel, Manfred Stamm, Dirk Kuckling, and Petra Uhlmann. "Temperature responsive polymer brushes with clicked rhodamine B: synthesis, characterization and swelling dynamics studied by spectroscopic ellipsometry." Royal Society of Chemistry, 2012. https://tud.qucosa.de/id/qucosa%3A27823.
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Here, we report on a new temperature responsive polymer brush system with a terminal “click” functionality. Bifunctionalized poly(N-isopropylacrylamide) (PNiPAAm) with distinct functional end groups was synthesized by atom transfer radical polymerization (ATRP) and grafted to a modified silicon substrate. The presence of the active terminal alkyne functionality is validated using an azide-modified rhodamine B (N3-RhB) via copper(I) catalyzed alkyne–azide cycloaddition (CuAAC). The optical properties and swelling dynamics of an N3-RhB modified PNiPAAm brush are analyzed in dry state and in situ by VIS-spectroscopic ellipsometry (SE). The best-fit results are obtained using a Gaussian oscillator model and are confirmed by UV/VIS-spectroscopy. We observed evidence of interactions between the aromatic residues of the dye and the PNiPAAm amide groups, which significantly affect the swelling behavior of the modified polymer brush.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Naidoo, Kevin Jonathan. "Metal ion cages with stabilized conformations : synthesis, characterization and properties." Master's thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/18805.
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The stabilized conformers lel₃, lel₂ob, ob₂lel and ob₃ of the [Co(±pn)₃]³⁺ system have previously been synthesized giving relative equilibrium (100 °C) amounts of 35.0 : 41.1 : 18.0 : 4.0. With the recent encapsulation of the resolved lel₃-[Co(pn)₃₋]Cl₃ complexes, and their subsequent interconversion to the ob₃-[Co(NH₃)₂-pnsar]Cl₅ diastereomers, the synthesis of the lel₂ob and ob₂lel cage complexes is a necessity to complete the investigation of the [Co(NH₃)₂-pnsar]⁵⁺ system. Consequently the eight Δ, Λ isomers of the lel₂ob-[Co(NH₃)₂-pnsar]Cl₅ complex were synthesised in a one pot reaction and resolved into the respective isomers having the following relative yields: lel₂ob-1 (~ 24% ), lel₂ob-2 ( ~ 43% ), lel₂ob-3 ( ~ 28%) and lel₂ob-4 (~ 5%). The resolution order of these isomers on SP Sephadex C-25 was used to tentatively identify the (3 mer and 1 fac) geometrical isomers of these lel₂ob diastereomers. For the first time ob character was directly encapsulated with the synthesis of the ob₂lel-[Co(NH₃)₂-pnsar]Cl₅ conformers ( ~ 1 % ). NMR analysis showed these to consist almost entirely (80%) of the ob₂lel-1 isomer. The geometry of this isomer was later discovered to be most favourable when the lel₂ob-1 isomer gave a 50% conversion to the ob₂lel-1 isomer, unlike the poor conversions of the ob₂lel-2 (~ 5%) and the ob₂lel-3 (~ 15%). The electronic spectra (UV /VIS and CD) of the individual isomers were investigated and revealed the presence of large distortion of the CoN₆ chromophore from regular Oh geometry. This was confirmed by the crystal structural analysis of the lel₂ob-2 isomer which reveal a trigonal twist angle (ф) of ~ 45°. The crystal structure also proved the identification logic right, which was based on the relation between chemical and resolution order evidence. The infrared spectra of [Co(NH₃)₂-sar]⁵⁺ ion was analysed using isotopic labelling methods in combination with the results of group theory calculations. These assignments make it possible to study the spectra of the [Co(NH₃)₂-pnsar]⁵⁺ isomeric complexes. Comparison of the infrared spectra of these complexes reflect the increase in number of bands on progressing from D₃, through C₃ to C₁ symmetry. Even the orientation of methyl groups in the different conformer types affect the symmetry of the CoN₆ core.
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Mossuto, Maria Francesca. "Protein amyloidogenesis: characterization of aggregation prone conformations and fibrils structure." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425566.
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Current interest in studying amyloid fibrils arises from their involvement in different fields (Chiti and Dobson, 2006). First, they play a crucial role in disorders such as Alzheimer's and Parkinson's diseases. Second, since it has been demonstrated that all polypeptide chains form fibrils under appropriate conditions, the understanding of why and how this process happens has become central problem in protein knowledge. Last, the ordered ultrastructure characterizing amyloid fibrils may be thought as a basis for nanomaterials with possible technological applications. However, despite the ability of most proteins to form amyloid fibrils, very little is known about their structures and the factors that govern their formation.
The process of amyloid formation requires the partial unfolding of protein molecules into such conformations able to interact to each others and reorganize into well-ordered structured aggregates, named amyloid fibrils.
In this Thesis the amyloid formation by globular proteins has been analyzed from different points of view, focusing in the first part of the research work on the elucidation of some conformational features promoting protein aggregation. The second part was concentrated on the characterization of the final supramolecular structure of amyloid fibrils.
In order to study the partially folded state, two globular proteins have been analyzed, alpha-lactalbumin (LA) and HypF-N. Indeed, under specific conditions, these proteins populate a not fully folded state previously shown to play an important role in the amyloid formation (Uversky, 2002; Chiti et al., 2001).
The study conducted on LA has been based on the effects of the proteolytic dissection of the molecule on its conformational features and aggregation properties. It was previously shown that LA is able to form fibrils morphologically indistinct from the pathological ones (Uversky et al., 2002). Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the ?-subdomain of the native protein (1-40/53-123, named des?-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. The main conclusion of this work was that the inherent flexibility of the LA derivatives allows the large conformational changes required to form the cross-?-structure of the amyloid fibrils. It has been emphasized that proteolysis can be considered a causative mechanism of protein aggregation and fibrillogenesis (Polverino de Laureto et al., 2005).
In the other case, the conformational characterization of an amyloidogenic state of HypF-N has been performed at acid pH, in order to allow the protein to populate a partially unfolded ensemble. Combining different biophysical and biochemical techniques, it has been shown that this partially unfolded structure has all the hallmarks of a pre-molten globule state, i.e. it is more compact than a random coil-like state but less organized than a native-like intermediate or a MG state (Uversky, 2002). Furthermore, it is shown that a modulation of the total ionic strength of the solution allows enhancing the apparent rate of aggregation of HypF-N under these conditions. This increased rate of aggregation has been shown to be mediated by the interaction of monomers to form initial oligomers, through a particular region in the sequence, corresponding to the sequence part having highly hydrophobicity, the highest beta-sheet propensity and with no net charge at acid pH, representing the ideal segment suitable to mediate protein oligomerization.
From all these studies, it is clear that, except the unique native state of globular proteins wherein the side chains pack together in a unique manner, every state of a polypeptide molecule is a broad ensemble of often diverse conformations. It is not surprising, therefore, that even the fibrillar products of aggregation processes are characterized by morphological and structural diversity, representing variations on a common theme.
The second part of my PhD Thesis deals with the structural characterization of fibrils. Many studies have been conducted on amyloid aggregates formed under different conditions by peptides, such as A?, TTR and prion fragments (Kodali and Wetzel, 2007). Indeed, the problem of amyloid formation by a full-length protein is more complex, since the dense packing reachable in amyloid fibrils made of peptides (10-40 residues) could not be accomplished in all the amino acid residues of a full-length protein, except in the core regions (Chatani and Goto, 2005). The object of my study was human lysozyme due, most of all, to the fact that some natural variants of human lysozyme (HuL) are responsible for the formation of amyloid plaques in vivo, in a so called familial non-neuropathic systemic amyloidosis (Pepys et al., 1993; Booth et al., 1997). Moreover, it has been possible to exploit the available wealth of structural and folding information about wild type HuL, since it has been shown to be able to form fibrils quite similar to the pathological ones, under acidic conditions and high temperature (Morozova-Roche et al, 2000). With respect to fibrils made of peptides, besides, studying amyloid fibrils conformations from HuL is more challenging because it is a 130 amino acid chain with the structural constrains given by the four disulfide bridges present in the lysozyme molecule. This study can also give some insights into the complex problem of strains diversity, such as for prion diseases, helping the clarification of the structural principles of amyloid fibrils which can produce multiple and distinct amyloid conformations from one protein sequence.
In the presented study, fibrils of wild-type HuL formed at low pH have been analyzed by limited proteolysis experiments and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates (Frare et al., 2006). After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive ?-sheet structure and substantial elements of non ?-sheet or random structure that are reduced significantly in the fibrils after digestion. The sequence 32-108 includes the ?-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme (Frare et al., 2004). The present data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain.
HuL variants, however, aggregate in a physiological environment, roughly at pH 7-7.5 at 37 °C, because of their instability (Dumoulin et al., 2005). In my work, it has been demonstrated that also HuL is able to aggregate under conditions similar to the pathological ones, presumably neutral pH and 37 °C. Considering that HuL forms amyloid fibrils in such different conditions (pH 2.0 50°C and pH 7.5, 60°C), a comparison of the structure and the stability of fibrils obtained under these different conditions has been conducted. In this study HuL fibrils were produced at acidic and at neutral pH, leading both to the formation of fibrils having the three hallmarks of amyloid, that are cross-beta structure, binding of ThT and an overall amyloid fiber morphology. These fibrils have been studied by means of ANS binding, FTIR and X-ray fiber diffraction in order to characterize the differences in the structure. Guanidinium-induced fibrils dissociation, instead, has been applied in order to test the chemical stability of the two kinds of fibrils. The results clearly indicate that the solution conditions used for lysozyme aggregation promote the formation of fibrils with different structural features and stability properties, due to the diverse rearrangements of the lysozyme polypeptide chain into the fibril structure.
In conclusion, the research work conducted in this Thesis allowed the comprehension of important aspects of the unfolding of some globular proteins leading to amyloid fibrils. In addition, original data have been obtained on the structural polymorphism of amyloid fibrils.
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Kallberg, Yvonne. "Bioinformatic methods in protein characterization /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-370-8/.
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Banks, Jennifer Dawn. "Characterization of a minimal avian leukosis-sarcoma virus packaging signal /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/11528.
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Park, Sunho 1976. "Characterization of nanoparticle-DNA conjugate and control of DNA conformation on particle surface." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/49761.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2009.Includes bibliographical references.
Nano-science has exploited the hybridization and de-hybridization phenomena of DNA which are one of its fundamental functions. In particular, conjugates of gold nanoparticles and DNA (Au NP-DNA) have been extensively explored for their potential in biological applications such as DNA delivery for gene therapy and disease detection. However, DNA strands are known to adsorb onto the Au NP surface, which can severely limit the hybridization ability of Au NP-DNA conjugates. Therefore, methods of chemical modification of Au NP surfaces and evaluating DNA conformation via Ferguson analysis of gel electrophoresis are proposed in the thesis. Conjugates of DNA with Au NP of different sizes and coverages are evaluated with Ferguson analysis to characterize important parameters such as hydrodynamic size and zeta-potential. Surface modified Au NP exhibits enhanced stability and hybridization specificity in the system, which infers the effectiveness of those methods towards biological systems where non-specific adsorption is problematic. To confirm the validity of the concept, Au NP-antisense DNA experiments for gene silencing are performed in the work. Antisense DNA is designed to inhibit ribosomal activity on mRNAs and cooperatively works with Au NPs to enhance physical blocking mechanisms. However, the result shows that Au NP-DNA conjugates can enhance in vitro gene expression depending on DNA sequence and coverage of the conjugates. Suggestions are made for further investigation on proof and improvement of the translation enhancer concept.
by Sunho Park.
Ph.D.
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Spuhler, Philipp S. "High-throughput detection of DNA orientation and conformation for characterization of protein-DNA interactions." Thesis, Boston University, 2012. https://hdl.handle.net/2144/31605.
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Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
A deep understanding of disease processes requires understanding of DNA, RNA and protein function on a molecular level. Protein-DNA interactions play a crucial role in many of these processes, including in gene expression. The high-throughput capability of micro-array based technologies enables studies on the sequence dependence to determine the functional roles of protein-DNA binding. In vitro protein binding to double stranded DNA microarrays has proven to be an effective complementary technique to the in vivo methods, as this allows rapid characterization of the protein-DNA binding sequence specificity and much higher resolution of the binding site. In this dissertation, we apply Spectral Self-Interference Fluorescence Microscopy (SSFM) to develop a platform that improves upon existing in vitro protein binding microarray technologies. The platform permits measurement of the protein binding site without the need for protein tagging with radiolabels or fluorophores. The technology enables precise quantification of DNA conformation changes that are induced by protein binding. There are currently no viable high-throughput techniques available to investigate the sequence dependence on protein induced DNA bending. While they are not well understood, such structural changes are thought to play a critical role in transcription. A crucial component of the technique is the measurement of DNA orientation, as this permits the detection of the protein binding location and DNA conformation changes. We apply a novel polymeric scaffold to control the surface charge of the sensor and, thus, the orientation of surface immobilized oligonucleotides. The orientation is precisely quantified through sub-nanometer height localization of fluorophore labels on either end of the oligonucleotide probes. The DNA binding protein Integration Host Factor (IHF) is used as the model system to demonstrate a proof of principle for the platform. IHF is known to bind DNA with high sequence specificity and to induce a 160° bend. The binding position of IHF to the dsDNA probes is resolved to three nucleotides and the conformation changes that result due to a single nucleotide polymorphism in the consensus binding sequence are observed. The platform is scalable to permit observation of protein binding to hundreds of thousands ofunique DNA sequences on a single chip.
2031-01-01
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Tseng, Hui-yuan [Verfasser], and Reinhard [Akademischer Betreuer] Fässler. "Identification and characterization of conformation-specific cytoplasmic integrin interactors / Hui-yuan Tseng ; Betreuer: Reinhard Fässler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1124395806/34.
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Farooqi, Mohammed Junaid. "METHODS FOR IN SITU PIEZOPHYSIOLOGICAL STUDIES: OPTICAL SECTIONING VIA STRUCTURED ILLUMINATION AND FLUORESCENCE BASED CHARACTERIZATION OF NADH CONFORMATION." Oxford, Ohio : Miami University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1249225952.
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Xia, Nan. "Time-of-flight secondary ion mass spectrometry (ToF-SIMS) characterization of conformation and orientation of adsorbed protein films /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/9869.
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Holder, Isabelle T. [Verfasser]. "Non-Canonical Nucleic Acids in Bacteria -Structural Characterization and Functional Properties of Quadruplex and Triplex Conformations- / Isabelle T. Holder." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1110771606/34.
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Meyer, Gordon Joel. "Synthesis, Characterization, and Mixed-Valence Studies of Conformationally Constrained Bisferrocenyl Complexes for the Study of Through-Space S***π; Interactions." Diss., The University of Arizona, 2014. http://hdl.handle.net/10150/337289.
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A series of conformationally constrained 2,6-bisferrocenylphenyl thioethers were synthesized via Suzuki-Miyaura cross coupling reactions. Structural information was obtained using X-ray crystallography and dynamic ¹H NMR spectroscopic studies, showing highly constrained m-terphenyl systems. Interaction of the ferrocene moieties through space mediated by the sulfur were studied by ultra-violet photoelectron spectroscopy (UPS), cyclic voltammetry, differential pulse voltammetry, UV-Vis-NIR spectroscopy and DFT computations. Electrochemical results show two, fully reversible 1e⁻ redox processes for the ferrocenes where the separation of peaks is affected by both solvent and supporting electrolyte, suggesting significant electrostatic interaction which is further confirmed in the gas phase by UPS studies. To determine if these interactions could be observed at greater distances, extended m-terphenyl complexes were shown in which 2-sulfur and 3-aromatic moieties were synthesized using a developed selective Suzuki-Miyaura monocoupling procedure in good yields. In these systems, interaction was not observed by electrochemistry or UPS. This suggests the distance between redox centers (~16 Å) is too great for electrostatic interaction, even though there is enhanced interactions observed in the truncated systems. Two new bisferrocenylphenylsulfoxides were also synthesized and studied to determine the effect of the polar sulfoxide bond on through space interaction between the ferrocene moieties. The electronic and redox properties of these compounds were studied by ultra-violet photoelectron spectroscopy, cyclic voltammetry, differential pulse voltammetry, and DFT computations. Electrochemical results for 2,6-bis(ferrocenyl) thioanisole S-oxide show two, fully reversible one electron redox processes. The initial oxidation shows a 62 mV negative shift compared to the sulfide analog 2,6-bis(ferrocenyl)thioanisole, and an increased peak separation for the oxidations of 160 mV. No peak separation is observed in the extended sulfoxide system. No intervalence charge transfer band was observed in the truncated sulfoxide complex by monitoring the UV-Vis/NIR spectroscopy of the mixed valence complex, ruling out electronic communication. Thus, the through space electrostatic interactions of the sulfoxide causes the non-equivalent ferrocenes in the truncated system to have different oxidation potentials. Synthesis was developed towards the synthesis of 1,8-bisferrocenyl-9-(alkylthio) anthracene complexes. It was observed that due to steric congestion at the C9 position of the anthracene scaffold, standard thionation reactions did not proceed as expected. Instead, the reaction of 1,8-dibromo-9-anthrone with Lawesson reagent afforded the intramolecular nucleophilic aromatic substitution cyclization product in quantitative yields. The reaction of the same anthrone under studied dithioketal formation conditions led to sulfur-rearrangement, giving the undesired 1,8-bisferrocenyl-10-(ethylthio)anthracene derivative, as confirmed by X-ray crystallography. Attempted Newman-Kwart rearrangement of 1, 8-dibromoanthracen-9-yl) dimethylcarbamothioate afforded no significant observed product formation, and decomposition of starting materials when heated for extended times. 1,8-bisferrocenyl-9-(methoxy)anthracene was synthesized and structurally characterized by dynamic X-ray crystallography to confirm connectivity. Electrochemical experiments show 2 reversible redox processes separated by 115 mV. Chemical oxidation experiments show unexpected, strong electronic coupling in the mixed valence complex. This coupling was characterized by near-IR absorption at 941 nm, indicating intervalence charge transfer (IVCT). Single electron reduction of 1,8-bisferrocenyl-9-(methoxy)anthracene, followed by quenching with various electrophiles afforded an inseparable mixture of products, one of which was identified by mass spectrometry as the desired 1,8-bisferrocenyl-9-(methylthio)anthracene product. However, this complex was not separable from the mixture and further characterization was not possible. All other routes attempted to incorporate sulfur into the system afforded no conversion of starting materials or decomposition of the reaction mixture.
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Agrahari, Aditya. "Synthesis and Characterization of Di- Aryl Pentanes and Mechanistic Study of Aldol Reaction of 9-Acetylanthracene with Paraformaldehyde." Cleveland State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=csu1440082002.
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[Verfasser], Felix Helge Deluweit, and Christiane [Akademischer Betreuer] Ritter. "Conformation based in vitro selection of mammalian prion seeds – Propagation and characterization of prion strains / Felix Helge Deluweit ; Betreuer: Christiane Ritter." Braunschweig : Technische Universität Braunschweig, 2015. http://d-nb.info/1175819646/34.
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Han, Zhong. "Characterization of the mechanisms of transcription termination by the helicase Sen1." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS202/document.
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La transcription cachée est un phénomène répandu aussi bien chez les eucaryotes que chez les procaryotes. Elle se caractérise par une production massive d’ARNs non-codants au niveau de régions non-annotées du génome et est potentiellement dangereuse pour la cellule car elle peut interférer avec l’expression normale des gènes. Chez S. cerevisiae, l’hélicase Sen1 induit la terminaison précoce de la transcription non-codante et joue ainsi un rôle clé dans le contrôle de la transcription cachée. Sen1 est très conservée et des mutations dans son homologue humain, senataxin (SETX), ont été associées à des maladies neurodégénératives. Malgré de nombreuses recherches menées sur ces protéines, leurs propriétés biochimiques ainsi que leurs mécanismes d’action restent peu connus. Durant ma thèse, j’ai étudié le mécanisme de terminaison par Sen1.Premièrement, j’ai caractérisé les activités biochimiques de Sen1 et analysé comment elles permettent d’induire la terminaison. Pour cela, j’ai utilisé un ensemble de techniques in vitro, notamment un système de transcription-terminaison qui contient uniquement des composants purifiés : Sen1, l’ARN polymérase II (Pol II) et les ADN matrices. Ce système permet de modifier les différents éléments de façon contrôlée afin de comprendre leur rôle précis dans la terminaison. J’ai tout d’abord analysé la fonction des différents domaines de Sen1 dans la terminaison. Sen1 est une protéine de taille importante qui possède un domaine central catalytique flanqué par deux domaines impliqués dans l’interaction avec d’autres facteurs. J’ai montré que le domaine hélicase est suffisant pour déclencher la terminaison de la transcription in vitro. Ensuite, j’ai montré que Sen1 utilise l’énergie de l’hydrolyse de l’ATP pour se déplacer sur des acides nucléiques simple bras (ARN et ADN) dans le sens 5’ vers 3’. J’ai alors étudié le rôle des différents acides nucléiques du système dans la terminaison par Sen1 et j’ai montré que l’interaction de Sen1 avec l’ADN n’est pas nécessaire; en revanche Sen1 doit s’associer à l’ARN naissant et se déplacer vers la polymérase. J’ai aussi montré qu’une fois que Sen1 entre en collision avec la Pol II, elle y exerce une action mécanique qui conduit à la terminaison uniquement quand la Pol II marque une pause. Cela indique que la terminaison est fortement dépendante de la pause transcriptionnelle. Deuxièmement, en collaboration avec le groupe d’E. Conti, nous avons réalisé une analyse structure-fonction du domaine hélicase de Sen1. Nous avons observé que Sen1 présente une organisation similaire à celle d’autres hélicases proches avec un core composé de deux domaines de type RecA avec plusieurs domaines auxiliaires. En général, le core est très conservé au sein des hélicases proches, alors que les domaines accessoires ont des caractéristiques distinctes qui confèrent des propriétés spécifiques aux différentes hélicases. En effet, nous avons identifié un sous-domaine spécifique à Sen1 mais conservé au cours de l’évolution que nous avons appelé le “brace”. Nous avons également détecté des différences notables au niveau d’un autre domaine accessoire que nous avons nommé le “prong”. Nous avons pu montrer que le “prong” est essentiel pour la terminaison par Sen1. Nos données suggèrent que les caractéristiques structurales spécifiques de Sen1 que nous avons révélées sont des déterminants majeurs de son activité dans la terminaison de la transcription. Finalement, nous avons utilisé Sen1 comme modèle pour étudier des mutations dans SETX qui sont associées à des maladies neurodégénératives. Nous avons introduit chez Sen1 une partie des mutations liées à des maladies et nous avons réalisé une caractérisation biochimique complète de chaque mutant. Nous avons ainsi montré que toutes les mutations sont fortement délétères pour la terminaison de la transcription. En conclusion, nos résultats ont permis d’améliorer la compréhension de l’origine des maladies provoquées par des mutations dans SETXPervasive transcription is a common phenomenon both in eukaryotes and prokaryotes that consists in the massive production of non-coding RNAs from non-annotated regions of the genome. Pervasive transcription poses a risk that needs to be controlled since it can interfere with normal transcription of canonical genes. In S.cerevisiae, the helicase Sen1 plays a key role in restricting pervasive transcription by eliciting early termination of non-coding transcription. Sen1 is highly conserved across species and mutations in the human Sen1 orthologue, senataxin (SETX), are associated with two neurological disorders. Despite the major biological relevance of Sen1 proteins, little is known about their biochemical properties and precise mechanisms of action. During my PhD I have studied in detail the mechanisms of termination by Sen1.In a first project, I have characterized the biochemical activities of Sen1 and investigated how these activities partake in termination. To this end I have employed a variety of in vitro approaches, including a minimal transcription-termination system containing only purified Sen1, RNA polymerase II (RNAPII) and DNA transcription templates that allows modifying the different elements of the system in a controlled manner to understand their role in termination. First, we have analysed the function of the different domains of Sen1 in termination. Sen1 is a large protein composed of a central catalytic domain flanked by additional domains with proposed roles in protein-protein interactions. We have demonstrated that the central helicase domain is sufficient to elicit transcription termination in vitro. Next, we have shown that Sen1 can translocate along single-stranded nucleic acids (both RNA and DNA) from 5’ to 3’. Then, we have analysed the role of the different nucleic acid components of the elongation complex (i.e. nascent RNA and DNA transcription templates) in termination. Our results indicate that termination does not involve the interaction of Sen1 with the DNA but requires Sen1 translocation on the nascent RNA towards the RNAPII. Importantly, we show that upon encountering RNAPII, Sen1 can apply a mechanical force on the polymerase that results in transcription termination when RNAPII is paused under certain conditions. This indicates that RNAPII pausing is a strict requirement for Sen1-mediated termination. In a second project, in collaboration with the group of E. Conti we have performed a structure-function analysis of the helicase domain of Sen1. Comparison of Sen1 structure with that of other related helicases has revealed an overall similar organization consisting in two tandem RecA-like domains from which additional accessory subdomains protrude. In general, the core RecA-like domains are very well conserved among related helicases and most variation is found in the accessory subdomains, that often confer specific characteristics to different helicases. Indeed, we have found that Sen1 contains a unique but evolutionary conserved structural feature that we have dubbed the “brace”. In addition, Sen1 is different from other helicases in an auxiliary subdomain that we have named the “prong”. Importantly, we have shown that the integrity of this subdomain is critical transcription termination by Sen1. We propose that the specific features identified in our structural analyses are important determinants of the transcription termination activity of Sen1. Finally, we have used Sen1 as a model to investigate the molecular effect of SETX mutations linked to neurodegenerative diseases. We have introduced disease-associated mutations in Sen1 and performed a complete biochemical characterization of the different mutants in vitro. Importantly, we found that all mutants were severely affected in transcription termination. Taken together, our results elucidate the key structural determinants of the function of Sen1 and shed light on the molecular origin of the diseases associated with SETX mutations
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Raphela, Mashikoane Pinky Jane. "Molecular characterization of Mycoplasma synoviae in chickens in South Africa using single-stranded conformation polymorphism and high-resolution melting curve analysis of the vlhA gene." Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/26196.
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Mycoplasma synoviae (Ms) causes respiratory infection and synovitis in chickens and turkeys and is an economically important pathogen of poultry worldwide. It is critically important to characterize Ms strains, especially in countries in which poultry flocks are vaccinated with the live attenuated Ms strain MS-H. Vaccination with this vaccine may cause sero-conversion and persistence of the vaccine strain in the respiratory tract and will frequently result in positive Ms cultures and PCR results. Vaccination of flocks therefore complicates the diagnosis of Ms by the presence of detectable antibodies in the blood. Many diagnostic techniques cannot distinguish between the vaccine strain and wild type strain. Single stranded conformation polymorphism (SSCP) and real-time PCR with high melting curve (HRM) analysis can discriminate between the different Ms strains obtained from the field and also distinguish them from the live vaccinestrains. These techniques provide effective tools for the further study of the epidemiology and spread of Ms strains in chickens in South Africa. This project was undertaken to establish whether SSCP and HRM analyses can be used effectively to discriminate between Ms field isolates and the vaccine strain. Mycoplasma synoviae DNA was extracted from samples and conventional PCR was performed using oligonucleotide primers complementary to the single-copy conserved 5’ end of the variable lipoprotein and haemagglutinin encoding gene (vlhA). Twenty six samples were separated by agarose gel electrophoresis and prepared for SSCP and real-time PCR and HRM curve analysis. Results obtained from SSCP were compared to real-time PCR/HRM. Differences obtained by SSCP and melting curve analysis between different isolates were confirmed by sequencing. Results obtained from the different techniques differentiated the strains from the vaccine strain (isolate Ms10), which had a different melting temperature to the others and a different band pattern on the SSCP gel. These results confirmed that HRM and SSCP methods can be used to detect and discriminate between Mycoplasma synoviae field isolates and the vaccine strain.Dissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
unrestricted
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Francou, Bruno. "Contribution à la caractérisation de nouveaux gènes impliqués dans les hypogonadismes hypogonadotropes : caractérisation des mécanismes moléculaires et cellulaires." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS101/document.
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Les hypogonadismes hypogonadotropes congénitaux (CHH) sont des maladies héréditaires caractérisées par un déficit de sécrétion des gonadotrophines par l’hypophyse, à l’origine d’une infertilité ou d’une absence complète de puberté. On distingue les formes isolées avec olfaction normale (nCHH) et les formes syndromiques associant au déficit gonadotrope d’autres signes, tel qu’un défaut d’olfaction dans le cas du syndrome de Kallmann (SK), la forme plus fréquente de CHH. Les gènes identifiés dans le SK participent au développement embryonnaire et les gènes des nCHH sont impliqués dans la régulation de la sécrétion de la GnRH ou de son action. A ce stade, deux populations de neurones hypothalamiques gonadotropes sont connues, le neurone à GnRH et le neurone KNDy, sécrétant les Kisspeptines et la Neurokinine B. On estimait que l’ensemble des gènes identifiés couvraient moins de 20% des étiologies génétiques. L’objectif de ce doctorat était d’étudier prévalences et mécanismes physiopathologiques des gènes connus et d’identifier de nouvelles étiologies génétiques de CHH. Dans la première partie, nous avons caractérisé la fonctionnalité de tous les variants identifiés sur les gènes KISS1R, TACR3 et TAC3. Cela a permis de préciser les prévalences chez 600 patients, d’identifier un profil neuroendocrinien propre à l’altération de la signalisation Neurokinine B et de démontrer l’implication des Kisspeptines au cours de la vie embryonnaire. Enfin, nous proposons un modèle d’interaction entre le neurone à GnRH et le neurone KNDy. Dans la seconde partie, nous avons identifié deux nouveaux gènes, SEMA3A dans une forme familiale de SK et PNPLA6 dans une forme familiale rare de CHH syndromique. En conclusion, notre connaissance accrue des formes génétiques de CHH, a permis de développer un panel d’exome ciblé dédié au diagnostic par séquençage nouvelle génération permettant l’analyse simultanée de gènes candidats et de gènes connusCongenital hypogonadotropic hypogonadism (CHH) is characterized by deficient or absent pubertal development due to deficient or absent secretion of the pituitary gonadotropins. The many known genetic causes are generally classified into distinct nosological groups. One comprises abnormalities that affect the pre-natal development or migration of GnRH neurons, the paradigm of which is Kallmann syndrome. The other encompasses molecular abnormalities that only affect hypothalamic GnRH synthesis, GnRH release or GnRH signaling at pituitary level. At this stage, two populations of hypothalamic neurons implicated in a gonadotrop function are identified, GnRH neurons and KNDy neurons secreting kisspeptins and neurokinin B. All of the identified genes would represent less than 20% of genetic etiologies.The aim of this PhD was to study the prevalence and pathophysiology mechanisms of known genes and to identify new genetic etiologies of CHH.In the first part, we characterized the function of all molecular events identified on KISS1R, TACR3 and TAC3 genes. Prevalences were estimated in 600 patients. A particular neuroendocrine profile was identified in patients presenting an alteration of neurokinin B signaling. Importance of Kisspeptins during embryonic life was validated. According to these data, a model of interaction between GnRH and KNDy neurons was proposed.In the second part, we identified two new CHH genes using various molecular genetics approaches. SEMA3A was identified in a familial form of Kallmann syndrome and PNPLA6 in a rare familial form of CHH.Finally, our increased knowledge of the various genetic forms of CHH allows proposing a new genetic approach based on next generation sequencing to test together all known and several candidate genes
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Coty, Jean-Baptiste. "Caractérisation des nanomédecines pour la clinique : développement de méthodes évaluant les interactions nanoparticules-protéines plasmatiques pour une application en contrôle qualité." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS432/document.
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Les nanomédecines injectées par voie intraveineuse interagissent avec les éléments biologiques qui les entourent dans le compartiment sanguin. Parmi ces interactions, celles avec les protéines sanguines se révèlent être très importantes dans le devenir de ces nanovecteurs, leur conférant une identité biologique influençant leur chemin jusqu’au tissu et aux cellules cibles. La compréhension et le contrôle de ces phénomènes reste un enjeu crucial dans le développement des nanomédecines. Des méthodes permettant une étude facilitée de ces interactions sont nécessaires à cet égard. Les travaux de cette thèse ont eu pour but de développer des méthodes, utilisables en routine, permettant une caractérisation fine des nanomédecines et de leurs interactions avec les protéines plasmatiques, applicables dans un contexte clinique. Ils s’inscrivent dans un projet intitulé « Nano Innovation for CancEr » (NICE, BPI France) regroupant un consortium de partenaires industriels en développement clinique de nanomédecines.Dans un premier temps, un travail bibliographique sur les méthodes actuellement mises en œuvre pour une telle caractérisation ont pu mettre en avant deux limitations majeures. (i) D’une part, la complexité des méthodes actuelles disponibles pour lesquelles la spécificité des équipements et l’expertise requise limitent une utilisation à large échelle. (ii) D’autre part, les propriétés aujourd’hui caractérisées en routine (taille, morphologie globale, charge) ne sont que grossières comparées à la finesse des processus biologiques qui interagissent et « analysent » les nanovecteurs une fois introduits dans le milieu biologique. Ces deux aspects limitent aujourd’hui un développement plus sûr des nanomédecines pour une bonne reproductibilité en clinique et garantir des essais de contrôle qualité fiables.Au cours de nos travaux, nous avons développé des méthodes permettant de répondre en partie à la problématique posée par la caractérisation des nanomédecines. Une méthode d’analyse à haut débit de l’activation du système du complément par immunoélectrophorèse en deux dimensions a été développée et validée. Elle permet l’analyse reproductible de l’activation de la protéine C3. Elle est applicable à l’étude de l’effet de la présence de nanoparticules dans le sérum humain et leur degré d’action sur la cascade du complément. Cette méthode a été utilisée pour mener une étude plus fondamentale du mécanisme de l’activation du système du complément en regard de l’architecture de la surface de nanoparticules.Une deuxième méthode d’étude de l’activation du complément produit par des nanomédecine a été proposée sur la base de la résonnance plasmonique de surface (SPR). Une puce permettant un screening automatisé de l’activation du complément a été développée. L’application de cette méthode comparée à d’autres méthodes d’études de l’activation du système du complément (Immunoélectrophorèse 2D, ELISA) a permis d’identifier des biais lors de leur application à l’évaluation des nanomédecines.Enfin, une approche originale de caractérisation de la surface de nanoparticules a été proposée utilisant des protéines pour sonder la capacité de la surface des nanoparticules à adsorber ou repousser ces dernières. Dans cette méthode, l’électrophorèse capillaire est utilisée comme outil analytique permettant une analyse directe de l’échantillon sans séparation préalable des nanomédecines.Les méthodes développées au cours de ces travaux peuvent être appliquées à la caractérisation de nanomédecines et proposées comme des méthodes de contrôle en routine de façon plus générale. Un développement de la caractérisation dans ce sens constitue l’un des leviers pour une translation plus fructueuse des nanomédecines entrant en phase cliniqueNanomedicines injected intravenously interact with surrounding biological elements in the bloodstream. Among these interactions, those with blood proteins turn out to be very important regarding the becoming of the nanovectors. They acquire a biological identity upon interaction with proteins which influence their path to target tissue and cells. The understanding and mastering of these phenomena remains a crucial issue in nanomedicine development. Methods allowing an easier study of these interactions are needed. The aim of these PhD thesis was to develop such methods, usable on a routine basis in a clinical context, allowing a fine characterization of nanomedicines and their interactions with plasmatic proteins. This PhD is part of the project “Nano Innovation for CancEr” (NICE, BPI France), gathering a consortium of industrials partners developing clinical nanomedicines.In a first time, a bibliographic study about current methods used for such a characterization could identify two major limitations. (i) On one hand, the complexity of current available methods for which the equipment specificity and required expertise prevent their use at a large scale. (ii) On the other hand, properties today characterized on a daily basis (size, morphology, charge) are too rough compared to the sharpness of biological processes who interact and “analyze” the nanovectors introduced in biological media. These two aspects are limiting a safer development of nanomedicines as well as a good reproducibility of their action in clinics.During this thesis, we developed methods allowing a beginning of answer to the wide problematic of nanomedicine characterization. A method for a high throughput analysis of complement activation by nanomedicines via 2D immunoelectrophoresis was developed and validated. It allows the reproducible analysis of protein C3 fragmentation. This method is applicable to the study of the impact of nanoparticles in human serum and their degree of action on the complement cascade. This method has been used for a more fundamental study on complement activation pathways activated according to the architecture of nanoparticles surface.A second method for the study of complement activation produced by nanoparticles has been proposed using surface plasmon resonance (SPR). A chip allowing an automated screening of complement activation has been developed. This method was compared to other methods for complement activation study (2D immunoelectrophoresis, ELISA) and allowed the identification of bias during nanomedicine evaluation.Finally, an original approach for the characterization of nanomedicine’s surface architecture using proteins as molecular probes has been proposed. In this method, capillary electrophoresis has been used as analytical tool to allow a direct analysis of sample without preliminary nanoparticle removal step.Methods developed during this work can be applied to the characterization of nanomedicines and proposed as routine methods for quality control. A development of nanomedicines characterization in this direction constitute one of the lever for a more fruitful translation of nanomedicines entering in clinical phase
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Fleischmann, Matthias [Verfasser], Ruth M. [Akademischer Betreuer] Gschwind, Kirsten [Akademischer Betreuer] Zeitler, and Manfred [Akademischer Betreuer] Scheer. "NMR investigations on catalysts and conformations in organo- and photocatalytic reactions, and characterization of electrolytes and supramolecular switchable container molecules / Matthias Fleischmann. Betreuer: Ruth M. Gschwind ; Kirsten Zeitler ; Manfred Scheer." Regensburg : Universitätsbibliothek Regensburg, 2011. http://d-nb.info/1026165520/34.
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Xiao, Jianxi. "NMR conformational and dynamic characterization of triple helical peptides." 2009. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051923.
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Weng, Shao-En, and 翁紹恩. "Conformational and dynamic characterization of Na+-dependent bile acid transporter ASBTNm." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/q2dsnz.
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碩士國立中興大學
生物化學研究所
106
Apical Sodium-dependent Bile acid Transporter (ASBT), a Na+-dependent secondary active transporter, is localized at the apical membrane of enterocytes and mediates the reuptake of bile acid from ileum back to liver. The structural information of ASBT is attributed to the crystal structures of bacterial homologs ASBTNm and ASBTYf, revealing the snapshots at inward- and outward-facing states, and the putative mechanism of substrate translocation via an “elevator-like” movement. Nevertheless, the triggers of conformational changes and the dynamics at different substrate conditions remain undetermined. In this project we employed Site-directed Alkylation monitored by in gel Fluorescence (SDAF) to probe the in situ alteration of substrate binding pocket accessibility. We tried to apply SDAF assay in topology determination of membrane proteins, and the results demonstrated it is valid and much more efficient than previous methods. In our earlier SDAF studies, a series of residues located at the substrate pathway in ASBTNm were substituted with cysteine, but the functional effects of these point mutations are still unknown. We performed whole cell [3H]-taurocholate uptake assay using ASBTNm-overexpressed E. coli C43 to monitor the transportation activities of these mutants. Although most ASBTNm mutants have lower activities than WT ASBTNm, they still reserve transport activity at certain level. In order to validate our dynamics data obtained by SDAF, we spin-labelled Y39 and T303 for Double Electron-Electron Resonance (DEER) using Electroparamagnetic Resonance (EPR) spectroscopy. The DEER results indicated that DDM-solubilized ASBTNm is populated as two discrete conformational species in K+ buffer, representing outward- and inward-facing states, and one major conformational species in Na+ buffer, and in taurocholate added Na+ buffer, representing inward-facing and occluded states. In order to obtain soluble ASBTNm in lipid bilayer for DEER spectroscopy, we tried to purify ASBTNm in styrene maleic acid lipid particles (SMALP). The C-terminal GFP fusion facilitates tracking of ASBTNm-SMALP, although the purity requires further optimization. The soluble membrane protein particles in SMALP will enable us to preserve the native behavior of ASBTNm for structural and dynamical studies. We also analized surface charge of ASBTNm using APBS, and performed SwissDock for prediction of possible substrate binding sites. The predicted results are of our expectation. The studies will provide practical methods in obtaining crystal structure of ASBTNm bound with taurocholate at outward-facing state.
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Simão, Alcides Pinto. "Gas-Phase Conformational Characterization of Trace Amine Neurotransmitters: a rotational spectroscopy study." Doctoral thesis, 2020. http://hdl.handle.net/10316/91053.
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Tese no âmbito do Doutoramento em Química, ramo de Espectroscopia
Molecular apresentada ao Departamento de Química da Faculdade de
Ciências e Tecnologia da Universidade de CoimbraA presente dissertação trata das preferências conformacionais de uma série de aminas residuais (octopamina, sinefrina, fenilglicinol e fenilefrina), bem como de um precursor aminoácido (ácido pipecólico) e de um aminossacarídeo (glucosamina), estudadas em condições de isolamento em fase gasosa recorrendo ao uso de uma combinação de técnicas de espectroscopia rotacional hifenada com ablação laser. Livre de qualquer tipo de interacção intermolecular, torna-se possível a caracterização f física das interacções intramoleculares para cada confórmero, bem como o seu papel no panorama conformacional de cada biomolécula. Os resultados experimentais foram posteriormente comparados com os resultados teóricos, de modo a se proceder a uma caracterização completa dos confórmeros observados. Esta correlação de dados permitiu ainda estabelecer a natureza das principais interacções intramoleculares envolvidas em cada conformação.
In the present dissertation, the conformational preferences of a series of trace amines (octopamine, synephrine, phenylglycinol and phenylephrine), as well as an aminoacid precursor (pipecolic acid) were studied in gas phase isolation conditions using a combination of rotational spectroscopy tech- niques together with laser ablation. Free from any kind of intermolecular interaction, the physical characterization of the intramolecular interactions for each conformer and their role in the conformational panorama of each biomolecule became possible. Experimental results were then compared with theoretical results, in order to fully characterize the observed conform- ers. This data correlation also allowed to establish the nature of the major intramolecular interactions involved in each conformation.
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Hsiao, Chun-Che, and 蕭均哲. "Characterization of the conformational and functional properties of two in vitro refolded Bacillus subtilis SigB." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/73692598406431862636.
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碩士國立中興大學
生物化學研究所
104
Transcription initiation is the first step of gene expression and a major point for gene regulation.σ factor of RNA polymerase (RNAP；α2ββ''σ) confers the specificity of promoter recognition and initiates transcription. It is also involved in open complex, abortive transcription and regulation of gene expression. Recent works in our lab demonstrated that the N-terminally truncated primary σA factor, SND100-σA, and σB of Bacillus subtilis, both of which lack region 1.1, are able to specifically interact with promoter DNA in the absence of core RNA polymerase in vitro. Moreover, SND100-σA factors with different functional properties were achieved through the use of slightly different refolding protocols. Among them, SND100-σA1 is capable of exhibiting specific and core RNAP-independent promoter-binding activity; whereas SND100-σA3 is able to melt DNA independent of core RNA polymerase in vitro. Present study is aimed to investigate whether the B. subtilis σB can also adopt different functional properties through protein refolding. To fulfill this goal, the σB protein was overexpressed in E. coli BL21 (DE3) harboring a σB-expressing plasmid. The overexpressed σB in inclusion bodies were denatured with guanidine hydrochloride and refolded through mild serial dilution of the guanidine hydrochloride solution. The refolded σB were soluble and can be purified using Superdex G-200 and Superdex G-75 columns. Buffers with or without both PMSF and DTT were used for protein refolding and purification and the σB proteins thus obtained were named σB1 and σB3, respectively. Results of circular dichroism reveal that σB1 and σB3 have similar contents of secondary structures albeit with different overall conformations. Moreover, both σB1 and σB3 are able to bind promoter DNA as analyzed by electrophoretic mobility shift assay (EMSA). However, the two σB-promoter DNA complexes are different in sensitivity to heparin challenge, suggesting that they possesss different DNA-binding properties.
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Baldwin, Richard Kimo. "Preparation, characterization and conformational variability of [eta]6-Hexaethylbenzene and other arene complexes of ruthenium." Phd thesis, 1998. http://hdl.handle.net/1885/144675.
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ZHANG, JIANMING. "Conformational and Mechanical Characterization of Organic Thin Films on Surfaces by Neutron Reflection and Atomic Force Microscopy." Diss., 2014. http://hdl.handle.net/10161/9400.
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Engineering thin, organic materials with tailored properties requires both the understanding of the conformation of thin organic films and their conformational response to changes in the environment, and the accurate characterization the mechanical properties of the materials as a thin layer on surfaces. These issues have not yet been sufficiently addressed due to the paucity of appropriate tools and data interpretation approaches to reveal the nanometer scale conformation and mechanics of surface-grafted, thin, organic films. In this dissertation, I report on the characterization of conformational and mechanical properties of thin organic films, and the development of techniques that allow more detailed and reliable measurement of these material properties. First, I co-developed a novel approach to evaluate neutron reflectivity data and to simulate the conformational structure for thin stimulus-responsive polymer brushes. In this approach, we used a molecular-based lattice mean-field theory, augmented with experimentally obtained parameters to describe the polymer chains. The approach and fitting results required fewer fitting parameters, and captured the thermal response of the sample self-consistently.
Second, I demonstrated the capability of force-modulation microscopy in imaging surface-grafted, organic thin films in aqueous environments, with high spatial resolution and sensitivity to conformational details that affect the contact mechanics. To this end, I developed a new parameter-selection approach. This approach allowed both highly sensitive mapping of subtle differences in the molecular packing of thiol molecules on the substrate surface, and generation of high-contrast contact-stiffness images of end-grafted protein patterns on a surface. Finally, I discussed model selection and error estimation in calculating the reduced Young's modulus of soft materials on surfaces. I found that the detailed characterization of probe apex profiles, using probe-reconstruction techniques, provide only marginal improvements in calculating the reduced Young's modulus of thin films, compared with analytical models of equivalent probe radii; however, I found that a hybrid worn-cone model is appropriate for large indentations on soft materials, and benefits from the characterization of the probe apex profile. Additionally, we rendered error maps of several common scenarios, referenced to indentation and probe radius values, in the determination of the reduced Young's modulus.
Dissertation
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Hansen, Jeffrey Craig. "Characterization of the conformational transitions of the estrogen receptor monomer by partition in aqueous two-phase systems." 1986. http://catalog.hathitrust.org/api/volumes/oclc/15244978.html.
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Thesis (Ph. D.)--University of Wisconsin--Madison, 1986.Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 153-162).
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Ujvari, Andrea. "Characterization of DNA structural elements and conformational changes accompanying the binding of T7 RNA polymerase to its promoter." 1999. https://scholarworks.umass.edu/dissertations/AAI9920662.
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The first step of transcription initiation, promoter binding, is characterized in the T7 RNA polymerase model system. A previous two-domain model for the T7 RNA polymerase promoter is tested by the use of different fluorescence methods. The increase in the fluorescence anisotropy of a rhodamine dye linked to the upstream end of the promoter provides a very sensitive measure of enzyme binding in simple thermodynamic titrations (best fit value of Kd is calculated to be 4.0 nM), and is the first quantitative assay to reliably measure both increases and decreases in the dissociation constant. The assay is used to follow polymerase binding to a series of synthetic oligonucleotides representing truncations of the consensus promoter sequence, and results show that the duplex region of the promoter upstream of and including position −5 is both necessary and sufficient for tight binding, and represents the core binding element of the promoter. In separate assays, 2-aminopurine, a fluorescent nucleotide analog was placed at different locations within the upstream and downstream domains of the promoter. The fluorescence intensity of 2-aminopurine reports on local base stacking and is used both to measure binding and also to provide direct evidence for local melting. Results with this probe confirm that the promoter region upstream of position −5 is recognized as a duplex, in contrast to the downstream promoter region, which appears to bind in a distorted or melted conformation, in the static polymerase-promoter complex. Our kinetic experiments also indicate that polymerase binding to its promoter and the structural transition in the DNA near the transcription start site is very fast, with binding close to the diffusion limit. Finally, gel mobility shift assays detect a small intrinsic (∼9°) and a larger (40–60°) protein-induced DNA bend for the T7 RNA polymerase. The protein-induced bend is estimated to be centered near the transcription start site. We propose a model in which part of the upstream binding energy is used by T7 RNA polymerase to melt the downstream initiation region of the promoter. Furthermore, the protein-induced bend may provide the mechanism which drives melting near the start site.
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Teklebrhan, Robel Berhe. "Conformational preferences and structural characterization of prolyl cis/trans isomerization of carbohydrate-templated proline mimetics of some model peptides using computational methods." 2009. http://hdl.handle.net/1993/21590.
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