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1
Lindgren, Joel. "Chemical Engineering of Small Affinity Proteins." Doctoral thesis, KTH, Proteinteknologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-141014.
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Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering.QC 20140207
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Zourna, Kalliopi. "Smart magnetic affinity adsorbents." Thesis, University of Birmingham, 2009. http://etheses.bham.ac.uk//id/eprint/511/.
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As the focus of research on ‘adaptive/responsive’ surfaces has in recent years contributed strongly towards the design of surface materials with ‘intelligent’ or ‘smart’ behaviour, current superparamagnetic adsorbents being employed both in small and large scale operations can be surface modified and improved by gaining dual functionalities. In this work, modification of M-PVA supports with polymer brushes of dual properties has been explored for their intended use in bioseparation technology, i.e. for both selectively protein binding and enhanced temperature elution of especially difficult to elute species such as haemoglobin. Tethering of polymer brushes was achieved by employing two different ‘grafting from’ routes, i.e. cerium (IV) initiated polymerisation and Atom Transfer Polymerisation Reaction (ATRP). By identifying the optimum cerium (IV) reaction conditions, the said chemistry was further utilised to attach different polymers (thermoresponsive and affinity ligands) and their combination (thermo-affinity) at fixed positions onto M-PVA supports, either as di-block or mixed functionality polymer brushes. The configuration of introduced polymer chains as well as the haemoglobin binding characteristics of the above materials was evaluated, and their efficiency for haemoglobin and GFP desorption via sequential temperature transitions was demonstrated. Mixed polymer brushes manufactured using sequential ATRP after partial bromination of AGE activated magnetic supports were characterised and tested likewise. Protein binding and release efficiency was dependent on brush configuration (length and spacing between the graft sites of polymers), pNIPAAm content, type of affinity ligand and type of protein employed. From the above materials those with polymer chains of sufficient pNIPAAm length and at such spacing allowing their ‘free’ expansion/collapse upon temperature change (especially those grafted via cerium (IV) route) were found efficient, as brush behaviour favour enhanced desorption of difficult to elute species.
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Beard, Hester Annie. "Affinity-guided chemical probes for the study of protein interactions." Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/20637/.
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Chemical methods that allow for the targeted labelling of a specific protein within a complex biological environment can enable valuable information regarding the structure and function of proteins to be gained. This thesis explores two different projects where affinity-guided chemical probes were used to study the interactions of proteins, both with small molecules (Chapter 2) and interacting protein partners (Chapter 3). Firstly, chemical labelling methods based on a recognition unit for the protein of interest are reviewed in Chapter 1. Then, Chapter 2 describes how a combination of chemical tools (including photoaffinity, biotinylated and fluorescent probes) were used to study the interaction of a small molecule inducer of human glioblastoma cell death and its relevant target. This work resulted in the identification of HSPD1 as a potential therapeutic target for the treatment of glioblastoma. Chapter 3 details the development of a method for traceless labelling of B-cell lymphoma 2 (BCL-2) family proteins, using a ruthenium-bipyridyl modified peptide. Myeloid cell leukaemia 1 (MCL-1) was rapidly and selectively labelled with fluorescent and biotinylated tags, in vitro, facilitated by the interaction with a peptide mimicking a binding partner. Overall, this thesis demonstrates how affinity-guided labelling of proteins can be used for understanding molecular mechanisms of disease and mapping protein interactomes.
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Tarasova, Anna Optometry UNSW. "Fabrication and characterisation of affinity-bound liposomes." Awarded by:University of New South Wales. Optometry, 2007. http://handle.unsw.edu.au/1959.4/29114.
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In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.
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Zulqarnain, Kamran. "Scale-up of affinity separation based on magnetic support particles." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313426.
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Stewart, David Johnston. "Immobilisation of triazine dyes on inert hydrophobic supports for affinity chromatography." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315974.
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Miller, Eric Alexander. "Development of thermostable affinity reagents for low-cost, paper-based medical diagnostics." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122849.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019Cataloged from PDF version of thesis.
Includes bibliographical references.
The timely diagnosis and treatment of disease in resource-constrained settings requires the development of robust point-of-care (POC) diagnostics, which provide accurate results and can be employed by users with minimal medical training and limited access to basic infrastructure. One of the most common POC diagnostic formats is the immunochromatographic rapid diagnostic test, which traditionally uses nitrocellulose-immobilized IgG antibodies as binding proteins for the capture of disease biomarkers from patient samples. However, these antibodies are expensive to produce and structurally complex, and are prone to thermal denaturation. In contexts where continuous cold chain storage may be infeasible, the resulting loss in binding activity can manifest as diminished assay sensitivity, leading to adverse clinical outcomes and eroding patient trust in the diagnostic format.
In the interest of replacing diagnostic antibodies with a more cost-effective, robust class of binding proteins, this thesis explores the development of thermostable affinity reagents based on the hyperthermophilic scaffold protein rcSso7d. Given its native microbial host, minimalist structure, and high wild-type melting temperature (98°C), rcSso7d represents a viable alternative to antibodies in in vitro POC assays. To assess the applicability of the rcSso7d scaffold in this context, protein engineering techniques were used to rapidly select analyte-specific binding variants from a yeast surface display library of >109 members. A high-affinity rcSso7d binder was identified, produced in high yield in a bacterial host, and readily purified in a single chromatographic step.
The in vitro activity and thermal stability of this engineered binder were characterized in the context of a low-cost, paper-based assay, and significant improvements in stability and production economics were observed for rcSso7d-based assays, relative to assays featuring a representative antibody reagent. Additionally, general strategies were developed to improve the diagnostic performance of paper-based assays employing rcSso7d-based reagents. In one instance, chimeric protein constructs in which rcSso7d variants are fused to a cellulose-binding domain were found to bind to unmodified cellulose in an oriented fashion and with high efficiency. This substrate anchoring approach permits the rapid, high-density immobilization of the rcSso7d species in paper-based assays, and yields significant sensitivity enhancement by enabling both the depletion of the soluble analyte from the sample, and the processing of large sample volumes within clinically relevant timescales.
Detection reagents incorporating rcSso7d binders were also developed, using novel fusion constructs which enabled in vivo labelling while preserving analyte binding activity. These techniques were applied in the context of a urine-based tuberculosis biomarker, and may one day permit the development of multiplexed assays targeting a suite of these analytes. Such a development would enable point-of-care diagnostic testing without requiring the production of sputa, facilitating disease detection in otherwise inaccessible patient populations (e.g. children under five, the elderly, and immunocompromised patients).
"People who have financially supported this thesis: the NIH Biotechnology Training Program, the Tata Center for Technology and Design, the Deshpande Center, the Sandbox program, and the Singapore- MIT Alliance for Research and Technology"
by Eric Alexander Miller.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering
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Hsu, Kuang-Hsin. "Scale-up of affinity chromatography for protein purifications." Ohio : Ohio University, 2000. http://www.ohiolink.edu/etd/view.cgi?ohiou1172002240.
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Lam, Hei Ning Henry. "Electrostatic and affinity enhancements of protein partitioning in two-phase aqueous micellar systems." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33700.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Includes bibliographical references (p. 175-188).
This thesis was motivated by the practical need to develop a scalable and cost-effective separation method for low-cost, high-volume protein products. This unmet challenge can potentially be addressed by extraction in two-phase aqueous micellar systems, in which biomolecules can be partitioned in mild, predominantly aqueous environments. The goal of this thesis was to explore various ways of enhancing protein partitioning in two-phase aqueous micellar systems, by the incorporation of electrostatic and affinity interactions, to obtain satisfactory yield and specificity for the purification of industrially relevant hydrophilic proteins. The electrostatically-enhanced partitioning of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated experimentally and theoretically. The successful enhancement, up to 22-fold, of the partitioning of the negatively-charged G6PD was attained by adding the positively- charged surfactant alkyltrimethylammonium bromide (CnTAB) to form charged mixed micelles with the phase-forming nonionic surfactant, decyl tetra(ethylene oxide) (C₁₀E₄).
(cont.) The effects of the tail length of the positively-charged surfactant on protein denaturation and protein partitioning behavior were also studied. Furthermore, the experimental results were used to validate a predictive theory for electrostatic enhancement. In the area of affinity enhancement, the affinity-enhanced partitioning of an engineered affinity-tagged protein, CBM9-GFP (Green Fluorescent Protein linked to a carbohydrate- binding module), in two-phase aqueous micellar systems was investigated experimentally and theoretically. The experimental results showed that the partition coefficient of the target protein, CBM9-GFP, can be improved more than 6-fold, by virtue of the affinity interactions, and that the enhancement is specific to the target protein. The system utilized requires only one surfactant, decyl [beta]-D-glucopyranoside (C₁₀G₁), which acts simultaneously as the affinity ligand and as the phase-forming surfactant, and as such, has important practical advantages. A novel theoretical framework to describe affinity- enhanced protein partitioning in two-phase aqueous micellar systems was developed and validated experimentally. In addition, the separation method developed was successfully applied to a real cell lysate.
(cont.) It was found that the protein impurities in the cell lysate do not interfere with the partitioning of the target protein (CBM9-GFP) at industrially relevant concentrations, and that the protein impurities were concentrated away from the target protein. Lastly, the theoretical description developed was used to identify various strategies for improving the affinity-enhanced partitioning of the target protein in two-phase aqueous micellar systems. Although more work remains to be done before the separation methods studied in this thesis can reach their full potential and be eventually commercialized, this thesis nevertheless represents an essential starting point for future efforts to improve, extend, and commercialize this promising bioseparation method.
by Hei Ning Henry Lam.
Ph.D.
10
Liebenberg, Liesl Eileen. "Non-covalent immobilisation of a ligand system : a new approach to affinity separation." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53522.
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Thesis (MSc)--Stellenbosch University, 2003.ENGLISH ABSTRACT: Advances in pharmacology, biochemistry and biotechnology are increasingly dependant upon affinity chromatography as a preferred separation technique for the purification and characterisation of specific biomolecules. In the past few years avidin-biotin technology has been widely and successfully used in the fields of medicine, pharmacy, biology and biochemistry. The avidin-biotin complex (ABC) has been used as a mediator for affinity chromatography, affinity cytochemistry, immunoassay, histopathology, bioaffinity sensors, erosslinking and immobilisation studies. The main reason for the popularity of the ABC and its growing usefulness in biotechnology is the exceptionally high affinity (1015 M-l) and stability of the noncovalent interaction between avidin and biotin. The use of the ABC is broadening as different biotin derivatives and avidin-containing conjugates are becoming commercially available. The aim of this work was to evaluate the usefulness of a plutonic" FI 08 and the ABC conjugate to effect affinity separation. Towards this aim, the adsorption of plutonic" F108 onto hydrophobic polysulphone membrane surfaces was studied. This information was used to determine the theoretical maximum amount of pluronic" FI08 that will adsorb onto a unit surface area of the membrane. It is known that the polypropylene oxide (PPO) centre block ofthe pluronic" F I08 surfactant molecule governs the concentration of pluronic" F I 08 molecules that will adsorb onto a given hydrophobic surface. If the maximum coating concentration of plutonic" FI08 is known, one can assume that the maximum coating concentration of any pluronic derivative, with the same PPO centre block size, will be the same. Adsorption studies were carried out, the Langmuir adsorption isotherm was determined, and subsequently the fractional coating was calculated. The end-groups of plutonic" FI08 were modified as follows and the substituted pluronic was adsorbed onto a membrane that was to act as the solid support matrix in the development of an affinity system: Amino pluronic was synthesised by first tosylating pluronic" FI08, followed by azidation with NaN3 then reduction with LiAI~. The synthesised amino pluronic was then biotinylated using N-hydroxysuccinimide biotin ester. The suitability of this synthetic route was first assessed on a model compound, 2-methoxyethylamine, and validated by NMR (Nuclear Magnetic Resonance) spectroscopy. The synthetic protocol was then used to derivatise the larger pluronic molecule. The affinity system was tested on two different hydrophobic surfaces: polystyrene and polysulphone membranes (PSMs). Avidin-conjugated horseradish peroxidase was obtained and used to interact with the immobilised biotin. The enzymatic reaction of the coupled peroxidase converted the substrate, 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) to a coloured product. The colour developed is proportional to the amount of biotin that was immobilised on the hydrophobic surfaces studied. Non-covalent immobilisation of the synthesised biotin-pluronic molecule was successfully obtained onto the hydrophobic polystyrene as well as the polysulphone membrane surfaces.
AFRIKAANSE OPSOMMING: Vooruitgang in die farmakologie, biochemie en biotegnologie word al meer afhanklik van affiniteits chromatografie as die verkose tegniek vir die suiwering en karaterisering van spesifieke biomolekules. Oor die afgelope jare het die avidien-biotien tegnologie homself as baie bruikbaar bewys in die mediese, farmakologiese, biologiese en biochemiese velde. Toepassings waar die avidien-biotien kompleks betrokke was sluit in die toepassing as 'n mediator vir affiniteits chromatografie, affiniteits sitologie, immuno bepalings, histopatologie, bioaffiniteits sensors sowel as kruisbinding en immobiliserings studies en vele meer. Die hoofrede vir die gewildheid van die avidien-biotien kompleks en die groeiende bruikbaarheid in die biotegnologie is die buitengewone hoë affiniteit (l015 M-I ) en stabiliteit van die nie-kovalente interaksie tussen avidien en biotien. Die toepassingsveld van die avidien-biotien kompleks word wyer met die verskeidenheid biotien derivate en avidien-bevattende konjugate wat kommersiëel beskikbaar is. Die doel van die werk wat hier gedokumenteer word is om die bruikbaarheid van Plutonic" FI08 en die avidien-biotien kompleks, vir gebruik in 'n affiniteits chromatografie sisteem, te evalueer. Om hierdie doel te bereik is die adsorpsie van Pluronic" FI08 aan hidrofobiese polisulfoon membraan oppervlaktes bestudeer. Die eksperimentele data wat gegenireer is, is gebruik om die teoretiese maksimum hoeveelheid Pluronic wat per eenheids oppervlakte membraan adsorbeer te bepaal. Dit is reeds bekend dat die polipropileen (PPO) middel blok van die Pluronic emulgant die konsentrasie van die geadsorbeerde Pluronic molekules op 'n gegewe hidrofobiese oppervlakte bepaal. Indien die maksimum bedekkingskonsentrasie VIr maksimum oppervlakbedekking van Plutonic" FI08 bekend is, kan teoreties aanvaar word dat die bedekkingskonsentrasie vir enige Pluronic derivaat met dieselfde grootte PPO blok dieselfde sal wees. Adsorpsiestudies was uitgevoer om die Langmuir adsorpsie isoterm te bepaal. Daaropvolgend was die fraksionele bedekking bereken. Amino-pluronic was gesintetiseer deur die eindpunte van Pluronic te derivatiseer. Hierdie Pluronic derivaat was gevolglik geadsorbeer aan 'n membraan wat gedien het as die soliede oppervlakte vir die ontwikkeling van 'n affiniteits chromatografie sisteem. Amino-pluronic was gesintetiseer deur Pluronic eers te tosileer en daarna te asideer met NaN3 en laastens te reduseer met LiAI~. Die produk was gebiotinileer deur gebruik te maak van N-hidroksisuksinimied-biotien-ester. Die bruikbaarheid van hierdie sintetiese roete is eers bepaal deur van 'n model verbinding, 2-metoksiëtielamien, gebruik te maak en dit met behulp van KMR (Kern Magnetiese Resonans) spektroskopie te karakteriseer. Die affiniteits sisteem is getoets op twee verskillende hidrofobiese oppervlaktes naamlik polistireen en polisulfoon membraan oppervlaktes. Avidien gekonjugeerd met 'n peroksiedase ensiem is gebruik om met die geïmmobiliseerde biotien te assosieer. Die ensiematiese reaksie van die gekoppelde peroksiedase het die substraat 2, 2' -azino-di-(3-etiel-benzthiazolien-6-sulfoonsuur) (ABTS) omgesit na 'n gekleurde produk, waar dit teenwoordig is. 'n Reeks wasstappe is gebruik om die gemodifiseerde peroksidase ensiem wat nie aan die hidrofobiese oppervlakte gekoppel nie, weg te spoel. Hierdeur is die mate van binding aan die hirofobiese oppervlakte gekwantifiseer deur die kleur te kwantifiseer wat ontwikkelomdat die kleurontwikkeling direk proporsioneel is aan die hoeveelheid peroksidase wat nog aan die membraan gekoppel is. Nie-kovalente immobilisasie van die gesintetiseerde biotien-pluronic molekule is suksesvolop beide die hidrofobiese polistireen oppervlakte sowel as die polisulfoon membraan verkry.
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陳磊碩 and Lui-sek Chan. "Chemical modification of immunoglobulins and the effects on antigen binding site affinity." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1993. http://hub.hku.hk/bib/B29913378.
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Chan, Lui-sek. "Chemical modification of immunoglobulins and the effects on antigen binding site affinity /." [Hong Kong] : University of Hong Kong, 1993. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13731506.
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Heimer, Brandon Walter. "Methylated DNA detection using a high-affinity methyl-CpG binding protein and photopolymerization-based amplification." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/98796.
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Thesis: Sc. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 123-132).
Methylation at the 5 position of the cytosine base, when followed by guanine (CpG) in the promoter region of a protein-coding gene, is an epigenetic modification that has been shown to be involved in gene silencing. While important for regulating many normal, cellular processes, aberrant DNA methylation has been implicated in the development of human diseases such as cancer. Medical research has begun to identify hypermethylated gene promoters that can be used as predictive, prognostic, and diagnostic biomarkers. Current technologies for DNA methylation detection, however, rely on sodium bisulfite conversion of unmethylated cytosine bases to urasil in the target DNA. Bisulfite treatment is time consuming to perform, degrades >90% of the DNA, and is susceptible to incomplete conversion which can lead to false results. As such, a method that eliminates both chemical conversion and PCR amplification of DNA would enable significantly lower-cost and rapid DNA methylation analysis in the clinic. Methyl binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them excellent transducers for DNA methylation in biosensing applications. I displayed three of the core members MBD1, MBD2, and MBD4 on the surface of Saccharomyces cerevisiae yeast cells. Using the yeast display platform, I determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9+/-1.3 nM for binding to singly methylated DNA. I further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wildtype protein to 3.1+/-1.0 nM. Further, concatenating the high-affinity MBD variant from 1x to 3x and expressing it in Escherichia coli as a green fluorescent protein (GFP) fusion improved affinity 6-fold for interfacial binding. After optimizing the expression and purification of engineered MBD-GFP proteins in E. coli, I developed a simple method for detecting methylated DNA fragments from the MGMT gene promoter. A defining feature is that target oligonucleotides from the test sample hybridize directly to capture probes printed in 300 pm diameter spots on an inexpensive biochip without requiring bisulfite conversion. Sub-nanomolar concentrations (0.3 nM) of methylated DNA duplexes are detected using an MBD-GFP protein to transduce binding to either a fluorescent or colorimetric (visible hydrogel) signal formed by photopolymerization with short (<2 min) reaction times.
by Brandon Walter Heimer.
Sc. D.
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Lippow, Shaun Matthew. "Computational analysis, design, and experimental validation of antibody binding affinity improvements beyond in vivo maturation." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38886.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (leaves 98-110).
This thesis presents novel methods for the analysis and design of high-affinity protein interactions using a combination of high-resolution structural data and physics-based molecular models. First, computational analysis was used to investigate the molecular basis for the affinity improvement of over 1000-fold of the fluorescein-binding antibody variant 4M5.3, engineered previously from the antibody 4-4-20 using directed evolution. Electrostatic calculations revealed mechanistic hypotheses for the role of four mutations in a portion of the improvement, subsequently validated by separate biochemical experiments. Next, methods were developed to computationally redesign protein interactions in order to rationally improve binding affinity. In the anti-lysozyme model antibody D1.3, modest binding improvements were achieved, with the results indicating potentially increased sucesss using predictions that emphasize electrostatics, as well as the need to address the over-prediction of large amino acids. New methods, taking advantage of the computed electrostatics of binding, yielded robust and significant improvements for both model and therapeutic antibodies.
(cont.) The antibody D44.1 was improved 140-fold to 30 pM, and the FDA-approved antibody cetuximab (Erbitux) was improved 10-fold to 52 pM, with an experimental success rate of greater than 60% for single mutations designed to remove undersatisfied polar groups or improve misbalanced electrostatic interactions. Finally, a physics-based improvement to the calculation of the nonpolar component of solvation free energy was implemented and parameterized to address the over-prediction of large amino acids. These results demonstrate novel computational capabilities and indicate their applicability for enhancing and accelerating development of reagents and therapeutics.
by Shaun Matthew Lippow.
Ph.D.
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Tellez, Rodriguez Carlos Mario 1967. "Studies of metal affinity interactions in metal recovery and bioremediation." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/288710.
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The primary goal of this dissertation project has been the study of metal affinity interactions in metal recovery and bioremediation. During the first part of this research a mathematical model that describes the affinity partitioning of metal ions in aqueous two-phase systems was derived. The model has been used to calculate complex formation constants between metal ions in solution and affinity ligands, satisfactorily describing their partition behavior. Simulation using this model shows the great effect that pH has on the partitioning of metal ions suggesting better conditions for the separation. Work on metal affinity interactions has led to the pursuit of characterization studies of metal uptake by microorganisms of relevance in bioremediation. The methanotrophic bacterium M. trichosporium 0B3b a mutant culture (PP358) that expresses soluble methane monooxygenase (sMMO) independent of the external copper concentration have been the subject of this research. Knowledge of substances and/or mechanisms that are involved in the copper uptake by M. trichosporium 0B3b will greatly facilitate application of this or like species to the bioremediation of hazardous waste. Specifically, the role of an extracellular copper-binding biochelator (CBL) in copper uptake by Methylosinus trichosporium 0B3b has been investigated. Experiments included the identification and physical characterization of the biochelator and elucidation of the environmental factors that affect its production. The biochelator is apparently an aromatic, low-molecular weight, hydrophobic molecule with high affinity and selectivity for copper. Results indicate that the mutation in PP358 is unrelated to possible defects in biochelator functionality and strongly suggest that the CBL is directly involved in the copper acquisition mechanism of this methanotroph. Finally, an existing colorimetric method currently used in the qualitative determination of sMMO has been modified and improved to provide additional quantitative information. Until now, the instability of one of the products of the reaction on which the current method is based has precluded the effective use of the assay as a quantitative tool. Stabilization of the compound of interest has been achieved, allowing the successful quantification of sMMO activity from M. trichosporium 0B3b and propane monooxygenase activity from the propane oxidizer M. vaccae JOB5.
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Teruya, Kanae. "Development of Affinity-Based Chemical Probes for Fluorescence Detection of Human Carbonic Anhydrases." Thesis, Griffith University, 2016. http://hdl.handle.net/10072/367357.
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The development of small molecule affinity-based chemical probes as research tools for studying the role of carbonic anhydrases (CAs) in their wider biological context has become an active field of research owing to an increasing awareness of the therapeutic relevance of this enzyme family, particularly in diseases such as glaucoma (CA II) and solid tumors (CA IX, CA XII). High CA isozyme selectivity, low nonspecific labeling, and efficient labeling yield are the characteristics of an ideal chemical probe, however achieving an effective balance of all three properties is challenging. A panel of chemical probes for two-step labeling of CA II or CA IX has been designed to study the impact of probe structural features on the efficiency and specificity of CA-selective labeling when in a challenging environment, including protein mixtures, cell lysates, or live cells. The panel comprised Generation 1 probes (P1 and novel probes P2–P5), Generation 2 linear PAL probes (P6 and novel probes P7–P15), and Generation 3 branched PAL probes (novel probes P16–P20).Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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Laffey, Brian David. "Monoclonal antibodies to bovine serum albumin : affinity purification and physicochemical characterization dc by Brian David Laffey." Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/32640.
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Swers, Jeffrey Seth. "Isolation and engineering of a high affinity antibody against P-selectin glycoprotein ligand-1 (PSGL-1)." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/32323.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Vita.
Includes bibliographical references (leaves 87-91).
We aim to develop novel protein antagonists of P-selectin adhesion as anti- inflammatory therapeutics. Blocking P-selectin adhesion is particularly attractive because this adhesion mediates leukocyte rolling which occurs early in the inflammatory cascade before extensive tissue damage caused by the amplification of inflammation by proinflammatory cytokines. Currently, no subnanomolar antagonists of selectin adhesion are available. The low affinity of current antagonists results in the need for frequent administration and large doses in order to obtain inhibition. High affinity antagonists are desirable because they can be administered in smaller amounts thus reducing the risk of harmful side effects and reducing production costs. Our approach for developing high affinity antagonists is to combine error prone PCR and in vivo homologous recombination to mimic in yeast the broad spectrum of mutagenic strategies exploited by B cells such as somatic hypermutation, receptor revision (... CDR replacement), receptor editing (chain shuffling), and amino acid insertions and deletions. Together with yeast surface display and flow cytometric screening (FACS), this approach has been used to effect at least a five order of magnitude affinity improvement in a single chain antibody (scFv) directed against the N-terminal 19 amino acids of P-selectin glycoprotein ligand- 1 (PSGL- 1). Three rounds of engineering were performed after an initial pool of binders was isolated from a non-immune scFv library. Chain shuffling was found to be important for generating an improved mutant in the first round of engineering.
(Cont.) For the final round of engineering, four different libraries were generated: one with random mutations, one with preferential replacement of the ... CDR1, one with preferential replacement of the ... CDR1 and the ... CDR2, and one with preferential replacement of the light chain. All of these methods produced two order of magnitude affinity improvements except the light chain exchange library. However, the CDR exchange libraries gave equivalent affinity improvement despite the fact that they were 77 fold smaller than the random mutagenic library. In addition, an insertion in CDR2 of the VH was isolated in the best binder from both of the CDR exchange libraries and this mutation could not have been found through random mutagenesis. These results suggest that chain shuffling is best used when the affinity of the antibody to be matured is weak (> 1 [mu] M). In addition, receptor revision is an equally robust method as random mutagensis for the generation of ultra-high affinity binders. The best antibody from the library with preferential replacement of ... CDR1 and ... CDR2 was converted to an IgG and characterized. It was found to better inhibit P-selectin binding to PSGL-1 than the commercially available antibody KPLI in a static adhesion assay and an in vitro rolling assay. Our integrated approach, made possible by in vivo homologous recombination in yeast, decreases the likelihood of convergence upon a single high affinity solution and increases the probability of obtaining an antibody with desired secretory properties and therapeutic potential. This facile method for combining all the mutational strategies used in nature should prove as a valuable tool in the antibody engineering field.
by Jeffrey Seth Swers.
Ph.D.
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Balliu, Aleksandra. "Exploring molecular interactions between polypeptide conjugates and protein targets : Manipulating affinity by chemical modifications." Doctoral thesis, Uppsala universitet, Organisk kemi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-327121.
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In this thesis molecular interactions between polypeptide conjugates and protein targets were investigated. Polypeptides were derivatized with small organic molecules, peptides and oligonucleotides. New strategies were developed with the aim to increase affinities for proteins of biological interest. A 42-residue polypeptide (4-C15L8) conjugated to a small organic molecule 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid (PP1), was shown to bind glycogen phosphorylase a (GPa) in the presence of zinc ions. Under the assumption that hydrophobic interactions dominated the binding energy, the hydrophobic residues of 4-C15L8-PP1 were systematically replaced in order to study their contribution to the affinity enhancement. The replacement of the Nle, Ile and Leu residues by Ala amino acids reduced affinities. The introduction of non-natural L-2-aminooctanoic acid (Aoc) residues into the peptide sequence enhanced the binding affinity for GPa. A decreased KD of 27nM was obtained when Nle5, Ile9 and Leu12 were replaced by Aoc residues, in comparison to the KD value of 280nM obtained for the unmodified 4-C15L8-PP1. It is evident that there are non-obvious hydrophobic binding sites on the surfaces of proteins that could be identified by introducing the more hydrophobic and conformationally flexible Aoc residues. The downsizing of the 42-mer peptide to an 11-mer and the incorporation of three Aoc residues gave rise to a KD of 550 nM, comparable to that of 4-C15L8-PP1 suggesting that bioactive peptides can be downsized by the introduction of Aoc. Aiming to improve in vivo stability, the affinity for human serum albumin (HSA) of hydrophobic, positively and negatively charged polypeptide-PP1 conjugates was evaluated. Increased hydrophobicity due to the introduction of Aoc residues did not significantly increase the affinity for HSA. No binding was observed in the case of the most negatively charged polypeptides whereas the slightly negatively and positively charged polypeptides conjugated to PP1 bound HSA with affinities that increased with the positive charge. It was found that polypeptide-PP1 conjugates target the zinc binding site of the HSA. Affinity enhancement was obtained due to the incorporation of PP1 and increased by charge to charge interactions between the positively charged amino acids of the polypeptide and the negatively charged residues of HSA, in close proximity to the HSA zinc binding site. The survival times of the peptide-PP1 conjugates in human serum were extended as a result of binding to HSA. Zn2+ ion chelating agents can be incorporated in potential peptide therapeutics with a short plasma half-life, without increasing their molecular weights.
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Kuzmich, Oleksandra. "Metal Labeling for Low Affinity Binding Biomolecules." Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/18862.
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Unter den Techniken der chemischen Proteomik hat Capture Compound – Massenspektrometrie (CCMS) den Vorteil, Interaktionen von Molekülen mit geringer Affinität zueinander effektiv untersuchen zu können. CCMS beruht auf kleinen molekularen Sonden (Capture Compounds, CCs), die aus drei funktionalen Bestandteilen bestehen: die Selektivitätsfunktion, ist ein kleines Molekül, das mit einem Zielprotein eine schwache Wechselwirkung eingeht. Die zweite Funktionalität erlaubt kovalente Anhaftung der molekularen Sonde an Proteine. Der dritte Anteil erlaubt Detektion mit sehr guten Sensitivität; allerdings ist die Quantifizierung weiterhin ein Schwachpunkt dieser Technik.
Ziel dieses Projektes ist, eine in CCMS verwendbare Quantifizierungsmethode zu entwickeln. Heutzutage gibt es zahlreiche MS-basierte Quantifizierungsstrategien; unsere beruht auf der Einführung von Lanthanoid-haltigen Labels – Metal Coded Affinity Tagging (MeCAT).
In dieser Arbeit wurde erstmalig die erfolgreiche Verwendung mit Metall- Markern chemoproteomischer Sonden (CCs) zur Detektion und absoluten Quantifizierung von Zielproteinen mit schwacher Wechselwirkung etabliert. Mit den Experimenten an isolierten Enzymen und an lebenden Zellen wurde nachgewiesen, dass Metall-Marker keinen negativen Einfluss auf andere funktionelle Teile chemoproteomischer Sonden haben. CCs, die mit Lanthanoid-Chelaten funktionalisiert sind, zeigen ähnliche Affinität zu ihren Zielproteinen wie die Referenz-Sonden. Zudem erlauben Metall-Marker, die für diese Art molekularer Sonden verwendet werden, die Entwicklung einer element-basierten Technik zur Bilderzeugung. Der herausragende Vorteil der Metall-funktionalisierten CCs kombiniert mit ICP-MS ist, dass diese eine absolute Quantifizierung der Ausbeute der Quervernetzungen ermöglichen.Capture compound mass spectrometry (CCMS) is a chemical proteomics technique that has the advantage of addressing low abundant target proteins in lysates as well as in living cells. The CCMS is based on small molecule probes (capture compounds) that consist of three functionalities: a small molecule (quite often it is a drug), which interacts with the target protein; the moiety that allows covalent attachment of the molecular probe to the protein; the one that allows detection. The detection moiety utilized for CCMS can offer high sensitivity; however, the challenge of absolute quantification is still a bottleneck of this technique. Metal Coded Affinity Tagging (MeCAT) is a quantitative approach based on the chemical labeling with lanthanide; it allows obtaining both the structural and quantitative information. In this work for the first time the successful utilization of chemoproteomic probes functionalized with a metal tag for the detection and absolute quantification of target proteins was established. With the experiments both on isolated enzymes and living cells it was determined that MeCAT does not negatively influence other functional parts of the probes; therefore, capture compounds functionalized with lanthanide chelates demonstrate similar affinity to the target as the reference probes. Moreover, metal tags utilized for this type of molecular probes can offer a promising elemental imaging technique. However, to achieve the sufficient resolution multiple metal tags per molecular probe are needed. The striking advantage of the approach of utilization metal functionalized capture compound combined with ICP-MS detection is that it allows absolute quantification of crosslink yield, what cannot be performed with other detection methods applied for this technology.
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Taylor, Georgette Nicola Lewis. "Variations on a theme : patterns of congruence and divergence among 18th century chemical affinity theories." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/10723/.
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The doctrine of affinity deserves to be recognised by historians of chemistry as the foundational basis of the discipline of chemistry as it was practiced in Britain during the 18th century. It attained this status through its crucial structural role in the pedagogy of the discipline. The importance of pedagogy and training in the practice of science is currently being reassessed by a number of historians, and my research contributes to this historiographical endeavour. My analysis of the variety of theories sheltered under the umbrella term ‘affinity theory’ has emphasised the role of pedagogy in influencing both the structure and the content of knowledge. I have shown that there were wide ranging discrepancies between many of the components of individual affinity theories. Nevertheless, the scope of divergence was limited. This underlying organisation resulted from the unifying hub of affinity theory, the logical common ground. This was the essence of the doctrine of affinity, encompassing the law of affinity and the conceptualisation of the table that brought together the relations described in the law. The doctrine of affinity thus provided a disciplinary common ground between chemists, providing a mediating level of understanding and communication for all those who subscribed to the doctrine of affinity, in spite of their detailed differences.
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Al, Bittar Sheiraz. "3-Deoxyanthocyanins : Chemical synthesis, structural transformations, affinity for metal ions and serum albumin, antioxidant activity." Thesis, Avignon, 2016. http://www.theses.fr/2016AVIG0264.
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Ce travail porte sur la synthèse chimique d’analogues simples d’anthocyanes, une classe majeure de pigments naturels solubles dans l’eau. Onze ions flavylium substitués par des groupements hydroxyl,méthoxyl et beta-D-glucopyranosyloxyl en positions 4’, 5 et 7 ont été préparés en utilisant des procédures simples. De plus, les deux principales 3-désoxyanthocyanidines du sorgho rouge, l’apigéninidine (APN) et la lutéolinidine (LTN), ont été synthétisées en une seule étape. Les propriétés physico-chimiques ainsi que l’activité antioxydante ont été étudiées pour le chlorure de 3’,4’,7-trihydroxyflavylium (P1), son 7-O-beta-D-glucoside (P2) et le chlorure de 3’,4’,5,7-tétrahydroxyflavylium (LTN). Grâce à leur noyau catéchol, ces pigments complexent rapidement FeIII, AlIII and CuI et ne se lient que faiblement à FeII tout en stimulant son autoxydation en FeIII. Suite à la complexation de CuII, les pigments subissent une oxydation. Les aglycones P1 et LTN sont des ligands modérés de l’albumine de sérum humain (HSA) et leurs chalcones ont montré une plus grande affinité pour la HSA que leurs formes colorées. Leur capacité antioxydante a été démontrée par le test de réduction du radical stable DPPH et par l’inhibition de la peroxydation lipidique induite par le fer héminique, un modèle de stress oxydant postprandial dans l’estomac. Les aglycones P1 et LTN (particulièrement, dans leur forme incolore chalcone) sont plus efficaces que le glucoside P2This work deals with the chemical synthesis of simple analogs of anthocyanins, the main class of watersolublenatural pigments. Eleven flavylium ions with hydroxyl, methoxyl and beta-D-glucopyranosyloxylsubstituents at positions 4’, 5 and 7 have been prepared by straightforward chemical procedures.Moreover, the two main 3-deoxyanthocyanidins of red sorghum, apigeninidin (APN) and luteolinidin(LTN), have been synthesized in a one-step protocol. The physicochemical properties and antioxidantactivity are investigated for 3’,4’,7-trihydroxyflavylium chloride (P1), its 7-O-beta-D-glucoside (P2) and3’,4’,5,7-tetrahydroxyflavylium chloride (LTN). Owing to their catechol B-ring, they rapidly bind FeIII,AlIII and CuI, more weakly interact with FeII while promoting its autoxidation to FeIII. Following CuIIbinding, the pigments undergo oxidation. Aglycones P1 and LTN are moderate ligands of human serumalbumin (HSA) with chalcones having a higher affinity for HSA than the corresponding colored forms.The antioxidant activity of P1, P2 and LTN is investigated via two tests: reduction of the stable DPPHradical and inhibition of heme-induced lipid peroxidation (a model of postprandial oxidative stress inthe stomach). Aglycones P1 and LTN (especially in their colorless chalcone form) are more potent thanglucoside P2
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Ogata, Yuko. "Automated affinity measurement of biospecific interactions using a lab-on-valve apparatus coupled to electrospray ionization mass spectrometry /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/11607.
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Keymeulen, Flore. "Development and physico-chemical characterization of supramolecular systems for anion recognition in aqueous media." Doctoral thesis, Universite Libre de Bruxelles, 2016. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/223788.
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Anion recognition is a topical area of research warranted by the potential applications of anion receptors in environmental and biological monitoring. Anions are indeed widespread in nature, are involved in many biochemical processes and are also major aqueous pollutants. Molecular receptors able to recognize anions with high affinity and selectivity in organic solvents are well documented in the literature but only few systems are efficient in aqueous media. Water is undeniably a particularly challenging solvent to work with due to the competition of water in the recognition process. Moreover, most of the receptors that are known to bind anions with high affinity and selectivity in organic solvents are not soluble in water. One strategy used to make hydrophobic molecular receptors “water-compatible” is micellar incorporation. This strategy is straightforward as no synthetic modifications of the receptor are required and has furthermore been seen to enhance the apparent properties, in particular binding properties, of the receptors. Following previous work undertaken in the laboratory, this thesis was devoted to the study of the micellar incorporation of different anion receptors. The first part of this thesis focused on the potential of Nuclear Magnetic Resonance (NMR) and more precisely Paramagnetic Relaxation Enhancement (PRE) experiments to provide robust information on the localisation of receptors within micelles. We studied the effect of various parameters and were able to rationalize the effect of the nature and concentration of the counterion and of the surfactant concentration on the PRE values obtained with cationic cetyltrimetylammonium (CTAX) micelles. By applying a normalization procedure we were then able to compare different receptor/micelle systems. This work has been reported in the Journal of Physical Chemistry (“Paramagnetic Relaxation Enhancement Experiments: a Valuable Tool for the Characterization of Micellar Nanodevices”, F. Keymeulen, P. De Bernardin, A. Dalla Cort, and K. Bartik, J. Phys. Chem. B, 2013, 117, 11654–11659).The second part of our work consisted in the study of the role played by the surfactant on the efficiency of the supramolecular system formed. Using UV-vis and/or NMR titrations, we studied the impact of the nature of the surfactant (cationic, zwitterionic or neutral) as well as its concentration on the apparent binding affinity for fluoride of two uranyl-salophen receptors. We showed that the supramolecular systems formed with cationic micelles are the most efficient, due to the favourable electrostatic interaction between the positively charged micelle and the fluoride despite the competition of the surfactant counter-ion. The concentration of the cationic surfactant does however have an impact as the apparent affinity decreases with surfactant concentration as a consequence of this non-specific interaction of the guest with the micelles. PRE and DLS (Dynamic Light Scattering) experiments allowed us to better understand the differences between the different types of micelles. This work has been reported in Organic & Biomolecular Chemistry (“Fluoride binding in water with the use of micellar nanodevices based on salophen complexes”, F. Keymeulen, P. De Bernardin, I. Giannicchi, L. Galantini, K. Bartik, and A. Dalla Cort, Org. Biomol. Chem. 2015, 13, 2437–2443).The final part of our study was devoted to the investigation of the applicability of micellar incorporation to other receptors. Two other uranyl-based receptors were studied in cationic CTAX and neutral Triton X-100 micelles. One suffered from chemical stability issues and the other receptor did not perform any better than the ones previously studied. We also studied trimesitylborane which can bind fluoride via Lewis acid-base interactions. This system, which is highly efficient in organic solvents, was shown to be ineffective once incorporated into micelles, probably because the change in the hybridization of the boron atom upon fluoride binding is not favourable in the confined micellar environment. Indolocarbazole-based anion receptors, which recognize acetate and benzoate via hydrogen bonding, were successfully incorporated into DPC micelles, albeit at low concentrations, and were observed to be efficient as the apparent binding affinity measured in water is of the same order of magnitude or higher as the one observed in organic solvents.Doctorat en Sciences de l'ingénieur et technologie
info:eu-repo/semantics/nonPublished
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Yuan, Hongyu. "Optimization of an Innovative Npu-N Resin Production." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555591103161637.
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PUNZALAN, LOUVY LYNN CALVELO. "Chemoproteomic Profiling of a Pharmacophore-Focused Chemical Library." Kyoto University, 2020. http://hdl.handle.net/2433/259001.
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Bagchi, Pritha. "Expanding the metallomics toolbox: Development of chemical and biological methods in understanding copper biochemistry." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52160.
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Copper is an essential trace element and required for various biological processes, but free copper is toxic. Therefore, copper is tightly regulated in living cells and disruptions in this homeostatic machinery are implicated in numerous diseases. The current understanding of copper homeostasis is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level. Intracellular copper is primarily present in the monovalent oxidation state. Therefore, copper(I) selective fluorescent probes can be utilized for imaging exchangeable copper ions in live cells, but these probes are often lipophilic and hence poorly water soluble. To address this problem, water-soluble fluorescent probes with greatly improved contrast ratio and fluorescence quantum yield are characterized in this work. This work also describes a novel application of water-soluble fluorescent probes, in-gel detection of copper proteins with solvent accessible Cu(I) sites under non-denaturing conditions. Knowledge of copper(I) stability constants of proteins is important to elucidate the mechanisms of cellular copper homeostasis. Due to the high affinity of most Cu(I)-binding proteins, the stability constants cannot be determined directly by titration of the apo-protein with Cu(I). Therefore, accurate determination of Cu(I) stability constants of proteins critically depends on the Cu(I) affinity standards. However, the previously reported binding affinity values of the frequently used Cu(I) affinity standards are largely inconsistent impeding reliable data acquisition for the Cu(I) stability constants of proteins. To solve this problem, a set of water-soluble ligands are developed in this work that form colorless, air-stable copper(I)-complexes with 1:1 stoichiometry. These ligands can be applied as copper(I) buffering agents and affinity standards in order to study copper biochemistry. Copper(I) binding proteins are an integral part of the copper homeostatic machinery and they work in conjunction to regulate copper uptake, distribution, and excretion. However, available evidence indicates the existence of putative copper-binding proteins that are yet to be characterized. Therefore, several proteomics-based methods are developed in this work by employing the strategy to label Cu(I)-binding cysteines in a copper-dependent manner which lays the foundation for the identification of new copper proteins from cellular extracts.
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Chivers, Claire Elizabeth. "Investigating high-affinity non-covalent protein-ligand interaction via variants of streptavidin." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:631c65ed-08d9-484e-a8df-309a4c95df45.
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The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (Kd ~ 10-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.
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Garcia-Barron, Javier Enrique. "Synthesis and study of chelating polymers and their application to protein and metal separation from aqueous solutions using novel metal affinity interaction techniques." Diss., The University of Arizona, 1999. http://hdl.handle.net/10150/283931.
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The main objective of this research work was the development, synthesis, and study of polymeric chelating derivatives. These derivatives were characterized in terms of their specific metal affinity interaction with biomolecules and metal ions. These engineered materials were used to test their feasibility as tools for separation of proteins and heavy metal ions from aqueous solutions using different affinity separation techniques. Linear and branched polymers were synthesized to create a variety of materials. Among the linear polymers synthesized was the chelated monomethoxy poly(ethylene) glycol (PEG-IDA). This derivative was used in metal affinity partitioning and metal affinity electrophoresis for fast protein-metal interaction analysis. Also a linear heterobifunctional poly(ethylene) glycol (Biotin - PEG - IDA) was synthesized and used as a tool to develop a modified enzyme-linked immuno sorbent assay (ELISA). A multi-armed high molecular weight chelating poly(ethylene) glycol (Star PEG-IDA) was prepared to enhance the separation of protein mixture in gel permeation chromatography. Iminodiacetic poly(ethyleneimine) (PEI-IDA) was prepared and used as a soluble chelating polymer in complexation-ultrafiltration studies for heavy metal ion removal from aqueous solutions. Similar PEIs were also used as casting polymers for the synthesis of affinity adsorbents useful in chromatographic applications. Either as a soluble macromolecule or as a casting polymer for the preparation of adsorbents, PEI chelated derivatives were used for ultratrace metal ion preconcentration and metal ion separations. All polymeric materials prepared were characterized using analytical techniques which include elementary analysis, atomic absorption, UV and IR spectroscopy, high performance liquid chromatography and several colorimetric assays for the determination of end groups and product purity. Metal affinity separation techniques studied with the aforementioned derivatives included: affinity partitioning, affinity electrophoresis and affinity size exclusion for protein purification; affinity complexation-ultrafiltration and metal ion affinity chromatography for removal of heavy metal. Efficient separation of protein mixtures were achieved based on selective affinity by some of the chelated polymers here described and extremely high metal adsorption capacities were found for some of the PEI-based adsorbents prepared. Even though, some of these techniques are still in developmental stages, the results are very promising and encouraging for biotechnical and environmental applications.
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Authimoolam, Sundar Prasanth. "STABILITY OF AFFINITY BASED LAYER-BY-LAYER POLYMERIC SELF-ASSEMBLIES FOR ORAL WOUND APPLICATIONS." UKnowledge, 2011. http://uknowledge.uky.edu/cme_etds/3.
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Oral mucositis is a painful and debilitating chronic inflammatory condition that can result from chemo and/or radiotherapy. While current treatment strategies which provide temporary relief exist, there is still an unmet clinical need for a robust long active barrier strategy which can simultaneously provide protection and release drug to enhance the wound healing response. It is proposed that an affinity based layer-by-layer self-assembled barrier administered as a series of mouth rinses can allow for wound specific drug delivery, providing an effective regenerative therapy.
In this work, biotinylated poly(acrylic acid) is used to develop LBL assemblies based upon biotin-streptavidin affinity interactions. To explore the ability of developed LBL assemblies to resist the harsh intraoral environment, in vitro chemical and ex vivo mechanical tests are performed. The stability results demonstrate significant LBL barrier stability with wear resistance. From principal component regression analysis, factors such as polymer MW and number of layers in assemblies contributed significantly to chemical barrier stability. Also it is observed that the extent of biotin conjugation plays a significant role in LBL development and in mechanical stability. Thus, the proposed affinity based multilayered assemblies with their excellent barrier properties offer a modular treatment approach in oral mucosal injuries.
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Knave, Axel. "Production and characterization of alternative scaffold proteins for medical applications." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278838.
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Antibodies, as forerunners in the field of biological drugs, are originally an organism’s answer to the invasion of different pathogens. Today, antibodies are a common treatment for many chronic diseases such as the immune-mediated inflammatory diseases rheumatoid arthritis or psoriasis. It is suspected that the cytokines interleukin 17a (IL17a) and interleukin 17c (IL17c) are involved in those diseases and are commonly treated with antibodies that inhibit the cytokines. Even though antibodies have been a huge success as biological drugs they also have downsides when it comes to their production, size and stability. In quest of finding alternatives to antibodies in diagnostics and therapy, a novel class of biologics has been developed. So-called alternative scaffold proteins are small polypeptide chains that can be engineered to show affinity towards different biomarkers. ABD-Derived Affinity ProTeins or ADAPTs are one example of these alternative scaffolds that can be modified to bind a biomarker as target and keep their affinity to Human Serum Albumin (HSA) at the same time, making them bispecific. In this project, twenty-four previously selected ADAPT binder candidates that have shown good prospects towards IL17a and IL17c in previous experiments were cloned, produced, purified and characterized to determine if they show potential as tools in diagnostics or therapy of autoimmune diseases. The proteins were produced in E. coli, purified by affinity chromatography and characterized using Surface Plasmon Resonance (SPR), Circular Dichroism (CD) and Size Exclusion Chromatography (SEC). All candidates were successfully cloned into E. coli and out of these, 10 could be produced and 5 showed affinity towards their target using SPR. Examination by SEC and CD showed that the protein variants did not seem to be structurally stable and hints of impurities in the samples could be detected. This and a low yield could be further confirmed via SDS-PAGE. In conclusion, binders were produced that could theoretically be promising candidates as tools in diagnostics or therapy of chronic diseases were IL17a and/or IL17c are important. Nevertheless, in order to support these claims further investigations and developments are necessary.Antikroppar, som föregångare inom området biologiska läkemedel, är ursprungligen en organisms svar på invasionen av olika patogen. Idag är antikroppar en vanlig behandling för många kroniska sjukdomar, såsom de immunmedierade inflammatoriska sjukdomarna reumatoid artrit eller psoriasis. Cytokinerna interleukin 17a (IL17a) och interleukin 17c (IL17c) tros vara involverade i dessa sjukdomar och behandlas vanligtvis med antikroppar som hämmar cytokinerna. Trots att antikroppar har varit en stor framgång som biologiska läkemedel har de också nackdelar när det gäller deras produktion, storlek och stabilitet. För att hitta alternativ till antikroppar inom diagnostik och terapi har en ny klass av biologiska läkemedel utvecklats. Så kallade alternative scaffold proteins är små polypeptidkedjor som kan manipuleras för att visa affinitet gentemot olika biomarkörer. ABD-Derived Affinity ProTeins eller ADAPTs är ett exempel på dessa alternative scaffolds som kan modifieras för att binda en biomarkör som mål utan att påverka affiniteten till Humant Serum Albumin (HSA), vilket gör dem bispecifika. I detta projekt klonades, producerades, renades och karakteriserades tjugofyra tidigare utvalda ADAPT-bindarkandidater som har visat goda förutsättningar gentemot IL17a och IL17c i tidigare experiment. Proteinerna producerades i E. coli, renades genom affinitetskromatografi och karakteriserades med användning av Surface Plasmon Resonance (SPR), Circular Dichroism (CD) och Size Exclusion Chromatography (SEC). Alla kandidater klonades framgångsrikt i E. coli och av dessa kunde 10 produceras. Fem bindare visade affinitet till deras mål med SPR. Undersökning med SEC och CD visade dock att proteinvarianterna inte var strukturellt stabila och antydan till föroreningar kunde detekteras i proverna. Detta och ett lågt utbyte kunde ytterligare bekräftas via SDS-PAGE. Sammanfattningsvis kunde bindare producerades och dessa kan teoretiskt vara lovande kandidater till diagnostik eller terapi av kroniska sjukdomar där IL17a och/eller IL17c är viktiga. För att stödja dessa påståenden krävs dock ytterligare experiment och utveckling av bindarna.
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Bateson, Hannah. "Detection and enrichment of cytochrome P450s using bespoke affinity chromatography and proteomic techniques : development of chemical immobilisation and novel affinity chromatography methods, with subsequent proteomic analysis, for the characterisation of cytochrome P450s important in cancer research." Thesis, University of Bradford, 2012. http://hdl.handle.net/10454/5712.
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Introduction: Cellular membrane proteins, such as the cytochrome P450 enzyme superfamily (P450), have important roles in the physiology of the cell. P450s are important in metabolising endogenous molecules, as well as metabolising xenobiotic substances for detoxification and excretion. P450s are also implicated in cancer as they can act to 'negatively' de-activate or 'positively' activate cancer therapeutics. Identifying specific P450s that are highly up-regulated at the tumour site could be used to predict drug response and formulate targeted cancer therapy to help diminish systemic side-effects. Methods: Previous enrichment strategies have been unable to isolate the full complement of the P450 superfamily. To develop enrichment procedures for the P450s, a proteomic strategy was developed so that compounds could be screened for their effectiveness as general P450 probes. A standardised work-flow was created, encompassing affinity chromatography, protein concentration/desalting, followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography-mass spectrometry (HPLC-MS). A ketoconazole analogue and a 2-EN analogue, with known P450 inhibition, were immobilised on a solid support for comparison to immobilised histamine. Co-factor removal, competitive elution and DTT cleavage of disulfide bonds of probes were utilised to elute bound proteins. Results/Discussion: Inhibitor-beads bound a large range of proteins, including P450's, of which some were eluted by co-factor removal, some by competitive elution. Specificity of binding was improved by optimising buffer conditions and solid supports, however non-specific binding was not totally eradicated. All human P450s from spiked samples and 18 P450s from more complex mouse liver samples were recovered using one or more ligands.
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Poma, Alessandro. "Automatic solid-phase synthesis of molecularly imprinted nanoparticles (MIP NPs)." Thesis, Cranfield University, 2012. http://dspace.lib.cranfield.ac.uk/handle/1826/7911.
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Molecularly Imprinted Polymers (MIPs) are potential generic alternatives to antibodies in diagnostics and separations. To compete with biomolecules in these technological niches, MIPs need to share the characteristics of antibodies (solubility, size, specificity and affinity) whilst maintaining the advantages of MIPs (low cost, short development time and high stability). For this reason the interest in preparing MIPs as nanoparticles (MIP NPs) has increased exponentially in the last decade. Cont/d.
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Harper, Robert T. "Determination of the proton affinities of gas phase peptides by mass spectrometry and computational chemistry." Scholarly Commons, 2007. https://scholarlycommons.pacific.edu/uop_etds/673.
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Helices in proteins have substantial permanent dipole moments arising from the nearly perfect alignment of the individual dipole moments of each peptide bond. Interaction with this helix "macrodipole" is thought to perturb the pKa values of basic or acidic residues at the helix termini. The goal of this project is to investigate the effect of the helix confonnation on the proton affinities ofbasic amino acids placed at theN- or Ctenninus of helical model peptides in the gas phase. Several series of model peptides having a basic residue, lysine (K) or 2,3- diaminopropionic acid (Dap ), located at either terminus were synthesized by solid phase peptide synthesis using conventional techniques or the amino acid fluoride approach. Proton affinities were determined for several basic amino acids and peptides using mass spectrometry by applying the extended Cooks' kinetic method. Favorable conformations and theoretical proton affinities were probed using computational chemistry. The proton affinities determined for Na-acetyl-(L)-lysine, Ac-AK, Ac-KA, and Ac-KAA are 236.8 ± 1.9 kcal mol-1 , 249.4 ± 2.0 kcal mol-1 , 241.5 ± 1.9 kcal mol-1 , and 244.4 ± 2.0 kcal mol-1 respectively. The large negative entropy changes for each of the peptides upon protonation ( -11.2 to - 21.7 cal mol-1 K- 1 ) are consistent with globular confmmations adopted by the protonated peptides due to extensive intramolecular hydrogen bonding. The measured proton affinities of the peptides increased with the size of the peptide as expected. However, the measured proton affinity of the peptide with C-terminal lysine, Ac-AK, is substantially higher than that of the con·esponding peptide with N-terrninal lysine, Ac-KA, contrary to expectations. Proton affinities determined for these compounds using computational chemistry are in reasonable agreement with experimental results. Additionally, proton affinities calculated for helical polyalanine and Aib (aaminoisobutytic acid) modified polyalanine peptides with C-terminal basic residues (Ac AnK and Ac-(AibA)n-Dap) are much larger than proton affinities calculated for the corresponding peptides with N-terminal basic residues. These results indicate that the helix dipole has a substantial effect on the basicity of residues at the helix termini.
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Datta, Saurav. "FUNCTIONALIZED POLYMERIC MEMBRANES FOR BIOSEPARATION AND BIOCATALYSIS." UKnowledge, 2007. http://uknowledge.uky.edu/cme_etds/26.
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Functionalized polymeric membrane based techniques are becoming increasingly popular in biotechnology, food and pharmaceutical industries due to their versatility and hydrodynamic benefits over traditional materials and methods. This research work has been directed towards the development of functionalized polymeric membranes, extensive experimental and theoretical analyses of some of the fundamental aspects of accessibility, membrane fouling and enzyme catalysis, and applications in affinity based bioseparation and biocatalysis. In this research work, the impact of different types of functionalization techniques, such as functionalization of different membrane materials, covalent and electrostatic immobilization, on interaction of various biomolecules and active sites in membrane has been studied in detail.
Avidin was used as model biomolecule, and covalently immobilized within acyl anhydride derivatized nylon based membrane. Quantification of the accessibility of covalently immobilized avidin sites was carried out by model biotinylated probe molecules, such as biotin 4-amidobenzoic acid and biotinylated-BSA. This study has been further extended to separate and purify a target protein, HIV-Tat, from a complex mixture of proteins (97-99 % unwanted protein) using avidin-biotin affinity interaction. It has been demonstrated that covalent immobilization of avidin in membranes reduces the accessibility of active sites for probe molecules. Accessibility decreases further for the biotinylated target protein present in the mixture of other unwanted proteins. Affinity based membrane separation of proteins is also associated with decrease in permeate flux due to fouling in membrane structure. Fouling in the membrane has been discussed by analyzing the characteristics of adsorbed protein layer in membrane.
In order to improve the accessibility and fouling behavior of affinity separation of Tat protein, a pre-filtration step has been introduced prior to affinity separation. Significant enhancement in accessibility and reduction in fouling has been observed for pre-filtered cases as it removes unwanted proteins prior to affinity interaction. Contribution of the pre-filtration step in reduction of fouling has been elucidated by simple model equations. Improvement in accessibility and fouling behavior reflects in higher separation efficiency (protein recovery) and lower processing time for the pre-filtered cases. Quality of membrane purified Tat protein was examined by different analytical techniques, such as SDS-PAGE, Western Blot and biotin analysis, and then compared with that purified by traditional packed-bead column chromatography. It has been demonstrated that membrane based technique was able to isolate superior quality of pure monomeric Tat protein compare to column chromatographic technique.
The other study carried out as a part of this dissertation, has involved development of high capacity, highly active, stable and reusable functionalized membrane domains for electrostatic immobilization of enzymes. Glucose oxidase (GOX) was used as a model enzyme to study the oxidation of glucose to gluconic acid and hydrogen peroxide under convective flow condition. Two different approaches of functionalization of membranes have been presented. In the first approach, alternative electrostatic attachment of cationic and anionic polyelectrolytes was carried out using Layer-By-Layer (LBL) assembly technique within a functionalized nylon based membrane. In the second one, a hydrophobic PVDF membrane was functionalized by in-situ polymerization of acrylic acid. Kinetics of glucose oxidation, effect of pH and flow rate on the activity of GOX was discussed. A comparative study was presented between the activity of free GOX, electrostatically immobilized GOX and covalently immobilized GOX, along with the advantage of convective mode of operation over soaking mode. A novel study has also been conducted on detachment and reattachment of GOX in the same membrane matrix.
Further study has been directed towards implementation of the above mentioned immobilized enzymatic system for oxidative dechlorination of chloro-organics. A first time attempt was made to use a 2-stack functionalized membranes system for simultaneous enzymatic production of hydrogen peroxide in first membrane, and oxidative dechlorination of 2, 4, 6-trichlorophenol (TCP) in the Fe+2 immobilized (by ion exchange) second membrane by Fenton reaction. The technique was efficient in destruction of TCP as evident from the overall dechlorination of 70-80 %. This technique provides additional benefit of reusing the same membrane matrices by reattaching fresh GOX and Fe+2.
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Ku, Xin [Verfasser], Bernhard Akademischer Betreuer] Küster, and Dieter [Akademischer Betreuer] [Langosch. "Development and application of small molecule probes for kinase affinity purification and quantitative chemical proteomics / Xin Ku. Gutachter: Bernhard Küster ; Dieter Langosch. Betreuer: Bernhard Küster." München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/105346763X/34.
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Zhou, Yipin. "Synthesis and Biophysical Characterization of Polymerized Hemoglobin Dispersions of Varying Size and Oxygen Affinity as Potential Oxygen Carriers for use in Transfusion Medicine." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1321406529.
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Lo, Bryan. "Site-directed cysteine mutagenesis and chemical modification of the high affinity Na§+/glucose transporter (SGLT1), elucidation of structure/function relationships underlying Na§+ permeation through the transporter." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ35230.pdf.
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Cigler, Marko [Verfasser], Kathrin [Akademischer Betreuer] Lang, Kathrin [Gutachter] Lang, and Stephan [Gutachter] Hacker. "Genetically encoding unnatural amino acids: Novel tools for protein labelling and chemical stabilisation of low-affinity protein complexes / Marko Cigler ; Gutachter: Kathrin Lang, Stephan Hacker ; Betreuer: Kathrin Lang." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1220322318/34.
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Chalumeau, Céline. "Développement d’outils chimiques pour l’élucidation de la biosynthèse des flavonoïdes du raisin : anthocyanes versus proanthocyanidines." Thesis, Bordeaux 1, 2010. http://www.theses.fr/2010BOR14188/document.
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Ces dernières années, des progrès remarquables ont été accomplis afin d’élucider la biosynthèse des flavonoïdes. Cependant les dernières étapes menant à la formation des proanthocyanidines ou tannins condensés issus de la vigne, restent à ce jour inconnues. Dans le but de déterminer si une ou plusieurs enzyme(s) spécifique(s) sont impliquées dans cette voie de biosynthèse, nous avons développé une approche de protéomique chimique, impliquant des matrices d’affinité constituées de substrats de type flavanols greffés sur un support solide. La validation de ces outils à l’aide de LDOX, une enzyme issue de Vitis vinifera a pu être menée à bien dans le cadre de ces travaux de thèseRemarkable progress toward the complete elucidation of the biosynthesis of flavonoids has been accomplished during the last decade, but the final step leading to proanthocyanidins still remain to be elucidated, in particular, the exact nature of starter and extension units as well as the enzymatic or non enzymatic condensation process. In order to answer whether some specific enzymes are involved in the biosynthesis of grapevine proanthocyanidins, we have developped a chemical proteomics approach, with an affinity chromatography-based tool in which a flavanol type substrate is loaded on an appropriate solid support. The validation of these tools with the LDOX enzyme from Vitis vinifera was developped and performed in this Ph.D work
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Schmitt, Philippe. "Potentiels de l'électrophorèse capillaire dans l'analyse des pesticides et des substances humiques : application à l'étude des interactions pesticides - substances humiques." Vandoeuvre-les-Nancy, INPL, 1996. http://www.theses.fr/1996INPL117N.
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Différentes méthodes électrophorétiques ont été développées pour l'analyse de pesticides et de substances humiques. En électrophorèse capillaire de zone (CZE), des herbicides cationiques (s-triazines) et/ou anioniques (acides phénoxyacétiques) peuvent être séparés aisément avec une limite de détection de l'ordre de 10 g/l. La CZE est également efficace dans l'analyse du comportement électrophorétique de mélanges complexes de polyélectrolytes anioniques tels que les substances humiques. Un exemple d'application de la CZE est donné dans l'analyse des métabolites hydroxyles de l'atrazine lors de processus photochimiques en présence de substances humiques dissoutes. L’addition dans le tampon de séparation de différentes cyclodextrines substituées permet la séparation d'énantiomères de pesticides racémiques. L’utilisation de la CZE dans l'analyse d'échantillons réels est démontrée dans le suivi de la dégradation sélective d'un mélange racémique de dichlorprop, après son application aux champs. Les énantiomères de pesticides neutres (organophosphorés, DDT, DDD, DDE, acétamides, ester d'acides phénoxyacétiques) sont séparés en utilisant l'électrophorèse capillaire micellaire (MECC) avec addition de cyclodextrines. L’addition dans le tampon de séparation de substances organiques (ou inorganiques) à réactions spécifiques avec les échantillons permet d'orienter et d'optimiser les séparations ; le capillaire devient un réacteur. Cette propriété est utilisée dans la technique d'électrophorèse capillaire d'affinité (ACE) qui a été adaptée pour la mesure simultanée des états de liaison entre quatre s-triazines et des acides phénoliques (addition des ligands dans le tampon de séparation). L’adsorption des mêmes herbicides à des substances humiques est modélisée d'une manière similaire qu'en électrophorèse capillaire micellaire (MECC), démontrant de surcroit les propriétés micellaires des substances humiques ; une concentration micellaire critique peut être définie pour les substances humiques (micelles chargées). De cette manière les coefficients de partage de s-triazines entre la phase aqueuse et différentes phases humiques dissoutes peuvent être déterminés rapidement et simultanément
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Stimple, Samuel Douglas. "Recent Advances in Developing Molecular Biotechnology Tools for Metabolic Engineering and Recombinant Protein Purification." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1514494485801145.
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Bull, James. "Application of Quantum Mechanics to Fundamental Interactions in Chemical Physics: Studies of Atom-Molecule and Ion-Molecule Interactions Under Single-Collision Conditions: Crossed Molecular Beams; Single-Crystal Mössbauer Spectroscopy: Microscopic Tensor Properties of ⁵⁷Fe Sites in Inorganic Ferrous High-Spin Compounds." Thesis, University of Canterbury. Department of Chemistry, 2010. http://hdl.handle.net/10092/4292.
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As part of this project and in preparation for future experimental studies of gas-phase ion-molecule reactions, extensive modification and characterization of the crossed molecular beam machine in the Department of Chemistry, University of Canterbury has been carried out. This instrument has been configured and some
preliminary testing completed to enable the future study of gas-phase ion-molecule collisions of H⁺₃ and Y⁻ (Y
= F, Cl, Br) with dipole-oriented CZ₃X (Z = H, F and X = F, Cl, Br). Theoretical calculations (ab initio
and density functional theory) are reported on previously experimentally characterized Na + CH₃NO₂, Na + CH₃NC, and K + CH₃NC systems, and several other systems of relevance. All gas-phase experimental and theoretical studies have the common theme of studying collision orientation dependence of reaction under singlecollision
conditions. Experimental measurements, theoretical simulations and calculations are also reported on some selected ferrous (Fe²⁺) high-spin (S=2) crystals, in an attempt to resolve microscopic contributions of two fundamental macroscopic tensor properties: the electric-field gradient (efg); and the mean square displacement (msd) in the case when more than one symmetry related site of low local point-group symmetry contributes to the same quadrupole doublet. These determinations have been made using the nuclear spectroscopic technique of Mössbauer spectroscopy, and complemented with X-ray crystallographic measurements.
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Pinato, Odra. "Analysis of allergenic proteins by mass spectrometry." Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3427029.
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MS Analysis of Allergenic Proteins. Food allergy is a significant worldwide public health issue. Proteins from cow's milk, chicken eggs, soybean and peanuts are the most frequent allergens contained in the complex foods prepared by industrial processes.
Allergenic proteins can induce allergic reaction in their native structural state or upon chemical or conformational changes induced by the industrial treatments. Nowadays, the identification of allergenic proteins in foods is conducted by using immunochemical methods such as ELISA tests, but these techniques suffer from several limitations due to cross-reactivity and false negative results. Indeed, alterations in the allergen’s structure or chemical modifications can prevent the interaction with the antibody, thus causing misleading data.
Since allergens are toxic even in trace amounts, there is a need for reliable and sensitive analytical methods for allergenic proteins. The purpose of this PhD project was to develop procedures for the identification of these proteins in food samples by using mass spectrometry (MS), likely overcoming some limitations of the immunochemical assays.
Indeed, the MS approach for identifying proteins makes use of data pertaining to the amino acid sequence of the protein, while immunochemical methods are linked to the integrity of the three-dimensional structure of proteins.
In order to test immunochemical approaches, polyclonal antibodies raised against the main allergenic proteins of milk (α-lactalbumin and β-lactoglobulin) and eggs (ovomucoid, ovalbumin and lysozyme) were purchased. Preliminary studies were performed in order to check the quality of the antibodies, in terms of specificity of recognition and cross-reactivity. Moreover, the responses of the antibodies using as antigens the purified commercial proteins and the same proteins contained in complex food matrices after thermal treatment were checked.
Since allergenic proteins usually are contained in complex mixtures of huge amounts of other proteins, the methodology nowadays named “targeted proteomics” was considered very appropriate. By this approach, a protein contained in a complex mixture can be identified by a MS analysis of a peptide fragment that is specific for the protein of interest and contained in the very complex mixture of a tryptic digest of a protein sample.
The procedure involves specific labelling and isolation of the specific peptide, named “proteotypic”. To this aim, tryptophan (Trp) residues in proteins were modified by reaction with 2,4-dinitrophenyl-sulfenyl chloride (DNPS-Cl), that leads to a Trp-derivative with the DNPS label attached at 2-position of the indole nucleus. The selection of Trp(DNPS)- peptides from the complex mixture of a tryptic digest of a protein sample was achieved by exploiting the significant change in hydrophobicity and retention time of DNPS-modified peptides in a reverse-phase HPLC column. Moreover, DNPS-labelled Trp-peptides were isolated by hydrophobic interaction chromatography, as well as by immunoaffinity chromatography using a column prepared with anti-DNP antibodies. The “targeted proteomics” procedure was optimised using a mixture of model proteins and then applied to identify a protein allergen contained in a raw bakery product. Overall, it was demonstrated that the novel procedure of selective labelling and isolation of Trp-peptides allows a considerable simplification of the fingerprinting/MS approaches nowadays used for the identification of proteins in proteomics research.
Other Research Activities. During the PhD course I had the opportunity to collaborate with other members of the lab in a couple of additional projects, partly as a continuation of previous research conducted for the doctoral thesis. Documentation of this activity is herewith included as an Appendix at the end of this PhD Thesis.
The molecular properties of the complex formed by α-lactalbumin with oleic acid were investigated in detail. This complex appears to be very interesting, since it has been shown to display cellular toxicity specifically for cancer cells. It was shown that the protein in the complex is in an oligomeric state, at variance from previous statements that the protein was monomeric. Moreover, it was shown the oleic acid can interact also with other proteins, including apomyoglobin. The main conclusion of this work was that the protein moiety serves as a carrier of the otherwise poorly soluble fatty acid, thus leading to an enhancement of its water solubility and consequently of its intrinsic cytotoxic properties. A manuscript rescrubbing these results is in an advanced state of preparation.
Enterocin AS-48 is a 70-residue circular polypeptide produced by Enterococcus faecalis displaying a wide antibacterial activity. Limited proteolysis of AS-48 was used to prepare a linear form of this enterocin, as well as 38- and 55-residue fragments. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that the circularization phenomenon is not required for the antibacterial activity, but it is crucial for the stabilization of the native structural state. This research was published in FEBS Lett. (2008).Analisi di proteine allergeniche mediante spettrometria di massa. Le allergie alimentari rappresentano ormai una problematica clinica di livello mondiale. Tra i prodotti alimentari considerati pericolosi per il loro elevato contenuto in proteine allergeniche troviamo il latte bovino, le uova, la soia e le arachidi. Le proteine allergeniche possono scatenare reazioni allergiche sia mantenendo la loro struttura nativa, sia in seguito a modifiche chimiche e conformazionali indotte dai processi industriali. I metodi d’elezione applicati per l’identificazione di proteine allergeniche negli alimenti sono rappresentati dai saggi immunochimici come i test ELISA. Tali metodi presentano però numerose limitazioni causate da fenomeni di cross reattività e da falsi positivi. Inoltre, alterazioni nella struttura delle proteine allergeniche o eventuali modifiche chimiche possono modificare l’interazione con gli anticorpi specifici, invalidando i risultati. Dal momento che gli allergeni sono tossici anche in tracce, è necessario sviluppare dei metodi analitici efficaci e affidabili per la loro identificazione. Lo scopo di questo progetto di tesi è stato quello di sviluppare delle procedure per l’identificazione di proteine allergeniche mediante spettrometria di massa (MS) che possano superare i limiti metodici dei saggi immunologici. Oltretutto, l’identificazione delle proteine mediante MS si basa sull’analisi della sequenza amminoacidica di quest’ultime, mentre i saggi immunochimici sono strettamente dipendenti dall’integrità della struttura tridimensionale della proteina antigenica. Al fine di testare la validità dell’approccio immnuchimico, sono stati testati alcuni anticorpi policlonali diretti contro le principali proteine allergeniche di latte α-lattalbumina e β-lattoglobulina) e uova (ovomucoide, ovalbumina e lisozima). Sono stati condotti alcuni studi preliminari per validare la qualità di questi anticorpi, in termini di specificità di riconoscimento della proteina antigenica e della presenza di eventuali fenomeni di cross reattività. Inoltre, è stata valutata la risposta anticorpale usando come antigeni sia le proteine commerciali purificate, sia le stesse proteine contenute in prodotti alimentari prima e dopo trattamento termico. Dato che le proteine allergeniche sono contenute in miscele complesse costituite da altre proteine, è stata considerata estremamente appropriata l’applicazione di una tecnica detta “targeted chromatography”. Secondo questo strategia, è possibile identificare mediante MS una proteina contenuta in una miscela complessa attraverso l’analisi di alcuni frammenti peptidici derivati dalla digestione triptica, che sono specifici della proteina stessa. Questa procedura prevede la modifica chimica e il successivo isolamento di specifici peptidi detti “prototipici”. A tale scopo, i residui di triptofano contenuti nelle proteine sono stati chimicamente modificati mediante una reazione con il composto 2,4- dinitrofenilsulfenil cloruro (DNPS-Cl), che porta alla formazione di un derivato triptofanilico, con il DNPS legato in posizione 2 dell’anello indolico. La selezione dei peptidi modificati con il DNPS-Cl contenuti in una miscela triptica è stata effettuata sfruttando l’aumento di idrofobicità e del tempo di ritenzione di questi peptidi modificati in una colonna HPLC a fase inversa. Inoltre, gli stessi peptidi modificati con DNPS-Cl sono stati isolati mediante cromatografia per immunoaffinità utilizzando una resina derivatizzata con anticorpi monoclonali diretti contro il gruppo DNP. La strategia di ”targeted proteomics” è stata ottimizzata utilizzando una miscela modello di sette proteine e successivamente è stata applicata per l’identificazione di una proteina allergenica contenuta in un prodotto dolciario. È stato inoltre dimostrato che queste nuove procedure di modifica selettiva e di selezione dei peptidi triptofanilici permette di semplificare considerevolmente l’analisi di fingerprinting/MS che è solitamente utilizzata per l’identificazione di proteine nei protocolli di proteomica. Altre attività di ricerca. Durante il periodo di dottorato, ho avuto l’opportunità di collaborare con altri membri del laboratorio in due progetti addizionali come continuazione di un progetto di ricerca precedente. La documentazione relativa a queste attività è riportata in appendice alla tesi di dottorato. Sono state investigate le proprietà molecolari del complesso formato da α-lattalbumina con l’acido oleico. Il complesso appare interessante poiché ha mostrato avere tossicità cellulare diretta selettivamente contro cellule tumorali. È stato dimostrato che la proteina nel complesso ha una struttura oligomerica, diversamente da quanto riportato nelle prime osservazioni, che ipotizzavano fosse in uno stato monometrico. Inoltre, è stato osservato che l’acido oleico interagisce anche con altre proteine, come l’apomioglobina. La principale conclusione di questo lavoro è stata che il motivo oligomerico della proteina veicola l’acido oleico, normalmente poco solubile, favorendo quindi la solubilizzazione dell’acido grasso e conseguentemente della sua proprietà citotossica. È in preparazione un articolo riguardante questi risultati. L’enterocina AS-48, è un polipeptide circolare di 70 residui prodotto da Enterococcus faecalis che mostra a vere attività antibatterica. La proteolisi limitata è stata usata per preparare una forma lineare e due frammenti di questa enterocina. Misure di dicroismo circolare nel lontano ultravioletto hanno dimostrato che la proteina ha una bassa ellitticità e una ridotta stabilità alla denaturazione termica, ma mantiene la sua attività antibatterica., mentre i frammenti presentano un’attività ridotta. Questi risultati indicano che la circolarizzazione è un fenomeno che non è richiesto per l’attività antibatterica, ma è cruciale per la stabilizzazione della struttura nativa. Questa ricerca è stata pubblicata in FEBS Lett. (2008).
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Zaveckas, Mindaugas. "Rekombinantinio žmogaus granulocitų kolonijas stimuliuojančio faktoriaus pasiskirstymas ir renatūracija vandens dvifazėse sistemose, dalyvaujant chelatuotiems metalų jonams." Doctoral thesis, Lithuanian Academic Libraries Network (LABT), 2005. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2005~D_20051116_154617-68277.
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The contribution of Cys17 and surface-exposed histidine residues in rhG-CSF interaction with Cu(II), Ni(II) and Hg(II) ions chelated by Light Resistant Yellow 2KT-polyethylene glycol derivative was evaluated in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran. It was determined that His43, His52, His156 and His170 residues are involved in protein interaction with chelated Cu(II) ions. Protein interaction with chelated Ni(II) is governed by His52 and His170 residues, though Cys17 is also involved. The contribution of Cys17 side chain is dominant in the interaction between rhG-CSF and chelated Hg(II) ions. The direct interaction between chelated Hg(II) ions and the –SH group of protein was determined for the first time. Based on the study of the interaction between rhG-CSF and chelated metal ions, rhG-CSF was successfully refolded from inclusion bodies in aqueous two-phase systems PEG-dextran containing chelated Ni(II) or Hg(II) ions for the first time. The refolding of rhG-CSF (C17S) in these systems was more effective compared to that of intact rhG-CSF. The dependence of refolding efficiency of rhG-CSF (C17S) in two-phase systems containing chelated metal ions on the number of histidine mutations was evaluated. It was determined that the refolding efficiency of protein in the systems containing chelated Ni(II) is inversely proportional to the number of histidine mutations. The affinity of purified rhG-CSF (C17S) and its histidine mutants for... [to full text]
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Лыкова, Ю. А., and Yu A. Lykova. "Исследование электрохимического восстановления 2-замещеных хиноксалинов в апротонной среде. Количественное определение вольтамперометрическим методом : магистерская диссертация." Master's thesis, б. и, 2020. http://hdl.handle.net/10995/94640.
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Объектами исследования являются 2-замещеные хиноксалины. Целью данной работы является изучение химических свойств хиноксалина и его производных. В работе рассматривалось восстановление производных хиноксалина (окислительно-восстановительные свойства, потенциал восстановления, ЭПР спектр, квантово-химический расчеты). Сравнение восстановительных свойств синтезированного ряда производных хиноксалина. Определено количество электронов, участвующих в процессе восстановления производных хиноксалина. Смоделирован процесс восстановления. Доказан одноэлектронный переход хиноксалина. Далее приводится количественное определение производных хиноксалина вольтамперометрическим способом. Изучение свойств хиноксалина является важной задачей, так как вновь синтезированные производные хиноксалинов проявляют химическую и биологическую активность. Из-за значительного увеличения вирусов и необходимости поиска новых лекарственных препаратов, исследование производных хиноксалинов и их химической активности, а также количественное определение новых синтезированных хиноксалинов является нужной и важной задачей. В работе доказан одноэлектронный переход хиноксалина экспериментальными и расчетными методами (ЭПР спектр, квантово-химический расчеты, хроноамперометрия). Также был построен ряд восстановительной активности хиноксалина и его производных. После чего были выбраны производные для количественного определения вольтамперометрическим методом. Результаты показали, что одноэлектронный переход хиноксалина свойственен и для его производныхРазработаны методики количественного определения формальдегида в объектах фармации на примере ЛП «Эндофальк» и товарного уротропина от ПАО «Метафракс». Правильность полученных результатов подтверждена сравнением с результатами независимых методов анализа, прописанных в ФС РФ XIV издания на субстанции уротропина и «Макрогола 3350».The object of research is 2-substituted quinoxalines. The goal of this work is to study the chemical properties of quinoxaline and its derivatives. This goal is divided into the following tasks: 1) Study of literature sources on the use of quinoxaline derivatives, chemical and electrochemical properties of these compounds, possible published methods for quantitative determination of quinoxaline derivatives by voltammetric method. 2) Study of reducing properties of compounds of quinoxalin derivatives (redox properties, reduction potential, EPR spectrum, quantum chemical calculations). Comparison of reducing properties of a synthesized series of quinoxaline derivatives. 3) Determination of the number of electrons involved in the reduction of quinoxaline derivatives. Modeling the reduction process. Proof of a single-electron quinoxalin transition. 4) Quantitative determination of quinoxalin derivatives by voltammetric methods.
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Abou-Dalle-Messaikeh, Hana. "Polymères insolubles fonctionnels : affinité spécifique pour les anticorps anti VIIIc." Paris 13, 1989. http://www.theses.fr/1989PA132006.
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Recherche d'adsorbants synthétiques constitués de polymères fonctionnels capables d'adsorber les anticorps anti viii: c dans des systèmes d'épuration plasmatique. Substitution de fonction sulfonate et sulfamion d'un acide aminés ou de deux acides amines sur un polystyrène
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Tait, Timo. "The production and purification of functional steroid hormone receptor ligand binding domains towards the development of a biological endocrine disruptor detection system." Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/96811.
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Thesis (MSc)--Stellenbosch University, 2015.ENGLISH ABSTRACT: During the last two and a half decades a large body of research has accumulated indicating the presence of various natural and synthetic chemical compounds within the environment capable of inducing hormone-like responses in humans and animals. Such compounds, termed endocrine disruptors, have been implicated in a variety of developmental, reproductive and physiological abnormalities which have been shown to converge on the endocrine system. Given that endocrine disrupters are comprised of a diverse group of molecules with dissimilar chemical structures, general screening techniques are not feasible for effective environmental monitoring. A primary method of action by which these exogenous molecules affect the homeostatic regulation of the endocrine system is believed to be via the modulation of gene transcription. It is now well established that many endocrine disrupting compounds act upon a principal group of transcription factors, the nuclear receptors, by chance interaction with the ligand binding domains of these proteins. With a view to ultimately design a portable kit for the detection of endocrine disrupting compounds in water based on the bio-specific immobilisation of nuclear receptor ligand binding domains to a stationary membrane matrix, this study specifically describes: 1. The effects on recombinant protein expression by the addition of small molecules to the cultivation media of bacteria. 2. The optimisation of conditions for the lysis of bacterial cells to increase the solubility of heterologously expressed proteins. 3. The purification of recombinant proteins from bacterial cell lysates by means of a two-step chromatographic methodology. 4. The cloning of the genes for the human androgen and estrogen receptors’ ligand binding domains into baculovirus transfer plasmids. 5. Transfer of genetic material from the created baculovirus transfer plasmids to a linearised baculovirus genome for the generation of recombinant viruses. 6. The cultivation, and baculoviral infection, of Spodoptera frugiperda and Trichoplusia ni cell lines. 7. Expression and purification of N-terminal hexahistidine-tagged human nuclear receptor LBDs from insect cell lysates by means of immobilised metal affinity chromatography.
AFRIKAANSE OPSOMMING: Die teenwoordigheid van natuurlike en sintetiese chemiese middels wat oor die vermoë beskik om die aksies van hormone in die mens en dier na te boots het toenemend aftrek gekry in navorsing gedurende die laaste twee en ’n halwe dekades. 'n Verskeidenheid van ontwikkelings-, reproduktiewe- en fisiologiese abnormaliteite ontstaan as gevolg van die aksies van hierdie molekule, genaamd endokriene-ontwrigters, op die natuurlike funksionering van die endokriene-sisteem. Gegewe dat die groep chemiese middels waaruit endokriene-ontwrigters bestaan van diverse oorsprong afkomstig is lei dit daartoe dat algemene analitiese tegnieke nie in alle gevalle geskik is vir effektiewe omgewingsmonitering is nie. Die modulasie van geentranskripsie is een van die metodes wat voorgestel word as ’n metode waarop hierdie eksogene molekule die homeostatiese regulering deur die endokriene-sisteem omverwerp. ’n Algemene metode waarop vele endokrien-ontwrigtende stowwe geentranskripsie beïnvloed, is deur interaksie met die hormoon-bindende gedeeltes van ’n belangrike groep transkripsiefaktore, die nukluêre reseptore. Hierdie studie, met die uiteindelike ontwikkeling van ’n draagbare toetsstelsel vir die opsporing van endokrien-ontwrigtende-stowwe in water, gebasseer op die bio-spesifieke immobilisering van nukluêre reseptor ligand bindingsdomeins op ’n stasionêre membraanmatriks, het ten doel om die volgende te beskryf: 1. Die effek wat die byvoeging van klein molekule tot die groeimedium van bakteriëe het op die uitdrukking van rekombinante proteïene. 2. Die optimisering van bakteriese sel-lisering in terme van verhoging in die oplosbaarheid van heteroloë proteïene. 3. Die suiwering van rekombinante proteïen vanuit bakteriese sellisate deur middel van ’n twee-stap chromatografiese sisteem. 4. Die klonering van die gene vir die menslike androgeen en estrogeen reseptore se ligand bindingsdomeine in bakulovirus oordragplasmiede. 5. Die oordrag van genetiese materiaal vanaf hierdie bakulovirus oordragplasmiede na ’n gelineariseerde bakulovirus genoom deur middel van homoloë rekombinasie vir die produksie van rekombinante virusse. 6. Die groei en infeksie van Spodoptera frugiperda en Trichoplusia ni sellyne wat lei tot die uitdrukking van menssoortgelyke nukluêre reseptor ligandbindingsdomains. 7. Suiwering van N-terminaal heksahistidien-etiket-gekoppelde menslike nukluêre reseptor ligandbindingsdomeins vanuit inseksellisate deur middel van geïmmobiliseerde metaal affiniteitschromatografie.
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Jia, Fuchao. "Thermodynamic and structural study of the interaction between Ru(bpy)2dppz 2+ and DNA." Phd thesis, Université de Strasbourg, 2013. http://tel.archives-ouvertes.fr/tel-01062684.
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Dans une première partie, nous mesurons l'affinité de l'interaction entre [Ru(pby)2dppz]2+ et l'ADN en utilisant la luminescence induite lors de la complexation. Nous étudions l'évolution de l'affinité lorsque la force ionique de la solution augmente. Dans une deuxième partie, nous modifions les extrémités d'un double brin d'ADN en y greffant des fluorophores. De la mesure de transfert d'énergie non-radiative entre ces fluorophores, nous étudions l'évolution de la longueur du complexe. Nous effectuons un dosage d'un double brin de 15 paires de bases d'ADN par le complexe ruthéné. Nous nous servons de la luminescence induite par l'intercalation du groupement dppz. Cependant, l'incrément de luminescence par groupement intercalé n'est pas connu, et nous ne pouvons pas le mesurer en saturant le brin d'ADN. Nous utilisons alors une technique mise au point par Nishida [Method for Measuring the Binding of Small Molecules to Proteins from Binding-Induced Alterations of Physical-Chemical Properties], dans laquelle deux titrations de deux solutions d'ADN de deux concentrations différentes sont effectuées. En utilisant le fait que, lorsque deux solutions d'ADN complexé par le composé ruthéné, possèdent la même luminescence par paire de base , le taux de complexation de ces deux solutions doit être le même, nous pouvons alors déterminer, sans hypothèse supplémentaire, le taux de complexation de l'ADN. De l'évolution de ce taux en fonction avec la concentration de ligand, nous déduisons son affinité pour l'ADN. Nous étudions maintenant le changement de longueur d'un double brin d'ADN de 15 paires de bases, modifié à ses deux extrémités par deux fluorophores : Alexa488 et Alexa568. Lorsque Alexa 488 est porté dans un état excité, il peut se désexciter en transférant de l'énergie de manière non-radiative à Alexa568, qui se désexcite alors en émettant des photons de plus faibles énergie que ceux émis par Alexa488. L'efficacité de ce transfert d'énergie peut être quantifié à partir de la mesure des intensités émises à basse et haute énergie. Elle dépend a priori de l'efficacité couplage (et en conséquence de la distance) entre les deux fluorophores. Nous effectuons des mesures de temps de vie des états excités de chacun des fluorophores. Nous avons observé que l'addition de ligand a pour conséquence une forte inhibition quenching des fluorophores. De l'analyse de l'évolution du temps de vie du fluorophore donneur d'une part et de celui du fluorophore accepteur d'autre part, nous déduisons l'évolution de l'efficacité du transfert d'énergie en fonction de la concentration de ligand. Nous confrontons les résultats obtenus par chacune de ces analyses, et en déduisons finalement, en nous servant de l'analyse de l'équilibre effectuée dans la première partie, l'évolution de la longueur de la chaîne en fonction du taux de complexation
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Lima, Maria da ConceiÃÃo LÃbo. "ContribuiÃÃo ao conhecimento quÃmico de plantas do Nordeste do Brasil: Lippia affinis gracillis H. B. K." Universidade Federal do CearÃ, 2006. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=1577.
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FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgicoEste trabalho descreve a investigaÃÃo fitoquÃmica das raÃzes, folhas e talos de Lippia affinis gracillis H.B.K. (Verbenaceae), popularmente conhecida no Nordeste do Brasil como âalecrim de vaqueiroâ. A anÃlise cromatogrÃfica dos extratos hexÃnico e etanÃlico das raÃzes e folhas de Lippia aff. gracillis permitiu o isolamento e caracterizaÃÃo do lapachenol (1), dos triterpenos friedelinona (2), acetato de epifriedelanil (3), Ãcido 3-acetil oleanÃlico (6) e lantandeno-A (7), das mistura dos esterÃides -sitosterol e estigmasterol (4) e seus glicosÃdeos (5), do Ãcido graxo Ãcido tetraeicosanÃico (8), das quinonas tecomaquinona (9), microfila quinona (10) e 5-hidroxinafto[2,3b]furan-4,9-quinona (11), dos flavonÃides quercetina (12), hispidulina (13) e da 3-metilquercetina (14). O estudo da composiÃÃo quÃmica dos Ãleos essenciais das folhas e talos de Lippia aff. Gracillis por CGL/EM, apresentou pouca similaridade, sendo os constituintes majoritÃrios das folhas o carvacrol (54,4%) (59) e o p-cimeno (10,7%) (53), enquanto que nos talos foram o carvacrol (30,2%) (59) e o biciclogermacreno (16,7%) (63). Com os Ãleos das folhas e talos, o carvacrol e seus derivados metilado e acetilado foram realizados teste para determinaÃÃo das atividades: Larvicida (Aedes aegypti), moluscicida (Biomphalaria glabrata), antimicrobiana e antioxidante. Na determinaÃÃo estrutural dos metabÃlitos secundÃrios isolados, utilizou-se de tÃcnicas espectroscÃpicas como infravermelho e ressonÃncia magnÃtica nuclear de hidrogÃnio e carbono-13, incluindo tÃcnicas bidimensionais (COSY, HMQC e HMBC), e ainda comparaÃÃo com dados espectroscÃpicos de compostos autÃnticos descritos na literatura.
This work describes the phytochemical studies of the roots, leaves and stalks of Lippia affinis gracillis (Verbenaceae), popularly known in the Brazilian northeast as âalecrim de vaqueiroâ. Chromatographic analysis of hexanic and ethanol extracts of the roots and leaves of Lippia aff. gracillis allowed the isolation and characterization of lapachenol (1), of triterpenes friedelinone (2), epifriedelanyl acetate (3), oleanÃlic acid 3-acethyl (6) and lantandene-A (7), of the mixture steroids -sitosterol and stigmasterol (4) and their glycosides (5), of the fatty acid tetraeicosanoic (8), of the quinone tecomaquinone (9), microphyllaquinone (10) and 5-hydroxynaphto[2,3b]furan-4,9-quinone (11) and flavonoids quercetine (12), hispiduline (13) and 3-methylquercetine (14). The chemical composition of essential oils Lippia aff. gracillis was identified by gas chromatography coupled to mass spectrometry (GCL/MS) study from the leaves and stalks presented similarity small, were major constituents from the leaves the carvacrol (54,4%) (59) of the pcymene (10,7%) (53), while that in the stalks were the carvacrol (30,2%) (59) and biciclogermacrene (16,7%) (63). With the essential oils from the leaves and stalks, the carvacrol and derivative methyled and acethyled from the carvacrol had been made the larvicidal activitys (Aedes aegypti), molluscicide (Biomphalaria glabrata), antibacterial and antioxidant. Structures determinations of all compounds had been established on the basis of spectroscopic techniques such as IR, 1D and 2D NMR (COSY, HMQC, HMBC), as well by comparison with the published data for structurally related compounds.