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Studies on the differential specificity of protein kinases and its applications /Loog, Mart, January 2001 (has links)
Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 5 uppsatser.
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Investigation into an affinity precipitation separation system based on the thermally reversible solution behavior of poly(n-isopropylacrylamide) /Yang, Heung Joon, January 1989 (has links)
Thesis (Ph. D.)--University of Washington, 1989. / Vita. Includes bibliographical references (leaves [177]-185).
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Optimization of large beaded cellulose as a chromatographic support /Kaster, Jeffrey Allen, January 1993 (has links)
Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 106-107). Also available via the Internet.
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Characterization of drug interactions with [alpha₁]-acid glycoprotein using high performance affinity chromatographyXuan, Hai. January 1900 (has links)
Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed Aug. 1, 2007). PDF text: 1 v. : ill. Includes bibliographical references. Also available in microfilm and microfiche formats.
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Studies of drug-protein binding using a limited digestion method and immobilized drug affinity columnsBian, Min. January 2008 (has links)
Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Feb. 17, 2009). PDF text: ix, 137 p. : ill. (some col.) ; 2 Mb. UMI publication number: AAT 3325856. Includes bibliographical references. Also available in microfilm and microfiche formats.
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Evaluation of Phenomena that Determine the Performance of Immunoaffinity, Peptide-Based and Ion Exchange Affinity SorbentsSines, Brian James 04 January 2001 (has links)
Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents.
The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity.
This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance?
This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins. / Ph. D. / This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures.
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Non-covalent immobilisation of a ligand system : a new approach to affinity separationLiebenberg, Liesl Eileen 03 1900 (has links)
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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Application of an affinity chromatography toolbox to drug repurposing for cancer therapeuticsCruickshank, Faye Louise January 2016 (has links)
Phenotypic screening of drug molecules relies on the generation of a specific response; however the means by which this is elicited often remains unknown. Affinity chromatography is a valuable tool in the discovery of drug binding partners and may even allow the elucidation of the wider interactome of the initial drug target. The introduction of easily cleavable linkers and affinity-independent elution protocols to affinity chromatography is of current interest, since they render the technique much more adaptable with respect to the characterisation of biologically active species of interest. This thesis details the application of a novel azobenzene linker developed by the Hulme group for use in affinity-independent chromatography. The first chapter reviews recent developments in affinity chromatography and describes the synthesis of an affinity linker toolbox with both affinity-dependent and affinity-independent linkers. These linkers are functionalised with an azide moiety for use in CuAAC coupling to alkynyl derivatives of bioactive small molecules and have been modified to include photoreactive groups giving a series of linkers for use in the identification of less abundant, or low affinity, proteins. The first drug investigated, anisomycin (ANS), is a small molecule which was initially introduced as an antibiotic drug (Flagecidin). At nanomolar concentrations ANS has been shown to affect the mitogen activated protein kinase (MAPK) pathways; downstream effects of these pathways are thought to play a role in a range of pathological disorders such as Alzheimer’s disease, cancer and spinal muscular atrophy (SMA). ANS is thus a candidate for drug repurposing. Although the downstream effects of MAPK/SAPK pathway activation induced by anisomycin are well-documented, the cellular target has yet to be revealed. Previous work by the Hulme group has shown that the N-propargyl anisomycin derivative (I) retains the biological activity of the lead compound ANS. Thus to evaluate the cellular protein targets, N-propargyl ANS (I) was coupled onto the linker toolbox to create an ANS affinity probe library as described in chapter 2. The second drug investigated, fingolimod, was introduced as an immunomodulating drug (Glienya) for the treatment of multiple sclerosis (MS). This small molecule has also been shown to have anti-cancer properties in a range of cancer cell lines; however the precise mechanism by which this is effected is unknown. Literature precedent shows that terminal modification of fingolimod generates analogues which still retain biological activity. Thus a novel fingolimod alkyne derivative (II) was synthesised and used to create an affinity probe library as described in chapter 3. Chapter 4 describes affinity pull-down experiments conducted with the aim of finding the protein target(s) of ANS and fingolimod, using the affinity probe libraries generated in chapters 2 and 3. This chapter concludes with a discussion of the implications of these findings and directions for future study.
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Synthetic strategies for the preparation of affinity label dynorphin A(1-11)NH��� analoguesLeelasvatanakij, Leena 22 April 1996 (has links)
Graduation date: 1996
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Relationship between Key Variables in Penile Plethysmograph and Viewing Time Measures of Sexual Arousal in Sex Offending Adult MalesCloyd, Lisa Loewinger 11 December 2007 (has links)
Penile plethysmography is amongst the best measures for prediction of sexual recidivism and has been extensively researched. However, there are a variety of criticisms regarding use of penile plethysmography (PPG), including financial investment, significant training needs, lack of standardization, intrusive nature of the measure, extensive time for testing, and inability to use PPG with all groups of sexual offenders (e.g., women and adolescents). Viewing time has been studied, though not as extensively as PPG, regarding detection of sexual interest. This study examined the relationship between Monarch 21 PPG and Affinity viewing time variables, including responses to individual stimuli, a sex deviance differential, and a sex deviance ratio. It was predicted that there would be a significant positive correlation between key variables of the Monarch 21 PPG and Affinity. Overall, the associations found between the Monarch 21 PPG and the Affinity were significant, although the magnitudes of the associations were modest. When considering sexual deviance ratios and differentials (as is typically utilized in PPG literature), there was a small, but significant association between the Monarch 21 PPG and the Affinity. Consequently, it is recommended that more research be done comparing these two sets of measures, further evaluating the Affinity and its' predictive validity, and examining the complex arousal pattern shapes, rather than focusing only on a series of discrete variables.
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