The design of existing beaded adsorbent materials for column-mode protein purification has emphasized the impact of diffusional transport phenomena upon adsorbent capacity. A design model is presented that optimizes molecular accessibility of proteins relative to the mechanical stability of the material by manipulation of size and solids content for uncross-linked cellulose beads. Cellulose beads of various sizes ranging from about 250 to 1000 pm diameter and having different solids contents were evaluated. Cellulose beads (1.2 mm diameter) gave pressure drops of less than 1 psi per cm of bed at superficial fluid velocities of 100 cm/min in a 1 5 cm bed. Solids content of greater than about 9% cellulose greatly reduced the permeability of large proteins such as thyroglobulin and p-Amylase into the beaded matrix at bead contacting times of 5 and 50 seconds. The amount of permeation in 3% cellulose beads by thyroglobulin at bead contacting times of 5 seconds was about tenfold larger than predicted by diffusion models using the diffusivity of the protein in water. The utility of a low solids content, large bead cellulose support was shown with immobilized IgG (Mr 155 kDa) capturing recombinant human Protein C (M, 62 kDa). The amount of immobilized antibody was varied and immunosorptive capacity of 1 mm cellulose beads was found to be equivalent to that of 0.1 mm cross~linked agarose beads. The immobilization of antibodies to these supports was studied by photomicroscopy of cross-sectioned beads containing immobilized fluorescent labeled antibodies. While 75% of the antibody was immobilized within 0.07 mm of the cellulose bead surface at an antibody density of 1 mg antibody per ml of beads, an appreciable amounts of antibody immobilized deeper into the bead may have been utilized in order to yield capacities equivalent to the smaller agarose beads. The beaded cellulose supports derivatized to form either immunoaffinity or anion exchange matrices exhibited very low non-specific binding. Thus, the particle size, solids content, and extent of derivatization of cellulose matrices can be engineered so as to create matrices that provide high flow rates with low pressure drops while also having desirable adsorptive capacity for proteins. / Ph. D.

The purpose of this thesis is to take the individual on a journey about what it is like to be engaged in radical anti-systemic activism in the 21st Century.  Along this journey the reader will learn about the experiences of what it was like to join the Occupy movement"an anti-systemic movement that began in 2011"through an empirical analysis of learning about and practicing the anarchist(ic) characteristics of the movement"horizontal, non-hegemonic, affinity and consensus-based ways-of-being as a part of your everyday lifeworld.  This journey is not only informed by my own personal experience joining the Occupy movement, but it is also informed by my simultaneous experience of maintaining the role of a radical activist-scholar throughout the process.  Accordingly, I will explore how this impacted my lifeworld both within and outside of academia, which informed the very framework, analysis, and outcomes produced in this thesis.  This project was thus also designed to inform social science research"particularly that on social movements"by reflecting on both social roles experienced in this journey in order to cohesively make sense of the paradoxes created by engaging in discourses about, within, and for the Occupy movement.  Of most importance, from an empirical and ontological experience as an Occupier and activist-scholar, this project will help to raise key questions about the frameworks to seek social justice utilized by contemporary anti-systemic social movements in the 21st Century"social movements that are now spreading around the globe. / Master of Science

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

Therapeutic antibodies dominate the pharmaceutical market and improve the lives of millions of people annually. One important step when developing new medicines is to kinetically characterise the drug candidates. For antibodies this is difficult since many antibody reactions are extremely slow. By combining a mathematical formula that was recently published with the well-established technology from Gyros Protein Technologies, a new method for full kinetic characterization was developed and tested in this master thesis. The method provided precise data for five antibodies while also proving to be highly reproducible. By using small sample volumes, unlabelled reagents and having the reaction proceed in solution, this method offers advantages compared to many conventional approaches.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 118-128).

No description available.

Purification of lipase from Pseudomonas aeruginosa (from both a washed cell suspension and crude culture supernatant as the enzyme source) was performed utilizing affinity chromatography. Affinity chromatography was carried out using n-dodecylamine bound to Sepharose 4B. Chromatography of the concentrated crude culture supernatant resulted in a 65 to 95 fold purification with 5.8% recovery. Washed cells collected from a ten hour culture suspended in water also produced enzyme. Activity of the washed cell suspension supernatant was found to be 4.5 fold higher than the activity of the culture supernatant. A thirty percent recovery was obtained using the washed cell suspension supernatant. The washed cell suspension provides a cleaner preparation for use with the dodecylamine-agarose chromatography in purifying the enzyme.

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC50 16¿640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a ¿gene-like sequence¿ of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC50 = 6.25 ¿M in a susceptible cell line, compared with > 100 ¿M for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes. / Yorkshire Cancer Research, BMSS, School of Life Sciences and the Frank Hudson Memorial Fund

The aim of this research is to establish the bond between Friedrich Nietzsche and the anarchists, through the apparatus of elective affinity , and to challenge the boundaries of several anarchist trends especially 'classical' and 'post' anarchism and 'ideologies' like anarchism and libertarian Marxism. Moreover, it highlights the importance of reading Nietzsche politically, in a radical way, to understand his utility for the contemporary anarchist movement. The review of the literature concerning the Nietzsche-anarchy relationship shows the hitherto limited bibliography and stresses the possibility of exploring this connection, with the methodological help of Michael Löwy s concept of elective affinity . The research opens with a discussion of anarchism, following the dominant model for categorizing anarchist traditions, presenting its basic features and currents and drawing on its historical development. This leads to the introduction of two points (the questioning of the anarchist canon and the exposure of the diversity that basic anarchist concepts bear among different anarchist currents) which contest the rigid ideological perception of anarchism in favour of a fluid and dynamic anarchy. There emerges the elective affinity with Nietzsche, serving a double goal: the unification of the distinct anarchist tendencies and the definition of the anarchist parameters in relation to other ideologies. The following section of the thesis examines Nietzsche, by presenting the evolution of his philosophical thought and the fundamental theses of his perception of politics. It, then, continues with a detailed analysis of the main concepts of his philosophy based on the interpretation made by Gilles Deleuze, Alexander Nehamas and Keith Ansell-Pearson, thus structuring its interpretative context for establishing the Nietzsche-anarchy connection. This establishment is realized in a dual way. Firstly, by exploring the elective affinity through the presence of Nietzsche in the thought and politics of anarchist/libertarian thinkers (Goldman, Landauer, Benjamin) and currents (post-anarchism), and secondly by recognizing the anarchist worldview in the Nietzschean philosophy. The first path (Nietzsche in anarchism) shows how Nietzsche has interacted with or has been absorbed by the anarchist way of thinking, whereas the second path (anarchism in Nietzsche) reveals the affinal worldview of the two parts by extensively using the interpretation context mentioned above. The final section of the thesis applies the whole analysis above on a Nietzschean reading of the December 08 revolt in Athens based on the Of the Three Metamorphoses discourse from Thus Spoke Zarathustra. What has been found is the existence of a clear bond, between Nietzsche and the anarchists, which even reaches the upper levels of Löwy s elective affinity , that is Nietzschean Anarchism as a result of the two parts interactive fusion. The significance of this finding is that the relevant affinity may contribute to an alternative, to the dominant, perception of anarchism as an ideology. It may also designate its special features together with its weaknesses, meaning the objections of Nietzsche to certain aspects of the anarchist practices and worldview (violence, resentment, bad conscience), thus opening a whole new road of self-criticism for the anarchists of the twenty first century. In addition, the location and analysis of the elective affinity serves the debunking of the Nietzschean concepts used by conservative and right-wing readings in order to appropriate Nietzsche, and of the accusations that the German philosopher had unleashed against anarchists, which reveals his misunderstanding of anarchist politics.

Antibodies specific for insulin and human immunoglobulin G (HlgG) were attached to controlled pore glass (CPG) particles which had been silanized with a diol-bearing silane. Up to 20 mg of antibody protein could be attached covalently to 1 gram of CPG. Such immobilized antibodies, or immunosorbents, would bind specific antigens, but not unrelated proteins, when used in a high pressure liquid chromatographic configuration. This technique was given the name "high performance immunoaffinity chromatography" (HPIC). The HPIC properties of these immunosorbents were evaluated by an equilibrium theory and were found to be comparable to batch values. An immunosorbent for HIgG antigen showed an HPIC association constant of 10⁷·⁶; the batch equilibrium constant for the same immunosorbent was 10⁷·⁸. Two different anti-insulin immunosorbents retained the intrinsic affinity (10⁶ and 10⁹) of the antibody used to make them. The total active antibody concentrations of these immunosorbents were evaluated by HPIC and batch methods with good agreement between the two. The immobilization reaction was seen to result typically in the loss of 90% of the original antibody activity. HPIC was shown to be applicable to the rapid analysis of antigens at levels as low as ng/mL. This was found to be possible in part because of the rapid forward kinetics which were assessed by HPIC. A forward rate constant of 3 X 10⁷ L·mol⁻¹·sec⁻¹ for the binding of insulin by a specific HPIC column could be determined. The possibility of HPIC fluorescence immunoassays was investigated using a highly sensitive fluorescence detector. An Eimac collimated xenon arc lamp provided sufficient power to detect picomolar levels of fluorescamine labeled insulin and other compounds. The limitations of HPIC in performing picomolar immunoassays were thus shown to be immunochemical rather than instrumental. The ability of immunoaffinity purifications to overcome these limitations was demonstrated.