M.A. / The shift from the Old to the New Economy has developed due to four key trends, namely globalisation, consumerism, environmentalism and corporate governance. Globalisation and the Internet has resulted in consumers being able to track the behaviour of corporations (Vise, 2006:119) and consequently, a New Consumer has emerged, where emphasis on corporate transparency and the environment has become a key concern for these New Consumers. New Consumers are characterised by Lewis and Bridger (2000:21) as independent, sophisticated, involved and well informed about the production of goods and services, where these New Consumers are feeling the pressure to confront and act upon the fact that unbridled production and consumption, which was proliferate in the Old Economy, comes with escalating pollution at a significant human/animal/earth cost (Trendwatching, 2007). Moreover, in the world of globalisation and information overload, Salzer-Mörling and Strannegård (2004:224) argue that the proliferation of brands as well as a cluttered marketplace has meant that corporations now need to not only be differentiated in the marketplace, but also be distinct and one of the ways which corporations in the New Economy are achieving this is by focusing on the corporate brand as the point of differentiation.

The actin and microtubule cytoskeleton plays a critical role in the establishment of cell polarity. Cell processes like mitosis and migration rely on the reorganization of the cytoskeleton to properly function. One driver of cell polarity is the formin, Inverted Formin-1 (INF1). INF1 is able to induce F-actin formation, activate the Serum Response Factor (SRF) pathway, stabilize microtubules, associate with microtubules, and disperse the Golgi body. Regulation of INF1 is unique, since it does not possess conserved formin regulatory domains. However, INF1 does possess many potential phosphorylation sites. In this study, we demonstrate that INF1’s ability to induce F-actin stress fibers and activate SRF is inhibited by Microtubule-Affinity Regulating Kinase 2 (MARK2). Inhibition of INF1’s actin polymerization activity by MARK2 likely occurs near INF1’s C-terminus. However, MARK2 was unable to inhibit INF1’s ability to stabilize microtubules, associate with microtubules, and disperse the Golgi. Furthermore, we show that INF1 overexpression is associated with primary cilium absence and in some cases, the presence of long cilia, suggesting that INF1 plays a role in primary cilium formation.

Transglutaminases (TGases) are a group of enzymes that catalyze the formation of an amide bond between the γ-carboxamide group of a glutamine residue and an amine donor, usually an ε-amino group of the lysine residue, leading to the formation of ε-(γ-glutamyl)lysine crosslinks. Owing to the roles that transglutaminases such as tissue transglutaminase (TG2) and Factor XIIIa (FXIIIa) have been found to play in a wide range of disease states, efforts have been directed towards the study of these proteins. The study of enzymes to better understand their function and mode of action is facilitated through the use of tools such as protein labelling, enzyme inhibition, and substrate analogue kinetic studies among others. Transition state analogues have been effective inhibitors in the study of enzyme activity. Sulfoxide inhibitors can efficiently mimic transition states leading to the tetrahedral intermediate of an acyl transfer reaction and we discuss the synthesis towards sulfoxide transition state analogue inhibitors of TG2 in chapter 2. Novel sulfoxide compounds were synthesized, though the desired target compounds proved difficult to isolate due to their instability. Fluorescent probes are effective in protein labelling as a means of discerning activity. This technique was applied in order to elucidate intracellular TG2 activity, which is a topic of controversy. To that end, the synthesis of a fluorescent, TG2-specific, cell permeable probe is discussed in chapter 3. However, preliminary in vivo results show that while the probe is cell permeable and fluorescent, it was not TG2-specific. Molecular modelling suggests that the hexa-arginine tag, designed to improve cell permeability, decreases the affinity of the probe for its intended target. Finally, FXIIIa has become a new addition to the study of transglutaminases in the Keillor group. Given our interest in this enzyme, we had three goals for this work as explained in chapter 4. Firstly, owing to the anticipated high demand for FXIIIa required for later experiments, our primary aim was the development of an optimized method for the expression and purification of recombinant FXIIIA. After evaluating different conditions for FXIIIA expression, the Studier auto-induction ZYP media1 at 20 °C for 24 h was found to provide the optimal conditions for the expression of recombinant GST-tagged FXIIIA, typically giving a total of 1.5 mg of protein/L of culture. Secondly, a variety of different peptides were synthesized and tested using a glutamate dehydrogenase (GDH)-based assay to identify a high affinity sequence for a substrate of FXIIIa. The two peptides with the highest affinity for FXIIIa were Ac-DQMMMAF-OH and Ac-DQMML-OH. Testing with TG2 displayed negligible reactivity, confirming their use as orthogonal peptides, results reinforced by modelling studies of the peptides with both FXIIIa and TG2. This discovery represents the first time peptides orthogonal to TG2 with affinity for FXIIIa have been kinetically characterized with both transglutaminase enzymes. Lastly, our work towards a fluorogenic activity assay by incorporating a coumarin ester through attachment to a glutamic acid residue into a peptide sequence recognized by FXIIIa, will be discussed.

Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), 19.3 ± 3.54 µM (n=10) and 43.5 µM (n=2), respectively (mean ± SD). Muscimol titration in the presence of 10, 20, 40 µM of gabazine resulted in IC50s values of 20.1 (n=2), 37.17 (n=4), and 120.45 (n=2), respectively. In the presence of 20, 80, and 160 µM of TMPP (trimethylolpropane phosphate) the IC50s were 0.86 (n=2), 3.07 (n=3), 6.67 (n=2) µM, respectively. Increasing concentrations of GABAA antagonists shifted agonist log concentration-response curves to the right with identical efficacies, indicating direct competition for the GABAA receptor. A Schild plot analysis with linear regression resulted in slopes of 1.18 ± 0.18, 1.29 ± 0.23 and 1.05 ± 0.03 for bicuculline, gabazine and TMPP, respectively. The potency of antagonists was determined in terms of pA2 values. The pA2 values were 6.63 (gabazine), 6.21 (bicuculline), and 5.4 (TMPP). This suggests that gabazine has a higher binding affinity to the GABAA receptor than bicuculline and TMPP. Hence, using spike rate data obtained from population responses of spontaneously active neuronal networks, it is possible to determine key pharmacological properties of drug-receptor interactions.

Orientador: Sônia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-18T14:28:20Z (GMT). No. of bitstreams: 1 Tsai_YuanMing_M.pdf: 3416327 bytes, checksum: d37231cf12f3af7bab5f7826907d5180 (MD5) Previous issue date: 2011 / Resumo: O presente trabalho teve como objetivo principal, o estudo comparativo dos agentes quelantes CM-Asp e IDA no processo da purificação de IgG humana por cromatografia de afinidade por íon metálico cobalto imobilizado em diferentes sistemas tamponantes na presença e ausência do sal NaCl. Observou-se, para todos os sistemas tamponantes estudados, a adição de sal foi desfavorável em termos de capacidade e seletividade de adsorção de IgG para ambos os agentes quelantes. De acordo com eletroforeses SDS-PAGE e análises nefelométricas, os melhores resultados foram observados em presença de Tris-HCl 25 mmol/L, pH 7,0 para o quelato CM-Asp-Co2+ e fosfato de sódio 25 mmol/L com imidazol 2 mmol/L, pH 7,0 para o quelato IDA-Co2+, obtendo-se fatores de purificação de 8,1 e 4,1 e pureza de 100% e 96%, respectivamente. Foram determinadas as curvas de ruptura por meio de experimentos cromatográficos realizados a diferentes diluições do soro humano, os resultados obtidos evidenciaram uma maior eficiência em termos da capacidade de adsorção nas diluições a 5 e 10 vezes (0,89 e 1,03 mg de proteína total/mL gel) comparado a soro diluído a 2,5 vezes (0,40 mg de proteína total/mL gel), tendo estes demonstrado comportamentos similares ao apresentado em etapa cromatográfica. Os dados de adsorção de IgG foram bem representados pelo modelo de Langmuir-Freundlich, a 25ºC, fornecendo valor de Qm igual a 94,44 mg IgG/mL gel e constante de dissociação de 8,18 x 10-5 mol/L, ordem de grandeza característica para ligantes pseudobioespecíficos / Abstract: In this work, we targeted the comparative study between chelating ligands agarose-CM-Asp and agarose-IDA in the purification of human IgG by Immobilized Metal-ion Affinity Chromatography (IMAC) with immobilized cobalt ion in different buffering systems in the presence or absence of salt. For all buffering systems the addition of salt was unfavorable in terms of adsorption capacity of IgG for both chelating ligands. The best results were obtained in the absence of salt. According to SDS-PAGE electrophoresis and nephelometric analysis, the best conditions observed were Tris-HCl 25 mmol/L, pH 7,0 for the agarose-CM-Asp and sodium phosphate 25 mmol/L with imidazole 2 mmol/L for the agarose-IDA (purification factor of 8,1 and 4,1 and purity of 100% and 96%, respectively). Chromatographic experiments were carried out with different dilutions of human serum to determine the breakthrough curves. The results showed a higher efficiency in terms of adsorption capacity at dilutions of 5 and 10 times (0.89 and 1.03 mg of protein Total / mL gel) compared to serum diluted 2.5 times (0.40 mg total protein / mL gel), these results had similar behaviors as those showed in the chomatografic stage. The adsorption data of IgG at 25ºC were well represented by Langmuir-Freundlich model. Values of Qm equal 94,44 mg of IgG/mL gel and dissociation constant of 8,18 x 10-5 mol/L, characteristic magnitude of pseudospecific ligands / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Le cœur du travail de notre groupe est l'étude de la réaction de photodétachement, qui consiste en l'expulsion de l'électron excédentaire d'un ion négatif lors de l'absorption d'un photon. Ce travail de thèse s'articule autour de deux projets : la microscopie de photodétachement, technique d'interférométrie électronique permettant de produire des données spectroscopiques sur les ions négatifs ; et le projet SIPHORE qui envisage la neutralisation d'un jet rapide d'ions négatifs à partir de la réaction de photodétachement, dans le but de servir la maîtrise de la fusion thermonucléaire contrôlée. Les évolutions de ces deux projets se recoupent dans la nécessité d'augmenter le nombre d'événements de photodétachement produits en un temps donné. Ce travail a permis d'étudier et de mettre en place différentes techniques expérimentales pour réaliser l'amplification de la réaction de photodétachement. Notre montage nous permet de produire cette réaction dans une zone d'interaction formée par l'intersection d'un jet d'ions et d'un faisceau laser. Nous envisageons d'une part la modification de la section efficace de photodétachement lorsque la réaction est produite en présence d'un champ magnétique, d'autre part l'amplification du flux de photons dans la zone d'interaction par stockage de lumière en cavité optique. Les avancées réalisées ouvrent de nouvelles perspectives sur les études fondamentales et les applications techniques liées aux ions négatifs. / The core of the work of our group is the photodetachment reaction, which consists in the expulsion of the extra electron of a negative ion by the absorption of a photon. This thesis work is organised around two projects: the photodetachment microscopy, an electron interferometric technique which produces spectroscopic data on negative ions; and the SIPHORE project which considers the neutralization of a fast negative ions beam by the help of the photodetachment process, for the purpose of controlled thermonuclear fusion. The evolutions of these two projects are overlapping in the need of increasing the number of photodetachment events produced per unit of time. This work has led to the study and the implementation of several experimental techniques to realise the amplification of the photodetachment reaction. Our setup permits to produce this reaction in an interaction area formed by the intersection of a negative ions beam with a laser beam. On the one hand we investigate the modification of the photodetachment cross section when the reaction is produced under a magnetic field. On the other hand we consider the amplification of the photon flux inside the interaction region using light storage with optical cavities. The results obtained pave the way towards new prospects for the fundamental studies and the technical applications affiliated with negative ions.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22686号 / 医博第4630号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The past few years have seen a rapid expansion in interest in the characterization of the entire complement of proteins, or proteome. Micro total analysis systems (μTAS) are an emerging promising method, offering rapid, sensitive and low sample consumption separations. I have demonstrated microchip capillary electrophoresis (CE) devices made of CaF2. New methods have been developed for micromachining enclosed capillaries in CaF2. CE analysis of fluorescently labeled amino acids was used to illustrate bioanalytical applications of these microdevices. Initial on-chip infrared spectroscopy results for qualitative analyte identification were achieved in microfluidic CaF2 channels. I have also shown the evaluation of poly(methylmethacrylate) (PMMA) and thermoset polyester (TPE) microchips for use in protein profiling. To improve separation efficiency and reduce protein adsorption, dynamic coating and poly(ethylene glycol) (PEG) grafting using atom transfer radical polymerization (ATRP) have been used in PMMA microdevices. Proteins, peptides and protein digests have been separated electrophoretically in these PMMA microchips. My results demonstrate that PMMA microdevices should be well suited as microfluidic systems for high performance separations of complex biological mixtures. In-channel ATRP has been developed for the surface modification of TPE microdevices. Characterization indicates that PEG-modified microchannels have much lower and more pH-stable electroosmotic flow, more hydrophilic surfaces and reduced nonspecific protein adsorption. CE of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. My results show that PEG-grafted TPE microchips have broad potential application in biomolecular analysis. Cancer marker analysis is important for medical research and applications. I report a method that can covalently attach appropriately oriented antibodies of interest on monolith surfaces. To reduce nonspecific adsorption, protein solutions were used to effectively block the monolith surface. Selective preconcentration and elution of human chorionic gonadotropin have been performed in my affinity columns, demonstrating that this type of system should have promising applications in cancer marker detection.

<p>ZMPSTE24 is a seven transmembrane domain zinc metalloprotease that resides in the ER and inner nuclear membranes of mammalian cells. The crystal structures of both the mammalian and yeast homologs, ZMPSTE24 and Ste24, respectively, were solved recently and revealed a common novel structure. Both structures contain a large chamber of mixed hydrophobicity that is capped on both sides. The canonical catalytic HExxH zinc-binding motif lies inside the chamber. Defects in the enzymatic function of human ZMPSTE24 have been shown to cause premature aging disorders. In addition to the well-defined role ZMPSTE24 and Ste24 play in the maturation of prelamin A in mammals and <b>a</b>-factor in yeast, both proteins have been proposed to play protective roles in Type 2 diabetes and viral infections by interactions with the cellular translocon. ZMPSTE24 can also be inhibited by several common HIV aspartyl protease inhibitors, possibly causing the frequent and common side-effects of these prescribed drugs. As of now, no precise location for substrate binding has been identified in either ZMPSTE24 or Ste24. Thus, the goal of this project is to localize residues in the enzyme that are important for substrate binding. The yeast homolog Ste24 was used as a model system as it functionally complements the mammalian enzyme and can be reliably cloned, overexpressed, and purified in an active form. </p> <p>Three approaches were taken to directly determine the <i>K_D </i>values for substrates of Ste24. The ability to perform a direct analysis of <i>K_D</i> values of Ste24 mutations was successfully optimized using microscale thermophoresis. Through <i>K_D</i> analysis, the Ste24 mutation G255A, while completely inactive, does not prevent substrate binding. Alternatively, L441A and L410A mutations showed both an increase in thermal stability and a decrease in binding affinity, that could explain their lower activity levels. A photoaffinity labeling-based proteomics experiment was utilized to precisely locate the site of the prenyl group to a hydrophobic patch lying just under a side portal of Ste24, near K234, during the -aaXing cleavage of <b>a</b>-factor maturation. To assess the method of inhibition of HIV protease inhibitors on Ste24 the conserved aspartate mutants were explored. All mutations of these aspartate residues resulted in a severe loss of Ste24 function and instability of the protein.</p>