The importance of interactions between transmembrane domains of integral membrane proteins has been well-established in a range of essential cellular functions. Most integral membrane proteins also possess regions that lie on the exterior of the membrane that may influence the ability of these transmembrane domains to interact. We sought to test this hypothesis by quantifying the energetics of transmembrane helix self-association in the absence and presence of an amphipathic helix that can bind to the membrane surface. The model chosen for this study was the major coat protein (MCP) of M13 bacteriophage, which has an N-terminal amphipathic helix linked to its single transmembrane segment via a flexible linker. Dimerization of both full-length MCP and a peptide containing only the transmembrane domain (MCPTM) was studied by solution NMR in SDS micelles. We found that there was an increase in the apparent dimerization affinity in the absence of the N-terminal helix. However, this increase in apparent affinity could be attributed to differences in detergent-binding properties of the two polypeptides in monomeric versus dimeric states when the empty micelle was considered to be a participant in the dimer dissociation. Preliminary results from the integral membrane protein, p7 of the hepatitis C virus are also presented in this thesis. It has been demonstrated that p7 enhances viral infectivity and accumulation, and that this function may require oligomerization in the membrane. While we encountered limitations due to challenges in the generation of sufficient quantities of pure p7 samples, we were able to perform circular dichroism spectroscopy under conditions that may favor different oligomeric states. These studies suggest that there is a change in the degree of helicity upon oligomerization, and suggest that SDS could be a suitable system to characterize the interactions of the p7 oligomer in the future.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22291号 / 理博第4605号 / 新制||理||1660(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 高田 彰二, 教授 平野 丈夫 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

The master's thesis is focused on study of specific protective proteins from animal products. Two different types of antimicrobial egg white proteins were studied in detail - antimicrobial protein ovotransferrin (conalbumin) and enzyme lysozyme. Ovotransferrin belongs to transferrin group of proteins and exhibits activities similar to milk protective protein lactoferrin. The main effects of ovotransferrin are antiviral, anticancer and immunomodulatory. Antimicrobial activity of ovotransferrin based on the possibility to bind iron is still a subject of interest. For comparison the second egg protein lysozyme (N-acetyl muramidglycan hydrolase) was used. Lysozyme is a hydrolytic enzyme which primary attack cell wall of bacteria. In the theoretical part of the thesis an overview of the specific antimicrobial proteins in selected animal products was introduced mainly focused on ovotransferrin and lysozyme. The experimental part of this work was focused on optimization of methods for the determination of antimicrobial activity, protein concentration and purity. For quantitative analysis of total proteins, optimized Hartree – Lowry spectrophotometric method was used. For the determination of molecular weight and purity SDS-PAGE was used and stained by Coomassie Brilliant Blue G250 and silver. In experimental part the real sample of egg white was compared with samples of lyophilized antimicrobial proteins and therapeutical pills supplied by industrial partner. Protein composition and purity of these preparative has been determined. Antimicrobial activity of ovotransferrin was studied on cultures of G+ bacterium Bacillus subtilis and for comparison on G– E. coli. Ovotransferrin showed antimicrobial effect only at very high concentrations of about 75 mg/ml (Bacillus subtilis) and 50 mg/ml (E coli) even with addition of high amount (100 mM) of hydrogen carbonate ions. The inhibitory effect was most evident in liquid media. On the other hand, lysozyme exhibited significant inhibitory activity from 0.3 mg/ml on gram positive bacteria. Inhibitory effect on E. coli was not observed. Another part of study was focused on isolation of ovotransferrin from egg white using gel permeation chromatography on Sephadex G100. As mobile phases 0.1 M phosphate buffer and 0.05 M Tris-HCl buffer were tested. By SDS-PAGE the purity of ovotransferin comparing to standard was evaluated. Finally, the encapsulation of ovotransferrin and lysozyme was tested. Ovotransferrin and lysozyme was encapsulated into liposome and chitosan particles. Particles stability, distribution and average size distribution were studied by dynamic light scattering and zeta potential measurement. The stability of particles in the model physiological conditions was studied too.

This thesis The Failure of the New Left in the US: The Case of SDS analyzes the causes and reasons of the failure of the New Left in the United States. The left-leaning students who were discontent with the social order and reality of the country gathered under the idea of participatory democracy in a group called Students for a Democratic Society. Their aim was to change and improve the system through universities being agents of social change, thus making a clear difference with the Old Left. The worker no longer stood in the center of social progress, but the student did. SDS promptly plunged into several burning issues of the era, such as civil rights movement and inferior position of the blacks and poor in the society. The Vietnam War and antiwar protest movement have also been great issues in which SDSers directed their energy. As the Vietnam War escalated in terms of American soldiers being sent overseas, the intensity of student protests grew as well. Inevitably SDS resorted to usage of violent means of expressing dissent and clashed with the forces of the establishment. The thesis seeks to answer what reasons, events and realities led them to finally adopting revolutionary Marxism as their flag ideology. Soon after that SDS broke up and its once great influence waned away.

Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.

Fusarium virguliforme (Aoki, O’Donnell, Homma & Lattanzi), the causal agent of sudden death syndrome (SDS) of soybean (Glycine max [L.] Merrill), is responsible for major soybean yield losses in North America and South America. Despite the importance of SDS, few agronomic practices have been used to manage SDS successfully. Understanding the pathogen and the mechanisms it uses to cause disease is vital to devise effective disease control strategies. However, our knowledge of the pathogenicity mechanisms used by F. virguliforme is limited. The identification of pathogenic genes will shed light on the molecular basis of the interaction between F.virguliforme and soybean, which may ultimately lead to better management of SDS. Therefore, the studies presented in this thesis were aimed at identifying and characterizing candidate pathogenicity genes in F. virguliforme.To fulfill this objective, 40 candidate pathogenicity genes of F. virguliforme were identified based on a combined approach, which included hands-on literature and database mining, functional genomics as well as transcriptome analyses. From these genes, the FvSNF1gene (a sucrose non-fermenting protein kinase 1 ortholog), the Fvstr1 gene (a striatin protein ortholog) were functionally characterized through a gene knock-out strategy. Targeted disruption of the FvSNF1 locus in wild type F. virguliforme strain reduced virulence significantly on soybean and abolished galactose utilization. In addition, the FvΔSNF1 mutant displayed significant reduction in expression of several CWDEs genes and was defective in colonizing the vascular system of the roots. To identify putative target genes regulated by FvSNF1, transcriptome analyses were performed in the FvSNF1 deletion mutant and in the wild-type. Disruption of FvSNF1 affected the level of transcription of 393 genes and a majority of the genes were involved in carbohydrate metabolism, lignin degradation, and cellular signaling pathway. The disruption of Fvstr1, a striatin ortholog in F. virguliforme, resulted in a complete loss of virulence as well as impaired conidiation, conidiophore development and pigmentation in the fungus. The FvΔstr1 mutant also failed to colonize the vascular tissues of roots of inoculated soybean plants. The results suggest that FvSNF1and Fvstr1 have critical roles in pathogenicity. Another part of the study was to investigate the efficacy of ILeVO®, a succinate dehydrogenase inhibitor fungicide from Bayer CropScience, against F. virguliforme. Our results showed that ILeVO® was very active against F. virguliforme in vitro and was very effective in minimizing F. virguliforme infection thus providing yet another tool to combat SDS.

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. / Bruker Daltonics

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