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付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24466号 / 理博第4965号 / 新制||理||1709(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 古市 剛史, 准教授 Huffman Michael Alan, 教授 今井 啓雄 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

<p> Proteins vary tremendously in many of their physical and chemical properties. In order to perform in vitro application or analysis, one protein must be separated from other cellular components. This process is called protein purification. With the advances of modem science and technology, many protein purification schemes have been developed. Among them, the ELP-intein protein purification system has recently attracted an increasing amount of attention because of its positive characteristics: it is simple, inexpensive, scalable, with a high throughput, protease-free, etc. However, although the scientific literature reports all those good aspects of the system, several bad responses to it still exist. In this thesis, through comparing expression and purification of four different self-cleavage ELP fusion proteins, we propose a general solution to these problems for the first time. This makes a significant contribution to increased utility of the method of protein purification using self-cleavable stimulus responsive tags. </p> <p> When ELP-intein fusion proteins are expressed in bacteria, formation of non-native cytoplasmic aggregates (inclusion bodies) is a common problem which affects the yield of target protein. Inclusion bodies are generally assumed to contain misfolded or partially folded protein through exposure of hydrophobic patches and the consequent intermolecular interactions. Despite a loss of total expression yield and the need for more time, culturing at a lower temperature was reported to promote the expression of genes into soluble proteins and alleviate IB formation. Directly motivated by previous reports, we have applied a low-temperature expression strategy to solve the problems in this research. As expected, most of the T4-ELP inclusion bodies disappeared, and were transformed into a soluble expression, when culturing at lower temperatures. </p> <p> Inverse transition cycling (lTC), as the core method for the system we investigated has proved successful in the past with proteins that were expressed to high levels. However poor level ELP-intein tagged protein expression happens from time to time. It is hypothesized that if an ELP tagged molecule is present in a solution at a very low concentration, adding an excess amount of free ELP to the sample would form hybrid aggregates via the interaction of ELP moieties of the two molecules. We used this efficient and reversible capture system for low yield recombinant protein purification, and found it is perform very well. </p> / Thesis / Master of Applied Science (MASc)

<p> Studies examining the characteristics of membranes that facilitate and affect fusion are central to understanding the intricacies of inter- and intra-cellular fusion processes and expanding the existing knowledge of other roles membranes may have. In this thesis a model membrane system, using Sendai Virus and Egg phosphatidylcholine (EggPC) liposomes containing the receptor glycophorin A (proteoliposomes), was used in examining different fusion with proteoliposomes prepared by different methods. For the first time glycophorin A was incorporated into EggPC liposomes vectorially. This was accomplished separately with two detergents: octylglucoside and CHAPS. Fusion of Sendai Virus with the reconstituted proteoliposomes revealed that octylglucoside reconstituted proteoliposomes exhibited lower fusion compared with CHAPS reconstituted proteoliposomes. Efforts to determine the basis for this difference, using either proteinase K or O-glycosidase digestion and subsequent fragment analysis using SDS-PAGE and silver-staining, were inconclusive. A separate study examined the ability of large membrane-anchored biopolymers (chosen in virtue of their large hydrophilic domains) to sterically stabilize Egg PC liposomes. Glycophorin A, the lipophosphoglycan (LPG) from Leishmania donovani, and a polyethyleneglycol-conjugated phospholipid (PEG5000-PE) were incorporated into Egg PC liposomes. In each case, binding of a soluble fluorescent probe, NeutrAvidin Oregon Green, to liposomes containing biotin-conjugated lipid was restricted. This supports the idea that large membrane-anchored biopolymers are able to sterically stabilize liposomes.</p> / Thesis / Master of Science (MSc)

Intermediate lipid structures such as inverted micelles and interlamellar attachment are thought to play a crucial role in different biological processes like viral infection. Lysophosphatidylcholine has been shown to inhibit membrane fusion at stabilizing concentrations (between 1 and 10% with respect to membrane lipids). Studies in this thesis looked at the effects of Lysophosphatidylcholine (LPC) properties on the inhibition of Sendai viral fusion. The effects of bilayer sidedness preference as well as flip-flop of Lysophosphatidylcholine (lyso PC) were examined. Octadecylrhodamine (R₁₈) lipid mixing assays were used to measure the fusion of Sendai virus with different biological, erythrocyte ghosts, and artificial systems consisting of different lipids and different viral receptor compositions. The data showed that external addition of LPC exhibits a dependency between the incubation time of the lysolipids and the inhibition of viral fusion. The results also demonstrate a relationship between the location of LPC in the bilayer and its ability to inhibit lipid mixing. LPC present only on the outer monolayer plays a role in the inhibition of viral fusion. Reorientation of LPC was also measured for the same incubation periods. A method using Bovine Serum Albumin (BSA) and radioactivity labelled LPC, was applied to measure the flip-flop. Significant transbilayer reorientation of Lyso PC in the bilayer was shown to take place. The rate of flip-flop was measured at 0.32 ± 0.08% LPC /min. Such reorientation can explain the time dependency observed earlier. The conclusions of this thesis lend support to stalk intermediate mechanism of viral membrane fusion. The ability of LPC to inhibit only when present on one side of the bilayer supports the idea of a negatively curved stalk intermediate. Moreover, it showed that the shape and curvature tendencies of the bilayer stabilizer determine its effects on viral fusion. / Thesis / Master of Science (MS)

Simulated sensor data from active and passive sensors has numerous applications in target detection and tracking. Simulated data is particularly useful in performance evaluation of target tracking algorithms where the ground truth of a scenario must be known. For real sensor data it is impossible to know the ground truth so simulated data must be used. This paper discusses existing methods for simulation of data from active sensors and proposes a method that builds on existing techniques from the field of computer graphics. An extension to existing methods is proposed to accommodate the simulation of active sensor data for which timing and frequency information is required in addition to intensity. Results from an existing method of active sensor data simulation are compared to the results of the proposed method. Additionally, a cloud computing framework is proposed and its scalability evaluated to address the fairly large computational load of such a simulation. / Thesis / Master of Applied Science (MASc)

This thesis explores the concept of how a drummer can approach soloing over written rhythmic backgrounds that the rest of the band provides. The ambition is to discover what different approaches there are to this type of improvisation, as well as to find ways how one can practice this specific solo format. To answer these questions, I have transcribed three different drummers, analyzed some of their phrases and created exercises based on them. I have in addition to this done research on what has already been written and said on this subject. The result was a concert that was held at the Royal College of Music in Stockholm on May 8th, 2024, and all the compositions that were performed are written by me. This project gave me the realization that this solo format is a great alternative to other types of improvisation but demands a high level of rhythmic understanding, confidence, and timing from both the soloist and the band. It is also important to practice this type of improvisation in a nuanced way to get nuanced results from the improvisations. / <p><strong>Musik:</strong></p><p><em>Remains</em><em></em></p><p><em>Faces</em></p><p><em>Priceless</em></p><p><em>Out Of Bounds</em></p><p><em>Sidenote</em></p><p><em>57</em></p><p>All musik är skriven av Felix Kling</p><p><strong>Medverkande musiker:</strong></p><p>Felix Kling - Trummor</p><p>Eskil Larsson - Tenorsaxofon</p><p>Milos Lindegren - Piano</p><p>Jonathan Jessen - Kontrabas</p><p>Svante Qvennerstedt - Elgitarr</p><p></p>

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