京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20812号 / 医科博第83号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 斎藤 通紀, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Stockholm växer och det kräver i sin tur nya bostäder. De fria ytorna i Stockholm city minskar och Stockholms stad försöker på nya sätt förtäta och förbättra staden konstant. Fastigheten Gulbetan är centralt belägen på Södermalm i anslutning till Ringvägen och Vitabergsparken. Idag används platsen till parkering och återvinning vilket gör att platsen är dyster och inte fyller sin fulla potential. Detta examensarbete leder till färdiga förslagshandlingar för fastigheten. Handlingar tas fram utefter en noggrann platsanalys som i sin tur resulterar i en byggnad med butik i hörnet av plan 0, trygghetsboende på plan 1-2 och bostäder på plan 3-7. Även ett gångstråk skapas mellan parkerna Vitabergsparken och Lilla Blecktornsparken. Tanken med arkitekturen under hela projektet har varit att skapa ett nytt avslut av Ringvägen, med en stilren byggnad som står sig i tiden. / Stockholm is growing and that in turn requires new buildings. The free surfaces of Stockholm decreases and Stockholm City are trying to find new ways to improve the city. The property Gulbetan is located on Södermalm in connection with Ringvägen and Vitabergsparken. Today the site is used for parking and recycling which makes the place dismal and it does not fulfill its full potential. This thesis leads to the final draft documents for the property. Documents are produced along a thorough site analysis, which in turn results in a building with a shop on the corner of plane 0, sheltered housing on floors 1-2 and apartments on floors 3-7. Even a pedestrian area created between the parks Vitabergsparken and Lilla Blecktornsparken. The idea of the architecture throughout the project has been to create a new clearance of Ringvägen, with a stylish building that is timeless.

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Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

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Proteins are among the most versatile organic macromolecules essential for living systems and present in almost all biological processes. Cancer is associated with mutations that either enhance or disrupt the conformation of proteins. These mutations have been shown to accumulate in specific regions of a proteins three dimensional structure. In this thesis, the aim is to find connections that secondary structure elements make and explore them using a self-organizing map (SOM). The detection of these connections is done by first mapping the three-dimensional structure onto a novice type of distance matrix that also incorporates chemical information, and then deploying a density-based clustering algorithm. The connections found are mapped onto the SOM and later analyzed in order to see if highly mutated connections are more common among certain SOM-nodes. This was tested with an ANOVA that indicated that there are indeed mutational asymmetries among the nodes. By further analyzing the map it could also be stated that certain nodes were to a large extent activated by connections from genes associated with cancer. / Proteiner tillhör några av de mest mångsidiga organiska makromolekylerna, och är direkt nödvändiga för alla levande system och biologiska processer. Cancer orsakas av mutationer som antingen förstärker eller stör strukturen hos proteinet. Dessa mutationer tenderar att att samlas i specifika områden av proteinets tredimensionella struktur. I den här rapporten är målet att hitta kopplingar som sekundärstrukturselement skapar, och utforska dem med hjälp av en självorganiserande karta. Dessa kopplingar finnes genom att först skapa en tvådimensionell representation av proteinets tredimensionella struktur, och sedan använda en densitetsbaserad klustringsalgoritm. De funna kopplingarna mappas till de olika neuronerna i kartan och analyseras sedan för att se om kopplingar med hög mutationsnivå är mer vanliga hos vissa neuron. För att undersöka detta användes ett ANOVA-test som visade att så var fallet. Genom att ytterligare studera kartan upptäcktes fynd som indikerade att vissa neuron i högre utsträckning var aktiverade av kopplingar som härstammar från gener vi vet är associerade med cancer.

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Pin1 (protein interacting with NIMA 1) is a peptidyl-prolyl isomerase involved in mitosis. As a potential anti-cancer drug target, Pin1 interacts and regulates the activity of an increasing number of cell cycle enzymes by an unknown mechanism. These cell cycle enzymes include Cdc25, Cdc27, Cyclin D1, Myt1, Wee1, NIMA, Cdc2, Plk1 and c-Myc. Recent research has revealed that Pin1 is overexpressed in a variety of cancer cell lines and Pin1 inhibitors inhibit proliferation activity of several cancer cells overexpressing Pin1. The most potent Pin1 inhibitors identified so far are in the micromolar range and no pharmacophore has been identified. In order to assist the understanding of the biological function of Pin1 using molecular probes, two amide isosteres of Ser-<i>trans</i>-Pro and Ser-<i>cis</i>-Pro dipeptides were designed and stereoselectively synthesized. The conformationally locked Ser–<i>trans</i>–Pro mimic, Boc-SerΨ[(<i>E</i>)CH=C]Pro–OH, was synthesized through the use of an Ireland-Claisen [3,3]-sigmatropic rearrangement in nine steps with 13% overall yield from a serine derivative. The Ser-<i>cis</i>-Pro mimic, Boc-SerΨ[(<i>Z</i>)CH=C]Pro–OH, was synthesized through the use of a Still-Wittig [2,3]-sigmatropic rearrangement in 11 steps with an overall yield of 20% from the same starting material. Conformationally locked peptidomimetics, including two exactly matched peptidomimetics, Ac–Phe–Phe–pSer–Ψ(<i>E</i>)CH=C]Pro–Arg–NH2 and Ac–Phe–Phe–pSer–Ψ[(<i>Z</i>)CH=C]Pro–Arg–NH2, were synthesized from these Ser-Pro isosteres using Fmoc SPPS. A protocol for in vitro Pin1 inhibition assay was established for measuring the inhibition constant for these peptidomimetics. A conformationally locked cis peptidomimetic inhibits Pin1 with a <i>K</i>_i of 1.7 <i>μ</i>M, 23-fold more potent than its trans counterpart, illustrating the preference of Pin1 for a cis amide bond in its PPIase domain. The A2780 ovarian cancer cell antiproliferation activity of these peptidomimetics parallels their respective Pin1 inhibition data. This research provides a start toward more drug-like Pin1 inhibitor design. Gly–<i>trans</i>–Pro isosteres were synthesized using the Ireland-Claisen route. The construction of a non-peptidic (Z)-alkene library for Pin1 inhibition was attempted using the Ser-<i>cis</i>-Pro mimic, Boc—SerΨ[(Z)CH=C]Pro–OH as the core. / Ph. D.

Pin1 is a peptidyl prolyl isomerase (PPIase) enzyme with two domains, the catalytic domain and the WW domain. Both domains specifically bind pSer/pThr–Pro motifs. Pin1 plays an important role in regulating the cell cycle, and it is involved in many diseases, such as cancer, HIV-1, Alzheimer's disease, asthma, hepatitis B, and rheumatoid arthritis. Pin1 is a very promising target for new drug development. Three stereoisomers: (R,S)-, (S,R)- and (S,S)-Ac–pSer–Ψ[(Z)CH=C]–Pip–2-(2-naphthyl)ethylamine were synthesized as inhibitors binding to the Pin1 catalytic domain. The (R,S)- and (S,R)-isomers were synthesized via a 13-step route, with overall yields of 2.0% and 1.4%, respectively. The newly formed stereogenic center in the piperidyl ring was introduced by a Luche reduction, followed by a stereoselective [2,3]-Still-Wittig rearrangement. The configuration of the stereocenter was determined by NOESY of a bicyclic derivative. The (Z)- to (E)-alkene ratio in the rearrangement was (5.5:1). The (S,S)-isomer was obtained as the epimerized by-product resulting from the (S,R)-isomer in the Na/NH3 deprotection step. The IC50 values for Pin1 inhibition were: 52, 85, and 141 μM, respectively. We concluded that in this Z-alkene isostere, the R-configuration would be preferred at both stereogenic centers, as mimics of L-Ser and L-Pip, to improve the affinity. Combinatorial chemistry is a powerful method to discover biologically active compounds, and solid-phase synthesis is most commonly used to synthesize combinatorial libraries. To identify ligands for the Pin1 WW domain, a library, R1CO–pSer–Pro–NHR2, was designed. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite function was synthesized in one step from commercially available Wang resin. The SPPR was applied in the preparation of a designed library through parallel synthesis. The library contained 357 members (17 Ã 21), and was screened by an enzyme-linked enzyme binding assay (ELEBA). The best hits were resynthesized, and the competitive dissociation constants, Kd-rel, were measured by ELEBA, with a Kd-rel value of 130 μM for the best ligand. The absolute dissociation constants will be measured by our collaborator, Prof. Jefferey Peng, University of Notre Dame, using NMR methods. Besides the identification of the Pin1 WW domain ligands, I created a practical method for solid-phase synthesis of phosphopeptides. / Ph. D.