京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20304号 / 薬科博第73号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

TRiC (TCP-1 ring complex), a type II chaperonin, facilitates protein folding, and we previously showed that TRiC crosslinks to ribosome-bound actin and luciferase nascent chains. Here, it was found that actin and luciferase nascent chains were adjacent to more than one TRiC subunit at different stages of translation. Six and seven out of the eight TRiC subunits were photocrosslinked to the luciferase and actin nascent chains, respectively. Actin nascent chains with widely-spaced, site-specific probe locations were adjacent to the same three TRiC subunits (a, b and e) at different stages of translation. The exposure of other TRiC subunits to nascent chains varied with the length and identity of the nascent chain. In addition, the presence or absence of ATP influences the photocrosslinking yields. This suggests that ATP alters the conformation of the subunits and/or their affinity for the nascent chain. Photocrosslinking also revealed that TRiC is in close proximity to the exit site of the ribosomal tunnel, presumably to create a protected folding environment for the nascent chain. Immunoprecipitations under native conditions revealed that prefoldin photocrosslinks to the actin nascent chain and that these prefoldin-containing photoadducts are coimmunoprecipitated with antibodies specific for the TRiC a subunit. This result suggests that prefoldin and TRiC bind simultaneously to the same actin nascent chain. Photocrosslinking studies with probes at position 68 in the actin nascent chain revealed that prefoldin binds to the nascent chain subsequently to TRiC binding. An unknown protein with an apparent molecular mass of 105 kDa was shown to photocrosslink to the luciferase nascent chain in a length-dependent manner at specific probe locations close to the N-terminus of the nascent chain. Thus, the nascent chain sees a variety of proteins in its immediate environment as it emerges from the ribosomal tunnel and undergoes its chaperonin-assisted folding.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18211号 / 薬博第801号 / 新制||薬||237(附属図書館) / 31069 / 京都大学大学院薬学研究科生命薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

La chaperonine CCT/TriC tient un rôle central dans le maintien de la protéostase cellulaire. Elle compte parmi ses clientes un grand nombre de protéines impliquées de près ou de loin dans la régulation du cycle comme l’actine, la tubuline et la cycline E. afin de mieux comprendre l’implication de la CCT/TriC dans la cancérogenèse, nous avons quantifié, dans 18 lignées cellulaires tumorales, une lignée issue d’un tissu sain et un tissu sain, ses taux d’expression et son activité. Nos résultats indiquent que l’expression de la CCT/TriC n’est pas toujours corrélée à son activité. Nous avons dans un second temps, documenté l’expression de ces partenaires/modulateurs d’activité, préfoldine, PhLP3, Hop/p60, Hsp/c70 et leur influence sur son activité. Nos résultats montrent que l’activité de la CCT/TriC pourrait être régulée à travers les variations des niveaux d’expression de la préfoldine et/ ou Hsp/c 70. Enfin, nous avons montré que dans les cellules qui se divisent le plus lentement, l’activité de la CCT/TRiC est la plus faible. Ces observations montrent que les variations de l’activité de la CCT/TriC pourraient constituer un point de contrôle dans la prolifération cellulaire maligne. / The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis. It mediates the folding of lot of proteins involved in cell cycle regulation as the major cytoskeletal proteins tubulins and actins and the oncoprotein cyclin E. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsp/Hsc70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Finally, our study reveals that cells with the longest doubling time host the smallest CCT/TRiC activity suggesting that CCT/TRiC-mediated client protein folding constitutes a bottle-neck in cancer cell proliferation. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.

Excitation-contraction (EC) coupling is the process by which Ca²⁺ is released from the sarcoplasmic reticulum (SR) and is fundamental to cardiac and skeletal muscle function. The SR contains many uncharacterised ion channels and proteins which may influence EC coupling and in this thesis I have investigated the biophysical properties of some of these channels. I have demonstrated that the single-channel gating and conducting properties of SR K⁺ channels from various mammalian species (rabbit, sheep and mouse) are very similar. I investigated the actions of possible physiological regulators of these channels and demonstrated that luminal Ca²⁺ and Mg²⁺ can block K⁺ flux in a voltage-dependent manner, while luminal Ca²⁺, Ni²⁺, and alkaline pH can reduce Po by additional mechanisms. I also characterised the single-channel properties of the various SR anion channels that are observed after incorporating mammalian SR vesicles into artificial membranes. The trimeric intracellular cation channels (TRIC-A and TRIC-B) and Mitsugumin 23 (MG23) are suggested to be SR cation channels. I have therefore utilised Tric-a KO and Mg23 KO mice to study SR membranes devoid of TRIC-A and MG23. Additionally, I have begun to investigate the single-channel properties of purified c. elegans TRIC-B1 and human TRIC-A. I found that SR K⁺ channel function was altered in SR from Tric-a KO or Mg23 KO tissue, however the underlying mechanisms for the observed changes appear to be complex. My initial studies of the purified TRIC-A and TRIC-B proteins show that they are permeable to K⁺, Ca²⁺, choline, and Cl^-, properties which deviate from those of SR K⁺ channels from rabbit, mouse and sheep. This may reflect species differences or alterations to protein function caused during the purification process or that SR K⁺ channels remain an unidentified class of ion channel.

Made available in DSpace on 2014-12-17T14:57:44Z (GMT). No. of bitstreams: 1 AndreaCSC.pdf: 2806475 bytes, checksum: a164123a8cfe29ac9b333a265d0521fa (MD5) Previous issue date: 2007-09-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The tricalcium phosphate ceramics has been widely investigated in the last years due its bioresorbable behavior. The limiting factor of the application of these materials as temporary implants is its low strength resistance. The tricalcium phosphate presents an allotropic transformation β→α around 1250 ?C that degrades its resistance. Some studies have been developed in order to densify this material at this temperature range. The objective of this work is to study the influence of the addition of magnesium oxide (MgO) in the sintering of β-TCP. The processing route was uniaxial hot pressing and its objective was to obtain dense samples. The samples were physically characterized through density and porosity measurements. The thermal behavior was studied through dilatometric, thermal differential and thermogravimetric analysis. The mechanical properties were characterized by three point flexure test and Vickers microhardness measurements, analyzed of the microstructure. The addition of magnesium oxide doesn t cause an improvement of the mechanical strength in relation to material without additive. / As cer?micas de fosfato de c?lcio (β-TCP) t?m sido intensamente investigadas nos ?ltimos anos devido as suas caracter?sticas bio - absorv?veis. Um fator limitante da aplica??o destes materiais em implantes tempor?rios ? a sua baixa resist?ncia mec?nica. O tricalciofosfato apresenta uma transforma??o alotr?pica β→α em torno de 1250 ?C, o que degrada significativamente sua resist?ncia mec?nica. V?rios estudos t?m sido realizados com o intuito de densificar este material nesta faixa de temperatura. O objetivo desse trabalho ? estudar a influ?ncia da adi??o do aditivo (MgO) na sinteriza??o do β-TCP. O m?todo de processamento utilizado foi prensagem uniaxial a quente, teve por objetivo obter corpos densos. As amostras foram caracterizadas por meio de medidas de porosidade aparente e densidade aparente e termicamente por dilatometria, an?lise termogravim?trica e t?rmica diferencial. Os corpos sinterizados foram caracterizados mecanicamente por resist?ncia a flex?o em 3 pontos, microdureza Vickers e an?lise da microestrutura. A adi??o do ?xido de magn?sio n?o ocasionou melhoria na resist?ncia mec?nica em rela??o ao material sem aditivo

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第19646号 / 薬博第816号 / 新制||薬||239(附属図書館) / 32682 / 京都大学大学院薬学研究科生命薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 岡村 均 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Das 20S Proteasom ist das Kernelement des Ubiquitin-Proteasom-Systems und baut fehlerhafte, nicht mehr benötigte und oxidierte Proteine ab, wobei drei katalytisch aktive Untereinheiten die Polypeptidkette schneiden. Das proinflammatorische Cytokin IFNg induziert die Expression und Inkorporation der alternativen katalytisch aktiven Immunountereinheiten, was in variablen Isoformen des 20S Proteasoms resultiert. Die zusätzliche Assoziation des 19S Regulators, bzw. des PA28 und des PA200 Aktivators an eine Isoform erweitert das Sortiment an proteasomalen Komplexsubtypen. Der zeitliche Verlauf einer IFNg Stimulation zeigte, dass die Aktivatoren PA28 und PA200 antagonistisch an das 20S Proteasom assoziieren und niedermolekulare Komplexsubtypen bilden, sodass in dieser Studie auch zum ersten Mal eine IFNg abhängige Assoziation des PA200 Monomers an das 20S Proteasom detektiert wurde. Ex vivo Versuche zeigten, dass die Defizienz der Immunountereinheit LMP7 mit der Assoziation des PA28-Aktivators an das 20S-19S Proteasom kompensiert wird, wobei die funktionelle Wirksamkeit aber offen bleibt. In einer monozytären Zelllinie wird ein sehr hochmolekularer, chymotryptisch aktiver Komplex assembliert und massenspektrometrische Analysen detektierten proteasomale Untereinheiten und viele Komponenten der Proteinbiosynthese, was für eine Assoziation des Proteasoms mit dem Polysom spricht. Diese Möglichkeit der kotranslationalen Degradation kann auch die Assoziation des detektieren Chaperonins TriC erklären, wobei dieser Komplex, der ATP abhängig Proteine faltet, möglicherweise auch direkt mit dem Proteasom interagieren könnte, wie elektronenmikroskopische Aufnahmen belegten. Neben den neuen strukturellen Ergebnissen, bestätigte die funktionelle Analyse den Abbau polyubiquitinierter Substrate durch 19S-Regulator assoziierte Komplexsubtypen, doch das 19S-20S-19S Proteasom konnte das Modellsubstrat HA-Ubi-IkBa-flag besser abbauen und deubiquitinieren als das 20S-19S Proteasom. / The 20S proteasome is the core element of the ubiquitin-proteasome-system, which degrades defective, unneeded and oxidized proteins, while three catalytically active subunits hydrolyze the peptide bonds of the polypeptide. The proinflammatory cytokine IFNg induces the expression and incorporation of three alternative, catalytically active immunosubunits resulting in variable isoforms of the 20S proteasome. The additional association of the 19S regulator, or the PA28 and PA200 activator, respectively, expands the range of proteasome complex subtypes. The time course of IFNg stimulation showed that the proteasomal association of PA28 and PA200 occurs antagonistically, forming low molecular weight complex subtypes. Furthermore, this study revealed for the first time an IFNg dependent association of the PA200 monomer to the 20S proteasome. Ex vivo experiments showed that the deficiency of the immunosubunit LMP7 is compensated by the association of the PA28 activator to the 20S-19S proteasome, whereas the functional efficacy remains elusive. In a monocytic cell line, a chymotryptic active complex with a very high molecular weight was detected, and mass spectrometry confirmed proteasomal subunits and components of the protein synthesis machinery, suggesting an association of the proteasome with the polysome. The fact of cotranslational degradation may also explain the association of the chaperonin TriC, an ATP dependent protein folding chaperonin. Electron micrographs could reveal that TriC possibly interacts directly with the proteasome. Next to the new structural results, the functional analysis confirmed the degradation of polyubiquitinated substrates by 19S regulator associated complex subtypes, and in addition to it, the 19S-20S-19S proteasome degraded and deubiquitinated the model substrate HA-Ubi-IkBa-flag better than the 20S-19S proteasome.

Made available in DSpace on 2014-12-17T14:57:45Z (GMT). No. of bitstreams: 1 EduardoGR.pdf: 1635946 bytes, checksum: 593244cdda721eea4684ae85323cfee4 (MD5) Previous issue date: 2006-06-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The calcium phosphate ceramics have been very investigated as material for bone implants. The tricalcium phosphate (β-TCP) had a great potential for application in temporary implants like a resorbable bioceramic. This material presents a limitation in its sintering temperature due to occurrence of the allotropic transformation β → α at temperatures around 1200?C, not allowing the attainment of dense ceramic bodies. This transformation also causes cracks, what diminishes the mechanical strength, limiting its use to applications of low mechanical requests. This work studies the influence of the addition of manganese oxide in the sintering of β-TCP. Two processing routes were investigated. The first was the powder metallurgy conventional process. The test bodies (samples) were pressed and sintering at temperatures of 1200 and 1250?C. The second route was uniaxial hot pressing and its objective was to obtain samples with high relative density. The samples were physically characterized through density and porosity measurements. The thermal behavior was studied through dilatometric, thermal differential and thermogravimetric analysis. The mechanical properties were characterized by three point flexure test and Vickers microhardness measurements. The microstructure was analyzed by scanning electron microscopy. The addition of manganese oxide caused an improvement of the mechanical strength in relation to the material without additive and promoting the stabilization of β-TCP to greater temperatures / As cer?micas de fosfato de c?lcio t?m sido intensamente investigadas como materiais para implantes ?sseos. O fosfato tric?lcico (β-TCP) possui um grande potencial para aplica??o em implantes tempor?rios por ser uma biocer?mica absorv?vel. Entretanto, este tipo de material apresenta uma limita??o na sua temperatura de sinteriza??o devido ? ocorr?ncia da transforma??o alotr?pica β → α em torno de 1200?C. Isto impede a obten??o de corpos cer?micos densos e provoca trincas, diminuindo a resist?ncia do material e limitando a sua utiliza??o a aplica??es de baixa solicita??o mec?nica. A influ?ncia da adi??o de ?xido de mangan?s na sinteriza??o do β-TCP foi estudada neste trabalho. Duas rotas de processamento foram investigadas. A primeira utilizou o processo convencional de metalurgia do p?. Os corpos de prova foram prensados, sendo posteriormente sinterizados nas temperaturas de 1200 e 1250?C. O segundo m?todo de processamento utilizou a rota de prensagem uniaxial a quente, e tinha como objetivo obter corpos de prova com alta densidade relativa. As amostras foram caracterizadas fisicamente por meio de medidas de porosidade e densidade e termicamente por dilatometria e an?lise termogravim?trica e t?rmica diferencial. Os corpos sinterizados foram caracterizados mecanicamente por resist?ncia a flex?o em 3 pontos e microdureza Vickers, sendo tamb?m sua microestrutura analisada por microscopia eletr?nica de varredura. A adi??o do ?xido de mangan?s ocasionou uma melhoria da resist?ncia mec?nica em rela??o ao material sem aditivo, al?m de promover uma estabiliza??o do β-TCP em temperaturas mais elevadas