Zur Charakterisierung der myokardialen SR Ca2+-Transportaktivität nach experimentellem Myokardinfarkt wurde 10-15 Wochen alten Ratten die linke Koronararterie unterbunden und sechs Wochen später die Funktion und Expression der Ca2+-ATPase des sarkoplasmatischen Retikulums untersucht. Ein Teil der Tiere wurde mit dem Carnitin-Palmitoyl-Transferase-1- Inhibitor Etomoxir behandelt, der die mitochondrale Oxidation von langkettigen Fettsäuren hemmt und dadurch verstärkt Glukoseoxidation bewirkt. Diese Stoffwechselumstellung sollte die Schädigung der myokardialen Funktion und die gestörte zelluläre Ca2+-Homöostase nach Myokardinfarkt vermindern. Die mit Etomoxir behandelten Tiere wiesen nach sechs Wochen eine deutlich verminderte Infarktgröße auf. Durch die CPT-1-Hemmung entstand eine starke biventrikuläre Hypertrophie. Darüber hinaus ergaben sich Hinweise auf den Erhalt einer normalen Wandspannung des linken Ventrikels und einer verbesserten Kontraktilität gegenüber der unbehandelten Gruppe. Unter Etomoxir zeigte die für die diastolische Ca2+ Senkung verantwortliche ATP-abhängige Ca2+-Rückbindung in das SR verbesserte Transportraten. Damit korrelierte der immunchemisch gemessene erhöhte SERCA2a-Proteinspiegel. Die Ergebnisse lassen vermuten, daß es infolge einer Behandlung mit Etomoxir nach Myokardinfarkt zu einer verbesserten Zellstoffwechsellage des ischämisch nicht irreversibel geschädigten Myokards kommt. Dafür scheint die verstärkte Glukoseoxidation bei Hemmung der Oxidation langkettiger Fettsäuren im Herzmuskel verantwortlich zu sein. Die Größe des infarzierten Areals wird begrenzt, dadurch lassen sich zum Teil verbesserte hämodynamische Parameter und gesteigerte SR Ca2+-Transportraten und SERCA2a-Proteinspiegel erklären. Auf die Hämodynamik hat sicherlich auch die durch Etomoxir erfolgte myokardiale Hypertrophie einen wesentlichen Einfluß. Für weitere Studien bleibt abzuklären, durch welchen Mechanismus Etomoxir seine verbessernde Wirkung nach Myokardinfarkt entfaltet. Außerdem ist zu prüfen, ob nach akutem Myokardinfarkt beim Menschen mit dem Prinzip der chronischen Verschiebung der myokardialen Substratverwertung von Fettsäure- nach Glukoseoxidation eine verbesserte Überlebensrate und das Hinauszögern bzw. Verhindern einer Dekompensation des Herzens zu erreichen ist. / To characterise the activity of sarcoplasmic reticulum (SR) Ca2+ after myocardial infarction the left coronary artery of 10-15 week old male rats was ligated; six weeks later function and expression of the SR Ca2+-pump ATPase (SERCA2a) transport in the surviving myocardium was investigated. Part of the animals were treated with the carnithin palmitoyltransferase-1 (CPT-1) inhibitor etomoxir (8mg/kg/d for six weeks) to decrease the oxidation of long chain fatty acids. Due to the drug-induced shift from fatty acid to carbohydrate utilization an attenuated myocardial dysfunction and an improved SR Ca2+ handling homeostasis in the surviving myocardium could be expected. The etomoxir treated rats showed a decreased infarct size six weeks after coronary ligation. Due to CPT-1 inhibition a significant biventricular hypertrophy was observed. In addition, the treatment normalized elevated LVEDP and improved contractility. Compared to sham-operated controls treatment with etomoxir caused an enhanced SR Ca2+ uptake activity that correlated with increased immunoreactive SR Ca2+-ATPase levels. The results suggest that chronic inhibition of CPT-1 after myocardial infarction improves the metabolism of reversible damaged tissue. It appears that increased oxidation of glucose and inhibition of long chain fatty acid oxidation is responsible for this effect. Limitation of the infarct size induces improvement of haemodynamic parameters, increases SR Ca2+ uptake and protein levels of SERCA2a. Further studies are required to find out whether etomoxir is able to delay the development of congestive heart failure in humans and whether it could decrease mortality in patients after myocardial infarction.

<p>As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-1, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain of YB-1 may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-1 binds to the RING finger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-1 as a means of targeting the oncogenic effects of YB-1. In order to identify E2 enzymes involved in the ubiquitination of YB-1, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-1 in conjuction with RBBP6, whereas Ubc13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6.</p>

<p>As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-1, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain of YB-1 may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-1 binds to the RING finger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-1 as a means of targeting the oncogenic effects of YB-1. In order to identify E2 enzymes involved in the ubiquitination of YB-1, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-1 in conjuction with RBBP6, whereas Ubc13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6.</p>

Philosophiae Doctor - PhD / Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.

>Magister Scientiae - MSc / In the past few decades, the studies of f-electron materials have revealed unusual physical properties such as Fermi-liquid, non-Fermi-liquid behaviour at low temperatures, heavy- Fermion behaviour, valence fluctuation, Kondo effect, superconducting and magnetic ordering. These materials include binary and ternary compounds and alloys with Cerium (Ce) or Ytterbium (Yb) based rare earth elements or Uranium (U) based actinide element. In these systems the localized magnetic moments formed by Ce, Yb or U ions transform the electronic properties of these compounds leading to quasiparticles with masses in excess to 1000 times the bare electron mass. These materials are known as heavy-fermion materials. Two well known heavy – Fermion compounds with Ce based rare earth elements of interest in this thesis are CePt₂Si₂ and CeCu₅In. The effect of substituting Ce with moment bearing Tb or Dy in these two compounds, are reported through measurements of electrical resistivity, magnetic susceptibility and magnetization. The three alloy systems (Ce₁₋ₓREₓ)Pt₂Si₂ (RE = Tb, Dy) and (Ce₁₋ₓTbᵪ)Cu₅In under investigation in the present thesis, was synthesized and characterized by x-ray diffraction. The alloy systems (Ce₁₋ₓREₓ)Pt₂Si₂ (RE = Tb, Dy, 0≤ ᵡ ≤1) formed a single phase in the P4/nmm tetragonal CaBe₂Ge₂ – type structure across the whole series while the (Ce₁₋ₓTbᵪ)Cu₅In alloy system formed a single phase in the Pnma orthorhombic CeCu₆ – type crystal structure up to 40% Ce substitution. The physical properties of these systems is reported and discussed through the measurements of electrical resistivity, magnetic susceptibility and magnetization. The variables of this study are: the doping concentration of Tb or Dy, the applied magnetic field and the sample temperature. Electrical resistivity studies for all the systems revealed coherence effect at Ce – rich alloys (0≤ ᵡ ≤0.2) and single-ion Kondo scattering with further increased RE concentration ( ᵡ ≥ 0.3). The magnetic property studies indicate antiferromagnetic ordering only for the (Ce₁₋ₓREₓ)Pt₂Si₂ alloy system in the concentration range 0.7≤ ᵡ ≤ 1. The present thesis is comprised of six chapters, which are arranged as follows: The first chapter deals with the theoretical background of the physical properties of Ce based intermetallics compounds and alloys. Experimental techniques constitutes chapter II and explains the techniques used in this study. The theoretical overview of the two parent compounds of interest in this thesis (CePt₂Si₂ and CeCu₅In) is presented in chapter III. The fourth and the fifth chapters of this study deals with results and discussion. The thesis is completed with a conclusion in chapter six.

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Magister Scientiae - MSc / As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-1, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain of YB-1 may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-1 binds to the RING finger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-1 as a means of targeting the oncogenic effects of YB-1. In order to identify E2 enzymes involved in the ubiquitination of YB-1, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-1 in conjuction with RBBP6, whereas Ubc13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6. / South Africa

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22659号 / 工博第4743号 / 新制||工||1741(附属図書館) / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 山田 啓文, 教授 川上 養一, 准教授 杉山 和彦 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Thesis advisor: Baldassare Di Bartolo / Thesis advisor: Pradip Bakshi / The search for multicolored light produced by some IR laser-excited luminescent nano-powders has revealed, for laser power exceeding a threshold value, the emission of white light (WL) with black-body characteristics. I am directing my research to the study of the physical parameters that may influence the threshold power of the laser and the efficiency of the WL emission. A typical compound that I will investigate will consist of nano-powders of SrZrO3 doped with Yb. The parameters of relevance may include Yb concentration, pressure, temperature, size of nano-crystals, exciting power and wavelength of the laser, dynamical parameters such as decay and build-up patterns. The aim of my research will be both theoretical and experimental: theoretical for I will try to uncover the mechanism of the WL production and experimental for the possible application as efficient light sources of systems similar to the ones that I will investigate (oxide nano-powders doped with lanthanide or transition metal ions). The “new” light sources in the market (fluorescence lights sources, and LED lamps) beat the Edison bulbs in efficiency, but they do not produce the black-body emission of the Edison bulbs that is most pleasing to the eye. The search for efficient black-body type of sources is still on and we want to be a part of it. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Physics.

>Magister Scientiae - MSc / Retinoblastoma binding protein 6 (RBBP6) is a 250 kDa RING finger-containing protein whose function is known to be mediated through interaction with other proteins. RBBP6 plays a role in the regulation of the tumour suppressor protein p53 and is also thought to be involved in mRNA splicing although its role has yet to be characterised. A recent study utilising a yeast 2-hybrid screen identified the cancer-associated protein known as YB-l as an interacting partner of RBBP6, and showed that RBBP6 ubiquitinates YB-I, leading to its degradation in the proteasome.Human Y-box binding protein 1 (YB-I) is member of the cold-shock domain family of proteins, which regulates a number of growth related genes through both transcriptional and translational mechanisms. YB-l is a cell-survival factor whose expression is increased in proliferating normal and cancer cells. It also protects cells against p53-mediated apoptosis by repressing the p53- promoter and down-regulating endogenous p53. The interaction between RBBP6 and YB-l involves the RING finger-like domain ofRBBP6 and the C-terminal62 amino acids ofYB-l. As a means of further localising the interaction, truncated fragments derived from the C-terminal region of YB-I, were tested for their interaction with the RING finger domain of RBBP6 using three different assays: a directed yeast 2-hybrid assay, co-immunoprecipitation and NMR chemical shift perturbation analysis. Our results suggest that the entire 62 amino acid region at the C-terminal domain ofYB-l may be involved in the interaction with RBBP6. Using chemical shift perturbation analysis, this study provides an indication of where YB-l binds to the RING fmger. This represents the first step towards the design of therapeutics aimed at modulating the interaction between RBBP6 and YB-l as a means of targeting the oncogenic effects ofYB-l. In order to identify E2 enzymes involved in the ubiquitination of YB-I, we examined the efficiencies of selected E2s in an in vitro ubiquitination assay. UbcH5c and UbcH7 were both found to catalyse the ubiquitination of YB-l in conjuction with RBBP6, whereas Ubc 13 was not. Finally, we show using NMR that two single-point mutations of the RING finger-like domain are sufficient to abolish homodimerisation of the domain. These will be used in future studies to investigate the requirement for homodimerisation on the ubiquitination activity of RBBP6. www.etd.