Sufficient and balanced pools of deoxyribonucleotide triphophates (dNTPs) is crucial for high-fidelity DNA replication as well as correct DNA repair. The enzyme RiboNucleotide Reductase (RNR) catalyses NDP to dNDP and is therefore an essential enzyme by providing the “building blocks” to the cells. dNTPs production needs to be tightly regulated in order to minimize mutation frequencies and prevent genome instability. RNR in S. pombe is composed of two proteins, Cdc22R1 and Suc22R2, and has been described as a heterotetramer with a dimer of each subunit: the big subunit Cdc22R1 and the small subunit Suc22R2. S. pombe also posseses an RNR inhibitor: Spd1, as well as a second RNR regulator Spd2 which has been newly discovered. Spd1 has been demonstrated to inhibit RNR and to regulate its activity throughout the cell cycle. The detailed mechanism of the RNR regulation during the cell cycle or after DNA damage is not entirely clear, as are the means of inhibition by Spd1. In order to shed some light on the RNR complex and its regulation, we used various microscopybased methods to study RNR in vivo as well as in vitro. The data of this thesis suggest there are different forms of active RNR heterocomplexes, found throughout the cell cycle in the cytoplasm as well as in the nucleus. We propose that the precise stoichiometry of subunits in the complexes may vary, or that the complex conformation may be modified in an Spd1-dependent manner. In addition, treatment of the cells with a UV mimetic agent, 4NQO, seems to promote RNR regulation in an Spd1-dependent manner. On the contrary, inhibition of RNR by HydroxyUrea (HU) affects the RNR in a possible structure-related manner, independently of Spd1 or Spd2. The in vivo observations correlate with structural and/or oligomerization modifications of the RNR, representing a novel RNR regulation in S. pombe.

The isoprenoid biosynthetic pathway is targeted in the treatment of several diseases, including hypercholesteremia and bone related disorders. Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are isoprenoid biosynthetic pathway intermediates that are utilized during post-translational modification of proteins termed farnesylation and geranylgeranylation, respectively, together known as prenylation. The Ras and Rho GTPase family members are examples of proteins that are prenylated. Prenylation is essential for proper membrane localization and function of these small GTPases. Activating mutations or over-expression of these proteins promote oncogenic events, such as increased proliferation and migration. Studies have demonstrated that farnesyl transferase inhibitors and geranylgeranyl transferase inhibitors possess anti-cancer effects in humans and animal models of cancer, respectively. An alternative way to impair protein prenylation is through the depletion of FPP and GGPP. Statins and nitrogenous bisphosphonates (NBPs) deplete FPP and GGPP leading to impaired protein prenylation by inhibiting HMG-CoA Reductase (HMGCR) and FPP synthase (FDPS), respectively. These drugs have been shown to induce apoptosis, inhibit cancer cell migration, and induce cell cycle arrest. The anti-cancer effects of statins and NBPs can be prevented by GGPP addition, suggesting that GGPP depletion may be the mechanism by which these agents interfere with cancer cell survival. We and our collaborators have developed bisphosphonate inhibitors of GGPP synthase (GGDPS), an enzyme that produces GGPP from the substrates FPP and isopentenyl pyrophosphate. The goal of this research was to identify novel GGDPS inhibitors and to assess the effects of specific inhibition of GGDPS on cancer cell survival and function. Two aromatic bisphosphonates were identified as potent inhibitors of GGDPS in enzyme and cellular assays. Apoptosis hallmarks such as PARP cleavage and DNA fragmentation demonstrated that GGDPS inhibition induces apoptosis in K562 chronic myeloid leukemia cells through GGPP depletion and FPP accumulation. Isobologram analysis and enhanced impairment of protein geranylgeranylation showed that GGDPS inhibition is synergistic with the inhibition of HMGCR. Migration assays, transwell assay and large scale digital cell analysis system microscopy, demonstrated that GGDPS inhibition interferes with MDA-MB-231 breast cancer cell migration. Increased LC3-II expression showed that FDPS and GGDPS inhibition induces autophagy in PC3 prostate and MDA-MB-231 breast cancer cells. Inhibition of autophagy enhances the toxic effects of GGDPS inhibition as measured by MTT assay. Propidium iodine staining of DNA and immunostaining of cell cycle proteins such as p27 did not show significant effects of GGDPS inhibition on cell cycle progression. Importantly, exogenous addition of GGPP prevented most of the effects observed with GGDPS inhibition, suggesting specific inhibition of GGDPS by our bisphosphonate inhibitors. The data obtained herein suggest that GGDPS can be targeted to interfere with the progression of cancer cells.

Abstract not available

Le Phosphate Diphosphate de Thorium (β-PDT) est considéré comme une matrice<br />céramique potentielle en vue de l'immobilisation des actinides en formation géologique<br />profonde. Il s'est avéré donc impératif d'étudier les effets de l'irradiation sur la structure de<br />cette céramique et les conséquences sur sa durabilité chimique.<br />Des échantillons de β-PDT et des solutions solides associées de β-PDTU ont été irradiés<br />puis altérés en solution aqueuse. Selon la valeur du TEL électronique, le β-PDT peut<br />s'amorphiser totalement ou partiellement. Par ailleurs, la capacité de recristallisation du<br />matériau amorphe par recuit thermique a été démontrée. Les tests de lixiviation menés sur ces<br />échantillons irradiés ont montré une influence significative de la fraction amorphe sur la<br />vitesse de dissolution normalisée qui augmente d'environ un facteur 10 entre le matériau non<br />irradié et le matériau amorphe. Corrélativement, la fraction amorphe modifie le temps requis<br />pour atteindre les conditions de saturation associées aux équilibres thermodynamiques. En<br />revanche, elle ne présente aucune influence ni sur d'autres paramètres cinétiques, tels que<br />l'énergie d'activation du processus de dissolution ou l'ordre partiel par rapport aux protons, ni<br />la nature de la phase néoformée identifiée comme le Phosphate HydrogénoPhosphate de<br />Thorium Hydraté (PHPTH).<br />Des échantillons de β-PDTU ont aussi été irradiés sous rayonnements γ et α pendant les<br />tests de lixiviation afin d'étudier les effets de la radiolyse du milieu lixiviant sur la vitesse de<br />dissolution du matériau. Il est apparu que les espèces radiolytiques intervenant dans le<br />mécanisme de dissolution étaient peu stables, disparaissant rapidement dès la fin de<br />l'irradiation. Leur caractère fortement oxydant vis-à-vis de l'uranium tétravalent permet d'expliquer la différence de comportement entre les cations métalliques (U et Th)

Platelet transfusions can be necessary during treatment of patients with thrombocytopenia or impaired platelet function. Platelet function in platelet concentrates (PC) deteriorate with storage time. Studying swirling is often used to control the quality of PC’s before transfusion but the method has some disadvantages. Therefore other methods can be useful, for example impedance aggregometry (IA, Multiplate® Analyzer) to measure platelet function.      In this study the change in platelet function over time was examined in buffy coat and apheresis platelets with IA where aggregation had been induced with adenosine diphosphate (ADP) and collagen. PC’s were tested on day 1, 4 and 7 after donation. One of the main aims of this study was to evaluate if dilution to a standardized platelet concentration (800x109 platelets/L) for IA of PC’s could be used, since platelet concentration has been shown to influence aggregation. The effect of pathogen inactivation (INTERCEPT) on platelet function and the importance of fibrinogen for aggregation were also studied.      The dilution of platelet samples reduced the range of measured values and was suitable to use with collagen but not ADP. The platelet function decreased significantly over time with both agonists. There was a significant difference between pathogen inactivated and gamma irradiated PC’s with collagen activation on day 1. Fibrinogen was shown to be of importance for platelet aggregation, but other factors in plasma seem to be necessary too.      In conclusion, IA is a suitable method for following change in aggregability over time in PC’s and sample dilution reduced variation in results.

At least three poorly soluble compounds can be formed in the CuSO4 – K4P2O7 – H2O system: a single salt Cu2P2O7.5H2O and two dimorph’s both having chemical formula Cu3K2(P2O7)2.3H2O. A previously known form of Cu3K2(P2O7)2.3H2O (we named it Dimorph A) transforms into a novel Dimorph B, which has a different structure. The similarities between the XRD patterns and vibrations spectra of copper–ammonium and copper–potassium Dimorph’s B imply that they are isostructural. The values of the angle for copper–potassium and copper–ammonium salts and Cu2P2O7.5H2O are relatively low and vary within a narrow range (123.1 – 127.1°). The formation of the compounds significantly depends on the conditions of the experiment. A double salt in the CuSO4 – K4P2O7 – H2O system may be formed after some time in the reaction mixture as a result of the following heterogeneous reaction: 3Cu2P2O7∙5H2O↓ + H2P2O72– + 4K+ → 2Cu3K2(P2O7)2∙3H2O↓ + 2H+ + 9H2O This reaction has not been previously described for any polyphosphate system. Therefore, novel results of scientific investigation are presented. The rate, duration and yield of heterogeneous reaction substantially depend on pH of the solution, [Cu2+ + P2O74–] and n ([P2O74–] / [Cu2+]). At a relatively low pH the induction time is high, the reaction rate is low and duration is long. The maximal yields close to 100% can be achieved when [Cu2+ + P2O74–] = 0.1 M. At increased pH values, the induction time, the yield and duration of the reaction diminish... [to full text] / Naudojant IR-spektrometrijos, rentgenofazinės analizės, atominės absorbcinės spektroskopijos, kolorimetrijos analizės metodus buvo ištirta sistemose CuSO4 – K4P2O7 – H2O ir CuSO4 – (NH4)4P2O7 – H2O susidarančių mažai tirpių junginių sudėtis, jų susidarymo dėsningumai ir vykstančios reakcijos, įvairių veiksnių įtaką nuosėdų sudėčiai ir struktūrai. Nustatyta, kad sistemoje CuSO4 – K4P2O7 – H2O, be jau žinomų mažai tirpių junginių (paprastosios druskos Cu2P2O7 ∙ 5H2O ir dvigubosios druskos Cu3K2(P2O7)2 ∙ 3H2O), susidaro ir naujas mokslinėje literatūroje neaprašytas junginys (Dimorfas B), dvigubajai druskai patiriant polimorfinį perėjimą. Polimorfinio perėjimo trukmė labai priklauso nuo pH ir reaguojančių jonų koncentracijų. FTIR duomenų analizė parodė, kad difosfato jonų energetinė būsena Cu – K ir Cu – NH4 Dimorfuose B panaši. Jų rentgenogramos yra panašios, tikėtina, kad šie junginiai – izostruktūriniai, jų kristalinės gardelės tipas – monoklininis. Naudojant Lazarevo lygtį, bei Rulmonto priklausomybę buvo apskaičiuoti Cu – K ir Cu – NH4 Dimorfų A ir B bei paprastosios druskos P – O – P kampai, gauta, kad jie mažai keičiasi siaurame 123,1 – 127,1 º intervale. Nustatyta, kad dviguboji druska susidaro iš paprastosios, vykstant heterogeninei reakcijai: 3Cu2P2O7 ∙ 5H2O↓ + H2P2O72– + 4K+ → 2Cu3K2(P2O7)2∙ 3H2O↓ + 2H++ 9H2O. Mokslinėje literatūroje tokia reakcija neaprašyta jokiai difosfatų sistemai, jos tyrimo duomenys yra nauji. Taip pat nustatyta, kad heterogeninės reakcijos... [toliau žr. visą tekstą]

Disertaciniame darbe naudojant IR-spektrometrijos, rentgenofazinės analizės, atominės absorbcinės spektroskopijos, kolorimetrijos analizės metodus buvo ištirta sistemose CuSO4 – K4P2O7 – H2O ir CuSO4 – (NH4)4P2O7 – H2O susidarančių mažai tirpių junginių sudėtis, jų susidarymo dėsningumai ir vykstančios reakcijos, įvairių veiksnių įtaka nuosėdų sudėčiai ir struktūrai. Nustatyta, kad sistemoje CuSO4 – K4P2O7 – H2O, be jau žinomų mažai tirpių junginių (paprastosios druskos Cu2P2O7 5H2O ir dvigubosios druskos Cu3K2(P2O7)2 H2O, susidaro ir naujas mokslinėje literatūroje neaprašytas junginys (Dimorfas B), dvigubajai druskai patiriant polimorfinį perėjimą. Polimorfinio perėjimo trukmė labai priklauso nuo pH ir reaguojančių jonų koncentracijų. FTIR duomenų analizė parodė, kad difosfato jonų energetinė būsena Cu – K ir Cu – NH4 Dimorfuose B panaši. Jų rentgenogramos yra panašios, tikėtina, kad šie junginiai – izostruktūriniai, jų kristalinės gardelės tipas – monoklininis. Naudojant Lazarevo lygtį bei Rulmonto priklausomybę buvo apskaičiuoti Cu – K ir Cu – NH4 Dimorfų A ir B bei paprastosios druskos P – O – P kampai, gauta, kad jie mažai keičiasi siaurame 123,1 – 127,1 º intervale. Nustatyta, kad dviguboji druska susidaro iš paprastosios, vykstant heterogeninei reakcijai: 3Cu2P2O7 5H2O↓ + H2P2O72– + 4K+ → 2Cu3K2(P2O7)2∙3H2O↓ + 2H++ 9H2O. Mokslinėje literatūroje tokia reakcija neaprašyta jokiai polifosfatų sistemai, jos tyrimo duomenys yra nauji. Taip pat nustatyta, kad heterogeninės... [toliau žr. visą tekstą] / At least three poorly soluble compounds can be formed in the CuSO4 – K4P2O7 – H2O system: a single salt Cu2P2O7.5H2O and two dimorph’s both having chemical formula Cu3K2(P2O7)2.3H2O. A previously known form of Cu3K2(P2O7)2.3H2O (we named it Dimorph A) transforms into a novel Dimorph B, which has a different structure. The similarities between the XRD patterns and vibrations spectra of copper–ammonium and copper–potassium Dimorph’s B imply that they are isostructural. The values of the angle for copper–potassium and copper–ammonium salts and Cu2P2O7.5H2O are relatively low and vary within a narrow range (123.1 – 127.1°). The formation of the compounds significantly depends on the conditions of the experiment. A double salt in the CuSO4 – K4P2O7 – H2O system may be formed after some time in the reaction mixture as a result of the following heterogeneous reaction: 3Cu2P2O7∙5H2O↓ + H2P2O72– + 4K+ → 2Cu3K2(P2O7)2∙3H2O↓ + 2H+ + 9H2O This reaction has not been previously described for any polyphosphate system. Therefore, novel results of scientific investigation are presented. The rate, duration and yield of heterogeneous reaction substantially depend on pH of the solution, [Cu2+ + P2O74–] and n ([P2O74–] / [Cu2+]). At a relatively low pH the induction time is high, the reaction rate is low and duration is long. The maximal yields close to 100% can be achieved when [Cu2+ + P2O74–] = 0.1 M. At increased pH values, the induction time, the yield and duration of the reaction diminish... [to full text]

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 3 uppsatser.

No description available.

Background: Peroxisome proliferator-activated receptor δ (PPARδ) is a versatile regulator of distinct biological processes and overexpression of PPARδ in cancer may be partially related to its suppression of its own co-regulators. Aims: To determine whether recruited suppressor proteins bind to and regulate PPARδ expression, activity and PPARδ-dependent cholangiocarcinoma proliferation. Methods: Yeast two-hybrid assays were done using murine PPARδ as bait. PPARδ mRNA expression was determined by qPCR. Protein expression was measured by western blot. Immunohistochemistry and fluorescence microscopy were used to determine PPARδ expression and co-localization with NDP Kinase alpha (NM23-H2). Cell proliferation assays were performed to determine cell numbers. Results: Yeast two-hybrid screening identified NM23-H2 as a PPARδ binding protein and their interaction was confirmed. Overexpressed PPARδ or treatment with the agonist GW501516 resulted in increased cell proliferation. NM23-H2 siRNA activated PPARδ luciferase promoter activity, upregulated PPARδ RNA and protein expression and increased GW501516-stimulated CCA growth. Overexpression of NM23-H2 inhibited PPARδ luciferase promoter activity, downregulated PPARδ expression and AKT phosphorylation and reduced GW501516-stimulated CCA growth. Conclusions: We report the novel association of NM23-H2 with PPARδ and the negative regulation of PPARδ expression by NM23-H2 binding to the C-terminal region of PPARδ. These findings provide evidence that the metastasis suppressor NM23-H2 is involved in the regulation of PPARδ-mediated proliferation.