Os efeitos de ribonucleosídeos de guanina substituídos na posição C-8 na proliferação de linfócitos B estão bem documentados na literatura. Esses compostos são análogos de adutos formados pela adição de radicais livres a ribonucleosídeos e a RNA. Neste trabalho, verificamos as propriedades proliferativas de dois desses adutos, 8-metilguanosina (8-MeGuo) e 8-oxo-7 ,8-di-hidroguanosina (8-OxoGuo) e comparamos com 8-bromoguanosina (8-BrGuo), o composto mais estudado como indutor da proliferação de linfócitos B. 8-MeGuo e 8-OxoGuo foram sintetisados em rendimentos de 28 e 55%, respectivamente, e foram caracterizados por UV, NMR e CG-massa. Seus efeitos sobre a incorporação de timidina radioativa ([3H] TdR) no DNA de células de baço, fibroblasto 3T3(A31) e melanoma B16F10 foram examinados. Os dois adutos foram mitogênicos para células de baço mas foram seletivos quanto as células imortalizadas. 8-MeGuo atuou sobre células 3T3(A31) e 8-OxoGuo sobre as células de melanoma B16F10. O análogo não fisiológico 8-BrGuo foi efetivo em todas as células testadas. Experimentos de contagem de células, citotoxicidade e citometria de fluxo, indicaram que a síntese de DNA induzida pelas guanosinas substituídas na posição C-8 refletia crescimento celular. Foi proposto que os compostos agem de dentro da célula uma vez que seus efeitos são bloqueados em presença de um inibidor de transporte de nucleosídeo, mas não foram inibidos por um antagonista de receptor purinérgico. Os resultados obtidos, junto com os descritos na literatura, sugerem que no caso dos fibroblastos 3T3(A31) e células de baço de camundongo os efeitos proliferativos dos compostos não são dependentes do metabolismo desses compostos via salvação das purinas. No caso das células de melanoma, entretanto, os compostos parecem fazer parte do \"pool\" de nucleosídeos. A demonstração de que adutos produzidos por ataques radicalares em ribonucleosídeos e RNA são capazes de induzir proliferação celular, abre novas perspectivas para a compreensão do papel de radicais livres em processos carcinogênicos. / The ability of CS-substituted guanine ribonucleosides to induce B cell proliferation has been well documented in the literature. These compounds are analogues of adducts formed from free radical attack on ribonucleosides and RNA. Here we examined the proliferative properties of two of these radical adducts, 8-methylguanosine (8-MeGuo) and 8-oxo-7 ,8-dihydroguanosine (8-OxoGuo) and compared them with those of the well studied B cell mitogen, 8-bromoguanosine (8-BrGuo). 8-MeGuo and 8-OxoGuo were synthesized in yields of 28 and 55 %, respectively, and were characterized by UV, NMR and CG-MS. Their effects upon [3H] thymidine uptake by Swiss mice splenocytes, mouse embryo 3T3 (A31) fibroblasts and mouse B16F10 melanocytes were examined. Both guanosine radical adducts were shown to increase [3H] thymidine uptake by mice splenocytes but displayed selectivity in regard to continuous cell lines. 8-MeGuo acted upon 3T3(A31) fibroblasts whereas 8-OxoGuo acted upon B16F10 melanocytes. The non physiological analogue 8-BrGuo acted upon all tested cells. Parallel experiments of cell counting, cytotoxicity, and cell sorting indicated that DNA synthesis induced by the C8-substituted guanosines reflected cell growth. It is proposed that the compounds act intracellularly because their proliferative effects were blocked in the presence of a nucleoside transport inhibitor but were not inhibited by an antagonist of the A2 purine receptor. The obtained results, taken together with data from the literature suggest that in the case of 3T3 (A31) fibroblasts and mice splenocytes the proliferative effects of the compounds are not dependent on metabolism through purine salvage pathways. In the case of melanocytes, however, the compounds are likely to become part of the purine nucleoside pool. The demonstration that adducts produced by free radical attack on ribonucleosides and RNA are able to induce cell proliferation opens new perspectives for the understanding of free radical mediated carcinogenesis.

Orientador: Pedro Oliver Dunstan Lozano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T07:28:40Z (GMT). No. of bitstreams: 1 Khan_AbdulMajeed_D.pdf: 3004508 bytes, checksum: 1d8c778094bf0cb1b93fa7bf80e19074 (MD5) Previous issue date: 2013 / Resumo: Os adutos MX2.nL (M = Mn, Fe, Co, Ni, Cu ou Zn, L = 3-cianopiridina, piperidina ou piperazaina, X = Br, n = 0,5, 0,75, 1, 1,5, 2 ou 4), foram sintetizados e caracterizados através de análise elementar, determinação de pontos de fusão, espectroscopia IV e eletrônica e análise termogravimétrica. Todos os adutos são sólidos e sensíveis à umidade atmosférica. Os dados espectroscópicos na região do infravermelho, para os adutos, indicam a presença de um mesmo padrão de bandas em comparação com os respectivos ligantes livres. No entanto, deslocamentos de algumas bandas são evidentes devido à coordenação dos íons metálicos aos ligantes. As entalpias de dissolução em HCl 1,2 molL dos adutos, brometos metálicos e ligantes, foram determinados. Através de ciclos termoquímicos apropriados, foram calculadas as variações de entalpia padrão para as reações: MBr2 (s) + nL (s,l) MBr2.nL (s) DrH MBr2.nL (s) MBr2 (s) + nL (g) DDH MBr2 (g) + nL (g) MBr2.nL (s) DMH Foram também calculadas as variações entálpicas padrão de formação dos adutos e as variações entálpicas padrão da ligação metal-nitrogênio. Os parâmetros termoquímicos permitiram determinar a ordem de acidez dos adutos: para os adutos de ligante 3-cianopiridina: NiBr2>FeBr2> CoBr2>MnBr2 e ZnBr2>CuBr2, para os adutos de ligante piperidina: FeBr2 > MnBr2, CoBr2 > NiBr2, para adutos de ligante piperazina: FeBr2 > MnBr2 > NiBr2 e CuBr2 > ZnBr2 > CoBr2. A ordem de basicidade das ligantes e (Piperidina) >(3-cianopiridina) para os adutos de ZnBr2 e (Piperidina) > (Piperazina) para os adutos de CuBr2. / Abstract: The adducts MX2.nL (M = Mn, Fe, Co, Ni, Cu or Zn, L = 3-cyanopyridine, piperidine or piperazaina, X = Br, n = 0.5, 0.75, 1, 1.5, 2 or 4), were synthesized and characterized by elemental analysis, melting point measurements of the adducts, vibrational and electronic spectroscopy and thermogravimetric analysis. All adducts are solid and sensitive to moisture. The spectroscopic data in the infrared region, indicate the presence of a similar band pattern as compared with the free ligands. However, dislocation of some bands are observed due to coordination of the metal ions to the ligands. The enthalpies of dissolution in HCl 1.2 molL of the adducts, metal bromides and ligands have been determined. Through appropriate thermochemical cycles, we calculated the standard enthalpy changes for the reactions: MBr2 (s) + nL (s,l) MBr2.nL (s) DrH MBr2.nL (s) MBr2 (s) + nL (g) DDH MBr2 (g) + nL (g) MBr2.nL (s) DMH Standard enthalpy of formation and standard mean enthalpy of Metal-Nitrogen bonds were also calculated. The thermochemical parameters allowed to determine the acidity order of metal bromides for the adducts of the same stoichiometry: For the adducts of ligand 3-cyanopyridine: NiBr2> FeBr2> CoBr2> MnBr2 and ZnBr2> CuBr2. for the adducts of ligand piperidine: FeBr2> MnBr2 and CoBr2> NiBr2. for the adducts of ligand piperazine: FeBr2> MnBr2> NiBr2 and CuBr2> ZnBr2> CoBr2. The order of basicity is (Piperidine)> (3-cyanopyridine) for the adducts of ZnBr2 and (Piperidine)> (Piperazine) for the adducts of CuBr2. / Doutorado / Quimica Inorganica / Doutor em Ciências

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22730号 / 医博第4648号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 遊佐 宏介, 教授 溝脇 尚志, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background: Glycation influences the ultrastructure and clot kinetics of fibrin clots due to the post-translational modifications in fibrinogen. Methylglyoxal (MG) is used to measure the level of glycation which has been associated with the pathogenesis of type 2 diabetes Melilites (T2DM) and coronary heart disease (CHD). The aim of the study was to determine the role of MG on clot kinetics and fibrin clot structure in CHD patients with and without T2DM to provide insight into the mechanism of pathogenesis of atherosclerosis in T2DM which results in the development of CHD. Methodology: Scanning electron microscopy (SEM) was used to evaluate the morphology of fibrin clots. Thromboelastography (TEG) was used to assess the physiological clot properties (kinetics). Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of methylglyoxal-adducts. Results: The morphology of clots from controls analysed using SEM showed thick and thin fibres which created an organised mesh of fibrin fibres. In T2DM, CHD with T2DM and CHD some alterations in the morphology were observed. The ultrastructure micrographs in CHD shows that some of the fibrin fibres formed have individual fibres with both thick and thin fibres as well as a thick mass of fibres with a net-like structure that forms dense-matted deposits. In addition, the fibrin fibres are not organised. The densitometry analysis between controls and patient groups’ (CHD: mean (standard deviation) 0.42±0.11; CHD+T2DM: 0.31±0.08 and T2DM: 0.29±0.08) was found to be significantly lower in all groups compared to the control which had a mean of 0.57±0.1, p<0.0001. There are no significant differences in the alpha angle between CHD, T2DM, CHD with T2DM and controls (60.88±2.321˚ vs. 60.81±2.385˚ vs. 59.09± 3.185˚ vs. 66.47±1.300˚, p=0.5279). There was no significant difference found in the K-value between T2DM, CHD with T2DM, CHD and control subjects (3.458±0.446mins vs. 5.118±1.589mins vs. 3.758±0.450mins vs. 2.839±0.2156mins, p=0.0102). The maximum amplitude was higher in T2DM patients compared to CHD, CHD with T2DM and controls (40.51±1.914mm vs. 34.10±2.127mm vs. 33.12±3.365mm vs. 33.60±1.525mm, p=0.0102). The MRTG was higher in CHD compared to T2DM, CHD 4 with T2DM and controls (10.74±3.335 dyn cm-2 s -1 vs. 4.268±0.690 dyn cm-2 s -1 vs. 5.046± 0.927 dyn cm-2 s -1 vs. 6.535±0.664 dyn cm-2 s -1 , p=0.0096). The reaction time was higher in CHD with T2DM patients compared to T2DM, CHD and controls (32.58±4.005min vs. 23.92±2.793min vs. 21.29± 2.383min vs. 8.322±0.886min, p<0.0001). There was no significant difference found in the TTG between T2DM, CHD with T2DM, CHD and control subjects (231.3±28.68 dyn cm-2 vs. 258.5±38.15 dyn cm2 vs. 343.7±71.92 dyn cm-2 vs. 287.7±21.37 dyn cm-2 , p=0.8421). The TMRTG was higher in T2DM patients compared to T2DM, CHD with T2DM, CHD and controls (23.91±2.409mins vs. 20.46±3.411mins vs. 14.14±1.287mins vs. 10.16±0.751mins, p<0.0001). To assess if an association between MG-adducts and clot kinetics exists, the Spearman r correlation was completed for each clot parameter. The reaction time (p=0.0047, 95% CI: 0.138 to 0.665) and time taken before maximum speed of the clot growth to be achieved (p=0.3958, 95% CI: 0.072 to 0.644) was significant. This indicates the relationship between the parameters i.e., the higher the level of MGadducts present, the longer it takes for clotting to begin and reach maximum speed of formation. Conclusion: This study showed that there are ultrastructural differences in fibrin fibres formed in CHD patients with T2DM. The viscoelastic parameters indicated that haemostasis was irregular in CHD and T2DM. The levels of MG-adducts were much higher in T2DM, CHD with T2DM and CHD and may be a contributing factor to the pathogenesis associated with altered coagulation in these patients. / Dissertation (MSc (Physiology))--University of Pretoria, 2020. / NRF / Physiology / MSc (Physiology) / Unrestricted

An O-methylated analog of protonated phenazine-di-N-oxide radical anion abstracts hydrogen from primary and secondary alcohols in a slow (k1 < 500 M-1 s-1) bimolecular reaction. No kinetic evidence has been found for the unimolecular release of free methoxyl radicals through the homolytic N-OMe bond cleavage in these species. DFT calculations at the UB3LYP 6-31G(d) level indicate that protonated and O-alkylated radical anions of pyrazine, quinoxaline and phenazine di-N-oxides are close analogues of aromatic nitroxyl radicals with the highest spin density localized on the oxygen and nitrogen of the nitrone moiety.

No description available.

We developed an approach to the β-thioenamide linkage contained in the S-(2-aminovinyl)cysteine (avCys) residue of thioviridamide.1,2 Kinetic and thermodynamic control of radical additions of thiols to ynamides were studied for the formation of β-thioenamide linkage. Thiyl radicals are electrophilic and ynamides are electron-rich alkynes. This complementary polarity of the radical and acceptor increases the likelihood of a successful radical addition reaction. Because little is known about these types of compounds (β-thioenamides), we were unsure what kinds of yields and stereoselectivities (cis vs. trans) to expect. The adduct stability is another issue to consider. Fortunately, under typical radical addition conditions, the two separable isomers (cis and trans) are formed in good yield. Selective formation of kinetic (cis) and thermodynamic (trans) isomers are controlled by reaction time and equivalents of thiol. We converted the kinetic isomer to the thermodynamic isomer to confirm that isomerization can occur under the reaction conditions. Alkyl and aryl thiols including cysteine-derived thiols with different ynamides were used in this process.

<p> The addition compounds of selenium tetrafluoride with sulfur trioxide, boron trifluoride, and arsenic, antimony, bismuth, vanadium, niobium and tantalum pentafluorides have been prepared. These compounds have been studied in the solid state using X-ray powder diffraction, and infra-red and Raman spectroscopy; in the molten state using Raman spectroscopy, nuclear magnetic resonance spectroscopy, and conductimetric and viscosity measurements; and in solution using Raman spectroscopy, cryoscopy, conductivity and nuclear magnetic resonance spectroscopy.</p> <p> The compound SeF4SO3 has a fluorosulfate-bridged polymeric structure. The remaining compounds have fluorine-bridged structures, this interaction being the strongest in SeF4BF3 and SeF4VF5 and decreasing in the order SeF4NbF5 SeF4TaF5 SeF4AsF5 SeF4SbF5 SeF4BiF5.</p> <p> The characteristics of the SeF3+ group are discussed. The properties of fluorine bridging and various methods of detecting such interactions are also considered.</p> / Thesis / Doctor of Philosophy (PhD)