Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographic references (leaves [144]-153).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 175-196).

The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.

Herein 47 2,4-disubstituted quinazolines were synthesized and tested against Leishmania donovani intracellular amastigotes. A structure-activity relationship was conducted and lead to the identification of quinazolines with EC50s in the single digit and high nanomolar range with favorable antileishmanial selectivity indexes. Quinazoline 2.6 and 2.31 underwent in vivo efficacy studies in murine models of visceral leishmaniasis, reducing liver parasitemia by 12 % and 24 %, respectively, when given by the intraperitoneal route at 15 mg/kg/day x 5 days. The antileishmanial efficacy and easy of synthesis make the 2,4-disubstituted quinazoline compound series a suitable platform for the future development of antileishmanial agents. A similar series of 50 N2,N4-disubstituted quinazoline-2,4-diamines has also been synthesized and tested against multi-drug resistant strains of Acinetobacter baumannii. Quinazolines with MICs in the single digit micromolar range were identified within the structure-activity relationship. The observed potencies of the top compounds and the easy of synthesis lend to the further investigation of in vivo efficacy studies and could be considered a suitable platform for the future development of anti-bacterial agents against A. baumannii.

Thesis (M.Sc.(Chemistry)) -- University of Limpopo, 2016 / Imidazolyl-salicylaldemine Schiff base ligands were prepared by the condensation of different substituted salicylaldehydes with histamine dihydrochloride: 2,4-di-tert-butyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L1; 4-methoxy-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L2; 2-ethoxy-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl]-phenol L3; 2-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L4; 3-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L5; 4-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L6; 5-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L7; 2-tert-butyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L8; 3-methoxy-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L9; 4-tert-butyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L10. The ligands were characterised by 1H, 13C and 15N NMR, FT-IR, UV-vis spectroscopic methods, mass spectroscopy and elemental analysis where possible. Reactions of L1-L9 with MnCl2 and CuCl2 yielded complexes C1–C18 all in a ratio of (1:1). C1 – C18 here, i.e. C18 2-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol Cu(II) chloride. The complexes that are paramagnetic were mainly characterised by FT-IR spectroscopy, UV-vis spectroscopy, mass spectroscopy and elemental analysis. Reaction of L9 with ZnCl2 yielded 2-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol Zn(II) chloride, C19 which is diamagnetic and was characterised by 1H, 13C NMR spectroscopy Mass spectroscopy and Elemental analysis. Condensation of o-phenylenediamine and glyoxylic acid yielded 2-quinoxalinone Q1, reaction of 2-quinoxalinone and benzenesulfonyl chloride yielded 2-benzenesulfonyloxyquinoxaline Q2, and the reaction of 2-benzenesulfonyloxyquinoxaline with ethynyltrimethylsilane yielded 2-ethynyltrimethylsilanequinoxaline Q3. Quinoxaline and its preceding starting compounds were characterised by 1H, 13C NMR spectroscopy. Attempts to cross couple Schiff base complex C19 and Q3 via Sonogashira cross coupling mechanism method was not successful after two attempts from following two different reaction routes and conditions. The trans-metalation route yielded quinoxaline derivative QA in good yield after the reaction of Q3 with ZnCl2 and, C19 in the presence of Et2NH which were characterised by NMR (1H, 13C and HMBC), Mass spectroscopy (HRMS) and Elemental analysis. iv These reactions showed hydroamination instead of coordination in yield of (90%) using ZnCl2 as catalyst and 60% using C19 as catalyst. Biological activity studies were done by quantitative antibacterial activity by assay of minimum inhibition concentration (MIC). Bacterial cells used as pathogenic microorganisms were gram negative: Escherichia coli and Pseudomonas aeruginosa, gram positive: Staphylococcus aureus and Enterococcus faecalis. Compound used for testing : L9, C19, C18, C9, Q3 and QA. All compounds were found to be active against bacterial cells of E. coli and E. faecalis with a minimum inhibition concentration of 0.004 mg/ml average and total activity of 500 mg/ml average. For all compounds used for testing bacterial activity against cells of P. aeruginosa and S. aureus, the lowest concentration was 0.063 mg/ml average and total activity of 266 mg/ml average for C18. P. aeruginosa and S. aureus were found to be less active compared to bioactive standards used for E. coli and E. faecalis.

Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2016 / Imidazolyl-salicylaldemine Schiff base ligands were prepared by the condensation of different substituted salicylaldehydes with histamine dihydrochloride: 2,4-di-tert-butyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L1; 4-methoxy-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L2; 2-ethoxy-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl]-phenol L3; 2-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L4; 3-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L5; 4-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L6; 5-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L7; 2-tert-butyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L8; 3-methoxy-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L9; 4-tert-butyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol L10. The ligands were characterised by 1H, 13C and 15N NMR, FT-IR, UV-vis spectroscopic methods, mass spectroscopy and elemental analysis where possible. Reactions of L1-L9 with MnCl2 and CuCl2 yielded complexes C1–C18 all in a ratio of (1:1). C1 – C18 here, i.e. C18 2-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol Cu(II) chloride. The complexes that are paramagnetic were mainly characterised by FT-IR spectroscopy, UV-vis spectroscopy, mass spectroscopy and elemental analysis. Reaction of L9 with ZnCl2 yielded 2-methyl-6-{[2-(1H-imidazol-4-yl)-ethylimino]-methyl}-phenol Zn(II) chloride, C19 which is diamagnetic and was characterised by 1H, 13C NMR spectroscopy Mass spectroscopy and Elemental analysis. Condensation of o-phenylenediamine and glyoxylic acid yielded 2-quinoxalinone Q1, reaction of 2-quinoxalinone and benzenesulfonyl chloride yielded 2-benzenesulfonyloxyquinoxaline Q2, and the reaction of 2-benzenesulfonyloxyquinoxaline with ethynyltrimethylsilane yielded 2-ethynyltrimethylsilanequinoxaline Q3. Quinoxaline and its preceding starting compounds were characterised by 1H, 13C NMR spectroscopy. Attempts to cross couple Schiff base complex C19 and Q3 via Sonogashira cross coupling mechanism method was not successful after two attempts from following two different reaction routes and conditions. The trans-metalation route yielded quinoxaline derivative QA in good yield after the reaction of Q3 with ZnCl2 and, C19 in the presence of Et2NH which were characterised by NMR (1H, 13C and HMBC), Mass spectroscopy (HRMS) and Elemental analysis. iv These reactions showed hydroamination instead of coordination in yield of (90%) using ZnCl2 as catalyst and 60% using C19 as catalyst. Biological activity studies were done by quantitative antibacterial activity by assay of minimum inhibition concentration (MIC). Bacterial cells used as pathogenic microorganisms were gram negative: Escherichia coli and Pseudomonas aeruginosa, gram positive: Staphylococcus aureus and Enterococcus faecalis. Compound used for testing : L9, C19, C18, C9, Q3 and QA. All compounds were found to be active against bacterial cells of E. coli and E. faecalis with a minimum inhibition concentration of 0.004 mg/ml average and total activity of 500 mg/ml average. For all compounds used for testing bacterial activity against cells of P. aeruginosa and S. aureus, the lowest concentration was 0.063 mg/ml average and total activity of 266 mg/ml average for C18. P. aeruginosa and S. aureus were found to be less active compared to bioactive standards used for E. coli and E. faecalis.

Human uric acid transporter 1 (hURAT1; SLC22A12) is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure-activity relationship fndings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK) studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5′-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing) or intravenously (orbital sinus) administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to compute noncompartmental model PK values. Mono-oxidation (Phase I) and glucuronidation (Phase II) pathways were observed in vitro and in vivo. The in vitro data were used to compute hepatic intrinsic clearance, and the in vivo data were used to compute peak blood concentration, time after administration to achieve peak blood concentration, area under the curve, and orally absorbed fraction. The experimental data provide additional insight into the hURAT1 inhibitor structure-activity relationship and in vitro-in vivo correlation. Furthermore, the results illustrate that one may successfully prepare potent inhibitors that exhibit moderate to good oral bioavailability.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20065号 / 農博第2194号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5021(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)准教授 赤松 美紀, 教授 植田 和光, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM