京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19032号 / 農博第2110号 / 新制||農||1031(附属図書館) / 学位論文||H27||N4914(農学部図書室) / 31983 / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 谷 誠, 教授 北山 兼弘, 教授 本田 与一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods. Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.

No description available.

Worldwide, malaria is one of the deadliest diseases. In 2016 it sickened 216 million people and caused 445,000 deaths. In order to control the spread of this deadly diseases to human, we can either target the mosquito vector (Anopheles gambiae) or the parasite (Plasmodium falciparum). Due to recent emergence of resistance to current insecticides and antimalarial drugs there is a pressing need to discover and develop new agents that engage new targets in these organisms. To circumvent the effect of resistance to pyrethroid insecticides on the efficacy of insecticide treated nets (ITNs), the use of acetylcholinesterase (AChE) inhibitors on ITNs has drawn attention. In the first project, we explored a small library of γ- substituted oxoisoxazole- 2(3H)-carboxamides and isoxazol-3-yl carbamates, and nitriles as AChE inhibitors targeting wild- type (G3) and resistant (Akron) An. gambiae mosquito. In total 23 compounds were synthesized and evaluated. Both carbamates and carboximides with a 2-cyclopropylethyl side chain (1-87a and 1-88a) were extremely toxic to Akron mosquitos, yet these compounds did not exhibit appreciable selectivity between human and An. gambiae AChE. Unfortunately, none of the nitriles showed appreciable toxicity to G3 strain of the mosquitoes, nor did they inhibit An. gambiae AChE. In the second project, conducted in collaboration with Professor Michael Klemba, we focused on the mode of action of an established antimalarial drug, Mefloquine (MQ). Dr. Klemba's recently developed amino acid efflux assay was used to determine the effect of MQ and its open-ring analogs on hemoglobin endocytosis and catabolism in P. falciparum-infected erythrocytes. In total 26 MQ analogs were synthesized and 18 were studied in depth to determine their potency to inhibit leucine (Leu) efflux and parasite growth (SYBR Green). An excellent correlation (R² = 0.98) over nearly 4 log units was seen for these 18 compounds in the two assays. These data are consistent with the hypothesis that the antimalarial action of these compounds principally derives from inhibition of hemoglobin endocytosis. After this observation, a number of photo-affinity probes were designed and synthesized in hopes of isolating the molecular target of MQ. These analogs are currently being used by Dr. Klemba in pull-down experiments. In the third project, conducted in collaboration with Professor Belen Cassera, we sought to optimize a new antimalarial drug lead that would circumvent current resistance mechanisms. In Plasmodium parasites, the methylerythritol phosphate (MEP) pathway is known to be essential for its growth. This pathway is absent in humans, presenting the opportunity to develop potentially safe and effective therapeutic candidates. Previous work in the Cassera and Carlier lab had established that MMV008138 was the only compound in the Malaria Box that targeted the MEP pathway and that it was (1R,3S)-configured. My research expanded previous efforts in the Carlier group and produced synthesis of 73 analogs of MMV008138 (3-21a'1) that were tested for growth inhibition. These analogs featured variation at the A-, B-, C- and D-ring. In the process, a novel Pictet-Spengler ring expansion reaction of ortho-substituted acetphenones was discovered. The ring-expanded products were identified by means of 1D and 2D NMR experiments, HRMS, and X-ray crystallography. Among the 73 analogs prepared, four compounds showed similar growth inhibition potency to the lead 3-21a'1. In particular, the methoxyamide 3-80a, and the fluorinated A-ring analogs 3-124a, 3-124c and 3-124d all showed excellent (500-700 nM) growth IC₅₀ values against P. falciparum. All four showed full rescue upon co-application of IPP (200 μM), confirming that they target the MEP pathway. ADME-Tox evaluation of these new analogs will soon be underway. / PHD

No description available.

No description available.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered. / AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF) en isoniasied, wat deel van die eerstelyn anti-tuberkulose behandeling vorm. Mutasies in die rpoB geen, wat die β-subeenheid van die RNA polimerase enkodeer, word geassosieer met RIF weerstandigheid. S531L en H526Y rpoB mutasies kom die algemeenste voor. RIF weerstandigheids vlakke verskil egter tussen isolate met identiese rpoB mutasies, wat impliseer dat ander faktore ook 'n rol in RIF weerstandigheid speel. 'n Toename in transkripsie van die Rv1258c geen, wat 'n multi-middel effluks pomp enkodeer, is waargeneem met blootstelling aan RIF, slegs in sommige M. tuberculosis H37Rv RIF mono-weerstandige mutante and MDR kliniese isolate, maar nie in ander nie. Die faktore wat die induksie van die Rv1258c effluks pomp beïnvloed is nie goed nagevors nie. Die studie ondersoek die effek van die rpoB mutasies op die uitdrukking van die Rv1258c en whiB7,'n transkripsionele regulator van Rv1258c, gene in M. tuberculosis H37Rv in vitro gegenereerde RIF weerstandige mutante, in die teenwoordigheid en afwesigheid van RIF. Die promotor area van die M. tuberculosis H37Rv Rv1258c geen is in 'n posisie stroomop van 'n lacZ geen, wat vir β-galaktosidase enkodeer, in multi-kopie episomale en integreerende vektors ingekloneer. Die funksionaliteit van die vektor en effek van rpoB mutasies op Rv1258c promotor aktiwiteit is ondersoek in die naverwante nie-patogeniese spesies, M. smegmatis en daarna in M. tuberculosis deur β-galaktosidase essais te doen. qRT-PCR is gedoen om die effek van rpoB mutasies op die vlak van transkripsie van die natuurlike Rv1258c geen en die whiB7 geen te bestudeer. Beide die episomale en integreerende vektors was funksioneel en daar is besluit om die integreerende vektor vir daaropeenvolgende β-galaktosidase essais in M. tuberculosis te gebruik. Rv1258c promotor aktiwiteit van die S531L mutant was ongeveer 1.5 keer minder as en die van die H526Y mutant 1.5 keer hoër as die van die ongemuteerde bakterië in M. smegmatis. Soortgelyk was die Rv1258c promoter aktiwiteit van die S531L mutant ongeveer die helfde van en die van H526Y mutant ongeveer dubbel die van die ongemuteerde bakterië in M. tuberculosis 'n Soortgelyke neiging in die vlakke van Rv1258c en whiB7 transkripte van die natuurlike geen is gedurende qRT-PCR waargeneem alhoewel die verskille nie beduidend was nie. Blootstelling aan sub-inhibitoriese konsentrasies van RIF het geen effek op Rv1258c uitdrukking in die M. smegmatis of M. tuberculosis rpoB mutante gehad nie. Die rpoB mutasies het net 'n effense effek op Rv1258c en whiB7 transkrip vlakke in M. tuberculosis rpoB mutante, maar transkrip vlakke het 'n soortgelyke neiging as die Rv1258c promoter aktiwiteit getoon. Of die waargenome verskille biologies betekenisvol is, moet nog bepaal word. Indien effluks pompe as teikens vir bahandeling gebruik sou word, moet in ag geneem word dat effluks pompe moontlik verskillend uitgedruk word in verskillende rpoB mutante. / The DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University / DAAD-NRF in Country Scholarship and Ernst and Ethel Eriksen Trust / Harry Crossley Foundation

This research investigates soil respiration (Rs) in a boreal jack pine (Pinus banksiana Lamb.) fire scar chronosequence at Sharpsand Creek, Ontario, Canada. During two field campaigns in 2006 and 2007, Rs was measured in a chronosequence of fire scars in the range 0 to 59 years since fire. Mean Rs adjusted for soil temperature (Ts) and soil moisture (Ms) (Rs T,M) ranged from 0.56 μmol CO2/m2/s (32 years post fire) to 8.18 μmol CO2/m2/s (58 years post fire). Coefficient of variation (CV) of Rs adjusted for Ts and Ms ranged from 20% (16 years post fire) to 56% (58 years post fire). Across the field site, there was a significant exponential relationship between Rs adjusted for soil organic carbon (Cs) and Ts (P = 1.24*10-06; Q10 = 2.21) but no effect of Ms on Rs adjusted for Cs and Ts for the range 0.21 to 0.77 volumetric Ms (P = 0.702). Rs T,M significantly (P = 0.030) decreased after burning mature forest, though no significant (P > 0.1) difference could be detected between recently burned and unburned young forest. Rs was measured in recently burned boreal jack pine fire scar age categories that differed in their burn history and there was a significant difference in Rs T,M between previously 32 v 16 year old (P = 0.000) and previously 32 v 59 year old (P = 0.044) scars. There was a strong significant exponential increase in S R T,M with time since fire (r2 = 0.999; P = 0.006) for the chronosequence 0, 16 and 59 years post fire, and for all these age categories, Rs T,M was significantly different from one another (P < 0.05). The Joint UK Land Environment Simulator (JULES) was used to model vegetation re-growth over successional time at Sharpsand Creek, though it appeared to perform poorly in simulating leaf area index and canopy height. JULES probably over estimated heterotrophic Rs at Sharpsand Creek when Ts corrected simulated values were compared with measured Rs T,M. The results of this study contribute to a better quantitative understanding of Rs in boreal jack pine fire scars and will facilitate improvements in C cycle modelling. Further work is needed in quantifying autotrophic and heterotrophic contributions to soil respiration in jack pine systems, monitoring soil respiration for extended time periods after fire and improving the ability of JULES to simulate successional vegetation re-growth.

α-Tocopherol has a well known antioxidant action but is also considered likely to exert significant non-antioxidant effects in cell membranes. Due to its lipophilic nature α-tocopherol inserts into biological membranes where it influences the organisation of the component lipids and may therefore influence biophysical parameters including the membrane dipole potential. The dipole potential has been demonstrated to modulate the function of several membrane associated proteins and perturbation of this physical parameter by α-tocopherol may prove to be a significant non-antioxidant mechanism underlying several of its cellular effects. This study investigates the influence of α-tocopherol, and the non-antioxidant structural analogue α-tocopherol succinate, on the membrane dipole potential employing fluorescence spectroscopy techniques with the dipole potential sensitive probe Di-8-ANEPPS. Similar techniques are utilised with the surface potential sensitive probe FPE to investigate the interaction of the charged α-tocopherol succinate molecule with membranes. α-Tocopherol and α-tocopherol succinate are shown to decrease the dipole potential of egg-phosphatidylcholine vesicles and Jurkat T-lymphocyte cell membranes. This effect is placed in the context of the significant influence of membrane cholesterol oxidation on the dipole potential. 7-ketocholesterol, an oxidised form of cholesterol, significantly influences several cellular processes and is thought to mediate these effects, in part, through its physical effects on the cell membrane. These include altering the composition, and therefore biophysical properties, of rafts; structures which are considered to support the function of a host of membrane proteins. This study attempts to correlate the effect of 7-ketocholesterol on the dipole potential of microdomains with the influence of the oxysterol on the function of two microdomains associated receptors: P-glycoprotein and the insulin receptor, assessed by determining the extent of ligand binding using flow fluorocytometry. α-Tocopherol has been suggested to inhibit the raft-mediated effects of 7-ketocholesterol and the influence of this molecule on the effect of 7-ketocholesterol on the dipole potential are investigated as a potential mechanism for this inhibition. It is hypothesized that α-tocopherols may protect against the deleterious effects of cholesterol oxidation in cell membranes by excluding 7-ketocholesterol from specific microdomains, of which rafts are a subset, acting to preserve their dipole potential and maintain the function of the proteins they support. However, where significant cholesterol oxidation has previously occured the concurrent changes in the microdomain landscape of the membrane is suggested to prevent α-tocopherol succinate from eliciting this protective effect.

This work was aimed at developing novel tools that utilize HINT, an empirical forcefield capable of quantitating both hydrophobic and hydrophilic (hydropathic) interactions, for implementation in theoretical biology and drug discovery/design. The role of hydrophobicity in determination of macromolecular structure and formation of complexes in biological molecules is undeniable and has been the subject of research across several decades. Hydrophobicity is introduced, with a review of its history and contemporary theories. This is followed by a description of various methods that quantify this all-pervading phenomenon and their use in protein folding and contemporary drug design projects – including a detailed overview of the HINT forcefield. The specific aim of this dissertation is to introduce our attempts at developing new methods for use in the study of antibacterial drug resistance and antiviral drug discovery. Multidrug efflux is commonly regarded as a fast growing problem in the field of medicine. Several species of microbes are known to have developed resistance against almost all classes of antibiotics by various modes-of-action, which include multidrug transporters (a.k.a. efflux pumps). These proteins are present in both gram-positive and gram-negative bacteria and extrude molecules of various classes. They protect the efflux pump-expressing bacterium from harmful effects of exogenous agents by simply evacuating the latter. Perhaps the best characterized mechanism amongst these is that of the AcrA-AcrB-TolC efflux pump. Data is available in literature and perhaps also in proprietary databases available with pharmaceutical companies, characterizing this pump in terms of the minimum inhibitory concentration ratios (MIC ratios) for various antibiotics. We procured a curated dataset of 32 β-lactam and 12 antibiotics of other classes from this literature. Initial attempts at studying the MIC ratios of β-lactam antibiotics as a function of their three dimensional topology via 3D-quantitative structure activity relationship (3D-QSAR) technology yielded seemingly good models. However, this methodology is essentially designed to address single receptor-ligand interactions. Molecules being transported by the efflux pump must undoubtedly be involved in multiple interactions with the same. Notably, such methods require a pharmacophoric overlap of ligands prior to the generation of models, thereby limiting their applicability to a set of structurally-related compounds. Thus, we designed a novel method that takes various interactions between antibiotic agents and the AcrA-AcrB-TolC pump into account in conjunction with certain properties of the drugs. This method yielded mathematical models that are capable of predicting high/low efflux with significant efficiency (>93% correct). The development of this method, along with the results from its validation, is presented herein. A parallel aim being pursued by us is to discover inhibitors for hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 3 (HPIV3) by in silico screening. The basis for targeting HN is explored, along with commentary on the methodology adopted during this effort. This project yielded a moderate success rate of 34%, perhaps due to problems in the computational methodology utilized. We highlight one particular problem – that of emulating target flexibility – and explore new avenues for overcoming this obstacle in the long run. As a starting point towards enhancing the tools available to us for virtual screening in general (and for discovering antiviral compounds in specific), we explored the compatibility between sidechain rotamer libraries and the HINT scoring function. A new algorithm was designed to optimize amino acid residue sidechains, if provided with the backbone coordinates, by generating sidechain positions using the Dunbrack and Cohen backbone-dependent rotamer library and scoring them with the HINT scoring function. This rotamer library was previously used by its developers previously to design a very successful sidechain optimization algorithm called SCWRL. Output structures from our algorithm were compared with those from SCWRL and showed extraordinary similarities as well as significant differences, which are discussed herein. This successful implementation of HINT in our sidechain optimization algorithm establishes the compatibility between this forcefield and sidechain rotamer libraries. Future aims in this project include enhancement of our current algorithm and the design of a new algorithm to explore partial induced-fit in targets aimed at improving current docking methodology. This work shows significant progress towards the implementation of our hydropathic force field in theoretical modeling of biological systems in order to enhance our ability to understand atomistic details of inter- and intramolecular interactions which must form the basis for a wide variety of biological phenomena. Such efforts are key to not only to understanding the said phenomena, but also towards a solid basis for efficient drug design in the future.