M. Tech. Chemistry.

No description available.

One unique feature of cancer cells that can be exploited for anticancer drug discovery is their dependence on their own cellular stress responses to survive the stressful acidotic, hypoxic and nutrient-deprived conditions within the tumor. Reasoning that desert organisms surviving under stressful conditions may have evolved to produce small molecule metabolites capable of modulating heat shock protein 90 (Hsp90) function, and/or other cell stress responses, we employed the cellular heat shock response in a moderate-throughput phenotypic assay. This strategy has resulted in the isolation and characterization of a number of small molecule natural products with heat shock induction activity from these organisms. Three such natural products are the subject of this study.In a limited structure-activity relationship (SAR) study, a previously known Hsp90 inhibitor radicicol (RAD), and several structurally related molecules including the fungal metabolite monocillin 1 (MON) were found to interact with Hsp90. In addition, RAD and MON were shown to lead to the degradation of Hsp90 client proteins involved in the cancer cell survival the estrogen receptor (ER) and the insulin-like growth factor receptor 1 (IGF-1R).We further characterized MON and showed that by targeting the molecular chaperone Hsp90, this compound induces components of the heat shock response at the transcriptional and translational levels, and leads to the acquisition of a thermotolerant phenotype in seedlings of the plant Arabidopsis thaliana. These findings support our hypothesis that there is ecological significance to the elaboration of small molecules that target stress responses.A number of extracts active in our phenotypic assay contained small molecules with no apparent Hsp90 activity. One such extract afforded terrecyclic acid A (TCA) with significant anti-tumor activity against a panel of human cancer cell lines. To characterize the biological activities of TCA we examined three key stress responsesthe heat shock, oxidative, and inflammatory responsesand show that TCA destabilizes these pathways associated with cancer cell survival through induction of oxidative stress (ROS), and inhibition of NF-kappaB transactivation.The isolation of RAD, MON and TCA from Sonoran desert organisms provides proof of principle that we have developed an effective strategy for the discovery of small molecule modulators of cellular stress responses that can serve as leads for the development of new anticancer drugs with novel mechanisms of action.

The overall goal of this research is to understand roles of gut microbiota metabolites and adipocyte transcription factor (TF) network in health and disease by developing systematic analysis methods. As microbiota can perform diverse biotransformation reactions, the spectrum of metabolites present in the gastrointestinal (GI) tract is extremely complex but only a handful of bioactive microbiota metabolites have been identified. We developed a metabolomics workflow that integrates in silico discovery with targeted mass spectrometry. A computational pathway analysis where microbiota metabolisms are modeled as a single metabolic network is utilized to predict a focused set of targets for multiple reaction monitoring (MRM) analysis. We validated our methodology by predicting, quantifying in murine cecum and feces and characterizing tryptophan (TRP)-derived metabolites as ligands for the aryl hydrocarbon receptor. The adipocyte process of lipid droplet accumulation and differentiation is regulated by multiple TFs that function together in a network. Although individual TF activation is previously reported, construction of an integrated network has been limited due to different measurement conditions. We developed an integrated network model of key TFs - PPAR, C/EBP, CREB, NFAT, FoxO1, and SREBP-1c - underlying adipocyte differentiation. A hypothetic model was determined based on literature, and stochastic simulation algorithm (SSA) was applied to simulate TF dynamics. TF activation profiles at different stages of differentiation were measured using 3T3-L1 reporter cell lines where binding of a TF to its DNA binding element drives expression of the Gaussia luciferase gene. Reaction trajectories calculated by SSA showed good agreement with experimental measurement. The TF model was further validated by perturbing dynamics of CREB using forskolin, and comparing the predicted response with experimental data. We studied the molecular recognition mechanism underlying anti-inflammatory function of a bacterial metabolite, indole in DC2.4 cells. The indole treatment attenuated the fraction of cells that were producing the pro-inflammatory cytokine, TNFα and knockdown of nuclear receptor related 1 (Nurr1; NR4A2) resulted in less indole-derived suppression of TNFα production. The first discovery of NR4A2 as a molecular mediator of the endogenous metabolite, indole is expected to provide a new strategy for treatment of inflammatory disorders.

The total synthesis of a novel diarylheptanoid isolated from a South African medicinal plant, Siphonochilus aethiopicus, was investigated. S. aethiopicus (Indungulu in Zulu) is the only South African species of the Zingiberaceae plant family and is widely used in traditional medicine. One of the compounds isolated from this plant is a novel diarylheptanoid. Diarylheptanoids constitute a distinct group of natural plant metabolites characterized by two aromatic rings linked by a linear seven-carbon aliphatic chain, with varying functional groups on the aryl and the aliphatic chain. The target molecule for our synthesis contains two highly oxygenated aryl rings linked by an aliphatic chain with two stereogenic centres and a trans-alkene. In this study we present our investigation of different strategies to a viable synthetic method that could provide material to supplement the relatively small quantity of product that can be isolated from the plant extract. The major challenges of this synthesis were to develop procedures for the preparation of the homobenzylic trans-alkene, the stereogenic centres and to attach the electron-rich aromatic rings to the aliphatic chain. In this thesis the following aspects are described: • Various types of olefination reactions (including Wittig, Julia and organometalic-mediated type of olefination reactions) • Various types of alkylation reactions (including Grignard, Friedel-Crafts and organometalic-mediated type of alkylation reactions) • Incorporation of the stereogenic centres (including asymmetric hydroxylation and use of chiral starting materials) The synthesis will not only give a viable synthetic route to the target compound but is also versatile enough to allow the preparation of analogues. / Thesis (Ph.D. (School of Chemistry)) - University of KwaZulu-Natal, Pietermaritzburg, 2008.

The search for new antibiotics continues in a rather overlooked hunting ground. In the course of screening for new antibiotic-producing microorganisms, seventy-nine isolates showing antimicrobial activity were isolated from soil samples from various habitats in the KwaZulu-Natal midlands, South Africa. Existing methods of screening for antibiotic producers together with some novel procedures were reviewed. Both modified agar-streak and agar-plug methods were used in the primary screens. The use of selective isolation media, with or without antibiotic incorporation and/or heat pretreatment, enhanced the development of certain actinomycete colonies on the isolation plates. Winogradsky's nitrite medium (Winogradsky, 1949), M3 agar (Rowbotham and Cross, 1977), and Kosmachev's medium (Kosmachev, 1960), were found to be selective for actinomycetes. Statistical analysis showed highly significant interactions between isolates, assay media and the test organisms. The diameters of inhibition zones were found to be larger on Iso-sensitest agar (ISTA)[Oxoid, England] than in nutrient agar plates. Of the 79 isolates that showed antimicrobial activity, 44 isolates were selected for confirmatory screening. Of these, 13 were selected for secondary screening. Criteria for selection were based on significant inhibition of at least two test organisms and/or the inhibition of the specifically targeted organisms, Pseudomonas and Xanthomonas species. Following secondary screening eight isolates were considered for further investigation. The isolates were tentatively identified . on the basis of morphological features, using both light microscopy and scanning electron microscopy(SEM); their ability to utilize various carbon sources; and selected physiological and staining tests. Suspected actinomycetes were further characterized on the basis of selected chemical properties using thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) techniques. High pressure liquid chromatography analysis (Beckman 6300 analyzer) detected the presence of diaminopimelic acid (DAP) in whole-cell hydrolysates of six of the isolates while TLC analysis confirmed the type ofDAP present. The isolates N2, N12, N16, N19 and N35 were tentatively identified as Thermomonospora, Saccharopolyspora, Nocardiodes, Corynebacterium and Promicromonospora, respectively. Isolate N30 was identified as belonging to the coryneform group ofbacteria, possibly an Arthrobacter species. Isolate, N8, tentatively identified as Actinosynnema, was unique among the isolates tested as it showed good antimicrobial activity against all the Gram- positive and Gram-negative bacteria, and yeasts used as test organisms in the present investigation. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

The human brain consists of a myriad of chemical compounds critical to its functioning. A group of these compounds, collectively known as metabolites, have been a research interest for years because the pathogenesis of neurodegenerative diseases, a tumours classification, the effectiveness of a drug, etc., can be investigated via variations in brain metabolite concentration levels. Nuclear Magnetic Resonance Spectroscopy (NMRS) enables investigators to conduct non-invasive in vivo studies of metabolites in the human brain and the rest of the body. However a number of problems have hindered the usage of NMRS as a clinical diagnostic tool. One is the non-uniqueness of the most widely used analysis methods, i.e. as the parameters and/or prior knowledge data of an analysis method are changed, the results also change. A second problem is the lack of a method that can automatically classify the signal components estimated via signal decomposition based signal analysis methods. Additionally, some of the most widely used analysis methods, by virtue of their algorithms, intrinsically assume the nature of NMRS signals, e.g. stationary, linear, Lorentzian, etc. Hence, this thesis explores a new analysis approach, based on a theoretical and practical understanding of NMRS, that (a) avoids making assumptions about the nature of experimentally acquired NMRS signals, (b) relies on a unique decomposition analysis method, and (c) automatically classifies the estimated peaks of an analysis. Unique decomposition analysis was conducted via the rarely used unique and non-linear signal decomposition method − the Fast Pad´e Transform (FPT). The FPT is compared with the main decomposition based NMRS analysis methods via a detailed mathematical analysis, and a comparative analysis. Automatic classification was conducted via a novel classification method, which is introduced herein, and which is based on quantum mechanical predictions of metabolite NMRS behaviour.

Vemurafenib is a potent serine/threonine kinase inhibitor and is registered as Zelboraf® for the treatment of metastatic melanomas harboring BRAFV600E mutations. There is a large individual variation in drug response and the side effects observed among patients treated with Zelboraf® has proven to be severe.  LC-MS/MS methods were developed to measure vemurafenib and its metabolites in human plasma for prediction of treatment outcome and side effects in order to individualize treatment with Zelboraf®.  A novel, rapid quantification method was developed for vemurafenib using a stable isotope labeled internal standard. The method was validated according to international guidelines with regard to calibration range, accuracy, precision, carry-over, dilution integrity, selectivity, matrix effects, recovery and stability. All parameters met the set acceptance criteria.  The first method suitable for quantifying vemurafenib metabolites in human plasma is presented. Lacking commercially available reference substances, human liver microsomes were used to produce the metabolites. In patient samples at steady-state five previously in vitro identified metabolites were quantified for the first time.

This dissertation combines laboratory and field experiments to investigate the mechanisms of atropisomer enrichment for chiral polychlorinated biphenyls (PCBs) and their metabolites in organisms. Stereoselective biotransformation and bioaccumulation were identified as two major reasons for the different environmental fate of PCB atropisomers. Other affecting factors, such as presence of nanoparticles and changes in feeding ecology of organisms, also affect the fate of chiral contaminants. In vitro incubations of rat cytochrome P-450 2B1 (CYP2B1) isozyme with chiral PCBs indicated that different biotransformation kinetics and competition among PCB congeners or between atropisomers were two main factors affecting atropisomer enrichment. Different interactions between chiral PCB congeners or atropisomers with rat CYP2B1 may occur at the molecular level. Non-racemic meta-hydroxylated-PCBs (5-OH-PCBs) were the major metabolites. CYP-mediated stereoselective formation of dihydroxylated PCBs from OH-PCBs was observed. Gold nanoparticles affected biotransformation activity of rat CYP2B1 and changed PCB atropisomeric composition, directly by electrostatic interaction, or indirectly by changes to the surrounding ionic strength. Thus, stereoselective metabolism of chiral PCBs and OH-PCBs by CYPs is a major mechanism for atropisomer enrichment of PCBs and their metabolites in the environment, with the degree of enrichment dependent, at least in part, on charged nanoparticles and stereoselective interference of atropisomers with each other at the enzyme level. The atropisomer compositions of chiral PCBs were measured in the marine biota of Cumberland Sound (Canada) and Svalbard (Norway). High trophic level organisms, including harp seal, beluga, and narwhal reported for the first time, had species-specific atropisomer signatures, likely due to a combination of in vivo biotransformation and trophic transfer. PCB chiral signatures in Greenland sharks supported the hypothesis that some of these PCB atropisomer compositions shifted over time and space, possibly due to a change in feeding ecology. To our knowledge, this is the first report to investigate temporal trends of PCB atropisomer signatures in Arctic biota.

Lo scopo principale di questa ricerca è stato di monitorare la concentrazione di nitrati, dell'erbicida terbutilazina e di un suo metabolita, la desethylterbuthylazina, in acque sotterranee superficiali, secondo le normali pratiche agronomiche in differenti aree italiane rappresentative della coltura maidicola. Il primo passo è stato identificare i siti di monitoraggio, quindi è stata creata una procedura generale al fine di individuare aree rappresentative per il mais in Italia. Dentro tali aree sono stati selezionati dei siti di: 4 in Lombardia, 2 in Emilia Romagna, 2 in Veneto, 2 in Piemonte e uno in Friuli-Venezia Giulia. In Aprile 2005 il monitoraggio ha avuto inizio e i campionamenti sono stati eseguiti ogni due mesi. L'indice “IDPR”, strumento per la mappatura della vulnerabilità a livello regionale e nazionale, è stato applicato alle aree oggetto di studio. L'indice riflette l'influenza che le formazioni geologiche del sottosuolo hanno sullo scorrimento superficiale e sull'infiltrazione. L'analisi della probabilità di distribuzione dei risultati ha mostrato che una bassa probabilità di superare i limiti fissati per legge, questa probabilità è risultata più alta per il metabolita. Le concentrazioni dei nitrati nelle acque sotterranee sono risultate molto basse. Inoltre, è stato applicato un modello matematico (MACRO vs 5.1) di previsione che calcola la percolazione dei pesticidi. / The main aim of the research was to monitor the concentrations of nitrates, the herbicide terbuthylazine and its products of the environmental degradation, desethyl-terbuthylazine, in shallow groundwater, according to the normal agricultural practices in different Italian areas representative of maize crop. The first step was to identify the monitoring sites, therefore it was necessary to create a general procedure to individuate maize representative areas in Italy. Inside these area 11 monitoring sites were selected: 4 in Lombardy, 2 in Emilia Romagna, 2 in Veneto, 2 in Piedmont and one in Friuli-Venezia Giulia. In April 2005 the monitoring started. Groundwater samplings were carried out every two months. The IDPR index, a tool for national and regional vulnerability mapping, was applied. The index reflects the influence of the subsurface geological formations on the surface water runoff or infiltration. The distribution probability analysis of the results showed that there was a very low probability of exceeding the limit fixed by law, this probability was higher for the metabolite. The greater probability of exceed the 0.1 μg/L occurred in two sites, both irrigated with basin irrigation system. Nitrate concentrations resulted to be very low in shallow groundwater. Moreover, it was used a mathematical model which calculate the leaching of pesticides (MACRO 5.1).