tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).

No description available.

The thesis entitled “Studies on the Stereoselective Geminal and Vicinal Heterodifunctionalization of Alkenes” consists of three chapters. Chapter 1: Part A: Bromonium Ion Mediated Stereoselective Geminal Aminooxygenation of Vinylarenes In this part (part-A) of Chapter 1, the development and mechanistic studies of the first method for the non-Wacker intermolecular geminal aminooxygenation of vinylarenes (styrenes) has been presented (Scheme 1). Sheme 1 The role of the substituent on controlling the competitive vicinal and geminal addition pathways has been studied. It was found that the unsubstituted amino alcohol takes both the vicinal addition pathways, whereas, the introduction of substituent on the aminoalcohols was found to favour only the geminal addition route (Scheme 2). Scheme 2 The diastereomeric alkenes were found to show stereoconvergence on the product formation. The migration of the phenyl group in the semipinacol rearrangement was confirmed by deuterium labeling studies. This highly stereoselective oxidative geminal addition is found to involve a semipinacol rearrangement (Scheme 3). Scheme 3 Chapter 1: Part B: Bromonium Ion Mediated Stereoselective Anti-Markovnikov Geminal Diamination and Dioxygenation of Vinylarenes In this part (part-B) of Chapter 1, the development of a facile straightforward method for the stereoselective intermolecular geminal diamination of vinylarenes under the bromonium ion mediated conditions is discussed (Scheme 4). Scheme 4 The addition of unsubstituted diamine was found to follow both geminal and vicinal addition routes, while the introduction of the substituent on the diamine was found to favour only geminal addition (Scheme 5). Scheme 5 The stoichiometric geminal dioxygenation of vinylarenes using 1,2 and 1,3 -diols was also found to work well. The substituent on the nucleophile and the nucleophilicity of the heteroatom was found to control the competitive geminal and vicinal addition pathways. The stereoselectivity of geminal dioxygenation is dependent on the ring size of the product formed and on the position of the stereo-inducing substituent. Unlike the unsubstituted diamine and the unsubstituted aminoalcohol, irrespective of the substituents attached to it, the 1,2-diols furnished only the geminal addition product (Scheme 6). Scheme 6 Interestingly, the α-methyl substituted 1,3-diols provided the corresponding 2,4-disubstituted 1,3-dioxanes with very high stereoselectivity. The β-propyl substituted 1,3-diol gave the 2,5-disubstituted 1,3-dioxane as a mixture of diastereomers (Scheme 7). Scheme 7 The phenyl migration in the semi-pinacol rearrangement in the geminal addition process was confirmed from the deuterium labelling studies (Scheme 8). Scheme 8 Chapter 1: Part C: Straightforward Synthesis of 1,3-Dioxolan-4-ones through Geminal Difunctionalization of Vinylarenes The development of a straightforward method for the synthesis of important chiral synthon, 1,3-dioxolan-4-ones by the geminal addition of α-hydroxy carboxylic acids to vinylarenes has been presented in the final part of this chapter (part-C, Chapter 1) (Scheme 9). Scheme 9 The effect of substituents on the α-hydroxy carboxylic acid on controlling the stereoselectivity of the reaction has been studied. In the case of α-hydroxy carboxylic acid derived from isoleucine containing the chiral substituent at the α position, it exclusively forms a single diastereomer of the corresponding 1,3-dioxolan-4-one (Scheme 10). Scheme 10 The reactions of α-hydroxy carboxylic acids with styrenes containing a variety of substituents have been found to work well, including the styrenes containing the electron withdrawing groups and the β-substituted styrenes. The migration of the phenyl group in the semi-pinacol rearrangement in the geminal oxidative reaction has been confirmed by deuterium labelling studies (Scheme 11). Scheme 11 Simple carboxylic acids are found to form only the vicinal addition products on reaction with styrenes. However, the alcohols under the same conditions formed only the geminal addition product, thereby demonstrating the role of nucleophilicty of heteroatom being added that control the competitive vicinal and geminal addition pathways (Scheme 12 Scheme 12 Chapter 2: Reagent-Switch Controlled Metal-Free Geminal Difunctionalization of Vinylarenes In this Chapter, the development of two new methods for the geminal oxyamination of vinylarenes and the detailed studies to understand their mechanism are presented. A novel reagent-switch for the control of migrating group by controlling the two independent, distinct pathways of the two reagent systems has been reported for this geminal addition (Scheme 13). Scheme 13 We have developed the first general method for the geminal diamination of vinylarenes with excellent stereoselectivity mediated by a hypervalent iodine reagent (Scheme 14). Scheme 14 This method is also found to be very efficient for the stoichiometric metal-free geminal dioxygenation of vinylarenes (Scheme 15). Scheme 15 The substituent on the nucleophile and the nucleophilicity of the heteroatom was found to control the competitive geminal and vicinal addition pathways. Chapter 3: Studies on the Synthesis of Enantiopure Morpholine Derivatives Mediated by Dimethyl (Methylthio) Sulfonium Triflate (DMTST) In this chapter, the development of a sulfonium ion mediated cylco-etherification methodology for the construction of biologically important molecules such as morpholines, morpholine carboxylates and morpholine methylthio ethers in good yields under mild conditions using DMTST has been presented. This method was also found to work well for the synthesis of 1,4-oxazepane (Scheme 16). (For figures pl refer the abstract pdf file)

No description available.

Gliotoxin ist ein ubiquitär vorkommendes Mykotoxin und wird unter anderem von Aspergillus sp. gebildet. Das Gliotoxin ist toxisch und stellt für Mensch und Tier ein gesundheitliches Risiko dar. Über die Aufnahme von kontaminierten Futtermitteln (FM) kann es in tierische Produkte und anschließend in die Nahrungsmittelkette gelangen. Dem Mykotoxin werden starke immunmodulatorische Eigenschaften zugeschrieben. Gliotoxin gilt als eine sehr reaktive Verbindung, die in der Umwelt schnell zu bis(methylthio)Gliotoxin (bmGliotoxin) umgesetzt werden kann. Dieser Metabolit ist sowohl atoxisch als auch biologisch inaktiv und wird aufgrund seiner Stabilität als ein wichtiger und zuverlässiger Diagnostikmarker beschrieben. In der Mykotoxinanalytik werden Konzentrationsbestimmungen primär durch chromato-graphische Verfahren durchgeführt. Mit diesen Verfahren konnten bereits Gliotoxin-Konzentrationen in verschiedensten FM bestimmt werden. Im Gegensatz zu anderen prominenten Mykotoxinen gibt es für das Gliotoxin kein Nachweisverfahren für routinemäßige Kontrolluntersuchungen. Daher war das Ziel der Dissertationsarbeit die Entwicklung eines ELISA, mit dem Gliotoxin in FM einfach und schnell bestimmt werden kann. Es wurden zunächst geeignete Protein-Gliotoxin-Konjugate für die Anti-Gliotoxin-Immunisierung hergestellt. Kaninchen wurden nach einem festgelegten Protokoll mit diesen Hapten-Konjugaten immunisiert (Kurzzeit- bzw. Langzeitimmunisierung). Anschließend erfolgte die Titerbestimmung in den Antiseren und die Überprüfung der Paratophemmbarkeit durch freies Gliotoxin. Aus dem Antiserum mit der höchsten IgG-anti-Gliotoxin-Konzentration wurden die Antikörper antigenaffinitäts-chromatografisch aufgereinigt. Zusätzlich wurde für den kompetitiven ELISA ein geeignetes Gliotoxin-Peroxidase-Konjugat hergestellt. Alle Testkomponenten wurden vorab geprüft und danach der kompetitive Gliotoxin-ELISA validiert. Mit dem optimierten kompetitiven Testsystem wurden die Gliotoxin-Konzentrationen in Schimmelpilz-Kulturüberständen (Aspergillus sp.) gemessen. Anschließend erfolgte die Untersuchung von Futtermittelproben (Silagen) auf Gliotoxin. Nach der entsprechenden Probenaufbereitung der FM wurde das Gliotoxin im kompetitiven ELISA ebenfalls quantitativ bestimmt. Die hier gemessenen Gliotoxin-Konzentrationen wurden mit Ergebnissen aus parallel durchgeführten Untersuchungen mit der LC-MS/MS verglichen. Mit dem entwickelten kompetitiven ELISA sind quantitative Aussagen zu erhöhten Gliotoxin-Konzentrationen in verschiedenen Probenmaterialien möglich. Das Mykotoxin kann sowohl frei in seiner nativen Form, gebunden an Proteine oder metabolisiert als bis(methylthio)gliotoxin detektiert werden. Mit dem Testsystem kann die quantitative Produktion von Gliotoxin durch verschiedene Schimmelpilzarten beurteilt werden. Die untersuchten FM (Silageproben) konnten im entwickelten ELISA alle erfolgreich gemessen und beurteilt werden.:1 Einleitung 1 2 Literaturübersicht 2 2.1 Schimmelpilze 2 2.1.1 Definition und Zuordnung 2 2.1.2 Vorkommen in der Umwelt 4 2.1.3 Bedeutung und Nutzen 5 2.1.4 Schadwirkung für Mensch und Tier 7 2.2 Mykotoxine 9 2.3 Gliotoxin 10 2.3.1 Allgemeine Merkmale 10 2.3.2 Bis(methylthio)Gliotoxin 16 2.3.3 Ausgewählte Pathogenitätsmechanismen 17 2.3.3.1 Redox-Zirkel und ROS-Produktion 17 2.3.3.2 Verschiebung des TH1/ TH2-Gleichgewichtes 18 2.3.3.3 Inhibition des Transkriptionsfaktors NF-κB 19 2.3.3.4 Einleitung von Apoptose und Nekrose 20 2.3.3.5 Inhibition von Mastzellen 21 2.3.4 Nachweisverfahren 22 2.3.4.1 Chromatographie 22 2.3.4.2 Enzyme-linked Immunosorbent Assay (ELISA) 23 2.3.5 Quantitative Mengenbestimmungen in Proben 25 2.3.5.1 Klinisches Probenmaterial 25 2.3.5.2 Belastete Futtermittelproben 27 2.3.5.3 Belastete Lebensmittelproben 33 3 Material und Methoden 34 3.1 Gliotoxin, Chemikalien und ausgewählte Laborgeräte 34 3.2 Gliotoxin-Hapten für die Immunisierung 35 3.3 Immunisierung 37 3.4 Untersuchung der Anti-Gliotoxin-Antiseren 37 3.4.1 Protein-Gliotoxin-Konjugate für ELISA 37 3.4.2 ELISA für Antiserumuntersuchung 39 3.4.3 ELISA für die Bestimmung der IgG-Konzentrationen anti-Gliotoxin 40 3.5 Etablierung des kompetitiven Gliotoxin-ELISA 42 3.5.1 Isolierung der IgG-anti-Gliotoxin 42 3.5.2 Herstellung des Gliotoxin-HRP-Konjugates 43 3.5.3 Prüfung der Testkomponenten 44 3.5.4 Validierung des kompetitiven Gliotoxin-ELISA 45 3.6 Untersuchung von Kreuzreaktionen 45 3.7 Untersuchung von Pilzkulturen auf Gliotoxin 45 3.8 Untersuchung von Futtermitteln auf Gliotoxin 47 3.8.1 Untersuchung von dotiertem Futtermittel 47 3.8.2 Futtermitteluntersuchung mit dem Gliotoxin-ELISA 48 3.8.3 Futtermitteluntersuchung mit der LC-MS/MS 49 3.9 Datenauswertung 49 4 Ergebnisse 50 4.1 Hapten für die Immunisierung 50 4.2 Untersuchung der Anti-Gliotoxin-Antiseren 50 4.2.1 Titerbestimmung 50 4.2.2 Bestimmung der Hemmungsrate durch freies Gliotoxin 51 4.2.3 IgG-anti-Gliotoxin-Konzentration in den Antiseren 53 4.3 Untersuchung der Antiseren 55 4.3.1 Isolierung des IgG-anti-Gliotoxin 55 4.3.2 Voruntersuchungen für die Etablierung des kompetitiven Gliotoxin-ELISA 57 4.3.3 Validierung des kompetitiven Gliotoxin-ELISA 60 4.4 Untersuchung von Kreuzreaktionen 64 4.5 Untersuchung von Pilzkulturen auf Gliotoxin 65 4.6 Untersuchung von Futtermitteln auf Gliotoxin 68 4.6.1 Untersuchung mit Gliotoxin dotierter Futtermittel 68 4.6.2 Futtermitteluntersuchung mit Gliotoxin-ELISA und LC-MS/MS 71 4.7 Auswertung und Bewertung 73 5 Diskussion 78 5.1 Bewertung der Gliotoxin-Antigen Entwicklung 78 5.2 Bewertung der Antiseren nach Immunisierung (28d- und 91d-Protokoll) 79 5.3 Etablierung des kompetitiven ELISA 83 5.4 Bewertung der Pilze und der Kulturüberstände 85 5.5 Bewertung der Futtermittel 88 6 Zusammenfassung 96 7 Summary 98 8 Literaturverzeichnis 100 9 Anhang 116

Made available in DSpace on 2017-07-10T17:48:29Z (GMT). No. of bitstreams: 1 Cleiton_Pagliari_Sangali.PDF: 810970 bytes, checksum: 721cfbdc17482672e30e45378a255317 (MD5) Previous issue date: 2012-06-14 / Fundação Araucária / Aiming to assess the relative bioefficacy of DL-2-hydroxy-4-methylthio butanoic acid (DL-HMBA) and of poly-herbal ingredient (PHI) relative to DL-methionine (DLM) in broilers, two experiments were conducted. In the first experiment, 1100 Cobb 500 males and females broilers were fed either, from 1 to 21 days of age with a methionine-deficient basal diet, or the basal diet with three levels (0.170, 0.340, 0.511%) of DL-HMBA or three levels (0.111, 0.221, 0.332%) of DLM in equivalent amount of 65% of the levels of DL-HMBA or still, three levels (0.111, 0.221, 0.332%) of PHI, in equivalent amount the levels of DLM. In the second experiment, 900 Cobb 500 male broilers were fed either, from 22 to 42 days of age with a methionine-deficient basal diet, or the basal diet with three levels (0.143, 0.286 e 0.429%) of DL-HMBA or three levels (0.093, 0.186 e 0.279%) of DLM in equivalent amount of 65% of the levels of DL-HMBA or still, three levels (0.093, 0.186 e 0.279%) of PHI, in equivalent amount the levels of DLM. Simultaneous regression analysis was used to determine the bioefficacy based on weight gain and in feed conversion the of birds of experiments I and II, being that, in experiment II the bioefficacy values were also determined in function of the carcass characteristics of broilers fed with each methionine source. Performance was improved with supplementation of DL-HMBA or DLM in equivalent amount to 65% (DLM-65) of the levels of DL-HMBA, relative to those broilers fed the basal diet. However these responses were not so evident in birds supplemented with PHI. In the first experiment (stage of 1 to 21 days of age), simultaneous linear regression analysis revealed relative bioefficacy of DL-HMBA relative to DLM 39% and 44% for weight gain and feed conversion, on a product basis, respectively, being that, the performance data of birds supplemented with PHI not adjusted the simultaneously regression models, thereby, was not possible determine the bioefficacy of PHI in relation to the DLM. In the second experiment (stage of 22 to 42 days of age), simultaneous exponential regression analysis revealed bioefficacy relative of DL-HMBA and of PHI relative to DLM of 52% and 5% for weight gain and of 57% and 4% for feed conversion, on a product basis, respectively. To breast yield, the simultaneous linear regression analysis revealed relative bioefficacy of DL-HMBA in relation to DLM the 65% on a product basis. The results of this study indicate that the relative bioefficacy of DL-HMBA relative to DLM for broilers in stages of 1 to 21 and from 22 to 42 days old are respectively 42% and 58% on a product basis on average across all criteria tested / Com o objetivo de avaliar a bioeficácia do ácido DL-2-hidróxi-4 (metil) butanoico (DL-HMB) e de um poli ingrediente de ervas (PIE) em relação à DL-metionina (DLM) em frangos de corte foram realizados dois experimentos. No primeiro experimento, 1100 pintos de corte, da linhagem comercial Cobb 500, machos e fêmeas, foram alimentados de 1 a 21 dias de idade com uma dieta basal, deficiente em metionina + cistina, ou a dieta basal suplementada com três níveis (0,170, 0,340, 0,511%) de DL-HMB ou três níveis de DLM (0,111, 0,221, 0,332%) em quantidade equivalente a 65% dos nível de DL-HMB, ou ainda três níveis de PIE (0,111, 0,221, 0,332%), em quantidade equivalente aos níveis de DLM. No segundo experimento, 900 frangos de corte machos da linhagem Cobb 500 foram alimentados dos 22 aos 42 dias de idade com uma dieta basal deficiente em metionina, ou a dieta basal suplementada com três níveis (0,143, 0,286 e 0,429%) de DL-HMB ou três níveis de DLM (0,093, 0,186 e 0,279%) em quantidade equivalente a 65% dos nível de DL-HMB, ou ainda três níveis de PIE (0,093, 0,186 e 0,279%), em quantidade equivalente aos níveis de DLM. A análise de regressão simultânea foi usada para determinar a bioeficácia baseada no peso corporal e na conversão alimentar das aves dos experimentos I e II sendo que, no experimento II os valores de bioeficácia também foram determinados em função das características de carcaça das aves alimentadas com cada fonte de metionina. O desempenho foi melhorado com a suplementação de DL-HMB ou DLM em quantidade equivalente a 65% (DLM-65) dos níveis de DL-HMB, em relação aos frangos alimentados com as dietas basais. No entanto estas respostas não foram tão evidentes nas aves suplementadas com PIE. Para o primeiro experimento (fase de 1 aos 21 dias de idade) a análise de regressão linear simultânea revelou bioeficácia relativa do DL-HMB em relação à DLM de 39% e 44% para ganho de peso e conversão alimentar, em base de produto, respectivamente, sendo que, os dados de desempenho das aves suplementadas com PIE não se ajustaram significativamente aos modelos de regressão simultânea, desta forma não sendo possível determinar a bioeficácia do PIE em relação à DLM. No segundo experimento (fase de 22 aos 42 dias de idade), a análise de regressão exponencial simultânea revelou bioeficácia relativa do DL-HMB e do PIE em relação à DLM de 52% e 5% para ganho de peso e de 57% e 4% para conversão alimentar, em base de produto, respectivamente. Em relação ao rendimento de peito, a análise de regressão linear simultânea revelou uma bioeficácia relativa do DL-HMB em relação à DLM de 65%, em base de produto. Os resultados do presente estudo indicam que a bioeficácia relativa do DL-HMB em relação à DLM para frangos de corte nas fases de 1 aos 21 e dos 22 aos 42 dias de idade são respectivamente de 42% e 58% numa base de produto, em média, em todos os critérios testados