Made available in DSpace on 2014-10-09T12:54:53Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:04Z (GMT). No. of bitstreams: 1 12797.pdf: 513559 bytes, checksum: 4b6e71bdbea803b32a3222d0b42f7b13 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo, Sao Paulo

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1. OH-FS wurden in vitro hergestellt, um als Standardsubstanzen zur gaschromato-graphischen Identifizierung von OH-FS in Pflanzenmaterial eingesetzt zu werden. 2. Für die Untersuchung der Oxylipin-Gehalte in A. thaliana wurden der virulente Pst-Stamm DC3000 sowie der avirulente Stamm avrRPM1 verwendet, um die kompatible Interaktion mit der inkompatiblen Interaktion vergleichen zu können. Die Konzentrationen der Oxylipine sowie SA wurden innerhalb einer Versuchsdauer von 60 h verfolgt. Dabei wurden PPF1 sowie 12- und 16-OH-FS, als Vertreter der nicht-enzymatisch entstandenen Oxylipine, 9- und 13-OH-FS, sowohl als enzymatisch als auch nicht-enzymatisch entstandene Oxylipine, sowie JA und deren Vorstufe OPDA als enzymatisch gebildete Phytohormone untersucht. Es wurden monophasische Konzentrationsanstiege, bei allen untersuchten Substanzen, in der kompatiblen Interaktion ermittelt, wohingegen die Konzentrationsanstiege in der inkompatiblen Interaktion biphasisch waren. In beiden Interaktionen wurden nach 48 bis 60 h Konzentrationsmaxima der freien sowie der veresterten OH-FS und PPF1 nachgewiesen, ein früher Konzentrationsanstieg nach 5 bis 10 h konnte ausschließlich in der inkompatiblen Interaktion ermittelt werden. Die gleichzeitige Akkumulation von 9-, 10-, 12-, 13, 15- und 16-OH-FS und PPF1 deutet auf eine parallel ablaufende Oxylipin-Synthese durch enzymatische, Photo-oxidative und über freie Radikale vermittelte Prozesse hin. Die Akkumulation veresterter OH-FS und PPF1 erfolgte in beiden Interaktionen 5 bis 12 h früher als die Konzentrationsanstiege der freien OH-FS und PPF1. Die Ergebnisse bestätigen die Hypothese, dass nicht-enzymatische Oxylipine in Membranen gebildet werden können und anschließend vermutlich durch eine Lipase frei gesetzt werden. In der inkompatiblen Interaktion konnte ein erstes frühes Konzentrationsmaximum von JA und OPDA nach 5 h beobachtet werden, während späte Maxima in beiden Interaktionen nach 24 bis 36 h erfolgten. Somit akkumulierten die OH-FS und PPF1 in der inkompatiblen Interaktion zeitgleich mit den Jasmonaten nach 5 h. 3. Bei einer Kälteexposition von A. thaliana bei 4°C über 2 h wurde jeweils ein 3,3-facher Konzentrationsanstieg der freien und der veresterten enzymatisch gebildeten 13-OH-FS nachgewiesen. Darüberhinaus wurde ein 4,6-facher Anstieg der enzymatisch entstandenen 9-OH-FS ermittelt. Die nicht-enzymatisch gebildeten 12- und 16-OH-FS zeigten dagegen keine signifikanten Konzentrationsanstiege über die basalen Konzentrationen hinaus. Die angewendeten Stressbedingungen bewirken demnach ausschließlich eine enzymatische Bildung von OH-FS in A. thaliana. 4. Zur Untersuchung der OH-FS-Synthese in der inkompatiblen Interaktion in Abhängigkeit von der bei der Pflanzenanzucht eingesetzten Lichtstärke wurden A. thaliana bei Licht und in Dunkelheit mit Pst avrRPM1 infiziert. Nach 10 h wurde eine 1,1- bis 3,7-fach stärkere Bildung der freien sowie eine 2,0- bis 3,4-fach stärkere Akkumulation der veresterten 9-, 10-, 12-, 13, 15- und 16-OH-FS bei den Pflanzen ermittelt, die bei Licht angezogen wurden. Die Lichtintensität, der Pflanzen während der Infektion mit Pst ausgesetzt sind, hat demnach große Bedeutung für die Entstehung enzymatisch und nicht-enzymatisch gebildeter OH-FS. Ein 4,9-facher Anstieg veresterter 15-OH-FS, ein Marker für eine photooxidative OH-FS-Entstehung, auch bei Dunkelheit widersprach der Hypothese, dass 15-OH-FS ohne Lichteinwirkung nicht gebildet werden können und deutet auf eine bisher unbekannte Licht-unabhängige Entstehung von 1O2 bzw. von 15-OH-FS hin. 5. Die Bestimmung von OH-FS in Blättern und Wurzeln von unbehandelten A. thaliana-Pflanzen ergab eine 13- bis 31-fach höhere Konzentration veresterter 9-, 10-, 12-, 13- und 16-OH-FS in den Blättern. Darüberhinaus wurde eine 111-fach höhere Konzentration von veresterten 15-OH-FS in Blättern im Vergleich zu Wurzeln nachgewiesen. 15-OH-FS wurden als selektiver Marker für eine Photo-oxidative OH-FS-Bildung durch 1O2 verwendet. Mit 0,57 µg/g TG kommt 15-OH-FS allerdings auch im Wurzelgewebe vor, was einen Hinweis darauf darstellt, dass neben einem Licht-abhängigen Hauptweg auch ein Licht-unabhängiger Entstehungsmechanismus von 15-OH-FS bzw. 1O2 existiert. Alternativ wäre es denkbar, dass ein Transport von 15-OH-FS von den Blättern in die Wurzeln stattfindet. 6. Eine Untersuchung der Gehalte an OH-FS und PPF1 in NahG-, lsd1-, atrbohD- und atrbohF-Mutanten ergab 48 h nach Infiltration von Pst avrRPM1 keine signifikanten Unterschiede im Vergleich zu den Pflanzen des jeweiligen Wildtyps Col-0 und WS. Unter den gewählten Versuchsbedingungen bewirken die genetischen Defekte der untersuchten Mutanten keine veränderte Akkumulation enzymatisch sowie nicht-enzymatisch gebildeter Oxylipine. / 1. To obtain reference-substances for the identification of OH-FA in plant material by GC-MS, OH-FA were made in vitro. 9- and 13-hydroxy-octadecadienoic acids were formed from linoleic acid, 9- and 13-hydroxy-octadecatrienoic acids from -linolenic acid by LOX-catalized reaction. 2. In order to compare the concentrations of oxylipins in A. thaliana during compatible and incompatible interaction, a virulent Pst-strain DC3000 and an avirulent strain avrRPM1 were utilized. The concentrations of oxylipins and salicylic acid were investigated within 60 h after inoculation. F1-phytoprostanes, 12- and 16-OH-FA were used as markers for non-enzymatically formed oxylipins. Concentrations of 9- and 13-OH-FA, either enzymatically or non-enzymatically formed, were measured as well as phytohormones, jasmonic acid and its precursor 12-oxo-phytodienoic acid. Within the compatible interaction an increase of the amount of all measured compounds was observed. In contrast, during incompatible interaction the increases of all measured substances was seperated into two phases. In both interactions, free and esterified OH-FA- and PPF1-concentrations exhibited maxima after 48 to 60 h, an early increase after 5 to 10 h was exclusively found in the incompatible interaction. The concurrent accumulation of 9-, 10-, 12-, 13, 15- und 16-OH-FA und PPF1 indicates that enzymatically, Photo-oxidative, and free-radical catalyzed synthesis of oxylipins takes place simultaneously. In both interactions the accumulation of esterified OH-FA and PPF1 occurred 5 - 12 h earlier than the increase of the concentrations of free OH-FA and PPF1. These results confirm the hypothesis that oxylipins are formed non-enzymatically from lipids inside cell membranes and are subsequently released by lipases. An early increase of JA- and OPDA-concentrations after 5 h was found in the incompatible interaction, while late maxima occurred in both interactions after 24 to 36 h. Therefore, OH-FA- and PPF1-accumulation took place at the same time as the increase of jasmonates after 5 h. 3. When A. thaliana plants were chilled for 2 h at 4°C an 3,3-fold incrceased formation of both, the enzymatically formed free and esterified 13-OH-FA was detected. The amount of enzymatically formed free 9-OH-FA increased 4,6-fold. In contrast, the amount of non-enzymatically formed 12- and 16-OH-FA did not increase significantly indicating that the applied mild stress conditions triggered exclusively enzymatical OH-FA-formation. 4. In order to get information about OH-FA-formation with regard to light intensity A. thaliana was infected by Pst avrRPM1 and cultivated either in the dark or in the light. When plants were cultivated in the light after 10 h an 1,1- to 3,7- fold increased formation of free and an 2,0- to 3,4-fold increased accumulation of esterified 9-, 10-, 12-, 13-, 15- und 16-OH-FA could be detected. Therefore, the light intensity during an infection with Pst avrRPM1 is an important factor for the formation of enzymatically as well as non-enzymatically formed OH-FA in planta. The 4,9-fold increase of esterified 15-OH-FA (a marker for photooxidative formation of OH-FA) in the dark contradicted the hypothesis that its formation is impossible without the impact of light. In contrast the accumulation of 15-OH-FA in the dark points to a light-independent 1O2- and subsequent 15-OH-FA-formation by a so far unknown mechanism. 5. Determination of the concentrations of OH-FA in leaves and roots of untreated plants of A. thaliana showed 13- to 31-fold higher amounts of esterified 9-,10-, 12-, 13 und 16-OH-FA in the leaves. Moreover, the amounts of esterified 15-OH-FA exceeded in leaves by the factor 111 in comparison to roots. 15-OH-FA was used as a selective marker for Photo-oxidative formation of OH-FA by 1O2. Though, 15-OH-FA occurred to an amount of 0,57 µg/g (dry weight) in roots as well. This indicates that, apart from an primarily used light depending mechanism, a second path for the formation of 15-OH-FA respectively 1O2 exists which does not depend on light strength. An alternative explanation could be the transport of 15-OH-FA from leaves to roots. 6. Compared to the wildtype plants no significant differences in the increase of OH-FA and PPF1 could be detected in NahG-, lsd1-, atrbohD and atrbohF-mutants 48 h after infiltration of Pst avrRPM1. Under the utilized conditions the genetic defects of the analyzed mutants did not cause a modified accumulation of both, enzymatically and not enzymatically formed oxylipins.

由於潔淨用水日漸短缺，科學家著力研究各種水淨化方法，其中以光催化技術作水淨化處理為可行的方法之一。光催化是以半導體光催化劑在光照射下所產生的活性物種（reactive oxidative species）進行消毒，其中的失活原理、各活性物種的作用和活性物種對細菌的攻擊方位，雖然已有廣範的研究，但當中仍有不清之處，比如說過氧化氫(H₂O₂)在光催化失活的作用便是其中之一，在光催化系統中所產生的H₂O₂濃度一般較低，因此其對細菌失活的效能仍然存有爭議。 / 本研究設計一種新的反應器去研究H₂O₂在連續供應模式中的失活動力學。在 8 mM 的H₂O₂下，10⁵的大腸桿菌(Escherichia coli)在8小時內完全失活。而在 2 mM 的H₂O₂ 下，並無出現顯著失活，由於該濃度遠遠高於一般光催化系統所產生的濃度（<50 μM），因此可以推斷，即使一般光催化系統所產生的H₂O₂是連續供應，也不會使細菌失活。然而在光照的情況下，其失活動力學大為不同，在強光照射(200 mW cm⁻²)下，H₂O₂的失活效率顯著增強，證明光照和過氧化氫之間存有協同效應。這現象亦出現於光預處理過(light pretreated)的大腸桿菌，進一步證實了光照改變細菌的生理機能，從而使其易於被H₂O₂失活。 / 其後我們使用RNA測序(RNA sequencing)去檢測的大腸桿菌的基因表達水平在光照下的變化，以便研究光照和H₂O₂之間的協同作用的機理。大多數涉及抵抗氧化的基因，包括過氧化氫酶(catalase, CAT)和超氧化物歧化酶(superoxide dismutase,SOD)的表達、DNA修復及細菌內的鐵含調控等等，其mRNA 水平沒有顯著的增加或減少，只有dps、fes和sodB有明顯的變化。此外，還有幾種調控細胞內的銅合量（cutA和cueR）和細胞膜組成（ompA、ompC、resx和gnsB）的基因在光照下產生顯著變化。 經RNA測序後，我們選定了10個目標基因，並選擇相對的大腸桿菌變異體(mutants)，對比他們和母體(E. coli BW25113)經過光預處理後被H₂O₂的失活效能。雖然這次研究並未找到相關基因，但研究結果表示，光照和H₂O₂的協同效應，應該是光照增加細胞膜的通透性和提高細菌內Fenton劑含量，使細菌內的羥基自由基(·OH)的濃度增加，因此加強對細菌DNA的損傷。 / 最後，我們亦比較了AgBr/Ag/Bi₂WO₆在不同的光源的照射下的對大腸桿菌的光催化失活效率。雖然發光二極管(light emitting diode)和熒光管都常用於室內照明，但AgBr/Ag/Bi₂WO₆的細菌失活效率在兩者的光照下表現出顯著的差異，而不同的發射波長下的細菌失活效率和AgBr/Ag/Bi₂WO₆光學吸收表現出良好的相關性。此外，相對其他顏色的發光二極管，綠色發光二極管照射下在犧牲劑研究(scavenger study)的結果大為不同，進一步表明了光照的發射波長(emissionwavelength)對光催化失活機制的影響。 / 本研究揭示了H₂O₂和光照在光催化失活中的重要性，並演示了H₂O₂和光照射之間的協同作用，也闡明了光照的屬性如何影響光催化下各活性物種的產生。本研究不僅提供了一個新的角度去探討的光照、H₂O₂和細菌的生理狀態在光催化失活中的重要性，也提供了新的方向和方法去研究光催化失活機制的。 / Due to the increasing concern for the need of clean drinking water, different methods for water purification have been developed. Photocatalysis, which makes use of semiconductor photocatalyst for the generation of reactive charged and oxidative species (ROSs) under light irradiation, is one of the most promising methods for water disinfection. The mechanisms of the photocatalytic inactivation have been extensively investigated. Different factors, including the roles of ROSs and the ROSs target site(s) of bacterial cell, were elaborated by different studies. However, there are still controversial issues on the role of H₂O₂ in photocatalytic inactivation. The effectiveness of the low concentration of H₂O₂ in the bacterial inactivation process is still under question. / This study designs a new reactor to study the kinetic of H₂O₂ inactivation in continuous supply mode. Complete inactivation of 5-log Escherichia coli within 8 h is achieved when 8 mM of H₂O₂ is applied. No significant inactivation was observed when 2 mM H₂O₂ is applied, this concentration of H₂O₂ is much higher than that detected in common photocatalytic system (< 50 μM). The results show that H₂O₂ produced by common photocatalytic system is not harmful to bacterial cell, even they are produced continuously. However, when light irradiation of 200 mW cm⁻² , using Xenon lamp as lighting source, was applied to the system, the inactivation efficiency of H₂O₂ was significantly enhanced, which demonstrate the synergistic effect between the light irradiation and H₂O₂. The enhancement of inactivation by H₂O₂ can also be observed with light pretreated E. coli K-12, further confirms that light irradiation alter the physiology of the bacterial cell which increases their sensitivity to H₂O₂. / In order to find out the mechanism(s) of the synergism between the light irradiation and H₂O₂, RNA sequencing (RNA-Seq) was used to reveal the change of gene expression level of the E. coli under light irradiation. The mRNA level of most of the genes involve in catalase (CAT) and superoxide dismutase (SOD) expression, DNA repairing and intracellular iron regulation did not have significant increase or decrease. Only dps, fes and sodB showed significantly changes. Moreover, some genes that related to regulation of intracellular copper (cutA and cueR) and membrane composition (ompA, ompC, resX and gnsB) also showed significantly changes under light irradiation. After the RNA-Seq, ten genes were chosen as the possible target genes that related to the mechanism(s). Then the inactivation of E. coli BW25113 (parental strain) and the isogenic deleted mutants by H₂O₂ with light pretreatment were conducted and compared. Although the gene(s) that directly involved in the mechanisms of the synergy between H₂O₂ and light irradiation are not identified in the study, the results show that genes that are important to bacterial defense of oxidative damages, such those responsible for CAT and SOD expression and DNA repairing, are not involved in the mechanism(s). Increase of cell permeability and intracellular Fenton’s reagent content should be the main causes for the enhancement of H₂O₂ under light irradiation. / Finally, the inactivation efficiency of E. coli K-12 using AgBr/Ag/Bi₂WO₆ under different lighting sources is compared. The results show that inactivation efficiency under different emission wavelength are highly correlated with the optical absorption of the AgBr/Ag/Bi₂WO₆. Photocatalytic inactivation under two indoor lighting sources, LED lamps and Fluorescence tubes, also showed significant difference. The result of scavenger study under green LED lamps is completely different from those under other colour of LED lamps, indicates that emission wavelength also has great influence in photocatalytic inactivation mechanisms. / This study reveals the roles of H₂O₂ and light irradiation in photocatalytic inactivation and demonstrates the synergism between the H₂O₂ and light irradiation. The influence of the properties of light irradiation, including the light intensity and major emission wavelength, on the ROSs production by photocatalyst is also reported as well. This study not only provides a new perspective to the importance of light irradiation, H₂O₂ and the physiology of bacteria in photocatalytic inactivation, but a new approach in the investigation of photocatalytic inactivation mechanisms as well. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ng Tsz Wai. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 111-131). / Abstracts also in Chinese.

"Tris-2-chlorethyl phosphate (TCEP) is a flame retardant and plasticizer that has been detected in drinking water sources and wastewater effluents in many countries. TCEP has been proven to be a recalcitrant compound that is also toxic and carcinogenic. The incomplete removal of TCEP in water and wastewater treatment plants necessitates that treatment processes be identified or developed that will completely remove TCEP from waters. Ozonation has been successfully used as an oxidant to degrade many problematic contaminants in water and wastewater. This research examined the effectiveness of ozone and ozone/hydrogen peroxide oxidation for removing TCEP from water. In laboratory experiments, batch reactions of TCEP solutions were conducted in purified water at different pH conditions and O3:H2O2:TCEP doses. The samples were tested at O3:TCEP molar ratios of 6:1, 15:1, and 30:1 and with O3:H2O2:TCEP molar ratios of 6:4.2:1, 15:10.5:1and to 30:21:1 respectively. Solution pHs of 4, 7, and 9 were investigated to cover the typical water and wastewater pH range. The Indigo Method was used to measure the starting ozone concentrations in sample water. TCEP concentrations were quantified by liquid-liquid extraction followed by gas chromatography (GC). Greater than 90% reduction of TCEP was achieved at equilibrium conditions with an O3:H2O2:TCEP molar ratio of 30:21:1 at all pH levels examined. Greater than 80% TCEP removal in diluted wastewater was observed at purified water to wastewater dilution of 4:1. The kinetics of oxidation of TCEP with ozone and ozone/hydrogen peroxide were explored in this research. It was found that the reaction rate constants of TCEP are lower than those of other contaminants typically found in water and wastewater. "

<p>New energy resources that can replace petroleum diesel are needed and biodiesel may be an excellent substitute. During storage and use, biodiesel can oxidize and form peroxides, leading to degradation of the fuel. It is therefore important to determine the peroxide content of the fuel. In this thesis the triphenylphosphine method, which involves GC-MS analysis has been compared to the standard iodometric method which involves titration with sodium thiosulfate. Working standards were made to see how well the iodometric method worked and also in order to make a calibration curve in GC-MS. Aged samples were then analyzed. The results showed that both methods gave good linearity and repeatability with limits of detection (LOD) and quantification (LOQ) of 0,06 mM and 0 17-0,65 mM respectively. However, the iodometric method seemed more circumstantial since it needed a lot of solvents and different reagents. The triphenylphosphine method was on contrary very simple to use and did not require too much preparation work. The latter also showed better reproducibility. In conclusions, both methods can be used in evaluating the peroxide content of biodiesel, but the triphenylphosphine method has the advantages of higher simplicity and better reproducibility.</p>