This thesis describes the synthesis of halogen bonding receptors for integration within interlocked anion host systems. Chapter 1 introduces the field of supramolecular chemistry, with a particular focus on anion recognition and sensing, halogen bonding, and the synthesis of mechanically interlocked structures. Chapter 2 describes the preparation and anion binding properties of carbazole-based receptor molecules. A systematic anion binding study on a series of halogen- and hydrogen-bonding 3,6-bis-triazolium carbazole acyclic receptors is described initially, followed by the development of a halogen bonding rotaxane. The anion and metal complexation properties of acyclic and macrocyclic systems incorporating the 1,8-bis-triazole carbazole motif are also presented. Chapter 3 details the synthesis and anion complexation investigations of halogen and hydrogen bonding naphthalene-based acyclic and interlocked rotaxane host molecules. Chapter 4 presents receptors based on the 4,4'-bis-triazole-2,2'-bipyridyl motif. A halogen bonding rhenium(I) bipyridyl complex is exploited in the development of a rotaxane host system which optically senses anions via luminescence purely through halogen bonding interactions. The anion recognition and sensing properties of diquat-based receptors are also investigated, and shown to exhibit optical and electrochemical responses to anions. Chapter 5 summarises the major conclusions from Chapters 2-4. Chapter 6 describes the experimental procedures used in the work, and includes characterisation data for the synthesised compounds. Supplementary information relating to crystallographic data, and absorption, luminescence and electrochemical studies, is provided in the Appendices.

This thesis investigates the design and synthesis of macrocycles and examines their ion-pair recognition, anion recognition and interpenetrative assembly capabilities. Chapter One introduces the field of supramolecular chemistry. Key concepts in host–guest complexation and self-assembly are outlined, with particular emphasis on the design of host systems for anion and ion-pair recognition. Chapter Two details the synthesis and binding properties of a heteroditopic 1,2,3-triazole- containing macrocycle that exhibits cooperative ion-pair recognition. Solution and solid-state evidence shows that the triazole motif simultaneously binds anions and cations. The exten- sion of this work towards the formation of interlocked structures containing an expanded heteroditopic macrocycle and threading components is explored. Chapter Three discusses the design and syntheses of series of electron-deficient macrocycles based on imidazolium, pyridinium and triazole. These are investigated for anion binding ability and assembly of interpenetrative assemblies with electron-rich threading components. Chapter Four describes research into the formation of interpenetrative assemblies using neutral components. Their design is based around aromatic donor–acceptor interactions and halogen bonding. Chapter Five presents some conclusions from the research undertaken. Chapter Six provides titration protocols, Job plot approximations, experimental procedures and characterisation of the compounds described in this thesis. Appendix details additional X-ray crystallographic data.

Einleitung: Die Kationen-Anionen Bilanz (DCAB) des Futters modifiziert den Säure-Basen Status von Tieren und findet Anwendung in der Prävention von Milchfieber bei Kühen, MMA bei Sauen und Urolithiasis bei Haustieren. Durch die Veränderung des Kationen-Anionen-Verhältnisses in Futterrationen können biologische Prozesse beeinflusst werden, der Stickstoffmetabolismus. Ziel der Untersuchung: Die vorliegende Studie befasst sich mit der Frage, ob eine kaliumbedingte Variation der DCAB des Futters für wachsende Schweine einen Einfluss auf bestimmte Parameter des Säure-Basen-Haushaltes und möglicherweise auch auf die Stickstoffbilanz hat. Materialien und Methoden: Dem Versuch standen insgesamt 38 männlich kastrierte Mastschweine (Dreirassen-Kreuzung von Pietrain x Deutsches Edelschwein x Deutsche Landrasse) mit einem Einstallungsalter von ca. 12 Wochen und einer Lebendmasse von 17,3 kg bis 30,3 kg zur Verfügung. In randomisierter Reihenfolge erfolgte die Zuteilung der Schweine zu den acht Versuchsfuttern; Rohproteingehalt von 140 g kg-1 Futter und 200 g kg-1 Futter, sowie je 4 g, 10 g, 14 g oder 20 g Kalium kg-1 Futter. Die Versuchsfutter wiesen eine konstante Konzentration an Natrium und Chlorid auf. Die Hauptfutterkomponenten waren Mais und Weizen. Die beiden Rohproteingehalte wurden durch unterschiedliche Sojaextraktionsschrot- und Maisklebermengen gewonnen. Über Kaliumhydrogencarbonat (KHCO3) und Kaliumchlorid (KCl) resultierte die Einstellung der genannten Kaliumkonzentrationen sowie der vier DCAB-Stufen von -125 mEq kg-1, 66 mEq kg-1, 168 mEq kg-1, und 342 mEq kg-1 Futter. In einer Adaptionsphase von 15 Tagen gewöhnten sich die Scheine an das Versuchsfutter und die Umgebung. Die Einstallung erfolgte in Einzelboxen und die Versuchstiere erhielten Wasser ad libitum. Während der anschießenden zwei Bilanzphasen von je fünf Tagen wurden die Schweine in Bilanzkäfigen gehalten. Zwischen den beiden Bilanzphasen kam es zu einer fünftägigen Pause ohne Änderung der Fütterung. In der Bilanzzeit wurden der gesamte Harn und Kot der Tiere gesammelt sowie der dazugehörige pH-Wert kontinuierlich bestimmt. Harn- und Kotaliquots wurden für Stickstoff- und Elektrolytanalysen einbehalten. Jede fünftägige Bilanz endete mit der Gewinnung einer Blutprobe von jedem Schwein aus der Vena jugulares zur Bestimmung von Kalium, Natrium, Chlorid, pH-Wert, Hydrogencarbonat, Basenüberschuss und Aminosäuren. Ergebnisse: Kalium hat einen Einfluss auf den Harn pH-Wert. Analog zur steigenden Kaliumaufnahme (DCAB↑) wurden die Harn pH-Werte basischer (-125 mEq kg-1 Futter = Ø 5,93; 342 mEq kg-1 Futter = Ø 8,37). Die Blut pH-Werte, die im Durchschnitt bei 7,21 lagen, wie auch die Hydrogencarbonat- und Basenüberschusskonzentration, reagierten aufgrund der renalen Kompensation nicht wesentlich auf die unterschiedlichen DCAB im Futter. Die dazugehörigen Kot pH-Werte waren bei -125 mEq kg-1 Futter und 66 mEq kg-1 Futter um 0,16 höher als bei den anderen beiden DCAB-Stufen. Die Stickstoffaufnahme variierte zwischen 0,90 g kg-1KM d-1 und 1,22 g kg-1KM d-1 aufgrund der beiden Rohproteingehalte (14 und 20 %) in den Versuchsrationen. Eine Senkung der DCAB im Futter bewirkte eine Verbesserung der Stickstoffverdaulichkeit von 86,1 % auf 89,9 % (p<0,05). Hingegen zeigten die Diäten mit der kaliumärmsten Konzentration die höchsten renalen Stickstoffexkretionen von 442 mg kg-1KM d-1 gegenüber den anderen drei Kaliumkonzentrationen (345 mg kg-1KM d-1). In Folge dessen ergibt sich eine Stickstoffretentionssteigerung mit zunehmender DCAB im Futter. Jedoch wurde bei 66 mEq kg-1 Futter (Kalium 10 g kg-1 Futter) die höchste Stickstoffretention von 643 mg kg-1KM d-1 festgestellt. Die Untersuchung der Blutproben ergab keine Beeinflussung der Summe aller Aminosäuren, die bei ø 44,66 mg dl-1 lag. Die Summe der essentiellen Aminosäuren war bei einer DCAB von 66 mEq kg-1 Futter im Blut geringer als bei den übrigen Variationen. Einige Parameter der Elektrolytbilanzen waren zwischen den Futtervariationen verschieden: Bei dem Versuchsfutter mit einer DCAB von -125 mEq kg-1 Futter (Kalium 4 g kg-1 Futter) schieden die Schweine Na: 2,83 mg kg-1KM d-1 und Cl: 1,54 mg kg-1KM d 1 weniger mit dem Kot und Na: 7,05 mg kg-1KM d-1 weniger mit dem Harn aus gegenüber den weiteren Versuchsgruppen. Die renale Chloridexkretion zeigte keine Variabilität. Die renale, als auch die fäkale Kaliumausscheidung nahm analog zur DCAB des Futters zu (DCAB im Futter: -125 mEq kg-1; 66 mEq kg-1; 168 mEq kg-1; 342 mEq kg-1; K-Abgabe in mg kg-1KM d-1, renal: 74,0; 273,3; 431,1; 609,1; fäkal: 24,5; 31,2; 32,6; 44,0). In der Gesamtheit betrachtet ergibt sich für die Natrium- und Chloridretention keine richtungsweisende Beeinflussung im Zusammenhang mit der DCAB der Versuchsrationen. Die Kaliumretention hingegen stieg von 66,5 mg kg-1KM d-1 (-125 mEq kg-1 Futter) auf 167,0 mg kg-1KM d-1 (342 mEq kg-1 Futter) an, was nicht von den Kaliumkonzentrationen im Blut wiedergegeben wurde. Entsprechendes gilt für die Natrium- und Chloridkonzentrationen im Blut. Schlussfolgerungen: In der Alkalisierung des Harns zeigt sich, dass der DCAB des Futters Einfluss auf den Säure-Basen Status nimmt. Der systemische pH-Wert blieb aufgrund der Puffersysteme des Organismuses weitestgehend unberührt. Durch die KHCO3-Zulagen wurde das intragastrale bzw. das intestinale pH-Milieu verändert, was sich in der schlechteren Verdaulichkeit von Stickstoff bei höherer DCAB wiederspiegelt. Die Stickstoffretention steht in keinem Zusammenhang mit der Stickstoffverdaulichkeit. Mit dem DCAB von 66 mEq kg-1 Futter bzw. K: 10 g kg-1 Futter wurde die beste Retention für Stickstoff beobachtet. Die täglichen Gewichtszunahmen und die Futterverwertungen der Versuchsschweine konnten dies allerdings nicht reflektieren. Anzumerken sei, dass für einen eindeutigen Effekt auf die tägliche Zunahme eine längere Beobachtungsphase notwendig wäre (Sprung der täglichen Zunahmen von 520 g für -125 mEq kg-1 Futter auf das Niveau von 692 g für 66 mEq kg-1 Futter und mehr). Eine Empfehlung in Anlehnung an diese Studie wäre ein DCAB-Wert um die 66 mEq kg-1 Futter. Wird dieser Wert erhöht sinkt die Stickstoffverdaulichkeit auf der anderen Seite verschlechtert sich die Stickstoffretention bei Verringerung der DCAB. / Initiation: The dietary cation-anion balance (DCAB) of the feed modifies the acid-base balance and is used in the prevention of milk fever in cows, MMA in sows and urolithiasis in pets. The modification of the cation-anion ratio in diets can take an impact on biological processes inducting nitrogen metabolism. Objectives of investigations: This study objectively clarifies, whether potassium-based variation of the DCAB of the food has an influence on certain parameters of the nitrogen balance and the acid-base balance. Materials and Methods: The trial covered a total of 38 male castrated pigs (three racial crossing Pietrain x Large White x German Landrace) with a housing-age of approximately 12 weeks and a live weight of 17.3 kg to 30.3 kg. In randomized order, the pigs were allocated to the eight experimental feed: crude protein content of 140 g per kg feed and 200 g per kg feed, as well as 4, 10, 14 or 20 g of potassium per kg feed. The sodium and chloride concentrations in the feed were kept constant. The main food components were corn and wheat. The two crude protein levels were determined by various soybean meal and corn gluten quantities. Potassium hydrogen carbonate (KHCO3) and potassium chloride (KCl) were used to establish the four DCAB levels of -125 mEq kg-1, 66 mEq kg-1, 168 mEq kg-1, and 342 mEq kg-1 feed. In an adaptation period of 15 days pigs were accustomed to food and environment. They were kept in individual pens and were given water ad libitum. During the following two trial phases of five days each, the pigs were kept in balance cages. Between the two trial periods, there was a break of five days (no diet change). During the trial period all urine and excrement of the animals was collected, and the respective pH-value was continuously measured. Aliquots of urine and faeces were used in nitrogen and electrolyte analyses. At the end of each five-day record a blood sample from the jugular vein was taken from each pig for determination of potassium, sodium, chloride, pH-value, hydrogen carbonate, base excess, and amino acids. Results: Potassium has a significant influence on renal pH values. Analogous to increasing potassium intake (DCAB ↑), the urine pH value turned more basic (-125 mEq kg-1 feed = 5.93; 342 mEq kg-1 feed = 8.37). The blood pH levels, which averaged at 7.21, as well as the hydrogen carbonate concentration and base excess concentration, did not respond to the different DCAB in the feed because of the renal compensation. The associated feces pH values at -125 mEq kg-1 feed and 66 mEq kg-1 feed were higher by 0.16 than at the other two DCAB levels. The nitrogen intake varied between 0.90 g kg-1BM d-1 and 1.22 g kg-1BM d-1, based on both crude proteins (14 % and 20 %) in the experimental feeds. A reduction of DCAB in the feed resulted in an improvement of the nitrogen digestibility from 86.1 % to 89.9 % (< 0.05). However, diets with the lowest concentration of potassium showed the highest renal nitrogen excretions of 442 mg kg-1BM d-1 compared to the other three concentrations of potassium (345 mg kg-1BM d-1). As a consequence, nitrogen retention increases with increasing DCAB in the feed. However, the highest nitrogen retention of 643 mg kg-1KM d-1 was found with a 66 mEq kg-1 diet (potassium 10 g kg-1 feed). The examination of blood samples revealed no influence on the sum of the amino acids, which was 44.66 mg dl-1. The sum of the essential amino acids was reduced at a DCAB of 66 mEq kg-1 in blood, similar to the other variations. Some parameters of the electrolyte balances were different between the feed variations: In the experimental diet with a DCAB of -125 mEq kg-1 diet (potassium 4 g kg-1 feed), the pigs eliminated Na: 2.83 mg kg- 1BM d-1 and Cl: 1.54 mg kg– 1BM d-1 less in the feces and Na: 7.05 mg kg- 1KM d-1 less in the urine with respect to the other experimental groups. Renal chloride excretion showed no variability. The renal and fecal excretion of potassium increased proportionally to the DCAB of the feed (DCAB in the feed: -125 mEq kg-1, 66 mEq kg-1, 168 mEq kg-1; 342 mEq kg-1; K output in mg kg- 1BM d-1, renal: 74.0; 273.3; 431.1; 609.1; fecal: 24.5; 31.2; 32.6; 44.0). When viewed against the totality of results for the sodium and chloride retention, there were no trend-setting influences in connection with the DCAB of the experimental diets. The potassium retention, however, increased from 66.5 mg kg- 1BM d-1 (-125 mEq kg-1 feed) to 167.0 mg kg- 1BM d-1 (342 mEq kg -1 feed), which was not reproduced from the potassium concentrations in the blood. The same applied to the sodium and chloride concentrations in the blood. Conclusions: The alkalization of the urine shows that the DCAB of the feed influences the acid-base status. The systemic pH remained largely unaffected due to the buffer systems of the organism. The intragastric, respectively the intestinal, pH medium was changed by the addition of potassium hydrogen carbonate, which is reflected in the poorer digestibility of nitrogen at higher DCAB. The nitrogen retention is not related to the nitrogen digestibility. The best retention of nitrogen was observed with the DCAB of 66 mEq kg-1 feed (K: 10 g kg-1 feed). The daily weight gain and feed utilizations of the pigs certainly could not reflect this. It should be noted, however, that a longer observation period would be necessary for a clear effect on daily gain (jump of the daily weight gain from 520 g of -125 mEq kg-1 feed to the level of 692 g for 66 mEq kg-1 feed and more). A recommendation based on this study would be a DCAB value of 66 mEq kg-1 feed. If this value increases, the nitrogen digestibility decreases; on the other hand, the nitrogen retention deteriorated with reducing DCAB.

Transition metal catalysis has emerged as one of the most versatile methods for the selective formation of carbon–carbon and carbon–heteroatom bonds. In particular, oxidative carbon–carbon bond forming reactions have been widely studied due to their atom economic feature. This thesis has been focused on the development of new palladium(II)-catalyzed carbocyclization reactions under oxidative conditions. The first part of the thesis describes the palladium(II)-catalyzed oxidative carbocyclization-borylation and -arylation of enallenes. In these reactions, the (σ-alkyl)palladium(II) intermediate, which was shown previously to undergo β-hydride elimination, could be trapped in situ by organoboron reagents (B2pin2 and arylboronic acids) to form new carbon–boron and carbon–carbon bonds. Through these two protocols, a range of borylated and arylated carbocycles were obtained as single diastereomers in high yields. The second part deals with a palladium(II)-catalyzed oxidative diarylative carbocyclization of enynes. The reaction was proposed to start with a syn-arylpalladation of an alkyne, followed by insertion of the coordinated alkene. Subsequent arylation afforded a series of valuable diarylated tetrahydrofuran and tetrahydropyran products. The final part of the thesis advances the previously developed palladium(II)-catalyzed oxidative carbocyclization-borylation of enallenes in an enantioselective manner. C2-symmetric chiral phosphoric acids were used as the novel co-catalyst to trigger the enantioselective formation of intramolecular carbon–carbon bonds. By using this chiral anion strategy, a number of enallenes were converted to the borylated carbocycles with high to excellent enantioselectivity. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Zusammenfassung in PDF Zielstellung: Der biologische Botenstoff N ist von signifikanter Bedeutung für die Funktionsfähigkeit des Herz-Kreislauf-Systems. Pathologische Bedingungen sind oft auf eine veränderte Verfügbarkeit von N zurückzuführen. Das Verständnis der Regulierung der N-Bildung durch Endothelzellen (EC) und die nachfolgende N-Chemie im gesunden und kranken Organismus ist deshalb essentiell, jedoch längst nicht vollständig. Das Ziel dieser Dissertation war aus diesem Grund Untersuchungen zum Schutz von N vor der Wechselwirkung mit S (durch SOD-mimetische Nitroxide), sowie zum Effekt des Membranpotentials (Em) und des Zellwachstums (Proliferation) auf die Aktivität der endothelialen N Synthase (eNOS) an Kulturen von Atrium-Endothelzellen des Rindes (BAtEC). Methoden: Zum empfindlichen und spezifischen Nachweis von N wurde die ozon-vermittelte N-Chemolumineszenz (N-CL) evaluiert, modifiziert und an verschiedene in vitro Systeme angepaßt. Die N-CL wurde auf Grundlage des durch SOD hemmbaren Abbaus von N durch S als ein neuartiger Ansatz zur Bestimmung physiologisch niedriger Konzentrationen an S vorgeschlagen. Ergebnisse: Die Nitroxide -hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 3-carboxy-proxyl und 3-ethoxycar-bonyl-proxyl erhöhten die N-Konzentration durch ihre SOD-mimetische Wirkung in Modellsystemen von 3-morpholinosydnonimine (SIN-1) und Kulturen von BAtEC. Mittels ESR-Spin-Trap Untersuchungen an Lösungen von SIN-1 wurde die Bildung von H-Addukten durch Dismutierung von S bestätigt, Peroxynitrit wurde als Quelle ausgeschlossen. Modulierung des Em (durch Veränderung der extrazellulären K + -Konzentration) beeinflußte die endotheliale N-Freisetzung; Hyperpolarisierung erhöhte, Depolarisierung verminderte die N Produktion. Inhibierung der elektrogenen Na + -K + ATPase induzierte synchrone Oszillationen der endothelialen N-Freisetzung. Die systematische Untersuchung zu EC-Wachstum/Proliferation zeigte keine Änderung der N-Produktion (in Gegenwart von SOD). Jedoch war die Menge von verfügbarem N (in der Abwesenheit von SOD) niedrig in präkonfluenten, stark proliferierenden BAtEC. Die Expression der eNOS erhöhte sich mit der Kultivierungsdauer und erreichte ein Maximum bei Konfluenz. Die relative eNOS-Aktivität (N, 2-, und L-citrulline-Produktion pro eNOS-Protein) war am größten in präkonfluenten, stark proliferierenden BAtEC. Schlußfolgerungen: Die Wechselwirkung mit S ist kritisch für die Halbwertszeit von N. Aus der durch diese Studie gezeigten Erhöhung von N in biologischen und chemischen Modellsystemen kann geschlußfolgert werden, daß Nitroxide unter Bedingungen einer S-bedingten Verminderung von verfügbarem N pharmakologische Wirksamkeit besitzen könn-ten. Untersuchungen ergaben den Nachweis zur Em-abhängigen Modulierung der endothelialen N-Freisetzung. Dies liefert eine Erklärung für die Em-bedingten Veränderungen des Gefäßto-nus, nachgewiesen während physiologischer Zellstimulierung, aber auch unter pathologischen Bedingungen. Untersuchungen zu EC-Wachstum/Proliferation lieferten den Beweis für die Ver-minderung von verfügbarem N in präkonfluenten, stark proliferierenden BAtEC durch endogen gebildetes S. Die Expremierung von eNOS war proliferationsabhängig. Die spezifische eNOS-Aktivität war in proliferierenden BAtEC erhöht (wahrscheinlich durch post-translationale Verän-derungen) und in ruhenden BAtEC erniedrigt (wahrscheinlich durch Substrat- oder Kofaktormangel). / abstract in pdf Objective: The biological messenger n is of significant importance for the functionality of the cardiovascular system. Pathological conditions are often attributed to an altered availability of n. The understanding of the regulation of n formation by endothelial cells (EC) and the concomitant chemistry of n in health and disease is therefore essential but far from being complete. The objective of this dissertation was therefore to study in culture systems of bovine atrial endothelial cells (BAtEC) the protection of n from the interaction with S by SOD-mimetic nitroxides, and the effect of the membrane potential (Em) and cell growth/proliferation on the activity of the endothelial nitric oxide synthase (eNOS). Methods: For the sensitive and specific detection of n, the ozone-mediated n chemiluminescence (n-CL) was evaluated, modified and adapted to various in vitro systems. Due to the SOD-inhibitable degradation of n by S , the n-CL was proposed as a novel approach to quantify the physiological low levels of S . Results: The nitroxides 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl increased the n concentration by their SOD-mimetic action in a model system of 3-morpholinosydnonimine (SIN-1) and cultures of BAtEC. EPR spin trapping in SIN-1 solution revealed the formation of H adducts due to dismutation of S and not via decomposition of peroxynitrite. Changes in Em (by alteration of the extracellular K + concentration) affected the endothelial n release; membrane hyperpolarization increased, membrane depolarization decreased the n production. Inhibition of the electrogenic Na + -K + ATPase induced synchronous oscillations in endothelial n liberation. The systematic investigations on EC growth/proliferation showed no change in total n production (in the presence of SOD). However, the bioavailable n (in the absence of SOD) was low in preconfluent, highly proliferating BAtEC. Expression of eNOS increased with culture duration with a maximum at confluence. Relative eNOS activity (n, 2, and L-citrulline production per eNOS protein) was highest in preconfluent, highly proliferating BAtEC. Conclusions: The interaction of S is critical for the half-life of n. With the increase of n in biological and chemical model systems shown by this study, nitroxides might exert pharmacological potential under conditions of S -mediated diminution of bioavailable n. Experimental evidence was shown for the Em-dependent modulation of endothelial n formation. It supports an explanation for the Em-mediated changes of vascular tone, as demonstrated for physiological cell stimulation but also under pathological conditions. Investigations on the EC growth/proliferation provided evidence that BAtEC-derived S decreases bioavailable n in preconfluent, highly proliferating BAtEC, whereas expression of eNOS is proliferation-dependent. The specific eNOS activity is upregulated in proliferating BAtEC (probably via post-translational modifications) and down-regulated in quiescent BAtEC (probably via substrate or cofactor limitations).

Os mecanismos oxidativos pelos quais macrófagos exercem atividade microbicida permanecem em discussão, e estudos com hospedeiros animais serão essenciais para elucidar tal questão. Nesse trabalho, estudamos os mecanismos microbicidas de macrófagos in vivo comparando parâmetros de infecção nas lesões de camundongos resistentes (C57Bl/6) e suscetíveis (BALB/c) ao protozoário Leishmania amazonensis. A comparação mostrou que o controle da infecção pelos camundongos resistentes é dependente da ativação de macrófagos com expressão da enzima óxido nítrico sintase induzível, síntese de óxido nítrico e extensa nitração e hidroxilação das proteínas dos parasitas dentro dos fagolisossomos dos macrófagos. O principal agente tóxico aos parasitas parece ser derivado do peroxinitrito porque a nitração dos parasitas ocorreu na ausência virtual de células polimorfonucleares e foi acompanhada de hidroxilação. Além disso, tempol um inibidor de reações de nitração mediadas por peroxinitrito, inibiu a nitração de proteínas da lesão e aumentou o número de parasitas nelas presentes. Também, estudos com parasitas em cultura confirmaram que o peroxinitrito é citotóxico aos parasitas enquanto o óxido nítrico é citostático. O camundongo suscetível se mostrou capaz de sintetizar óxido nítrico mas o fez em estágios tardios da infecção e, provavelmente, em resposta a uma infecção secundária por bactérias. Tomados conjuntamente, os resultados indicam que o peroxinitrito e radicais dele derivados são os principais agentes leishmanicidas produzidos por macrófagos in vivo. / Macrophage oxidative microbicidal mechanisms remain debatable and their elucidation is likely to depend on studies with mammalian hosts. To examine macrophage microbicidal mechanisms in vivo, we compared infection parameters in the lesions of resistant (C57Bl/6) and susceptible (BALB/c) mice to the protozoan Leishmania amazonensis. This comparison demonstrated that infection control by resistant mice relied on macrophage activation with inducible nitric oxide synthase expression, nitric oxide synthesis and extensive nitration and hydroxylation of the proteins of the parasites inside macrophage phagolysosomes. The toxic agent to the parasite is likely to be peroxynitrite-derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells and was accompanied by parasite hydroxylation. In addition, tempol, an inhibitor of peroxynitrite-mediated nitrations, inhibited protein nitration of the lesions and increased the number of parasites in them. Also, studies with parasite cultures confmed that peroxynitrite is cytotoxic to the parasites whereas nitric oxide is cytostatic. The susceptible mice were also able to synthesize nitric oxide but only at late infection time and, most likely, in response to a secondary bacterial infection. Taken together, the results indicate that peroxynitrite and derived radicals are the main leishrnanicidal agents produced by macrophages in vivo.

Este trabalho consistiu de estudos da viabilidade da geração e identificação de radicais livres envolvidos em processos oxidativos avançados por via eletroquímica. Para a geração e identificação de radicais livres em eletrodos modificados com organotióis via eletroquímica, escolheu-se uma técnica pouco aplicada para este fim; a espectroscopia de impedância eletroquímica, e o ajuste dos dados experimentais pela proposta de modelos de circuitos equivalentes. O eletrodo de trabalho utilizado (Au) foi caracterizado em meio ácido e básico e diversas monocamadas auto-organizadas conhecidas foram adsorvidas sobre o mesmo e também caracterizadas por esta técnica. Parâmetros importantes como constante dielétrica, capacitância de uma monocamada livre de defeitos e grau de recobrimento foram determinados com êxito. Também foram empregadas moléculas menos utilizadas como modificadoras de eletrodos (fenotiazinas e derivados). As mesmas foram caracterizadas no sistema Au/adsorvente/NaOH, pois, o pH alto garante a formação do radical superóxido e do ânion hidroperóxido. Os mesmos modelos de circuitos equivalentes puderam ser empregados na obtenção dos parâmetros físicos relativos a essas moléculas no sistema eletroquímico utilizado. Em uma etapa subsequente obteve-se a valiosa informação, se as moléculas adsorvidas sobre Au poderiam ser usadas para a geração de superóxido e outros radicais. As monocamadas de tióis, quando utilizadas para gerar radicais livres, se mostraram aptas a gerar superóxido em meio alcalino saturado com oxigênio molecular. Como se demonstrou na etapa de caracterização, as SAM-3 e SAM-6 não formaram um empacotamento totalmente hidrofóbico. Por este motivo, mesmo que superóxido tenha sido gerado, não pôde ser detectado. Já para a fenotiazina e seu metil-derivado, a cinética de adsorção e a utilização de soluções aquosas impediram a formação de um filme completo e homogêneo, imprescindível para a constituição de uma interface hidrofóbica e isolante. Além disso, possíveis reações de complexação entre as moléculas e superóxido, reações entre a fenotiazina cátion-radical e superóxido fazem necessários mais estudos acerca da natureza eletroquímica do sistema, que já é bastante conhecido fotoquimicamente. Posterior a modificação, todos os eletrodos se mostraram ser bastante sensíveis ao pH e aos eletrólitos utlizados, alterando-se a estrutura interna muito facilmente, o qual implica uma limitação no tempo de uso e reprodutibilidade quando da geração de um radical tão reativo quanto superóxido. / This work consisted in testing the viability of investigations into the electrochemical generation and identification of free radicals involved in advanced oxidative processes. In these studies, a technique which is rarely used for the electrochemical generation and identification of free radicals at organothiole-modified electrodes, electrochemical impedancy spectroscopy, was chosen and the experimental data obtained were fitted by equivalent circuit models. A working electrode (Au) was characterized in both acidic and basic media and several known self-assembled monolayers were adsorbed over this electrode and also characterized using this technique. Important parameters such as dielectric constants, capacitance of the monolayers free of defects and coverage were determined with success. Other lesser-known molecules such as phenothiazines and their derivatives were also used .The modified electrodes were characterized in NaOH solution, since the high pH assures the production of superoxide radicals and hydroperoxylate anions. The equivalent circuit model was employed to obtain physical parameters for these molecules in the electrochemical systems studied. In a subsequent step, a valuable piece of information was obtained; it was noted that the molecules adsorbed over Au could be used in order to generate superoxide and other radicals. The ability of thiol monolayers used in the generation of free radicals, to produce superoxides in alkaline media saturated with molecular oxygen was demonstrated. However, characterization of the self-assembled monolayers, SAM-3 and SAM-6, revealed that these were not able to pack in a completely hydrophobic manner. This may account for the fact that the superoxide could not be detected even when it was believed to have formed. For the phenothiazine-modified electrodes, the adsorption kinetics and the utilization of aqueous solutions prevented the formation of an intact and homogeneous film, which is essential in establishing a hydrophobic and isolating interface. Furthermore, possible complexation reactions between the molecules and the superoxide and reactions between the phenothiazine cation-radical and superoxide, point to the need for further studies regarding the electrochemical nature of this system, whose photochemical properties are very well known. Following modification, all the electrodes were shown to be too sensitive to pH and to the electrolytes used, since the inner structure was easily modified, which implies that they would have a limited time of use and in addition that the reproducibility of the rate of generation of reactive radical species such as superoxides could be compromised.

Doctor of Philosophy / Department of Chemistry / Paul Edward Smith / The activity and function of many macromolecules in cellular environments are coupled with the binding of ions such as alkaline earth metal ions and poly oxo anions. These ions are involved in the regulation of important processes such as protein crystallization, nucleic acid and protein stability, enzyme activity, and many others. The exact mechanism of ion specificity is still elusive. In principle, computer simulations can be used to help provide a molecular level understanding of the dynamics of hydrated ions and their interactions with the biomolecules. However, most of the force fields available today often fail to accurately reproduce the properties of ions in aqueous environments. Here we develop a classical non polarizable force field for aqueous alkaline earth metal halides (MX₂) where M = Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and X = Cl⁻, Br⁻, I⁻, and for some biologically important oxo anions which are NO₃⁻, ClO₄⁻, H₂PO₄⁻ and SO₄²⁻, for use in biomolecular simulations. The new force field parameters are developed to reproduce the experimental Kirkwood-Buff integrals. The Kirkwood-Buff integrals can be used to quantify the affinity between molecular species in solution. This helps to capture the fine balance between the interactions of ions and water. Since this new force field can reproduce the experimental Kirkwood-Buff integrals for most concentrations of the respective salts, they are capable of reproduce the experimental activity derivatives, partial molar volumes, and excess coordination numbers. Use of these new models in MD simulations also leads to reasonable diffusion constants and dielectric decrements. Attempts to develop force field parameters for CO₃²⁻, HPO₄²⁻ and PO₄³⁻ ions were unsuccessful due to an excessive aggregation behavior in the simulations. Therefore, in an effort to overcome this aggregation behavior in the simulations, we have investigated scaling the anion to water interaction strength, and also the possibility of using a high frequency permittivity in the simulations. The strategy of increasing relative permittivity of the system to mimic electronic screening effects are particularly promising for decreasing the excessive ion clustering observed in the MD simulations.

京都大学 / 新制・課程博士 / 博士(人間・環境学) / 甲第23995号 / 人博第1047号 / 新制||人||245(附属図書館) / 2022||人博||1047(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 内本 喜晴, 教授 中村 敏浩, 教授 陰山 洋, 教授 雨澤 浩史 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

<p>The aims of this thesis were to study the in vivo relevance of membrane transporters for intestinal absorption and the hepatic disposition of drugs in humans and preclinical models. Fexofenadine is a substrate for ABCB1 (P-glycoprotein) and members of the organic anion transporting polypeptide (OATP/SLCO) family. It is marginally metabolised in humans. </p><p>The influence of known inhibitors of ABCB1 and OATPs on the membrane transport and pharmacokinetics of fexofenadine was investigated in Caco-2 and porcine models and in humans. The permeability of fexofenadine remained low, even when significantly altered by the addition of an inhibitor. Using the Loc-I-Gut^® technique in vivo in humans, it was possible to see that the jejunal effective permeability of fexofenadine was unchanged when given with verapamil. However, the systemic exposure and apparent absorption rate of fexofenadine increased. This suggests that the first-pass liver extraction of fexofenadine was reduced by verapamil, probably through the inhibition of sinusoidal OATP-mediated and/or canalicular ABCB1-mediated secretion. The unchanged permeability can be explained by simultaneous inhibition of jejunal apical OATP-uptake and ABCB1-efflux, which would leave fexofenadine to be transported by passive trancellular diffusion. A Loc-I-Gut^® perfusion in the porcine model enabling blood sampling in the portal and hepatic veins and bile collection revealed increased jejunal permeability, but no subsequent verapamil-induced elevation in the systemic exposure of fexofenadine. This indicates a species-related difference in the localisation of and/or the substrate specificity of fexofenadine for the transporters involved. The absence of an effect on the first-pass liver extraction in the porcine model might be caused by the observed lower liver exposure of verapamil.</p><p>Finally, a novel intubation technique enabling dosing of fexofenadine in the jejunum, ileum and the colon showed that fexofenadine was absorbed less along the length the intestine in agreement with the properties of a low permeability drug.</p>