Abstract Not Provided / Thesis / Master of Science (MSc)

Doctor Pharmaceuticae - DPharm / Natural products have been an important source of drugs and novel lead compounds in drug discovery. Their unique scaffolds have led to the synthesis of derivatives that continue to give rise to medicinally relevant agents. Thus, natural product-inspired drugs represent a significant proportion of drugs in the market and with several more in development. Cancer is among the leading public health problems and a prominent cause of death globally. Chemotherapy has been important in the management of this disease even though side effects that arise due to lack of selectivity is still an issue.

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

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BARBOZA, Felipe Moreira. Espectroscopia Raman na L-valina deuterada a baixas temperaturas. 2012. 75 f. Dissertação (Mestrado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-04-23T21:25:11Z No. of bitstreams: 1 2012_dis_fmbarboza.pdf: 1400454 bytes, checksum: d614be224b439b279076ca7546ca4c23 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-04-29T17:45:05Z (GMT) No. of bitstreams: 1 2012_dis_fmbarboza.pdf: 1400454 bytes, checksum: d614be224b439b279076ca7546ca4c23 (MD5) / Made available in DSpace on 2015-04-29T17:45:05Z (GMT). No. of bitstreams: 1 2012_dis_fmbarboza.pdf: 1400454 bytes, checksum: d614be224b439b279076ca7546ca4c23 (MD5) Previous issue date: 2012 / Deuteration allows the identification of several species of vibrations, through the comparison of vibrational spectra of the deutered and hydrogeneted samples. In this work we base studied the vibrational properties of L-valine-d8 (99,8 % atom % D) through the Raman spectroscopy technique. At first, the assignment of all Raman active vibratonal mades of L-valine was revisited, and a comparison with a previous work was done. In particular, several bands associated to stretching of NH_3^+ and stretching of CH3, among others, which are observed in the interval 2000 – 2400 cm-1 were assigned. In the second part of the work, again using Raman spectroscopy, it was studied the vibrational modes of the crystal in the temperature range 100 – 300 K. It is known from literature that hydrogenated L-valine undergoes a phase transition at about 110 K. It also known that in deuterated crystals hydrogen bands - through Ubbehlode effect – tend to be less strange and, as a consequence, a comparative analyses between the deuterated and hydrogenated samples is very important. In a previous work on L-alanine it was observed that deutaration induces a new phase at low temperatures. In the investigation on L-valine, at least in the temperature range studied, it was not possible to note any change in the Raman spectra which could be associated to a structural phase transition. Both in the external modes region any great change is verified. As a consequence, we can infer that L-valine-d8 is stable between 100 and 300 K. A discussion about the difference behaviors at low temperatures of L-valine and L-alanine (both deuterated and hydrogenated) is also furnished in the present work. / A deuteração de uma determinada amostra permite fazer a identificação de vários tipos de vibrações, comparando-se o espectro vibracional com o de uma amostra hidrogenada. Neste trabalho estudou-se o comportamento vibracional da L-valina-d8 (99,8 átomo % D) através da técnica de espectroscopia Raman. Inicialmente revisitou-se o assinalamento de todos os modos vibracionais ativos no Raman, comparando-se com um estudo previamente realizado. Em particular foram identificadas diversas bandas associadas a vibrações do tipo estiramento do NH3+ e estiramento do CH3, entre outros, que são observadas na região entre 2000 e 2400 cm-1. Na segunda parte do trabalho foi realizado um estudo via espalhamento Raman dos modos vibracionais do cristal no intervalo de temperatura entre 100 e 300 K. Sabe-se da literatura que a L-valina hidrogenada apresenta uma transição de fase em torno de 110 K. Uma vez que nos cristais deuterados as ligações de hidrogênio via o efeito Uhbehlode tendem a ser mais fracas, uma análise comparativa entre as amostras hidrogenada e deuterada se faz necessário. Em particular, num estudo realizado na L-alanina descobriu-se que a deuteração induz a formação de uma nova fase em baixas temperaturas. No caso da L-valina, pelo menos no intervalo de temperatura investigado, não foi possível observar nenhuma mudança nos espectros Raman que pudessem ser associadas a uma transição de fase estrutural. De fato, tanto na região dos modos externos, quanto na região dos modos internos nenhuma grande modificação é verificada. Isso implica que a estrutura da L-valina-d8 é estável no intervalo de 100-300 K. Uma discussão acerca da diferença do comportamento a baixas temperaturas dos cristais de L-valina e de L-alanina nas formas hidrogenadas e deuteradas é também fornecida no presente trabalho.

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Wo und wie schnell nehmen Wurzeln Wasser auf? Obwohl diese Frage in Pflanzen- und  Bodenwissenschaften von großer Bedeutung ist, gibt es nur wenige experimentelle Daten darüber, an welcher Stelle der Wurzeln eine transpirierende Pflanze das Wasser aus dem Boden erhält. Die Antwort auf diese Frage erfordert direkte und in-situ Messungen des lokalen Wasserflusses in die Wurzel hinein. Ziel dieser Arbeit war es, eine neue Methode zu entwickeln und anzuwenden, um den lokalen Wasserfluss in unterschiedliche Segmente der Pflanzenwurzeln zu quantifizieren. Dabei wurde Neutronenradiographie eingesetzt um den Transport von deuteriertem Wasser (D2O) in die Wurzel von Lupinen zu untersuchen. Die Lupinen wuchsen in Aluminium Containern, die mit sandigem Boden gefüllt waren. Der sandige Boden wurde mit Hilfe von 1cm-dicken Schichten groben Sandes in verschiedene Bereiche eingeteilt. Diese Schichten reduzierten die Diffusion von D2O zwischen den verschiedenen Bereichen.  D2O wurde in ausgewählte Bereiche tagsüber (transpiriende Pflanzen) und nachts (nicht transpiriernde Pflanze) injiziert. Transport von D2O in die Wurzeln hinein wurde durch Neutronenradiographie mit einer räumlichen Auflösung von 100 µm in Intervallen von 10 Sekunden aufgezeichnet. Die Messungen zeigten: i) Transport von D2O in die Wurzel hinein war tagsüber schneller als nachst; ii) D2O wurde tagsüber schnell entlang der Wurzel in Richtung Spross transportiert, während dieser axiale Fluss nachts vernachlässigbar war. Die Unterschiede zwischen Tag- und Nachtmessungen wurden durch konvektiven Transport von D2O in den Wurzeln erklärt. Um den effektiven Wasserfluss in die Wurzeln hinein zu quantifizieren, wurde ein einfaches Konvektions-Diffusions Modell entwickelt, wobei die Zunahme der D2O Konzentration in Wurzeln vom konvektiven Transport abhängt und von the Diffusion des D2O in die Wurzeln. Die Ergebnisse zeigten, dass die Wasseraufnahme nicht gleichmäßig entlang der Wurzel stattfindet. Die Wasseraufnahme war in den oberen Bodenschichten höher als in den tieferen. Entlang einzelner Wurzeln war der radiale Fluss in nahen Teilen der Wurzel höher als in den weiter entfernten Teilen der Wurzel. In Lupinen fand die Wasseraufnahme im Wesentlichen in den lateralen Wurzeln statt. Die Funktion der Pfahlwurzel war es, das Wasser der lateralen Wurzeln zu sammeln und zum Spross zu transportieren. Diese Funktion wird durch eine geringe radiale und eine hohe axiale Leitfähigkeit sichergestellt.  Wir haben diese Technik auch angewandt um den Einfluss der Rhizosphäre auf die Wasseraufnahme zu untersuchen. Wie vor Kurzem auch in der Literatur berichtet wurde, wurde auch in dieser Arbeit beobachtet, dass der Boden in der unmittelbaren Nähe der Wurzeln, der sogenannten Rhizosphäre, hydrophob wird, wenn der Boden trocken wird. Zum ersten Mal konnte gezeigt werden, dass durch die Hydrophobizität der Rhizosphäre die Wasseraufnahme nach Trocknung und folgender Bewässerung reduziert wird. Es wurde die Schlussfolgerung gezogen, dass nach Trocknung die Rhizosphäre einen entscheidenden Wiederstand für den Wasserfluss zur Wurzel darstellt. Das beeinflusst vermutlich auch die Ausdehnung des Bereiches der Wurzeln, in dem Wasser aufgenommen wird. Die Bedeutung dieser Arbeit ist die Entwicklung einer neuen Methode, um Wasseraufnahme durch Wurzeln lebender Pflanzen lokal zu quantifizieren. Diese Methode macht es möglich quantitativ zu messen, wo und wie schnell Wurzeln Wasser im Boden aufnehmen. Diese Technik wird es erleichtern, die Funktionsweise der Wurzeln verschiedener Pflanzen zu verstehen und den Einfluss von Wurzelwachstum und wechselnder äußerer Bedingungen, wie Wassergehalt, Transpiration und Verfügbarkeit von Nährstoffen und vieler weiterer Faktoren zu untersuchen.  Die Antwort auf diese Fragen könnten einen weiten Bereich für landwirtschaftliche Anwendungen eröffnen, die darauf abzielen, Bewässerungsmethoden zu verbessern.

In part one, betulinic acid (1) was isolated from the American Sycamore (Platanus occidentalis) in 1.6% yield, while ursolic acid (3) was isolated from Fuji and McIntosh apple peels in 1.0% and 0.8% crude yields, respectively. Oleanolic (4) and dehydrocanophyllic (6) acids were previously available, along with several analogs. Additional analogs of 1, 3 and 4 were prepared, including 9 new compounds, for a total of 51 compounds. Compounds were initially screened for cortisol lowering properties in vitro using a fish head kidney cell assay. Platanic acid (43) was selected for in vivo study in rats, along with 1 and a blend of Platanus occidentalis and Souroubea sympetela. No significant cortisol lowering was observed in vivo. In part two, β-ionone-d6 (75) was synthesized in 6.5% yield from ethyl 2-oxo-cyclohexane carboxylate (77). Total deuterium incorporation was 99.85%, with 0.03% d0 analog. 75 was converted to retinoic acid-d6 (93) in 2.2% yield.