Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-05T14:42:32Z No. of bitstreams: 2 Dissertação Rafaela Damasceno Sá.pdf: 1935790 bytes, checksum: 15e763111ed57a5154fb97fb0918661c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T14:42:32Z (GMT). No. of bitstreams: 2 Dissertação Rafaela Damasceno Sá.pdf: 1935790 bytes, checksum: 15e763111ed57a5154fb97fb0918661c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02 / CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) / Chenopodium ambrosioides L., conhecida popularmente como mastruz, é uma erva amplamente distribuída no Brasil e muito utilizada como anti-helmíntica e para tratamentos de gripe. Como os estudos farmacológicos demonstram suas propriedades terapêuticas e devido ao amplo e reconhecido uso popular, a espécie possui grande potencial para se tornar um fitoterápico. Nesse contexto, o objetivo do trabalho foi realizar um estudo farmacognóstico da espécie. Foram realizados cortes transversais para análise microscópica de raiz, caule e pecíolo; nas folhas foram feitos cortes transversais, paradérmicos e testes histoquímicos. Através de cromatografia em camada delgada, investigou-se a presença de metabólitos secundários nas folhas, nas quais também foi determinado o teor de flavonoides totais utilizando espectrofotometria. Foi extraído ainda das folhas o óleo essencial, através de hidrodestilação, e calculado o rendimento. Para estabelecer o perfil quali-quantitativo e determinar a composição do óleo essencial, utilizou-se cromatografia gasosa acoplada à espectrometria de massas. Foram avaliados todos os parâmetros exigidos pela ANVISA para a validação de método desenvolvido para quantificação dos constituintes do óleo essencial por cromatografia gasosa com detector de ionização em chama. Os principais caracteres úteis na identificação da planta resultantes da análise microscópica foram: presença de idioblastos contendo areia cristalina no mesofilo, na nervura principal e no parênquima de caule e pecíolo; presença de tricomas tectores e glandulares capitados na folha e no caule; e crescimento secundário na raiz e no caule formado por uma sucessão de arcos cambiais. Através dos testes histoquímicos, ficou demonstrado que os cristais são de oxalato de cálcio, assim como se evidenciou a presença de substâncias lipídicas, esteroides, óleos essenciais e oleoresinas. Com relação à prospecção fitoquímica, foi demonstrada a presença de açúcares redutores, monoterpenos e sesquiterpenos, triterpenos e esteroides e flavonoides glicosilados. O teor de flavonoides totais calculado foi de 1,61 ± 0,02 (1,51%). A extração do óleo essencial das folhas teve um rendimento médio de 0,7% ± 0,0982. Considerando a análise CG-EM do óleo essencial, pode-se constatar a presença de 10 picos, com tempos de retenção entre 18,7 e 55,2 minutos. Os constituintes majoritários encontrados foram α-terpineno (42,14%) e α-terpinenil-acetato (31,57%). O conteúdo de ascaridol, geralmente relatado como majoritário, foi de 0,87%. Um teor elevado de ascaridol já foi relatado no Brasil e também em outros países, o que leva a conclusão de que o local de coleta da planta é um importante fator a ser considerado para a análise do óleo essencial de C. ambrosioides. O método proposto para a quantificação dos constituintes do óleo essencial mostrou-se linear, com alta sensibilidade de quantificação, além de ser considerado específico, preciso, exato e robusto. Os resultados obtidos fornecem informações relevantes para a identificação e controle de qualidade da espécie, já que a planta não apresenta monografia nos códigos atuais.

La pomme de terre produit des glycoalcaloïdes comme la plupart des Solanacées. Deux composés, en particulier, l’α-solanine et l’α-chaconine, sont produits en plus grande quantité lorsque le tubercule est exposé à la lumière et subit des dégradations. Ces molécules sont toxiques et peuvent représenter un danger pour le consommateur et des nuisances pour l’environnement. Dans le cadre de cette thèse, les teneurs en α-solanine et α-chaconine dans la chaire de pomme de terre verdie, en germination ou en décomposition retrouvées sur les marchés d’Abidjan (Côte d'Ivoire) ont été analysées. Les résultats ont montré que la chaire de ces pommes de terre contient des quantités élevées de ces deux composés, dépassant 2 à 5 fois la limite recommandée. Pour des raisons de sécurité sanitaire, ces pommes de terre doivent être proscrites de l’alimentation humaine. En revanche, la teneur élevée en glycoalcaloïdes dans ces pommes de terre représente une source de solanidine, un précurseur pour la synthèse d'hormones et de composés pharmacologiquement actifs, qui mérite d’être exploitée. Dans cet objectif, nous avons développé une méthode chimio-enzymatique simple, comprenant un traitement acide partiel suivi d’une hydrolyse enzymatique par la β-glycosidase de la blatte Periplaneta americana dont le gène a été isolé à partir d’une librairie génomique de cDNA afin de détoxifier ces composés et produire la solanidine. / Potato produce glycoalkaloids as most plants of Solanaceae family. The principal glycoalkaloids, α-chaconine and α-solanine are produced in greater quantities when potato tubers are exposed to light and are subject to deteriorations. These compounds are toxic and can represent a real danger for the consumer and the environment where they are discharged during their degradation. In this work, the estimation of glycoalkaloids in the flesh of different types of decayed potatoes usually found in different market places of Abidjan (Ivory Coast) was evaluated. The results showed that turned green and also sprouting or rotting potato flesh contain high amounts of toxic solanine and chaconine, exceeding by 2 to 5-fold the recommended limit. For safety consideration, these decayed potatoes should be systematically set aside. The accumulation of α-chaconine and solanine in potatoes can be seen as an attractive source of solanidine that is an important precursor for hormone synthesis and some pharmacologically active compounds. To this end, we proposed herein a simple chemo-enzymatic protocol comprising a partial acidic hydrolysis followed by an enzymatic treatment with the β-glycosidase from Periplaneta americana whose gene was isolated from a cDNA genomic library in order to detoxify these compounds and produce solanidine.

AGrâce à sa forte carbophilie, l’or est devenu en quelques années un métal de choix pour la formation de liaisons C-C et C-X (X = O, N, S). Par ailleurs, sa capacité à activer des hétéroatomes (acidité σ-Lewis) a été reconnue également et justifiée par de nombreux exemples de transformations de substrats carbonyles, imines, alcools π-activés et époxydes. Dans le contexte de la recherche de méthodes économiques et respectueuses de l'environnement dans le domaine de la chimie des ions N-acyliminiums, nous nous sommes intéressés à la capacité de l’or à promouvoir la formation des ions N-acyliminiums. Dans une première partie de ce travail, nous avons montré que l'acidité de type σ-Lewis de sels cationiques d’or (I) et (III) pouvait être exploitée pour catalyser efficacement la substitution nucléophile de divers alcoxy-et acétoxylactames, précurseurs d’ions N-acyliminiums. La réaction est compatible avec une large gamme de nucléophiles, incluant notamment l'allyltriméthylsilane, les éthers d'énols silylés, les arènes et les dérivés de méthylènes actifs. Dans un second temps, les deux caractères carbophile et oxophile des catalyseurs à l’or ont été combinés dans un processus cascade α-amidoalkylation/hydroarylation. Une seconde approche multicatalytique (acide de Brønsted – acide de Lewis) a ensuite été développée comme alternative pour pallier à certaines limites du système « tout or ». Ces séquences réactionnelles one-pot permettent d’accéder de façon rapide et efficace à un large panel de composés polycycliques fusionnés à partir de matières triviales. / AGold catalysts, as soft carbophilic Lewis acids, have rapidly develop and mature to become nowadays an outstanding tool for new C-C and C-X bonds (X = O, S, N) through π-bond activation. Moreover, its σ-Lewis acidity was also acknowledged and exemplified by numerous examples of carbonyl, imine, π-activated alcohol and epoxide activation. As part of our ongoing studies on N-acyliminium ion chemistry, we have been interested in using gold catalysts for N-acyliminium ion generation. First, we have shown that the σ-Lewis acidity of gold(I) and gold(III) could be exploited to efficiently catalyze the nucleophilic substitution reactions of various alkoxy- and acetoxylactams, precursors of N-acyliminium ions. The reaction was found to tolerate a wide range of nucleophiles, including allyltrimethylsilane, silyl enol ethers, arenes as well as active methylene derivatives. Next, gold catalysis was applied to cascade α-amidoalkylation/hydroarylation sequences unifying both the σ- and π-Lewis acid properties of gold complexes. Alternatively, Brønsted acid/Lewis acid multicatalysis approach has been alternatively developed to override some limitations featuring this unprecedented tandem intermolecular Friedel–Crafts/intramolecular hydroarylation sequence. This two complementary and highly efficient cascade sequences enable expedient access to complex fused polycyclic structures from trivial materials.

Abstract The Z-disk is a sophisticated structure that connects adjacent sarcomeres in striated muscle myofibrils. α-Actinin provides strength to the Z-disks by crosslinking the actin filaments of adjacent sarcomeres. α-Actinin is an antiparallel homodimer, composed of an N-terminal actin binding domain (ABD), the central rod domain, and two pairs of C-terminal EF-hands. The PDZ-LIM domain proteins interact with α-actinin at the Z-disk. Of these proteins, only the actinin-associated LIM protein (ALP), Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) and C-terminal LIM protein (CLP36) have a ZASP/Cypher-like (ZM) motif consisting of 26-27 conserved residues in the internal region between the PDZ and LIM domains. The aim of this work was to understand the molecular interplay between the ZM-motif containing members of the PDZ-LIM proteins and α-actinin. To unveil the biological relevance of the interaction between the PDZ-LIM proteins and α-actinin, naturally occurring human ZASP/Cypher mutations were analyzed. Two interaction sites were found between ALP, CLP36 and α-actinin using recombinant purified proteins in surface plasmon resonance (SPR) analysis. The PDZ domain of ALP and CLP36 recognized the C-terminus of α-actinin, whereas the internal regions bound to the rod domain. Further characterization showed that the ALP internal region adopts and extended conformation when interacting with α-actinin and that the ZM-motif partly mediated the interaction, but did not define the entire interaction area. ZASP/Cypher also interacted and competed with ALP in binding to the rod domain. The internal fragments containing the ZM-motif were important for co-localization of ALP and ZASP/Cypher with α-actinin at the Z-disks and on stress fibers. The absence of ALP and ZASP/Cypher in focal contacts indicates that other interacting molecules, for instance vinculin and integrin, may compete in binding to the rod in these areas or additional proteins are required in targeting to these locations. The co-localization of the ZASP/Cypher with α-actinin could be released by disrupting the stress fibers leading to an accumulation of α-actinin in the cell periphery, whereas ZASP/Cypher was not in these areas. This suggests that an intact cytoskeleton is important for ZASP/Cypher interaction with α-actinin. Earlier studies have shown that mutations in the ZASP/Cypher internal region are associated with muscular diseases. These mutations, however, did not affect ZASP/Cypher co-localization with α-actinin or the stability of ZASP/Cypher proteins. The Z-disk possesses a stretch sensor, which is involved in triggering hypertrophic growth as a compensatory mechanism to increased workloads. α-Actinin is a docking site of molecules that are involved in hypertrophic signaling cascades mediated by calsarcin-calcineurin and protein kinase C (PKC) isoforms. The internal interaction site may be involved in targeting PKCs, which bind to the LIM domains of ZASP/Cypher, to the Z-disks. The similar location of the internal interaction site with calsarcin on the rod suggests that ZASP/Cypher, ALP and CLP36 may regulate calsarcin-mediated hypertrophic signaling.

Of all of the cancer patients throughout the world, approximately 50% of them are affected to some degree by cachexia. This syndrome involves significant skeletal muscle wasting, loss of adipose tissue and overall decrease in body weight in patients, particularly those with lung, pancreatic and gastric cancers. Cancer-induced cachexia is characterized by the presence of increased cytokines, notably TNF-α, IL-1β and IL-6. Most patients suffering of cancer-induced cachexia experience increased toxicity in response to chemotherapy, leading to fewer rounds of treatment and thus impeding the patients’ chances for recovery. More research into effective treatments for cancer-induced cachexia would therefore be indispensable. The inhibitor of apoptosis proteins (IAPs) have emerged as important cancer targets, primarily because of their roles as caspase inhibitors and regulators of NF-κB signalling. Small molecule IAP antagonists known as Smac mimetic compounds (SMCs) are currently in stage I/II clinical trials. They function by targeting cIAP1 and cIAP2 (and to a lesser extent, XIAP) resulting in a cytokine mediated death response in cancer cells. SMCs induce the production of TNF-α, a cytokine with which SMCs can potently synergize. However, limited efficacy occurs in some cancer cell lines (presumably because TNF-α cannot be induced in an autocrine fashion) and an exogenous source of the cytokine, such as that induced by using an oncolytic virus, is required. Notably, TNF-α (initially known as “cachectin”) is known to play a significant role in the induction of skeletal muscle atrophy. We therefore wanted to examine the effects of TNF-α induction by SMC and oncolytic virus co-treatment on both tumour regression and skeletal muscle in tumour bearing mice. We investigated the effects of SMC treatment on Lewis Lung Carcinoma (LLC) and B16F10 melanoma cell lines, both of which have been shown to be established cachectic cancer cell lines. Our in-vitro analysis of LLC and B16F10 cells revealed that LLC cells are sensitive to SMC and TNF-α co-treatment whereas B16F10 cancer cells remain resistant. SMC treatment, in combination with an oncolytic virus, VSVΔ51, increased tumour regression and survival time in LLC tumour bearing mice. Based on findings from previous studies, we investigated the role of cellular FLICE-like inhibitory protein (c-FLIP) in the resistance of the B16F10 melanoma cell line to SMC treatment. We were able to determine that the down-regulation of c-FLIP sensitizes the B16F10 cells to SMC and TNF-α induced cell death. In extending these findings, we found that SMC treatment alone can cause skeletal muscle wasting in the tibialis anterior muscle of LLC tumour bearing mice. However, the atrophic response was observed to be minimal as documented by a slight but significant decrease (approximately 10%) in muscle fibre cross-sectional area. Moreover, no biochemical evidence of muscle atrophy, as visualized by changes in the expression of myosin heavy chain (MHC) and Muscle RING Finger protein 1 (MuRF1), was found. Regardless, we speculate that the impact of SMC treatment on muscle wasting would be transient and reversible, and propose that the benefits of such a combination immunotherapy would greatly outweigh the risks.

Magister Scientiae - MSc / To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. α-L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones. / South Africa

Cyanide was rapidly removed when added to culture supernatants of seven different Pseudomonas. The ability to remove cyanide was correlated with the accumulation of α-keto acids (pyruvate and α-ketoglutarate). These compounds react with cyanide forming less toxic cyanohydrins, thus conferring a mechanism for bacterial cyanide tolerance. When added to growth media the α-keto acids were shown also to serve as effective cyanide antagonists. While all bacteria tested accumulated α-keto acids, only those capable of utilizing cyanide as a nutritional nitrogen source were able to metabolize cyanohydrins. In P. fluorescens NCIMB 11764, the same enzyme (cyanide oxygenase) shown previously to be involved in cyanide metabolism appears responsible for cyanohydrin transformation. Keto acid excretion is believed to represent a new mechanism of bacterial cyanide detoxification with further enzymatic metabolism of the cyanohydrins helping to explain how cyanide can satisfy the nitrogen requirement in cyanide-utilizing bacteria.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23089号 / 医博第4716号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 浅野 雅秀, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Polymer based microelectromechanical systems (MEMS) micromachining is finding more interest in research and applications. This is due to its low cost and less time processing compared with silicon MEMS. SU-8 is a photo-patternable polymer that is used as a structural layer for MEMS and microfluidic devices. In addition to being processed with low cost, it is a biocompatible material with good mechanical properties. Also, amorphous silicon (α-Si) has found use as a sacrificial layer in silicon MEMS applications. α-Si can be deposited at large thicknesses for MEMS applications and also can be released in a dry method using XeF2 which can solve stiction problems related to MEMS applications. In this thesis, an SU-8 MEMS process is developed using amorphous silicon (α-Si) as a sacrificial layer. Electrostatic actuation and sensing is used in many MEMS applications. SU-8 is a dielectric material which limits its direct use in electrostatic actuation. This thesis provides a MEMS process with two conductive metal electrodes that can be used for out-of-plane electrostatic applications like MEMS switches and variable capacitors. The process provides the fabrication of dimples that can be conductive or non-conductive to facilitate more flexibility for MEMS designers. This SU-8 process can fabricate SU-8 MEMS structures of a single layer of two different thicknesses. Process parameters were tuned for two sets of thicknesses which are thin (5-10μm) and thick (130μm). Chevron bent-beam structures and different suspended beams (cantilevers and bridges) were fabricated to characterize the SU-8 process through extracting the density, Young’s Modulus and the Coefficient of Thermal Expansion (CTE) of SU-8. Also, the process was tested and used as an educational tool through which different MEMS structures were fabricated including MEMS switches, variable capacitors and thermal actuators.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22066号 / 工博第4647号 / 新制||工||1725(附属図書館) / 京都大学大学院工学研究科材料工学専攻 / (主査)教授 辻 伸泰, 教授 安田 秀幸, 教授 宇田 哲也 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM