Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-08T13:48:31Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE_DANILO FONTES_PPGCF_UFPE.pdf: 6750693 bytes, checksum: ec0d551dfa4fb27855a7caf940d95aab (MD5) / Made available in DSpace on 2016-09-08T13:48:31Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE_DANILO FONTES_PPGCF_UFPE.pdf: 6750693 bytes, checksum: ec0d551dfa4fb27855a7caf940d95aab (MD5) Previous issue date: 2016-03-18 / FACEPE / Os hidróxidos duplos lamelares (HDL), também conhecidos como compostos tipo hidrotalcita, são materiais capazes de incorporar espécies biologicamente ativas, negativamente carregadas, na sua região interlamelar, de modo a neutralizar as cargas po-sitivas das lamelas, através do mecanismo de troca iônica. Além deste mecanismo, os HDL possuem alta capacidade de adsorção de materiais não iônicos e carregados positivamente, através de interações eletrostáticas e ligações de hidrogênio na sua vasta área superficial. Os HDL possuem ocorrência natural e também podem ser sintetizados em laboratório por rotas simples e de baixo custo, que permitem o isolamento de sólidos de alta pureza. O presente trabalho tem por objetivo a síntese e caracterização de HDL (CaAl-HDL e MgAl-Cl-HDL), e sua aplicação junto aos fármacos antiretrovirais Efavirenz (EFZ) e Zidovudina (AZT), e do anti-chagásico Benznidazol (BNZ). Os materiais obtidos foram caracterizados pelas técnicas de difração de raios-X (DRX), termogravimetria (TG), calorimetria exploratória diferencial (DSC), análise térmica diferencial (DTA), espectroscopia no infravermelho (IV), microscopia eletrônica de varredura (MEV), microscopia de luz polarizada e análise elementar de metais e de carbono, hidrogênio e nitrogênio (CHN). Foi possível observar que em sistemas com HDL de cálcio e alumínio (CaAl-HDL), obtido pelo método de evaporação do solvente, contendo até 30% de EFZ e até 20% do BNZ, o fármaco tornou-se uma molécula com características amorfas, perdendo seu caráter cristalino. Este fenômeno pôde ser comprovado através da ausência de planos cristalinos do fármaco no DRX, e também do seu ponto de fusão no DSC. Nos testes de liberação, estes sistemas obtiveram destaque no incremento de solubilidade, sendo a proporção HDL-EFZ 30% a mais promissora, com aumento de 558% do EFZ solúvel em relação ao fármaco isolado; e o sistema HDL-BNZ 20%, proporcionando 702% de aumento na solubilidade do fármaco. Desta maneira comprovou-se que associação entre o CaAl-HDL e o BNZ e EFZ (fármacos de baixa solubilidade) confere incremento de solubilidade, promovendo uma maior taxa de dissolução nos estudos in vitro. A solubilidade aquosa de um fármaco constitui requisito prévio à absorção e obtenção de resposta clínica, para a maioria dos medicamentos administrados por via oral. No sistema contendo MgAl-Cl-HDL e AZT, obtido pelo método de síntese por co-precipitação a pH constante, foi possível notar que, o fármaco tornou-se amorfo através da interação com a superfície do HDL, como evidenciado através do DRX, TG, IV e MEV. No estudo de liberação, foi possível obter um perfil de liberação prolongada do AZT, de 90% de fármaco em 24 horas de estudo. Os sistemas CaAL-HDL:EFZ e MgAl-Cl-HDL:AZT tornaram-se menos tóxicos quando comparados a seus respectivos fármacos isolados, enquanto o sistema CaAL-HDL:BNZ, não alterou a toxicidade do BNZ, quando testados em linhagem de macrófagos humanos. / Layered double hydroxides (LDH), also known as hydrotalcite compounds, are materials able to incorporate biologically active species, negatively charged, into the interlayer region, to neutralize the positive charges of the lamellae through the ion exchange mechanism. Besides this mechanism, LDH have a high adsorption capacity for positively charged non-ionic materials, through electrostatic interactions and hydrogen bonds on its vast surface area. LDH are found in nature and can also be synthesized by simple and low-cost routes, that allow for the isolation of high purity solids. This paper presents the synthesis and characterization of the LDH (CaAl-LDH e MgAl-Cl-LDH), its applications with the antiretroviral drugs efavirenz and zidovudine, and the anti-chagas drug Benznidazol. The materials were characterized by the techniques of x-ray diffraction (XRD), thermogravimetry (TG), differential scanning calorimetry (DSC), differential thermal analysis (DTA), infrared spectroscopy (IR), scanning electron microscopy (SEM), polarized light microscopy, and elemental analysis of metals and carbon, hydrogen and nitrogen (CHN). It was observed that in systems with CaAl-HDL, obtained by the solvent evaporation method, containing up to 30% of EFZ and 20% of BNZ, the drug became a molecule with amorphous characteristics, losing its crystalline character. This phenomenon could be demonstrated by the absence of the drug crystal planes in the XRD analysis, and also by its melting point in the DSC analysis. In the release experiments, these systems stood out because they promoted an increase in solubility, with LDH-EFZ 30% being the most promising, with an increase in the EFV solubility of 558% compared to the drug itself; and the LDH-BNZ 20% system providing a 702% increase in solubility. Thus, the association between CaAl-LDH and BNZ and EFZ (low solubility drugs) was proven to increase the solubility of these drugs, promoting a higher dissolution rate in in vitro studies. The aqueous solubility of a drug is a prerequisite for obtaining absorption and clinical response for most drugs administered orally. In the system containing MgAl-Cl-LDH and AZT, obtained by the method of co-precipitation at constant pH, it was noticeable that, after the synthesis, the drug became amorphous due to the interaction with the surface of the LDH, as evidenced by the XRD, TG, SEM, and IR analyses. In the release study, it was possible to obtain a profile of the prolonged release of AZT, 90% of the drug in a 24-hour experiment. The cell viability experiment showed that the CaAl-LDH:EFZ and MgAl-Cl-HDL:AZT systems became less toxic than the isolated drugs and the CaAl-HDL:BNZ system did not alter the toxicity of the drug itself, when tested in human macrophage lineage.

Diferentemente do que muitos imaginam, o Brasil é detentor da maior diversidade de espécies de bambus dos países do Novo Mundo. O conhecimento sobre o potencial de aplicações de bambus nativos é extremamente subdesenvolvido em comparação ao de espécies asiáticas. Considerando que as espécies asiáticas são utilizadas na medicina popular e se têm relatos de várias atividades biológicas atribuídas à presença de flavonoides e outras substâncias fenólicas de interesse, espécies de bambu do Neotrópico são potenciais fontes de bioativos. Utilizando-se essa premissa, Merostachys pluriflora, uma espécie nativa de bambu, foi escolhida como modelo de estudo. Este trabalho teve como objetivos quantificar o teor de amido, carboidratos solúveis, lipídeos, fenóis totais, flavonoides, taninos totais e proantocianidinas de folhas e colmos de M. pluriflora; e avaliar o potencial biológico dos seus extratos, fases e substâncias isoladas através de ensaios in vitro da capacidade antioxidante, anti-HIV-1 e antibacteriana. Foi possível observar que o extrato bruto de folhas rendeu o dobro do extrato de colmo e que as fases obtidas com solventes mais polares, como a fase hidrometanólica, apresentaram maiores rendimentos. Dos metabólitos primários quantificados em M. pluriflora, os lipídeos se destacaram em conteúdo em ambos os órgãos estudados. Com relação as substâncias fenólicas, foi possível observar que o extrato bruto dos colmos apresentou uma maior abundância de fenilpropanoides e derivados do ácido clorogênico, enquanto o extrato bruto das folhas apresentou uma maior abundância de flavonoides, quando comparadas aos colmos. Das substâncias fenólicas presentes em M. pluriflora, foram identificadas duas flavonas, a vitexina e a isovitexina; e três fenilpropanoides, o ácido cafeíco, ácido ferúlico e o cafeato de metila. Das fases geradas por partição, a de acetato de etila e de diclorometano, para ambos os órgãos, foram as que apresentaram a maior parte dos constituintes fenólicos, sendo as fases de acetato de etila mais ricas em flavonoides e as de diclorometano em fenilpropanoides. No geral, os extratos brutos, assim como as fases de folhas e colmos de M. pluriflora, apresentaram um grande potencial antioxidante, principalmente antiradicalar e redutor de ferro, apresentando valores de EC50 de 16,30 μg/mL a 94,77 μg/mL no ensaio ABTS, no ensaio FRAP esses valores variaram de 27,92 μg/mL a 145,78 μg/mL. No ensaio antibacteriano, especialmente frente à P. pally, a fase de diclorometano de folhas se mostrou mais ativa, com MIC50 de 126,22 μg/mL o que pode indicar que as substâncias fenólicas de caráter lipofílico, nessa espécie, são promissoras para essa atividade. Embora o ensaio anti-HIV1 mostrou que as amostras não apresentam atividade antirretroviral, este estudo contribui para o conhecimento do potencial antiviral dos extratos de bambus brasileiros. M. pluriflora se mostrou uma espécie promissora para estudos de prospecção, com uma grande quantidade de substâncias fenólicas em sua composição / Brazil is the country with the highest diversity of bamboo species in the New World. Knowledge about the medicinal potential of native bamboos is extremely underdeveloped when compared to Asian species. Some Asian bamboo species are used in folk medicine and have reports of various biological activities attributed to the presence of phenolic compounds, so bamboo species of the Neotropics are potential sources of bioactive substances. Using this assumption, Merostachys pluriflora, a native bamboo species, was chosen as a model for this study. The aimed of this study was to quantify the contents of starch, soluble carbohydrates, lipids, total phenols, flavonoids, total tannins and proanthocyanidins in leaves and culms of M. pluriflora; and evaluate the biological potential of the extracts, phases and isolated substances through in vitro assays: antioxidant activity, anti-HIV1 and antibacterial activity. It was observed that the crude extract of leaves yielded twice more than the culm extract; phases obtained with more polar solvents, such as hydromethanolic phases, had the highest yields. Lipids were the class of primary metabolites that presented higher quantities on both organs studied. Regarding the phenolic substances, it was observed that the crude extract of culms presented higher abundance of phenylpropanoids and chlorogenic acid derivates, but the crude extract from leaves showed higher abundance of flavonoids (all of then derived from apigenin) when compared to culms. Were identified two flavones, vitexin and isovitexin, and three phenylpropanoids, caffeic acid, ferulic acid and methyl caffeate. Phases using ethyl acetate and dichloromethane as extraction solvents were those that retained the majority of phenolic constituents. Ethyl acetate phase presented flavonoids while dichloromethane phase presented phenylpropanoids as major contituients. In general, the crude extracts and phases from leaves and culms of M. pluriflora showed antioxidant activity, especially antiradical and iron reducer capacity, presenting EC50 values of 16.30 mg/mL to 94.77 mg/ml in ABTS assay. For FRAP assay these values ranged from 27.92 mg/mL to 145.78 mg/mL. In the antibacterial assay, especially for P. pally, the dichloromethane phase from leaves was more active, presenting MIC50 of 126.22 mg/mL. This might indicate that the lipophilic phenolic present in this species of bamboo are promising for antibacterial activity. Although the anti-HIV1 assay showed that the samples do not present antiretroviral potential, this study contributes to the knowledge of the antiviral potential of Brazilian bamboo species. M. pluriflora showed to be a promising species for prospecting studies, with a large amount of phenolic substances in its composition

A própolis é uma mistura complexa de substâncias com aspecto resinoso, elaborada majoritariamente por Apis mellifera. Possui composição química diversificada, que varia de acordo com a flora ao redor da colmeia. Os objetivos deste trabalho são a caracterização química de quatro própolis de diferentes localidades do Brasil (MG, CE, PR e SC) e o isolamento e testes das atividades antioxidante (métodos do DPPH e β-caroteno) e anti-HIV (atividade inibitória da transcriptase reversa) das substâncias presentes nestas amostras. A fração volátil da própolis verde de Viçosa (MG) foi extraída e analisada por CG-EM. Verificou-se a presença de mono- e sesquiterpenos e ácidos fenólicos, sendo este o primeiro relato da presença do ledeno, muuroladieno, β-copaeno, aloaromadendreno e nerolidol na fração volátil da própolis verde brasileira. Um de seus constituintes majoritários, o éster alílico do ácido 3-prenilcinâmico foi isolado e testado quanto às atividades biológicas, apresentando alta ação antioxidante no método do β-caroteno. Nos demais testes o éster não foi ativo. Sugere-se que esta falta de atividade está ligada à ausência de hidroxilas fenólicas livres nesta substância. As amostras do CE, PR e SC foram analisadas por várias técnicas cromatográficas, incluindo CLAE-EM-EM, e colorimétricas. A própolis do CE, com origem botânica desconhecida, possui flavonoides (ex. naringenina e isoramnetina) e ácidos fenólicos (ex. cafeico e ρ-cumárico). Sua composição química é diferente daquela previamente descrita para uma própolis do mesmo estado. Os flavonoides pinocembrina e galangina, típicos da própolis europeia, foram detectados na própolis de SC, sugerindo que a origem botânica desta própolis seja Populus deltóide, descrita anteriormente como fonte de resinas para própolis da região. A própolis do PR não possui esses flavonoides, e é composta por ácidos fenólicos altamente prenilados. Sua composição é diferente daquelas já descritas, sugerindo uma nova fonte de resina para as própolis do sul do Brasil. Estas três própolis foram submetidas ao isolamento biomonitorado de seus constituintes, sendo obtidas 19 substâncias, nove delas com alta ação antioxidante, uma com ação anti-HIV, e três com ação anti-HIV moderada. Sugere-se que a atividade antioxidante destas própolis seja conferida pelos seus componentes majoritários, como o ácido p-cumárico, presente nas três própolis; quercetina, isoraminetina e 7,4\',5\'-trimetilmiricetina-5,3\'-dihidroxi-3-O-cafeoil glucosídeo, na própolis do CE; a mistura dos ácidos diidrocafeoilquinico + dimetoxicinamoil-diidrocafeoilquinico, na própolis do PR; ácido cafeico, pinocembrina e uma substância desconhecida, identificada como 16, na própolis de SC. Substâncias com ação anti-HIV foram isoladas das própolis do CE (naringenina, isoraminetina e quercetina) e PR (ácido 4-acetil-5-carboxi cumárico), demonstrando que estas própolis possuem grande potencial na busca de substâncias ativas / Propolis is a complex mixture of substances with resinous aspect, prepared mostly by Apis mellifera honeybees. It has a diverse chemical composition, according to the flora around the hive. The aims of this work are to chemically characterize four samples of propolis from different regions of Brazil (MG, CE, SC and PR states) and to isolate and test the antioxidant (DPPH and β-carotene methods) and anti-HIV (inhibitory activity of HIV-1 reverse transcriptase) activities of the compounds present on those samples. The volatile fraction sample of green propolis from Viçosa (MG) was extracted and analyzed by GC-MS. We verified the presence of mono-and sesquiterpenes and phenolic acids. Among them ledene, muuroladiene, β-copaene, aloaromadendrene and nerolidol were detected for the first time in the volatile fraction of Brazilian green propolis. One of its major constituents, the allyl ester of 3-prenylcinnamic acid was isolated and tested for biological activities. It was shown to have high antioxidant activity by the β-carotene method, but showed no activity regarding the other tests. It is suggested that the lack of activity is linked to the absence of free phenolic hydroxyl on the compound. The samples from CE, PR and SC states were analyzed by various chromatographic, including HPLC-MS-MS, and colorimetric techniques. The sample from CE, with unknown resin source, contains flavonoids (eg, naringenin and isorhamnetin) and phenolic acids (e.g. ρ-coumaric acid and caffeic). Its chemical composition is different from a previously described sample from the same state. The flavonoids galangin and pinocembrin, typical from European propolis, were detected in the sample from SC, which suggests that its botanical source is Populus deltoide. The sample from PR does not have these flavonoids; instead it possesses prenylated phenolic acids. Its composition is different from samples previously described, suggesting that the sample corresponds to a new type of propolis from southern Brazil. The three propolis samples were subjected to bioguided isolation of their constituents. Nineteen substances were obtained, nine of them with high antioxidant activity, one with anti-HIV action, and three with moderate anti-HIV activity. It is suggested that the antioxidant activity of these propolis is conferred by their major constituents, such as p-coumaric acid, present in all three samples, quercetin, isorhaminetin and 7,4\',5\'-trimethylmyricetin-5,3\'-dihydroxy-3-O-caffeoyl glucoside in propolis from CE; a mixture of dihydrocaffeoylquinic and dimethoxycinnamoyl-dihydrocaffeoylquinic acids in propolis from PR; and caffeic acid, pinocembrin and a unknown compound named 16, in propolis from SC. Compounds with anti-HIV activity were isolated from propolis from CE (naringenin, quercetin and isorhaminetin) and PR (4-acetyl-5-carboxy-coumaric acid), indicating that these types of propolis have high potential in the search for active compounds

Flavonoids and other phenolics are groups of natural bioactive compounds widely distributed in edible plants and are well documented to possess biological potential. Hyptis (Lamiaceae) is used in Brazilian folk medicine to treat various diseases. The aim of this study was to evaluate the antioxidant, anti-acetylcholinesterase, cytotoxic, antiviral and antibacterial potential of Hyptis radicans and Hyptis multibracteata by isolating and characterizing major constituents and their biological activities. H. radicans and H. multibracteata were dried, powdered and macerated in 70% ethanol which resulted in a crude ethanol extract (EE) for each species. EE were dissolved in 50% methanol and then was fractionated by partition with hexane and ethyl acetate; were obtained three phases: hexane phase (HP), ethyl acetate phase (EAP) and hydromethanol phase (HMP). EAP from H. radicans was the sample that presented the highest levels of total phenolic content, especially flavonoids, and was the sample with the high antioxidant activity with promising values of EC50: DPPH (32.12 µg mL-1), ABTS (5.04 µg mL-1), Metal chelator assay (42.36 µg mL-1), TBARS (40.46 µg mL-1) and nonsite-Specific Hydroxyl Radical-Mediated 2-Deoxy-D-ribose Degradation (NS-Spe) with EC50 of 75.08 µg mL-1. EE from H. radicans showed high antioxidant activity for FRAP and ORAC with EC50 of 6.01 and 2.68 µg mL-1, respectively and had the highest amount of rosmarinic acid (17.64 mg ?-CE g-1). HMP from H. radicans showed high antioxidant activity in Site-Specific Hydroxyl Radical-Mediated 2-Deoxy-D-ribose Degradation (S-Spe) assay with EC50 of 0.32 µg mL-1 and had the highest content of chlorogenic acid derivatives. Regarding the results of cytotoxicity, HP from H. multibracteata induced the death of more than 80% of RAW 264.7 Cell Lines at 100 µg mL-1. Nepetoidin B, isolated from H. multibracteata had the best EC50 (52.73 µg mL-1) for anti-acetylcholinesterase activity. Antibacterial activity was evaluated in vitro against two Gram-negative bacteria, Pseudomonas aeruginosa and Escherichia coli, and a Gram-positive Bacillus subtilis. Phases from H. multibracteata were more effective on inhibiting B. subtillis with MIC50 of 23.6 ?g mL-1 and 12.13 ?g mL-1 for HP and EAP, respectively. HP was also activity against P. aeruginosa with MIC50 of 37.55 µg mL-1. EE and HMP phase from H. radicans showed moderate anti-HIV-1 activity (MIC50 159 µg mL-1; MIC50 180 µg mL-1). Contents of total phenolic were not the main sample feature to define this activity, but there was correlation between Rosmarinic acid contents and anti-HIV1 activity of H. radicans. Cirsimaritin and litospermic acid A were isolated for the first time, being the first time that they are described for the genus Hyptis. This study provides the first evidence of chemical and biological potential for these two Brazilian native species of Hyptis / Flavonoides e outros compostos fenólicos são grupos de compostos bioativos naturais amplamente distribuídos em plantas e estão bem documentados por possuírem potencial biológico. Hyptis (Lamiaceae) é usado na medicina popular brasileira para tratar várias doenças. O objetivo deste estudo foi avaliar o potencial antioxidante, anti-acetilcolinesterase, citotóxico, antiviral e antibacteriano de Hyptis radicans, e Hyptis multibracteata, isolar e identificar substâncias e correlacionar as atividades biológicas com a quantidade de compostos fenólicos e substâncias isoladas. H. radicans e H. multibracteata foram secas, pulverizadas e maceradas em etanol 70%, resultando em extrato etanólico bruto (EE). EE foi dissolvido em metanol 50% e depois foi fracionado por partição com hexano e acetato de etila, o que resultou em três fases: fase hexânica (HP), fase acetato de etila (EAP) e fase hidrometanólica (HMP). EAP de H. radicans foi a amostra que apresentou os maiores teores de conteúdo fenólico, principalmente flavonoides, e foi a amostra com a maior atividade antioxidante, com valores promissores de EC50: DPPH (32,12 µg mL-1), ABTS (5,04 µg mL- 1), Quelante de metais (42,36 µg mL-1), TBARS (40,46 µg mL-1) e Degradação da 2-deoxy-D-ribose de sitio não especifico mediada pelo radical hidroxil (NS-Spe) com EC50 de 75,08 µg mL-1. EE de H. radicans apresentou a maior atividade antioxidante para FRAP e ORAC com EC50 de 6,01 e 2,68 µg mL-1, respectivamente, e apresentou a maior quantidade de ácido rosmarínico (17,64 mg ?-CE g-1). HMP de H. radicans apresentou a mais alta atividade antioxidante no ensaio de Degradação da 2-deoxy-D-ribose de sitio especifico mediada pelo radical hidroxil (S-Spe) com EC50 de 0,32 µg mL-1 e apresentou o maior teor de derivados de ácido clorogênico. Em relação aos resultados da citotoxicidade, HP de H. multibracteata induziu a morte de mais de 80% das células do tipo RAW 264.7 com uma concentração de 100 µg mL-1. A Nepetoidina B isolada de H. multibracteata apresentou a melhor EC50 (52,73 µg mL-1) para atividade anti-acetilcolinesterase. A atividade antibacteriana foi avaliada in vitro contra duas bactérias Gram-negativas, Pseudomonas aeruginosa e Escherichia coli, e uma bacteria Gram-positiva. Bacillus subtilis,. Fases de H. multibracteata foram mais eficazes na inibição de B. subtillis com MIC50 23,6 ?g mL-1 e 12,13 ?g mL-1 para HP e EAP, respectivamente. HP também apresentou atividade contra P. aeruginosa com MIC50 de 37,55 µg mL-1. EE e HMP de H. radicans mostraram moderada atividade anti-HIV-1 (MIC50 159 µg mL-1; MIC50 180 µg mL-1). Não há correlação entre o conteúdo total de fenólicos e esta atividade biológica, mas sim entre a quantidade de ácido rosmarínico das fases e a atividade anti-HIV1 de H. radicans. Foram isoladas pela primera vez a Cirsimaritina e o ácido litospermico A, sendo esta a primeira vez que se descrevem para o gênero Hyptis. Este estudo fornece a primeira evidência do potencial químico e biológico para estas duas espécies nativas de Hyptis

Orientadores: Pedro Paulo Corbi, André Luiz Barboza Formiga / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T12:00:41Z (GMT). No. of bitstreams: 1 Abbehausen_Camilla_D.pdf: 19393479 bytes, checksum: 3e6aaec4c411ad2e24a5f26226d19220 (MD5) Previous issue date: 2014 / Resumo: Complexos metálicos inéditos de paládio, platina, ouro e prata com diferentes classes de ligantes foram desenvolvidos. Dentre os ligantes selecionados estão a L-aliina (ali) e a N-acetil-L-cisteína (nac) que compreendem a classe dos aminoácidos, a 2-mercaptotiazolina (mtz), dentro da classe das tiazolidinas, a sulfadoxina (sfx), representante da classe das sulfonamidas, e ligantes N-heterociclos, piridino derivados com diferentes valores de pKa. Complexos de Pd(II) com L-aliina ([Pd(C6H11NO3S)2]), Ag(I) com N-acetil-L-cisteína ([Ag(C5H9NO3S)]), Ag(I) com sulfadoxina ([Ag(C12H13N4O4S)]), Au(I) com 2-mercaptotiazolina ([Au(CN)(C3H5NS2)]) e uma série de complexos trifenilfosfinoouro(I) com ligantes N-heterociclos ([Au(PPh3)L]+) foram sintetizados e caracterizados por um conjunto de análises químicas e espectroscópicas. Estudos in vitro das sua atividades antibacterianas e antitumorais foram também reali-zados. Atividades antibacterianas e antitumorais significativas foram encontradas para o complexo de Pd(II) com ali e o DNA se mostrou um alvo provável. O complexo Au(I) com mtz apresentou atividade antitumoral e antibacteriana bastante expressiva e uma investigação preliminar de seus mecanismos de ação também demonstrou que o DNA não parece ser o alvo destes compostos nas células. Os complexos de Ag(I) com nac e sfx apresentaram atividades antibacterianas significativas sobre cepas Gram-positivas e Gram-negativas. Os ligantes N-heterociclos 4-picolina (pic), 2-amino-4-picolina (NH2pic) e dimetilaminopriridina (DMAP) possuem valores de pKa crescentes, e foram selecionados para investigar o efeito do pKa na atividade biológica de complexos trife-nilfosfinoouro(I) do tipo [Au(PPh3)L]+, onde L = N-heterocíclico. Esta investigação foi realizada por avaliações in vitro de suas atividades antitumorais, além de estudos do acúmulo celular, do bloqueio do ciclo celular e por interações com biomoléculas como o DNA e proteínas dedos de zinco (zinc fingers, ZF). A atividade antitumoral foi expressiva e os estudos de suas interações com os ZF mostraram que a inibição pode ser modulada com a variação do tipo de proteína e do ligante N-heterociclo selecionado / Abstract: Novel palladium, platinum, gold and silver complexes with different classes of ligands were designed. The selected ligands were L-alliin (ali) and N-acetyl-L-cysteine (nac) which are aminoacids, 2-mercaptothiazoline (mtz), which belongs to the class of thiazolidines, sulfadoxine that represents the class of sulfonamides and N-heterocyclic pyridine derivatives, with different values of pKa. Complexes of Pd(II) with L-alliin ([Pd(C6H11NO3S)2]), Ag(I) with N-acetyl-L-cysteine ([Ag(C5H9NO3S)]), Ag(I) with sulfa-doxine ([Ag(C12H13N4O4S)]), Au(I) with 2-mercaptotiazoline [(Au(CN)(C3H5NS2)]) and a series of complexes of triphenylphosphinegold(I) with N-heterocyclic ligands ([Au(PPh3)L]+) were synthesized and characterized by a set of chemical and spectroscopic analyses. Antibacterial and antitumor activities in vitro were also studied. Significant antibacterial and antitumor activities were found for Pd(II) with ali, and the DNA is the probable biological target. The Au(I) with mtz complex presented noteworthy antitumor and antibacterial activities, and preliminary investigations of its biological mechanism showed that, the DNA is probable not a target of this complex in the cells. The silver complexes with nac and sfx presented significant antibacterial activities. The series of N-heterocyclic ligands 4-picoline (pic), 2-amino-4-picoline (NH2pic) and dimethylaminopyridine (DMAP) shows crescent pKa values and they were selected to investigate the pKa effect in the biological activity of the complexes triphenylphosphinegold(I) [Au(PPh3)L]+, which L = N-heterocyclic. This investigation was performed by evaluation of its antitumor activities in vitro, and also by studies of cell uptake, cell cycle arrest and by interactions with biomolecules as DNA and zinc finger proteins (ZF). The antitumor activity was expressive and the studies with ZF showed that the inhibition is dependent of the kind of protein and of the N-heterocyclic ligand / Doutorado / Quimica Inorganica / Doutora em Ciências

Student Number : 9000987E - PhD thesis - School of Medicine - Faculty of Health Sciences / The generation of human monoclonal antibodies (mAbs) that are able to block HIV-1 infection in vitro would be useful reagents for studying virus neutralization, and assist in identifying neutralizing antibody (NAb) epitopes of HIV-1 envelope glycoprotein. This may provide important information for designing HIV-1 vaccine that aim to induce NAbs. HIV-1 subtype C individuals with high levels of NAb titres were identified, and peripheral blood mononuclear cells (PBMC) from these individuals were isolated and B-cells transformed with Epstein-Barr virus (EBV). Clones specific to HIV-1 gp120 using cell lysate preparations derived from HIV-1 subtype C infected cell lines were generated by performing limiting dilutions. Transformation efficiencies were estimated at over 80% by evaluating EBV-transformation cultures by microscopic visualization. Of these approximately 5% were HIV-1-specific. Five clones derived from the Du23 (1) sample secreting anti-HIV-1 antibodies were generated: 2.3C, 2.9D, 3.2C, 4.12E, and 1.5D. The 1.5D mAb could not be confirmed as anti-HIV-1 clone and it was probably lost during the process of subculturing. The remaining four Du23 mAbs were determined to be of IgG1 isotype lambda (λ) light chain. These mAbs bind to gp120, and 2.9D is probably a polyreactive clone. Clones 2.3C, 3.2C and 4.12E appear to be A32-like, but do not share the same epitope. We have determined that the binding sites for all four Du23 mAbs require at least the C1 region, and they also showed binding sites overlapping with F91 and 1.5E. All four Du23 mAbs required intact gp120 proteins for their binding, and soluble CD4 enhance their binding. Thus, their binding site is discontinuous and conformational. These mAbs are non-neutralizing as they showed limited activity of 30-59% when tested using T-cell line grown viruses or 0-30% when tested against pseudovirions. This activity is rather low when compared to over 80% shown by broadly neutralizing mAbs that have been described in the literature. The challenge in generating mAbs, in particular subtype C-derived, is to find those antibodies capable of suppressing viral replication in vivo and be capable of preventing infection. These reagents could be used to identify epitopes to guiding the design of HIV-1 subtype C envelope immunogens or vaccines. It is also envisaged that neutralizing antibodies used in therapeutic setting or in combination with antiviral drug therapy could reduce viral load and retard disease progression in infected people.

A alta prevalência da infecção pelo Vírus da Imunodeficiência Humana (HIV) arremete para a importância de novos e eficazes programas de intervenções na prevenção da infecção. Os efeitos adversos, não adesão ao tratamento e, principalmente, a resistência do HIV aos Antiretrovirais existentes são alertas para a busca de novos compostos efetivos contra esse vírus. Plantas medicinais tem se mostrado como um recurso promissor na identificação de novos compostos bioativos. Este estudo trata-se de uma revisão sistemática com finalidade de realizar um levantamento de artigos disponíveis nas principais bases de dados eletrônicas, LILACS, Medline (via PubMed), Web of Science, sobre plantas medicinais com potencial ação contra o HIV. A busca dos artigos para o estudo ocorreu no mês de outubro de 2017 e foram selecionados 42 artigos para a revisão. Dos 42 artigos selecionados foi evidenciado 50 plantas medicinais que apresentaram atividade anti-HIV, variando o mecanismo de ação desempenhado, seja por via inibitória da protease, integrase ou da transcriptase reversa. Houve relatos de novos compostos capazes de melhorar a atividade anti-HIV de antirretrovirais já comercializados e 19 manuscritos apresentaram plantas com atividade antiviral promissora desconhecida. Dessa forma, esta revisão reforça a necessidade da realização de estudos a fim de comprovar os meios pelos quais as plantas medicinais aqui estudadas podem vir a ser fonte de novos fármacos no combate ao vírus da imunodeficiência humana. / The high prevalence of Human Immunodeficiency Virus (HIV) infection raises the importance of new and effective intervention programs in infection prevention. Adverse effects, nonadherence to treatment and, especially, HIV resistance to existing Antiretrovirals are alerts for the search for new compounds effective against this virus. Medicinal plants have proved to be a promising resource in identifying new bioactive compounds. This study is a systematic review with the purpose of performing a survey of articles available in the main electronic databases, LILACS, Medline (via PubMed), Web of Science, on medicinal plants with potential action against HIV. The search for articles for the study occurred in October 2017 and 42 articles were selected for review. Of the 42 articles selected, 51 medicinal plants that showed anti-HIV activity were evidenced, varying the mechanism of action performed, either by protease inhibitor, integrase or reverse transcriptase. There were reports of new compounds capable of improving the anti-HIV activity of antiretrovirals already marketed and 19 manuscripts presented plants with promising antiviral activity unknown. Thus, this review reinforces the need to carry out studies in order to prove the means by which the medicinal plants studied here may be the source of new drugs in the fight against human immunodeficiency virus.

Translocation of viral integrase into nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). It has been considered as an important target for the drug development to treat AIDS. In order to understand the detailed mechanisms of integrase-host cell protein complex interactions, we cloned HIV-1 integrase-EGFP into pTRE2hyg as visible tag to monitor the translocation process. When transiently transfected this vector into Tet-off ready HeLa cells, the EGFP: integrase is mainly localized in the nucleus. It has been hypothesized that any drugs that can inhibit the translocation process are novel class of drugs for AIDS treatment. More than 30000 synthetic compounds and 80000 natural products were screened by virtual screening. A total of 34 compounds were obtained and screened for their ability to block the nuclear entry of HIV-1 integrase by monitoring the EGFP fluorescence in the cells by high-throughput live cell imaging. Eight synthetic compounds (DW-IN4, DW-IN5, DW-IN6, DW-IN9, DW-IN15, DW-IN16, DW-IN17, DW-IN21) and one natural product (DW-IN719) were found to block integrase translocation significantly. According to our screening result, six compounds (INNB-1, INNB-2, INNB-3, INNB-4, INNB-5, INNB-6) were designed and synthesized. INNB-1 and INNB-2 had significant inhibition on integrase nuclear translocation. DW-IN6, DWIN719, INNB-1, INNB-2, INNB-3 and INNB-4, showed significant inhibition on P24 production in live virus assay. DW-IN6, INNB-1, INNB-2, INNB-3 and INNB-4 showed significant syncitia formation inhibition in live virus assay. Six compounds (KM7, KM8, KM14, KM30, KM37, KM79) from Kunming were screened as integrase nuclear translocation inhibitors. Using similar cell imaging techniques, we have cloned the GFP-tagged chemokine receptor CXCR4 using the lentivirus transfection system. CXCR4 receptor is a critical co-receptor in CD4 positive lymphocytes mediating the fusion of HIV into the CD4 positive cells. CXCR4-GFP was over-expressed in 293T cells and the results showed that GFP:CXCR4 receptor is expressed at the plasma membrane of the cells. These cells have been used to monitor the blockage of CXCR4 receptor internalization for drug development. Four compounds (KX128, KX166, KX171, KX180) from Kunming showed CXCR4 internalization blockage in imaging assay. The interaction of these compounds with CXCR4 was predicted by molecular docking. KX128 showed significant HIV inhibition in live virus assays. / Gu, Wangang. / Advisers: Pang Chui Shaw; David Chi Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

核糖體抑活蛋白 (RIPs) 屬於糖苷酶的一種，能從23S或28S核糖體核糖核酸中的sarcin-ricin環(sarcin-ricin loop, SRL)移除一個特定的腺嘌呤，引致核糖體失效。由於核糖體蛋白協助RIP到達SRL，因此它們對RIP的核糖體特認性是極大的重要。雖然各RIPs的份子結構及催化活動非常相似，它們的核糖體特認性和效力存著很大的迥異。此外，現時還未能找出只有少數RIPs能同時抑制原核和真核生物的核糖體的原因。我們試圖從玉米核糖體抑活蛋白 (Maize RIP) 和真核生物的核糖體以及志賀毒素 (Shiga toxin) 和原核生物的核糖體的相互作用的研究中去解釋以上的現象。 / 我們發現Maize RIP提供一個前所未見的區域與核糖體蛋白P2結合，並展示RIPs的結構大大限制了它們與核糖體蛋白的相互作用的性質和強度，從而影響RIPs在核糖體上的效力。另外，我們發現志賀毒素跟細菌的核糖體的相互作用比跟真核生物核糖體的相互作用弱，並可能跟細菌核糖體蛋白L7/L10有交聯。我們在蓖麻毒蛋白 (Ricin) 的碳端 (C-terminus) 加上人類免疫缺陷病毒-(HIV-1) 蛋白酶特認的肽以增加 ricin 對HIV-1蛋白酶的特認性，並希望此研究結果有助於應用相類的策略到其他RIPs上。 / Ribosome-inactivating proteins (RIPs) are N-glycosidases that inactivate ribosome by removing a specific adenine from the sarcin-ricin loop (SRL) of 23S or 28S ribosomal RNA. Ribosomal proteins are critical for determining the ribosome specificity of RIPs as they assist RIPs to get access to the SRL. Ribosome specificity and potency of RIPs are highly varied although their tertiary structures and catalytic depurination are highly alike. Moreover, it is still unsolved why only a few RIPs acquiring the ability to inhibit both prokaryotic and eukaryotic ribosomes. We attempted to elucidate the phenomena by investigating the interactions of maize RIP with eukaryotic ribosome and shiga toxin with prokaryotic ribosome. / Here we showed maize RIP presents a novel docking site to interact with ribosomal protein P2 and demonstrated the structure of RIPs imposes a large constraint on the nature and strength of the interaction with ribosomal protein which in turn affect the potency of RIPs on the ribosome. Shiga toxin was found to interact with prokaryotic ribosome weaker than the eukaryotic ribosome and crosslinked to the bacterial ribosomal protein L7/L10. Additionally, we increased the HIV-1 specificity of ricin A chain by incorporating the HIV-1 protease specific peptide to the C-terminus of the toxin and hope our findings would help to extend similar scheme to other RIPs in the future. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Yuen-Ting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 146-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Contents --- p. iv - viii / Chapter Chapter One --- Introduction of ribosome-inactivating proteins / Chapter 1.1 --- Nomenclature and distribution of ribosome-inactivating proteins --- p.1 / Chapter 1.2 --- Enzymatic activity of ribosome-inactivating proteins and their biological role --- p.2 / Chapter 1.3 --- Structure and catalytic centre of ribosome-inactivating proteins --- p.3 / Chapter 1.4 --- Ribosome specificity of RIPs and their interaction with ribosome --- p.5 / Chapter 1.5 --- Cytotoxicity and antiviral activity of ribosome-inactivating proteins --- p.6 / Chapter 1.6 --- Antiviral activity of RIPs --- p.9 / Chapter 1.7 --- Cellular trafficking of ribosome-inactivating proteins --- p.10 / Chapter 1.8 --- Application and therapeutic use of ribosome-inactivating proteins --- p.10 / Chapter 1.9 --- Evolution of RIPs --- p.11 / Chapter 1.10 --- Other activities of RIPs --- p.12 / Chapter Chapter Two --- Characterization of the interaction between RIPs and rat liver ribosome and its correlation with the potency of RIPs / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Nature of interaction between RIPs and eukaryotic ribosome --- p.12 / Chapter 2.1.2 --- RIPs interact with specific ribosomal proteins --- p.15 / Chapter 2.1.3 --- RIPs demonstrate different specificity towards ribosomes --- p.16 / Chapter 2.1.4 --- Introduction of maize RIP --- p.20 / Chapter 2.1.5 --- Interaction between maize RIP and ribosome --- p.22 / Chapter 2.2 --- Objectives and significance --- p.22 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Cloning and site-directed mutagenesis of RIPs --- p.23 / Chapter 2.3.2 --- Protein expression and purification --- p.23-26 / Chapter 2.3.2.1 --- Maize RIP and variants / Chapter 2.3.2.2 --- His-myc-MOD and His-MOD / Chapter 2.3.2.3 --- Trichosanthin (TCS) / Chapter 2.3.2.4 --- Shiga toxin chain A [E167AE170A] (StxA) / Chapter 2.3.2.5 --- Ricin chain A (RTA) / Chapter 2.3.2.6 --- Pokeweed antiviral protein (PAP) / Chapter 2.3.2.7 --- C-terminal His-tagged MOD, TCS and RTA / Chapter 2.3.2.8 --- His-SUMO-protease / Chapter 2.3.2.9 --- P2 and its variants / Chapter 2.3.2.10 --- Protein concentration and storage / Chapter 2.3.3 --- Purification of rat liver ribosome --- p.26 / Chapter 2.3.4 --- In vitro pull-down assay with ribosome --- p.27 / Chapter 2.3.5 --- On-resin crosslinking and mass spectrometry --- p.27 / Chapter 2.3.6 --- Crosslinking assay and western blotting --- p.28 / Chapter 2.3.7 --- In vitro pull-down assay with P2 --- p.29 / Chapter 2.3.8 --- In vitro pull-down assay with P2 and its variants --- p.29 / Chapter 2.3.9 --- Surface Plasmon Resonance --- p.29 / Chapter 2.3.10 --- N-glycosidase activity assay and quantitative PCR --- p.30 / Chapter 2.3.11 --- Cytotoxicity on 293T --- p.31 / Chapter 2.3.12 --- Cellular uptake of RIPs and western blotting --- p.32 / Chapter 2.4 --- Results / Chapter 2.4.1 --- In vitro pull-down assay with ribosome --- p.32 / Chapter 2.4.2 --- On-resin crosslinking and mass spectrometry of crosslinked proteins --- p.37 / Chapter 2.4.3 --- Crosslinking assay and western blotting --- p.40 / Chapter 2.4.4 --- In vitro pull-down assay with P2 --- p.43 / Chapter 2.4.5 --- Sensorgram of binding between P2 and Maize RIP variants --- p.44 / Chapter 2.4.6 --- N-glycosidase activity of maize RIP variants --- p.45 / Chapter 2.4.7 --- Cytotoxicity of maize RIP variants --- p.48 / Chapter 2.4.8 --- In vitro pull-down assay with P2 and its variants --- p.49 / Chapter 2.4.9 --- Surface Plasmon Resonance of P2 and various RIPs --- p.52 / Chapter 2.4.10 --- N-glycosidase activity assay and quantitative PCR --- p.55 / Chapter 2.4.11 --- Cytotoxicity of RIPs to 293T --- p.57 / Chapter 2.5 --- Discussion --- p.59 / Chapter 2.6 --- Conclusion --- p.72 / Chapter Chapter Three --- Identifying prokaryotic ribosomal protein(s) interacting with shiga toxin / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Background of shiga toxin --- p.74 / Chapter 3.1.2 --- Trafficking and activation of shiga toxin --- p.75 / Chapter 3.1.3 --- Intoxication by Shiga toxin --- p.76 / Chapter 3.1.4 --- Dual specificity on ribosome --- p.77 / Chapter 3.2 --- Objectives and significance --- p.78 / Chapter 3.3 --- Materials and methods / Chapter 3.3.1 --- Cloning of Shiga toxin and ribosomal proteins --- p.79 / Chapter 3.3.2 --- Expression and purification --- p.79-80 / Chapter 3.3.2.1 --- His-SUMO StxA, His-StxA, and His-StxA [E167Q] / Chapter 3.3.2.2 --- Ribosomal proteins / Chapter 3.3.3 --- Isolation of E. coli ribosome and rat liver ribosome --- p.80 / Chapter 3.3.4 --- Pull-down assay of prokaryotic and eukaryotic ribosome --- p.81 / Chapter 3.3.5 --- Size-exclusion chromatography of RIPs and prokaryotic ribosome --- p.81 / Chapter 3.3.6 --- Pull-down assay of StxA with HepG2 and C41 lysate --- p.82 / Chapter 3.3.7 --- Two-dimensional electrophoresis --- p.82 / Chapter 3.3.8 --- Mass spectrometric analysis of pull-down assay --- p.83 / Chapter 3.3.9 --- Crosslinking of StxA with r-proteins --- p.84 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Cloning of wild-type shiga toxin --- p.84 / Chapter 3.4.2 --- Pull-down with prokaryotic and eukaryotic ribosome --- p.85 / Chapter 3.4.3 --- Size-exclusion chromatography of RIPs and prokaryotic ribosome --- p.88 / Chapter 3.3.4 --- Pull-down assay of StxA with HepG2 and C41 lysates --- p.90 / Chapter 3.4.5 --- Crosslinking of StxA with r-proteins --- p.97 / Chapter 3.5 --- Discussion and conclusion --- p.99 / Chapter Chapter Four --- Engineering ricin A chain for increasing its specificity toward Human Immunodeficiency Virus (HIV) / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.1.1 --- Human immunodeficiency virus --- p.104 / Chapter 4.1.2 --- Current drugs for HIV --- p.105 / Chapter 4.1.3 --- Anti-HIV mechanism of RIPs --- p.105 / Chapter 4.1.4 --- Engineering cytotoxic protein into HIV-1 specific toxin --- p.107 / Chapter 4.2 --- Objectives and significance --- p.109 / Chapter 4.3 --- Materials and methods / Chapter 4.3.1 --- Design and cloning of RTA HIV-1 specific variants --- p.109 / Chapter 4.3.2 --- Cloning, expression and purification of ricin variants --- p.112 / Chapter 4.3.3 --- Purification of HIV-1 protease --- p.112 / Chapter 4.3.4 --- HIV-1 protease induced cleavage of RTA variants --- p.113 / Chapter 4.3.5 --- Cytotoxicity on 293T and JAR --- p.114 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Purity check of RTA variants --- p.114 / Chapter 4.4.2 --- HIV-1 protease induced cleavage of RTA variants --- p.115 / Chapter 4.4.3 --- Cytotoxicity on 293T and JAR --- p.119 / Chapter 4.5 --- Discussion --- p.124 / Chapter 4.6 --- Conclusion --- p.126 / Concluding remarks and future prospect --- p.127 / Appendices / Appendix 1 --- p.128 - 132 / Appendix 2 --- p.133 - 134 / Appendix 3 --- p.135 - 138 / Appendix 4 --- p.139 - 145 / Bibliography --- p.146 - 159