Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T01:11:41Z (GMT). No. of bitstreams: 1 Micas_AndreFernandoDitondo_M.pdf: 1496595 bytes, checksum: c51897315ae5950ced334700741f31cf (MD5) Previous issue date: 2008 / Resumo: A violaceína é um composto indol, de intensa cor violeta que é produzido por Chromobacterium violaceum, uma bactéria ubíqua de zonas tropicais e subtropicais. Esta molécula apresenta inúmeras propriedades antimicrobianas, tais como agente antimicótico, antiviral e atividade contra certos protistas. Observações importantes apontam para uma atividade bactericida considerável, tornando premente um estudo mais aprofundado a cerca desta propriedade. No presente trabalho sondou-se a sensibilidade a este composto para algumas bactérias de interesse médico e um fitopatógeno, sua cinética de inibição do crescimento bacteriano, assim como se obtiveram importantes pistas sobre o mecanismo de ação. Foi encontrada atividade inibitória considerável para Staphylococcus aureus e Staphylococcus epidermidis, com uma maior inibição durante a fase estacionária, assim como para Xanthomonas axonopodis p.v passifloraceae. Através de micrografias eletrônicas pôde-se constatar profundas alterações na parede celular em S. aureus, indicando esta estrutura como sítio de atuação da violaceína, assim como através de espectrometria de massa (MALDI-TOF) a identificação de proteínas diferencialmente expressas por S. aureus na presença ou ausência de violaceína, indicando uma grande perturbação no metabolismo bacteriano, com inibição a alguns fatores de patogenicidade e também inibição da síntese protéica / Abstract: Violacein is a deeply violet indol compound produced by Chromobacterium violaceum, an ubique tropical and subtropical bacterium. This molecule presents many antimicrobial properties, like antimycotic, antiviral and activity against some protists. Important reports pointed to a considerable bactericide activity, which brings an extremely necessary quest. This work evaluated the resistance of many clinical bacteria, the violacein kinetic of bacterial growth inhibition and some cues about the mechanism of action of this molecule. It was obtained significant inhibition activity against Staphylococcus aureus, Staphylococcus epidermidis and Xanthomonas axonopodis p.v passifloraceae. From transmission electron micrographs it was observed profound disturbs on S. aureus cell wall, which indicates this structure as violacein site of action. By using mass spectrometry (MALDI-TOF) of proteins differently expressed from bidimensional gel electrophoresis of S. aureus treated and not-treated with violacein, it was found a strong metabolic disturb, with inhibition of some pathogenical factors and inhibition of proteic synthesis / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Orientador: Oswaldo Luiz Alves / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T15:38:00Z (GMT). No. of bitstreams: 1 Holtz_RaphaelDias_D.pdf: 5088635 bytes, checksum: d278014c22731df6df2370e7da3785ba (MD5) Previous issue date: 2012 / Resumo: Neste trabalho foram desenvolvidas nanoestruturas de vanadatos de prata, de cério e de bismuto e investigadas as suas propriedades antibacterianas. Tais nanoestruturas foram sintetizadas a partir de reações de precipitação entre o vanadato de amônio e os nitratos ou cloretos dos metais correspondentes, sendo posteriormente realizados tratamentos hidrotérmicos em autoclaves providas de agitadores mecânicos e medidores de pressão e de temperatura do meio reacional. Os materiais foram caracterizados por diversas técnicas de caracterização físico-química e morfológica, sendo sua atividade antibacteriana avaliada frente às cepas de bactérias Gram-Positivas e Gram-Negativas de interesse, incluindo cepas de Staphylococcus aureus resistente à meticilina e Enterococcus resistente à vancomicina. Foi observado que o vanadato de prata apresentou uma elevada atividade antibacteriana contra diversas cepas bacterianas. O elevado potencial antibacteriano do vanadato de prata despertou o interesse em avaliar a sua utilização como aditivo antibacteriano em uma tinta comercial à base de água. Os resultados de sua adição foram promissores uma vez que foram observados halos de inibição do crescimento bacteriano. Esses resultados levantaram questionamentos importantes acerca do seu comportamento frente à microorganismos aquáticos, uma vez que existe a possibilidade de, no futuro, este material ser utilizado comercialmente como aditivo antibacteriano para tintas e revestimentos. Foram realizados experimentos de toxicidade aguda frente ao microcrustáceo Daphnia similis, sendo que os valores de CE50 foram próximos aos valores apresentados para os compostos de prata / Abstract; In this work we report the development and characterization of silver-, cerium- and bismuth vanadate nanostructures as well as their antibacterial activity. These nanostructures were synthesized from simple precipitation reaction between ammonium vanadate and nitrates/chlorides of the corresponding metals. The obtained precipitates were treated by hydrothermal process in autoclaves with pressure and temperature gauges and under mechanical stirring. These nanomaterials were characterized by several physico-chemical techniques and morphological characterization, and their antibacterial activities were evaluated against several strains of Gram-positive and Gram-Negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). We observed that nanostructured silver vanadate presented a high antibacterial activity against a broad spectrum of bacteria. These results stimulated the evaluation of silver vanadate nanostructures as a potential antibacterial additive to commercial water-based paint. It was observed a zone of bacterial growth inhibition against MRSA. The possible use of this material as an additive to water-based paints lead us to investigate the toxicity of this material against aquatic microorganisms, once this material can be commercial used in future as antibacterial additive to paints and coatings. Acute toxicity assays against Daphnia similis showed that EC50 value was close to that observed in silver-based compounds / Doutorado / Quimica Inorganica / Doutor em Ciências

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Abstract Chlamydia pneumoniae is a common human pathogen worldwide, which causes both upper and lower respiratory tract infections. In addition, C. pneumoniae infections have been associated with atherosclerosis and other chronic diseases, and successful treatment and eradication of the organism from tissues would therefore be desirable. The purpose of this study was to assess the effects of C. pneumoniae inoculations on the development of chronic infection and atherosclerotic changes in normocholesterolemic, wild-type mice. We also aimed to elucidate the effects of antibiotic and other treatments on the eradication of chlamydia and on the reduction of the pathologic sequelae induced by these infections. Female C57BL/6J mice were fed either normal chow when assessing the effects of acute infection, or a diet supplemented with 0.2% cholesterol when evaluating the atherosclerotic changes. Primary or repeated inoculations with C. pneumoniae isolate K7 were given to the mice intranasally, and the effects of treatments with telithromycin, levofloxacin and erythromycin antimicrobial agents and with the phenolic compounds quercetin, luteolin and octyl gallate were evaluated. The following methods were used to measure infection and treatment effects and the presence of chlamydia in tissue: chlamydia culture, PCR and RT-PCR methods, histology of lung, heart and aortic tissue, serologic methods and measurements of aortic contractility responses. Repeated C. pneumoniae inoculations induced persistent chlamydial DNA and inflammation in lung tissue and development of mouse Hsp60 autoantibodies. Infection was shown to influence aortic endothelial function, and repeated inoculations significantly increased subendothelial lipid accumulation in the aortic sinus area. A flavonoid, luteolin, was shown to effectively decrease the chlamydial load and inflammatory reactions in lung tissue. All antimicrobial agents eradicated the presence of viable chlamydia effectively; however, PCR positivity persisted in lung tissue despite the treatments. Only immediate treatment after each inoculation was able to decrease aortic sinus lipid accumulation. In conclusion, these data support the role of C. pneumoniae in promoting atherosclerotic development via autoimmune responses and also via direct effects on aortic tissue. Conventional antimicrobial treatments may not effectively eradicate persistent infection, and further studies are warranted to seek for alternative treatment options.

Please read the abstract in the section 00front of this document / Thesis (PhD (Food Science))--University of Pretoria, 2006. / Food Science / unrestricted

Globally, methicillin resistant Staphylococcus aureus (MRSA) has become increasingly difficult to manage in the clinic and new antibiotics are required. The structure activity relationship (SAR) study presented in this thesis forms part of an international collaborative effort to identify potent and selective inhibitors of an MRSA pyruvate kinase (PK) enzyme target. In earlier work the known marine natural product bromodeoxytopsentin (1.6), isolated from a South African marine sponge Topsentia pachastrelloides, exhibited selective and significant inhibition of MRSA PK (IC₅₀ 60 nM). Accordingly bromodeoxytopsentin provided the initial chemical scaffold around which our SAR study was developed. Following a comprehensive introduction, providing the necessary background to the research described in subsequent Chapters, this thesis has been divided into three major parts. Part one (Chapter 2) documents the synthesis of two natural imidazole containing topsentin analogues 1.40, 1.46, five new synthetic analogues 1.58—1.61, 2.104. In the process we developed a new method for the synthesis of topsentin derivatives via selenium dioxide mediated oxidation of N-Boc protected 3-acetylindoles to yield glyoxal intermediates which were subsequently cyclized and deprotected to yield the desired products. Interestingly we were able to demonstrate a delicate relationship between the relative equivalents of selenium dioxide and water used during the oxidation step, careful manipulation of which was required to prevent the uncontrolled formation of side products. Synthetic compounds 1.40, 1.46, 1.58—1.61 were found to be potent inhibitors of MRSA PK (IC₅₀ 238, 2.1, 23, 1.4, 6.3 and 3.2 nM respectively) with 1000-10000 fold selectivity for MRSA PK over four human orthologs. In the second part of this thesis (Chapter 3) we report the successful synthesis of a cohort of previously unknown thiazole containing bisindole topsentin analogues 1.62—1.68 via a Hantzsch thiazole synthesis. Bioassay results revealed that these compounds were only moderate inhibitors of MRSA PK (IC₅₀ 5.1—20 μM) which suggested that inhibitory activity was significantly reduced upon substitution of the central imidazole ring of topsentin type analogues with a thiazole type ring. In addition in Chapter 3 we describe unsuccessful attempts to regiospecifically synthesize oxazole and imidazole topsentin analogues through a similar Hantzsch method. As a consequence of our efforts in this regard we investigated three key reactions in depth, namely the synthesis of 2.2, 3.38, 3.40, 3.41 via α-bromination of 3-acetylindole and the synthesis of indolyl-3-carbonylnitriles 2.13, 3.45—3.47 and α-oxo-1H-indole-3-thioacetamides 3.48—3.51. The investigation of the latter led to the isolation and elucidation of two anomalous N,N-dimethyl-1H-indole-3-carboxamides 3.52 and 3.53. Finally the third part of this thesis (Chapter 4) deals with in silico assessment of the binding of both the imidazole and thiazole containing bisindole alkaloids to the MRSA PK protein which initially guided our SAR studies. In this chapter we reveal that there appears to be no correlation between in silico binding predictions and in vitro MRSA PK inhibitory bioassay data. Superficially it seems that binding energy as determined by the docking program used for these studies correlated with the size of the indole substituents and did not reflect IC₅₀ MRSA PK inhibitory data. Although this led us to computationally explore possible alternative binding sites no clear alternative has been identified.

This thesis is divided into two parts, assessing marine and synthetic compounds active firstly against Plasmodium falciparum (Chapter 3 and 4) and secondly active against methicillin resistant Staphylococcus aureus (MRSA, Chapter 5). In Chapter 3 the synthesis of nine new tricyclic podocarpanes (3.203-3.207 and 3.209-3.212) from the diterpene (+)-manool is described. Initial SAR study of synthetic podocarpanes concluded that the most active compound was a C-13 phenyl substituted podocarpane (3.204, IC₅₀ 6.6 μM). By preparing analogues with varying halogenated substituents on the phenyl ring (3.209-3.212) the antiplasmodial activity was improved (IC₅₀ 1.4 μM), while simultaneously decreasing the haemolysis previously reported for this class of compounds. Inspired by the antiplasmodial activity of Wright and Wattanapiromsakul’s tricycle marine isonitriles (2.16-2.21 and 2.24-2.27) an unsuccessfully attempt was made to convert tertiary alcohol moieties to isonitrile functionalities in compounds 3.188, 3.204-3.207 and 3.209-3.212. Over a decade ago Wright et al. proposed a putative antiplasmodial mechanism of action for marine isonitriles (2.4, 2.9, 2.15, 2.19 and 2.35) and isothiocyanate (2.34) which involved interference in haem detoxification by P. falciparum thus inhibiting the growth of the parasite. In Chapter 4 we describe how we successfully managed to scale down Egan’s β-haematin inhibition assay for the analyses of small quantities of marine natural products as potential β-haematin inhibitors. Our modified assay revealed that the most active antiplasmodial marine isonitrile 2.9 (IC₅₀ 13 nM) showed total β-haematin inhibition while 2.15 (IC₅₀ 81 nM) and 2.19 (IC₅₀ 31 nM) showed partial inhibition at three equivalents relative to haem. Using contempary molecular modelling techniques the charge on the isonitrile functionality was more accurately describe and the modified charge data sets was used to explore docking of marine isonitriles to haem using AutoDock. In Chapter 5 we describe how a lead South African marine bisindole MRSA pyruvate kinase inhibitor (5.8) was discovered in collaboration with colleagues at the University of British Columbia (UBC) and how this discovery inspired us to design a synthetic route to the dibrominated bisindole, isobromotopsentin (5.20) in an attempt to increase the bioactivity displayed by 5.8. We devised a fast and high yielding synthetic route using microwave assited organic synthesis. We first tested this synthesis using simple aryl glyoxals (5.27-5.32) as precursors to synthesize biphenylimidazoles (5.21-5.26), which later allowed us to synthesize the ascidian natural product 5.111. This method was sucessfully extended to the synthesis of deoxytopsentin (5.33) from an N-Boc protected indole methyl ketone (5.89). We subsequently were able to effectively remove the carbamate protection via thermal decomposition by heating the protected bisindole imidazole (5.90) in a microwave reactor for 5 min under argon. The synthesis of 5.20 resulted in an inseparable mixture of monoprotected and totally deprotected topsentin products, and due to time constraints we were not able to optimise this synthesis. Nonetheless our synthesis of the marine natural product 5.33 which was faster and higher yielding than previously reported routes could be extended to the synthesis of other topsentin bisindoles (5.138-5.140). Work towards this goal continues in our laboratory.

Cyclic dipeptides have been well characterized for their multitude of biological activities, including antimicrobial and anticancer activities. Cyclo(His-Gly) and cyclo(His-Ala) have also recently been shown to possess significant anticancer activity against a range of cell lines, despite the limitations of these two molecules with respect to their physicochemical properties. Low Log P results in poor cell permeability which can often be problematic for drugs with intracellular mechanisms of action. It can also results in poor biodistribution, and theoretical Log P values for cyclo(His-Gly) and cyclo(His-Ala) were extremely low making them ideal candidates for inclusion into a nanoparticulate drug delivery system. The aim of this study was therefore to formulate and evaluate liposome-encapsulated cyclic dipeptides that increase the tumour-suppressive actions of the cyclic dipeptides, while showing a high degree of specificity for tumour cells. While liposomes are relatively simple to prepare, inter batch variation, low encapsulation and poor stability are often problematic in their production and this has lead to very few liposomal products on the market. This study aimed at using a comprehensive statistical methodology in optimizing liposome formulations encapsulating cyclo(His-Gly) and cyclo(His-Ala). Initial screening of potential factors was conducted using a 25-1 fractional factorial design. This design made use of two levels for each of the five factors and abbreviated the design to minimize runs. Although not much information is provided by these types of designs, the design was sufficient in identifying two critical factors that would be studies further in a more robust design. The two factors selected, based on the screening study, were cholesterol and stearylamine content. These two factors were then used in designing a response surface methodology (RSM) design making use of a central composite rotatable vii design (CCRD) at five levels (-1.5, -1, 0, 1, 1.5) for each factor in order to better understand the design space. Various factors influenced the measured responses of encapsulation efficiency, zeta potential, polydispersity index, cellular uptake and leakage, but most notable were the adverse effects of increasing stearylamine levels on encapsulations efficiency and cholesterol levels on leakage for both cyclo(His-Gly) and cyclo(His-Ala) liposomes. Optimized formulations were derived from the data and prepared. Fair correlation between the predicted and measured responses was obtained. The cytotoxic activity of the encapsulated cyclic dipeptides were assessed against HeLa and MCF-7 cells and found to have limited improvement in activity. However, modification of the polyethylene glycol (PEG) grafted to the liposome surface in order to target folate receptors showed good benefit in significantly decreasing the IC50 values recorded in all cells lines tested, particularly low folate HeLa cells with the lowest IC50 being recorded as 0.0962 mM for folate targeted cyclo(His-Ala). The results therefore indicate that hydrophilic cyclic dipeptides are ideal candidates for inclusion into targeted drug delivery systems such as liposomes. Key words: Liposomes, cyclo(His-Gly), cyclo(His-Ala), cyclic dipeptides, HeLa, MCF-7, folate receptors, factorial design, response surface methodology (RSM), central composite rotatable design (CCRD).

Please read the abstract in the section 00front of this document / Dissertation (MSc (Pharmacology))--University of Pretoria, 2007. / Pharmacology / unrestricted

Orientador: Glaucia Maria Pastore / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T02:22:20Z (GMT). No. of bitstreams: 1 Costa_GiselleAparecidaNobre_M.pdf: 32192539 bytes, checksum: 829ea1e46916c727a5cca642b084be51 (MD5) Previous issue date: 2005 / Resumo: Biossurfatantes são compostos anfipáticos de origem biológica que apresentam uma porção hidrofílica e outra hidrofóbica. Esta propriedade lhes confere a capacidade de diminuir a tensão superficial e interfacial. de um meio liquido. Na busca de fontes alternativas para a produção de compostos de interesse biotecnológico, o uso de subprodutos de processamento agrícola ou industrial apresenta um vasto campo a ser explorado. A industrialização da mandioca, em farinha e fécula, gera aproximadamente 240.000 L de resíduo/mês. Este resíduo, a manipueira, é altamente tóxico (devido à presença de glicosídeos cianogênicos) e poluente (dado a altas concentrações de matéria orgânica não esgotada que podem atingir 100 g DQO/L). A possibilidade da utilização deste resíduo como substrato para microrganismos e o interesse na produção de compostos biotecnologicamente relevantes nos incentivou a investigar a produção de biossurfatantes pela cepa Bacillus subtilis LB5a. Definidas as condições e cinética da fermentação, foi obtido uma produção de 2,5 g/L de surfatante em 36 horas de cultivo. O tensoativo se mostrou capaz de reduzir a tensão superficial do meio de 47,74 mN/m, para 25,96 mN/m, CMD-1 de 26,19 e CMD-2 de 29,47 mN/m, com tensão interfacial contra hexadecano de 0,76 mN/m, além de CMC de 10 mg/L. O extrato apresentou estabilidade a altas concentrações de sal (>15%), elevadas temperaturas (135ºC), ampla faixa de pH (6-12) e foi estável a variadas concentrações de enzimas do tipo cisteína-proteases. A análise de aminoácidos do produto purificado, revelou a presença de ácido glutâmico, ácido aspártico, valina e leucina na proporção 1:1:1:4, caracterizando o produto como um lipopeptídeo. Por espectrometria de massas foi confirmada a presença de uma série homóloga de lipopeptídeos com variações no tamanho da cadeia hidrocarbônica caracterizando o composto como o tensoativo surfactina. Além disso, verificou-se a predominância do homólogo de massa molecular 1036 g/Mol. O biossurfatante de B.subtilis LB5a, apresentou capacidade de formar emulsões estáveis em óleos, hidrocarbonetos e combustíveis; revelou-se como substância potencial para utilização na recuperação melhorada de petróleo (MEOR) e em biorremediação, avaliado a partir da remoção de óleo bruto de areias contaminadas. Além disso, apresentou atividade antimicrobiana contra bactérias gram positivas e negativas e apresentou ação antitproliferativa sobre células tumorais humanas. Os resultados descritos neste trabalho fornecem dados relevantes acerca do potencial do biossurfatante surfactina e suas possíveis aplicações. Além disso, evidenciam a manipueira como um excelente substrato para a produção de biossurfatante pela cepa estudada, possibilitando uma grande redução de custos de produção através da utilização de um resíduo como substrato, bem como pelo processo de obtenção do produto por métodos bastante simplificados / Abstract: Biosurfactants are amphypatic compounds from biologycal source showing both hydrophilic and lipophilic groups. Due to this feature, the biosurfactants acts decreasing the surface and interfacial tension of a liquid. The byproducts of agricola and industrial processing are an underexplored new sources to the production of biotechnological interesting compounds. The cassava industrialization yields c.a. 240.000L of residue by month. This residue, cassava wastewater (manipueira), is highly toxic (due to the presence of cianogenic glycosides) and poluent (due to high concentrations of organic matter, that may reach 100 gDQO/L). The possibility of the utilization of manipueira as substract for microorganism growth and the interest in the production of biotecnologically important aducts, encourage us to investigate the biosurfactants production by Bacillus subtilis LB5a strain. After the growing conditions optimizations, 2,5 g/L of surfactant was obtained in 36 hours. The biosurfactant have reduced the surface tension of the medium from 47,74 mN/m to 25,96 mN/m, CMD-1 of 26,19 and CMD-2 of 29,47 mN/m, with interfacial tension against hexadecane of 0,76 mN/m and CMC of 10 mg.L-1. The extract show stability with respect to high salt concentration, high temperature, wide range of pH (6-12) and to several concentrations of diferent cysteine proteases. The aminoacids analysis of purified product, revealed the presence of glutamic acid, aspartic acid, valine and leucine at a 1:1:1:4 ratio, characterizing the product as a lipopetide. Mass spectrometry analysis confirmed the presence of a lipopeptide homologous serie with variations in the hydrocarbon chain lenght, known as surfactin. Furthemore, was verified the predominance of the 1036 g.Mol-1 molecular mass homologous. The biosurfactant from LB5a strain was able to produce stable emulsions in oils, hydrocarbons and fuels, bioremediation and Microbial Enhanced Oil Recovery (MEOR), potential were observed by the remotion of crude oil from contaminated sand. The surfactin also showed antibacterial activity agains gram positive and negative bacterias and showed antiproliferative activity agains human tumoral cells. The results described herein give important data reflecting the surfactant potential of the surfactin and its applications. Moreover, they reveal the manipueira as an excellent substrate to the biosurfactant production by the studied strain. The utilization of an industrial residue as substrate allows a strong cost reduction to the sufactant production and consists a potentially alternative to the treatment of this effluent / Mestrado / Ciência de Alimentos / Mestre em Ciência de Alimentos