Understanding Human Carboxylesterase 2

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, Biotechnology

Lesões do trato gastrointestinal na síndrome da imunodeficiência adquirida: [title]estudo de 45 necrópsias consecutivas

Com objetivo de estudar afrequência e etiologia das lesões do tubo digestivo na Síndrome da Imunodeficiência Adquirida (AIDS), foram analisadas retrospectivamente 45 necrópsias consecutivas de pacientes adultos portadores do vírus da AIDS. Lesões macroscópicas e cortes histológicos de amostras da boca à região anal foram estudados, sendo as lâminas coradas por HE, métodos histoquímicos ou imunohistoquímicos. Trinta e sete (82,3 %)pacientes apresentaram lesões no tubo digestivo. O local mais freqüente de lesões foi a boca (73,3%), seguido do cólon (55,5%). Lesões múltiplas foram identificadas em 17 (37,7%) casos. O diagnóstico mais prevalente foi infecção pelo citomegalovírus (35,7%) identificado predominantemente no cólon. Candidíasefoi mais freqüente na boca (26,6%) e infecção herpética no esôfago (8,8%). Verificou-se leucoplasiapilosa oral em 16 (35,5%) eneoplasias emsete (15,5%). Asneoplasias incluíram quatro sarcomas de Kaposi, dois Carcinomas intramucosos anais e um linfoma gástrico. Os dados do presente estudo confirmam a importância do trato gastrointestinal como sede de alterações patológicas relacionadas à AIDS.

One-pot enzymatic resolution/separation of enantiomers using green solvents

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Peptidoglycan assembly machines: The Staphylococcus aureus Penicillin-Binding Proteins

Dissertation presented to obtain the Ph.D degree in Biology

Synthesis and characterization of sugar containing hydrophilic and hydrophobic polymers with potential application in medicine

Dissertation toobtaina Master of Science degree in Bioorganics

Expanding the magic circle in pervasive casual play

Dissertação para obtenção do Grau de Doutor em Informática

Potentiality of Achatina fulica Bowdich, 1822 (Mollusca: Gastropoda) as intermediate host of the Angiostrongylus costaricensis Morera & Céspedes 1971

Samples of Achatina fulica were experimentally infected with Angiostrongylus costaricensis larvae, etiological agent of abdominal angiostrongyliasis, showing that A. fulica is susceptible to the parasite. Achatina fulica may be a risk to urbanization of abdominal angiostrongyliasis presumably due to its high proliferation, continuous dispersion and remarkable adaptation in several Brazilian towns.

Prevalência, fatores de risco e caracterização genética dos vírus linfotrópico de células T humana tipo 1 e 2 em pacientes infectados pelo vírus da imunodeficiência humana tipo 1 nas Cidades de Ribeirão Preto e São Paulo

O objetivo deste estudo foi definir a prevalência dos vírus linfotrópico de células T humana tipo 1 e 2 em pacientes positivos para o vírus da imunodeficiência humana tipo 1 no Estado de São Paulo, Brasil. Avaliamos 319 indivíduos atendidos em clínicas de Ribeirão Preto e Capital. Os pacientes foram entrevistados e testados sorologicamente. Foram seqüenciadas as regiões tax e long terminal repeat para diferenciação e determinação do subtipo. A soroprevalência geral foi de 7,5% (24/319) e esteve associada somente com uso de drogas injetáveis e ao vírus da hepatite tipo C (p<0, 001). O genoma viral foi detectado em 13 das 24 amostras, sendo 12 caracterizadas como HTLV-2 subtipo 2c e uma como 1a. Nossos dados mostraram que o uso de drogas injetáveis é um importante fator de risco para a transmissão de HTLV-2 em populações infectadas pelo vírus da imunodeficiência humana tipo 1.

Technical optimization of the synthesis of flavours

The goal of this thesis is the investigation and optimization of the synthesis of potential fragrances. This work is projected as collaboration between the University of Applied Sciences in Merseburg and the company Miltitz Aromatics GmbH in Bitterfeld‐Wolfen (Germany). Flavoured compounds can be synthesized in different ways and by various methods. In this work, methods like the phase transfer catalysis and the Cope‐rearrangement were investigated and applied, for getting a high yield and quantity of the desired substances and without any by‐products or side reactions. This involved the study of syntheses with different process parameters such as temperature, solvent, pressure and reaction time. The main focus was on Cope‐rearrangement, which is a common method in the synthesis of new potential fragrance compounds. The substances synthesized in this work have a hepta‐1,5‐diene‐structure and that is why they can easily undergo this [3,3]‐sigma tropic rearrangement. The lead compound of all research was 2,5‐dimethyl‐2‐vinyl‐4‐hexenenitrile (Neronil). Neronil is synthesized by an alkylation of 2‐methyl‐3‐butenenitrile with prenylchloride under basic conditions in a phase‐transfer system. In this work the yield of isolated Neronil is improved from about 35% to 46% by according to the execution conditions of the reaction. Additionally the amount of side product was decreased. This synthesized hexenenitrile involved not only the aforementioned 1,5‐diene‐structure, but also a cyano group, that makes this structure a suitable base for the synthesis of new potential fragrance compounds. It was observed that Neronil can be transferred into 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid by a hydrolysis under basic conditions. After five hours the acid can be obtained with a yield of 96%. The following esterification is realized with isobutanol to produce 2,5‐dimethyl‐2‐vinyl‐4‐hexenoic acid isobutyl ester with quantitative conversion. It was observed that the Neronil and the corresponding ester can be converted into the corresponding Cope‐product, with a conversion of 30 % and 80%. Implementing the Cope‐rearrangement, the acid was heated and an unexpected decarboxylated product is formed. To achieve the best verification of reaction development and structure, scrupulous analyses were done using GC‐MS, 1H‐NMR and 13C‐ NMR.

Valorization of olive pomace through combination of biocatalysis with supercritical fluid technology

A supercritical carbon dioxide (scCO2) based oil extraction method was implemented on olive pomace (alperujo), and an oil yield of 25,5 +/- 0,8% (goil/gdry residue) was obtained. By Soxhlet extraction with hexane, an oil extraction yield of 28,9 +/- 0,8 % was obtained, which corresponds to an efficiency of 88,4 +/- 4,8 % for the supercritical method. The scCO2 extraction process was optimized for operating conditions of 50 MPa and 348,15 K, for which an oil loading of 32,60 g oil/kg CO2 was calculated. As a proof of concept, olive pomace was used as feedstock for biodiesel production, in a process combining the use of lipase as a catalyst with the use of scCO2 as a solvent, and integrating the steps of oil extraction, oil to biodiesel transesterification and subsequent separation of the latter. In the conducted experiments, FAME (fatty acid methyl ester) purities of 90% were obtained, with the following operating parameters: an oil:methanol molar ratio of 1:24; a residence time of 7,33 and 11,6 mins; a pressure of 40 MPa; a temperature of 313,15 K; and Lipozyme (Mucor miehei; Sigma-Aldritch) as an enzyme. However, oscillations of FAME purity were registered throughout the experiments, which could possibly be due to methanol accumulation in the enzymatic reactor. Finally, the phenolic content of olive pomace, and the effect of the drying process – oven or freeze-drying – and the extraction methods – hydro-alcoholic method and supercritical method – on the phenolic content were analysed. It was verified that the oven-drying process on the olive pomace preserved 90,1 +/- 3,6 % of the total phenolic content. About 62,3 +/- 5,53% of the oven-dried pomace phenolic content was extracted using scCO2 at 60 MPa and 323,15 K. Seven individual phenols – hydroxytyrosol, tyrosol, oleuropein, quercetin, caffeic acid, ferulic acid and p-coumaric acid – were identified and quantified by HPLC.

Biotransformation/separation routes to obtain pure enantiomers

The objective of the work presented in this thesis was the development of an innovative approach for the separation of enantiomers of secondary alcohols, combining the use of an ionic liquid (IL) - both as solvent for conducting enzymatic kinetic resolution and as acylating agent - with the use of carbon dioxide (CO2) as solvent for extraction. Menthol was selected for testing this reaction/separation approach due to the increasing demand for this substance, which is widely used in the pharmaceutical, cosmetics and food industries. With a view to using an ionic ester as acylating agent, whose conversion led to the release of ethanol, and due to the need to remove this alcohol so as to drive reaction equilibrium forward, a phase equilibrium study was conducted for the ehtanol/(±)-menthol/CO2 system, at pressures between 8 and 10 MPa and temperatures between 40 and 50 oC. It was found that CO2 is more selective towards ethanol, especially at the lowest pressure and highest temperature tested, leading to separation factors in the range 1.6-7.6. The pressure-temperature-composition data obtained were correlated with the Peng-Robinson equation of state and the Mathias-Klotz-Prausnitz mixing rule. The model fit the experimental results well, with an average absolute deviation (AAD) of 3.7 %. The resolution of racemic menthol was studied using two lipases, namely lipase from Candida rugosa (CRL) and immobilized lipase B from Candida antarctica (CALB), and two ionic acylating esters. No reaction was detected in either case. (R,S)-1-phenylethanol was used next, and it was found that with CRL low, nonselective, conversion of the alcohol took place, whereas CALB led to an enantiomeric excess (ee) of the substrate of 95%, at 30% conversion. Other acylating agents were tested for the resolution of (±)-menthol, namely vinyl esters and acid anhydrides, using several lipases and varying other parameters that affect conversion and enantioselectivity, such as substrate concentration, solvent and temperature. One such acylating agent was propionic anhydride. It was thus performed a phase equilibrium study on the propionic anhydride/CO2 system, at temperatures between 35 and 50 oC. This study revealed that, at 35 oC and pressures from 7 MPa, the system is monophasic for all compositions. The enzymatic catalysis studies carried out with propionic anhydride revealed that the extent of noncatalyzed reaction was high, with a negative effect on enantioselectivity. These studies showed also that it was possible to reduce considerably the impact of the noncatalyzed reaction relative to the reaction catalyzed by CRL by lowering temperature to 4 oC. Vinyl decanoate was shown to lead to the best results at conditions amenable to a process combining the use of supercritical CO2 as agent for post-reaction separation. The use of vinyl decanoate in a number of IL solvents, namely [bmim][PF6], [bmim][BF4], [hmim][PF6], [omim][PF6], and [bmim][Tf2N], led to an enantiomeric excess of product (eep) values of over 96%, at about 50% conversion, using CRL. In n-hexane and supercritical CO2, reaction progressed more slowly.(...)

Engineering bio-based polymers using alternative solvents and processes

The work presented in this thesis explores novel routes for the processing of bio-based polymers, developing a sustainable approach based on the use of alternative solvents such as supercritical carbon dioxide (scCO2), ionic liquids (ILs) and deep eutectic solvents (DES). The feasibility to produce polymeric foams via supercritical fluid (SCF) foaming, combined with these solvents was assessed, in order to replace conventional foaming techniques that use toxic and harmful solvents. A polymer processing methodology is presented, based on SCF foaming and using scCO2 as a foaming agent. The SCF foaming of different starch based polymeric blends was performed, namely starch/poly(lactic acid) (SPLA) and starch/poly(ε-caprolactone) (SPCL). The foaming process is based on the fact that CO2 molecules can dissolve in the polymer, changing their mechanical properties and after suitable depressurization, are able to create a foamed (porous) material. In these polymer blends, CO2 presents limited solubility and in order to enhance the foaming effect, two different imidazolium based ILs (IBILs) were combined with this process, by doping the blends with IL. The use of ILs proved useful and improved the foaming effect in these starch-based polymer blends. Infrared spectroscopy (FTIR-ATR) proved the existence of interactions between the polymer blend SPLA and ILs, which in turn diminish the forces that hold the polymeric structure. This is directly related with the ability of ILs to dissolve more CO2. This is also clear from the sorption experiments results, where the obtained apparent sorption coefficients in presence of IL are higher compared to the ones of the blend SPLA without IL. The doping of SPCL with ILs was also performed. The foaming of the blend was achieved and resulted in porous materials with conductivity values close to the ones of pure ILs. This can open doors to applications as self-supported conductive materials. A different type of solvents were also used in the previously presented processing method. If different applications of the bio-based polymers are envisaged, replacing ILs must be considered, especially due to the poor sustainability of some ILs and the fact that there is not a well-established toxicity profile. In this work natural DES – NADES – were the solvents of choice. They present some advantages relatively to ILs since they are easy to produce, cheaper, biodegradable and often biocompatible, mainly due to the fact that they are composed of primary metabolites such as sugars, carboxylic acids and amino-acids. NADES were prepared and their physicochemical properties were assessed, namely the thermal behavior, conductivity, density, viscosity and polarity. With this study, it became clear that these properties can vary with the composition of NADES, as well as with their initial water content. The use of NADES in the SCF foaming of SPCL, acting as foaming agent, was also performed and proved successful. The SPCL structure obtained after SCF foaming presented enhanced characteristics (such as porosity) when compared with the ones obtained using ILs as foaming enhancers. DES constituted by therapeutic compounds (THEDES) were also prepared. The combination of choline chloride-mandelic acid, and menthol-ibuprofen, resulted in THEDES with thermal behavior very distinct from the one of their components. The foaming of SPCL with THEDES was successful, and the impregnation of THEDES in SPCL matrices via SCF foaming was successful, and a controlled release system was obtained in the case of menthol-ibuprofen THEDES.

Aperfeiçoamento da técnica de eletroforese para análise isoenzimática de clones de seringueira (Hevea sp.)1

Este estudo foi conduzido no sentido de otimizar as técnicas de preparo de extratos de folíolos e identificar tampões e condições de corrida que proporcionassem uma boa definição dos perfis eletroforéticos de clones de seringueira. Foram testados 25 diferentes sistemas enzimáticos utilizando-se onze sistemas tamponantes. Não foram encontradas diferenças na qualidade dos zimogramas obtidos nos diferentes estádios fenológicos analisados, em amostras submetidas a diferentes condições de centrifugação e nas diferentes plantas de um mesmo clone. Extratos de folíolos liofilizados apresentaram resolução semelhante para Adh, Pgi, 6Pgdh, Lap-1, Skdh, Acp, Mdh, ßGlu, Pgm e Idh que homogenados de folíolos não liofilizados preparados até 2 dias pós-coleta. Os padrões de Got e Est apresentaram boa nitidez somente em extratos de folíolos frescos. As demais enzimas não apresentaram resolução satisfatória. Com o uso de diferentes substratos, foi possível detectar nove regiões de atividade esterásica nas condições empregadas, embora variação genética tenha sido caracterizada para apenas três locos nos clones analisados. Os fenótipos observados para esterase-7 utilizando-se ésteres derivados de naftol foram semelhantes aos padrões encontrados utilizando L-benzoil-ßnaftilamida como substrato, indicando que esta esterase seja, provavelmente, uma en-dopeptidase. A utilização de substratos fluorogênicos permitiu ainda a detecção de uma variante eletroforética de Acp em Hevea nítida e uma variante de ßGlu em Hevea rigidifolia.

Análise descritiva quantitativa do palmito de pupunheira

Este estudo foi conduzido para descrever o perfil sensorial do palmito de pupunha. A análise descritiva quantitativa (ADQ) foi utilizada para descrever os atributos sensoriais relacionados à aparência, aroma, e textura de três marcas comerciais de palmito de pupunha. As amostras foram avaliadas por uma equipe selecionada com onze provadores e foram definidos nove atributos sensoriais: cor amarela, aparência uniforme e úmida, aroma e sabor não característicos, sabor acido, residual acido e textura macia. Houve diferenças significativas em seis atributos entre as três marcas testadas. Os provadores descreveram os palmitos de pupunha como: cor amarela clara, aparência uniforme e úmida, aroma e sabor não característicos e sabor ácido com valores intermediários, residual acido e amargor final com valores de pouco e textura muito macia a uma textura intermediária.

Photolabile protection for amino acids: studies on the release from novel benzoquinolone cages

The synthesis of a novel fused nitrogen heterocycle, benzoquinolone, for evaluation as a photocleavable protecting group is described for the first time, by coupling to model amino acids (alanine, phenylalanine and glutamic acid). Conversion of the phenylalanine ester conjugate to the thionated derivative was accomplished by reaction with Lawesson’s reagent. Photocleavage studies of the carbonyl and thiocarbonyl benzoquinolone conjugates in various solvents and at different wavelengths (300, 350 and 419 nm) showed that the most interesting result was obtained at 419 nm for the thioconjugate, revealing that the presence of the thiocarbonyl group clearly improved the photolysis rates, giving practicable irradiations times for the release of the amino acids (less than 1 minute).