Syzygium cordatum Hoscht ex. C Krauss, also known as water berry, is normally used by the people of South Africa for respiratory ailments including tuberculosis, stomach complaints, treatment of wounds and as emetics. An extract of the leaves can be used as a purgative for diarrhoea treatment. The leaves of Syzygium cordatum Myrtaceae were obtained from the Eastern Cape Province of South Africa, air dried and sequential solvent extraction was done to obtain various non volatile crude extracts. The volatile extract, that is the essential oil was extracted from the leaves using hydrodistillation and analysis of compounds was done by GC/MS for composition. 32 compounds were obtained from the fresh leaves and 18 compounds were obtained from the dry leaves. The fresh oil contains caryophyllene (11.8 percent) and caryophyllene oxide (11.1 percent) as the main sesquiterpene component. α-Pinene(5.0 percent) was the only monoterpene compound identified in the fresh oil in substantial amount. The dry leaves oil had copanene (17.0 percent), β-Caryophellene (26.0 percent), cubenol (6.5 percent) and caryophellene oxide (14.2 percent) as the dominant constituent of the oil. Summary of the classes of compounds in the oil revealed that the chemical profile of both oils were dominated by sesquiterpenoid compounds. This is the first time that terpenoids compounds are being identified in both the fresh and dry leaf oil of S. cordatum. Hexane leaf extract was selected due to the interest in the terpenoid compounds. Column chromatography of the hexane crude gave five (5) of which two are fully reported. The isolates were fully elucidated using spectroscopic methods to be β-Sitosterol (HC3) and Friedela-3-one (HC1A/HC1D). Cytotoxicity analysis was carried out on the crude using the Brine shrimps assay. Isolates 1C and1D showed significant lethality using the brine shrimps assay with lethality values (LC50) of 4.105mg/ml for HC1C and 4.11mg/ml for 1D/1A respectively.

Orientadores: Everson Alves Miranda, Adriano Rodrigues Azzoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-18T15:34:36Z (GMT). No. of bitstreams: 1 Bonturi_Nemailla_M.pdf: 2076232 bytes, checksum: e3f4e56f400891ab77fe029e0c1f0899 (MD5) Previous issue date: 2011 / Resumo: O número de estudos em terapia gênica com vetores plasmídiais (pDNA) têm aumentado nestes últimos anos. Como resultado, a demanda para preparações de pDNA em conformidade com as recomendações das agências reguladoras (EMEA, FDA) também aumentou. O DNA plasmidial é frequentemente obtido através da fermentação de Escherichia coli transformada e purificada por uma série de operações unitárias, incluindo a cromatografia. Este trabalho teve como objetivo o desenvolvimento de um processo cromatográfico para a recuperação e purificação do pDNA superenovelado (sc pDNA) a partir do lisado neutralizado. Os ligantes fenil (hidrofóbico) e mercaptopirimidina (tiofílico) foram imobilizados em matrizes de agarose e celulose. A seletividade destes ligantes para com o sc pDNA foi determinada através de estudos de adsorção utilizando citrato de sódio 1,5 mol/L e fosfato de potássio 2,0 mol/L como tampões de adsorção. A cromatografia com o adsorvente fenil-agarose e o citrato de sódio 1,5 mol/L permitiu recuperar 58% do pDNA sem contaminação por gDNA, proteínas e endotoxinas, sendo uma alternativa potencial para a recuperação primária do sc pDNA. O resultado mais promissor foi obtido com a cromatografia com o adsorvente mercaptopirimidina-agarose e fosfato de potássio 2,0 mol/L como tampão de adsorção. Este sistema tampão de adsorção/adsorvente permitiu a obtenção de pDNA com 100% de pureza e dentro das recomendações das agências reguladoras no tocante à contaminação por RNA e endotoxinas. Assim, este trabalho lançou as bases para o desenvolvimento de dois métodos cromatográficos para a recuperação primária ou purificação de pDNA diretamente do lisado neutralizado, ambos potencialmente aplicáveis em larga escala / Abstract: The number of studies in gene therapy with plasmid vectors (pDNA) has witnessed an increase in the recent years. As result the demand for preparations of pDNA in compliance with recommendations of the regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is oftenly obtained through fermentation of transformed Escherichia coli and purified by a series of unit operations, including chromatography. This work aimed the development of a chromatographic process for the recovery and purification of supercolied pDNA (sc pDNA) directly from neutralized cell lysate. Phenyl (hydrophobic) and mercaptopyrimidine (thiophilic) molecules immobilized in agarose and cellulose matrices were the ligands used to capture the pDNA. Their selectivity towards sc pDNA was evaluated through adsorption studies using sodium citrate 1.5 mol/L and potassium phosphate 2.0 mol/L as the adsorption buffers. The chromatography with the adsorbent phenyl-agarose and sodium citrate 1.5 mol/L was able to recover 58% of sc pDNA without gDNA, proteins and endotoxins contamination, being an potential alternative for the primary recovery of sc pDNA. The most promising result was obtained with the chromatography with mercaptopyrimidine-agarose and potassium phosphate 2.0 mol/L adsorpition buffer. With the latter buffer/adsorbent system it was possible to obtain in a single step pDNA with 100% purity and within the recommendations of regulatory agencies with regard to contamination by RNA and endotoxins. Thus, this work laid the basis for the development of two chromatographic process for the recovery or purification of pDNA directly from the neutralized lysate, both potentially applicable in larger scale / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

A adição de vitaminas aos produtos industrializados tornou-se prática comum para as indústrias de alimentos e os teores adicionados devem obedecer à legislação brasileira durante toda a vida de prateleira dos produtos. Sabendo da sensibilidade das vitaminas a fatores como oxigênio, luz e calor, é essencial conhecer o comportamento destes compostos no alimento frente aos fatores críticos. Informações confiáveis sobre teores de vitaminas somente podem ser obtidas com métodos analíticos validados. Neste trabalho foi desenvolvido e validado um método de análise para vitaminas B1, B2, B6 e PP em leite em pó/fórmulas infantis, cereais e bebidas instantâneas por cromatografia líquida de alta eficiência com uma única etapa de extração que apresentou recuperações de 90 a 120% dependendo do teor e da matriz analisada. Após a validação do método analítico, foi avaliada a estabilidade das vitaminas em amostras submetidas a diferentes condições de estocagem durante 10 meses. Os resultados mostram que a principal causa de perda das vitaminas B2 e B6 é a exposição à luz, que pode ser agravada pela temperatura e/ou presença de oxigênio no meio, enquanto as vitaminas PP e B1 mostraram-se bem estáveis sob as diferentes condições e no tempo estudado. / Food fortification with vitamins is a very common practice in food industry and the added content must be in compliance with Brazilian Legislation during the whole product shelf life. Due to the vitamins sensibility to light, temperature and oxygen it\'s necessary to know the behavior of these compounds when submitted to these critical conditions. Trustful information about the vitamins content just can be obtained from validated analytical methods. In this work it was developed and validated a HPLC method for determination of vitamins B1, B2, B6 e PP in milk/infant formula, cereals and beverage powders with the same extract which presented recoveries within 90 and 120% depending on the matrix and the vitamins level. After method validation, the vitamins stability in different samples was evaluated during 10 months under different storage conditions. The results showed the high sensitivity of vitamins B2 and B6 to light exposure that can become worse when samples were exposed to high temperature and oxygen. Vitamins PP and B1 had very stable behavior under the studied conditions and for the period of this study.

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The purpose of this study was to compare the accuracy and reproducibility of two commercially available chromatography kits with Instant Thin Layer Chromatography-Silica Gel (ITLC-SG) and Whatman Chromatography Paper. The two commercial kits evaluated were the- MAC Klt- produced-General Radioisotopes Products, and the TECH Kit by Ackerman Nuclear.

MSc (Botany) / Department of Botany

Genetic discoveries have highlighted the role of gene expression dysregulation in both rare and common diseases. In particular, a large number of chromatin modifiers, transcription factors, and RNA-binding proteins have been implicated in neurodevelopmental diseases, including epilepsy, autism spectrum disorder, schizophrenia, and intellectual disability. Elucidating the disease mechanisms for these genes is challenging, as the encoded proteins often regulate thousands of downstream targets. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing (scRNA-seq) to characterize a mouse model of HNRNPU-mediated epileptic encephalopathy. This gene encodes a ubiquitously expressed RNA-binding protein, yet we demonstrate that reduction in its expression leads to cell type-specific transcriptomic defects. Specifically, excitatory neurons in a region of the hippocampus called the subiculum carried the strongest burden of differential gene expression. In Chapter 3, we use scRNA-seq to identify convergent molecular and transcriptomic features in four different organoid models of a cortical malformation called periventricular nodular heterotopia. In Chapter 4, we build on these successes to propose a high-throughput drug screening program for neurodevelopmental genes that encode regulators of gene expression. This approach—termed transcriptomic reversal—attempts to identify compounds that reverse disease-causing gene expression changes back to a normal state. Finally, in Chapter 5, we focus on the role of synonymous codon usage in human disease. Codon usage can affect mRNA stability, yet its role in human physiology has been historically overlooked. We use population genetics approaches to demonstrate that natural selection shapes codon content in the human genome, and we find that dosage sensitive genes are intolerant to reductions in codon optimality. We propose that synonymous mutations could modify the penetrance of Mendelian diseases through altering the expression of disease-causing mutations. In summary, the work in this thesis broadly focuses on the role of gene expression dysregulation in disease. We provide novel frameworks for interrogating disease gene expression signatures, prioritizing mutations that may alter expression, and identifying targeted therapeutics.

This study is concerned with the regulation of Lasparaginase (LA) in the cell-free crude extracts from Lactobacillus plantarum (ATCC8014). A previously reported finding that adenosine triphosphate (ATP) inhibits the action of LA in crude extracts was confirmed. The study was extended to include the mono-, di-, and triphosphates of adenosine, guanosine, cytidine, and uridine. These compounds were also shown to inhibit LA activity. These andother studies revealed that LA appears to be an allosteric type enzyme exhibiting positive homotropism with respect to substrate and heterotropism with respect to the nucleotides tested. The regulation of LA activity by high energy compounds, when coupled with asparagine synthetaseL suggests a relationship between amide synthesis-amide degradation and the energy levels of the cell.

The activity and wettability of raw fused silica capillary tubing was found to be widely variable which places severe limitations on the reproducibility of column deactivation and inertness. Hydrothermal treatment of the raw fused silica with nitric acid was proven to be very effective for cleaning and maximizing the degree of silanol coverage of the surface. The capillary rise method was used to obtain contact angle data for untreated fused silica and fused silica treated with a variety of deactivating reagents. This contact angle data was used in the construction of Zisman plots which allowed quantitative comparison of the wettability and degree of surface coverage obtained with the different deactivants. The thermal stability of the final column was related to the success of the deactivation procedure. The choice of cross-linking initiator was also found to have an affect on column inertness. In the synthesis of intermediate polarity polysiloxane stationary phases, mixtures of commercially available cyclic siloxanes were shown to be a viable alternative to the use of dichlorosilanes as starting material. The main advantages were the simplification of the synthesis procedure, simpler and better molecular weight control of the polymer, and the elimination of HC1 as a by-product of both the polymerization and endcapping steps. A new stationary phase, 7% cyanoethyl, 7% phenyl, 1% vinyl, methyl polysiloxane was synthesized and found to be more polar than OV-1701 with higher temperature stability, easily cross-linked, and suitable for use in supercritical fluid chromatography. / Ph. D.