Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2009. / Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus are all plants that are indigenous to South Africa. These plants are used in traditional medicine to treat various ailments. However, there is little or no scientific data to justify these traditional uses. Furthermore, it is difficult to reconcile traditional knowledge with scientific evidence because of the overwhelming targeting of signal-responsive systems by plant defensive compounds, multiple sites of action and the connectedness of the signaling pathways, which provide many cures and have pleiotropic effects. In order to evaluate the action spectrum of these plants, and validate its widespread use, this research evaluated the antibacterial, antioxidant, anti-inflammatory, anti-mosquito and immunomodulatory properties of these plants. Antimicrobial activity of the extract was determined by evaluating the bactericidal and fungicidal action using the agar disc diffusion assay. Anti-oxidative properties of the extracts were tested using the DPPH photometric assay. Anti-inflammatory properties were carried out using the 5-lipoxygenase assay. The larvicidal, repellency and insecticidal assay was determined against A.arabiensis. The safe use of these plant extracts was determined by evaluating toxicity, a brine shrimp lethality assay and an in vitro cell culture system using human myelogenous leukemia cell line. Potential carcinogenic activity was evaluated using the Ames Salmonella Mutagenecity assay. The immunomodulatory activity of the extracts on human peripheral blood mononuclear cells 6 was evaluated on freshly harvested lymphocytes using the MTT assay. Cytokine response was evaluated by measuring the secretion of interferon-gamma and interleukin-10. Elucidation of the B cells, T cells, activated T cells, CD 4+, CD 8+ and NK cells was performed by flow cytometry. The extracts showed anti-microbial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella typhimurium, Serratia marcescens, Bacillus cereus and Tricoderm sp. The highest activity was shown by methanolic and aqueous extracts of L. leonurus leaves followed by methanolic and aqueous extracts of D. cinerea. Extracts of C. tomentosa and D.cinerea demonstrated a higher degree of free radical scavenging than rutin, which was used as a standard indicating that these plants have strong antioxidant properties. None of the plants showed significant anti-inflammatory activity when compared to NDGA. In the anti-mosquito assays, the extracts showed strong repellency and insecticidal activity. L. leonurus extracts demonstrated the highest insecticidal and repellency activity against the mosquito, and was also found to cause ‗knockdown‘ and mortality. The extracts display no toxicity, cytotoxicity and mutagenicity. The immunological studies for immune modulation showed that the methanol extracts of these plants induce a Th1- predominant immune response because they significantly suppressed the secretion of IL-10 and augment IFN-γ production, which are hallmarks used to indicate a stimulation of the innate immune response. This study also provides new information, with respect to the potential use of these plants in producing a mosquito repellent and an immunostimulant. / National Research Foundation

Στην παρούσα μεταπτυχιακή εργασία ειδίκευσης περιγράφεται η σύνθεση και ο χαρακτηρισμός νέων μεσοπορωδών βιοενεργών υλικών. Παρουσιάζονται οι συσχετιζόμενες με τα πορώδη/μεσοπορώδη υλικά έννοιες, οι ιδιότητες τους και διάφοροι τρόποι σύνθεσης τους. Εν συνεχεία, αναλύονται θέματα βιουλικών, δίνοντας έμφαση στις βιοϋάλους και τις ιδιότητες τους, και ειδικότερα στη βιονεργότητα, που αποτελεί την πιο χαρακτηριστική ιδιότητα αυτών, ενώ δίνεται έμφαση, επίσης στις διάφορες μεθόδους που χρησιμοποιούνται για την σύνθεση τους. Στην συνέχεια αναλύονται οι τεχνικές που χρησιμοποιήθηκαν για το χαρακτηρισμό των προαναφερθέντων υλικών καθώς και ο τρόπος λειτουργίας της κάθε τεχνικής. Επίσης, γίνεται αναφορά στα διαφορετικά χαρακτηριστικά των υλικών που μπορούν να μελετηθούν για κάθε τεχνική ξεχωριστά. Στο τελευταίο κεφάλαιο παρουσιάζεται αναλυτικά η διαδικασία διεξαγωγής των πειραμάτων, οι διάφοροι τρόποι σύνθεσης που χρησιμοποιήθηκαν, καθώς και σχολιασμός των αποτελεσμάτων. Τέλος, παρουσιάζονται τα συμπεράσματα που προέκυψαν από τα πειραματικά αποτελέσματα για την παρούσα μεταπτυχιακή εργασία. Επίσης γίνεται αναφορά και σε μελλοντικές κατευθύνσεις που περιλαμβάνουν την χρήση αυτών των υλικών σε σύνθετα υλικά για παλμική εναπόθεση με Laser και τεχνικές εναπόθεσης λεπτού υμενίου. / In this master thesis the synthesis and characterization of novel bioactive mesoporous materials is being reported. The notions related to porous/mesoporous materials, their properties and various synthetic methods are being discussed. Subsequently, the notion of “biomaterials” is further analyzed, focusing on bioglasses and their properties. The meaning of bioactivity is also analyzed as the most characteristic feature of bioglasses while various methods used for the synthesis of these are discussed. Following the techniques used to characterize these materials as well as the operating mode of each technique is discussed. In the last chapter, the experimental processes are reported as well as the results are being discussed. Finally, the conclusions of all the experiments and analysis are reported. Also, future directions include the usage of these materials in composite materials for laser ablation and thin film deposition techniques are mentioned.

published_or_final_version / abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

No description available.

Orientador: Wagner Vilegas / Resumo: Organismos marinhos são importantes fontes de novos produtos naturais e compostos bioativos com funções variadas. Entre as principais fontes de novas moléculas esta o filo dos Cnidários, com uma importante contribuição no numero de compostos descritos nos últimos anos. Bunodossoma caissarum é uma espécie de anêmona endêmica do Brasil, cujo perfil químico foi analisado. Para essa análise foram realizados uma extração hidroalcoólica e o isolamento de determinadas moléculas com técnicas cromatográficas. Os compostos isolados foram aqueles que apresentaram absorbância no comprimento de onda no UV em 310 nm e foram identificados a partir de técnicas de espectrometria de massas e ressonância magnética. Entre as substâncias encontradas estão sais piridinicos, núcleos imidazóis, caissarona e a etilglicose, moléculas com importantes papeis fisiológicos / Abstract: Marine organisms are importante sources of new natural products and bioactive compounds with various functions. The phylum Cnidaria is among the main sources of new molecules, with na importante contribution to the number of compounds described in the last years. The anemone species Bunodossoma caissarum is endemic to Brazil and had it's chemical profile analyzed. For the purpose of this analysis were done a hydroalcoolic extraction and the isolation of certain molecules by chomatographic techniques. The isolated compounds were those that displayed absorbance in the 310nm wavelength of the UV spectrum and were identified on the basis of mass spectrometry and magnetic ressonance. Among the substances found are pyridinic salts, imidazole nucleus, caissarone and ethyl glucose, which are molecules with important physiological roles / Mestre

Los péptidos bioactivos son fragmentos que forman parte de muchas proteínas y que al ser liberados son capaces de regular una serie de procesos biológicos. Para liberarlos, las proteínas son degradas por medio de enzimas digestivas o procesos industriales. Estos péptidos mantienen propiedades antihipertensivas, antimicrobianas, antioxidantes, anticarcinógenas, antinflamatorias, entre otras, que podrían ser aprovechadas para el desarrollo de nuevos fármacos. En este trabajo de investigación, hemos resaltado la utilidad de los péptidos bioactivos como compuestos altamente específicos capaces de contribuir en la mejora de patologías tales como el cáncer, la hipertensión arterial y la enfermedad de Alzheimer. Particularmente, en estas enfermedades, mostramos experimentos in vitro e in vivo con resultados prometedores que motivan a continuar su estudio en humanos. Estos péptidos fueron extraídos del haba de la soja, la leche de vaca y el hígado de atún, evidenciando la disponibilidad de estos en la naturaleza y generando la incógnita de que otros compuestos podrían encontrarse en la flora y fauna del Perú. De la misma forma, se presentan una serie de tecnologías novedosas para el aislamiento de dichos compuestos, entre estas están la precipitación selectiva, la filtración por membrana, la cromatografía de exclusión por tamaño y el isoelectroenfoque. Éstas son escalables, de fácil aplicación, rápidas, no requieren mayor inversión en infraestructura u equipamiento y no necesitan de una gran cantidad de muestra. En conclusión, en este trabajo se demuestra que los péptidos bioactivos guardan gran promesa, en particular en la identificación de compuestos novedosos para el desarrollo de nuevas drogas. Si se genera una alianza en el Perú entre la comunidad científica, la industria y entes públicos financiadores, se podría explotar, responsablemente, la biodiversidad de nuestro país para proceder, en gran escala, con el aislamiento y la identificación masiva de compuestos bioactivos de gran interés a nivel mundial. / Bioactive peptides are fragments that are part of many proteins and which, upon release, are capable of regulating a series of biological processes. To release them, proteins need to be degraded by digestive enzymes or industrial processes. These peptides maintain antihypertensive, antimicrobial, antioxidant, anticarcinogenic, anti-inflammatory properties, amongst others, that could be used to develop new drugs. In this research work, we have highlighted the utility of bioactive peptides as highly-specific compounds capable to contribute in the improvements of pathologies such as cancer, high blood pressure and Alzheimer’s disease. Particularly, in these diseases, we show in vitro and in vivo experiments with promising results that motivate further studies in humans. These peptides were extracted from soy bean, cow milk and tuna liver, evidencing the availability of these in nature and posing the question of all the other compounds that could be found in the flora and fauna of Peru. Moreover, we present a serious of novel technologies to isolate said compounds, amongst these are selective precipitation, membrane filtration, size-exclusion chromatography and isoelectric focusing. These techniques are scalable, easily applied, rapid, do not require significant investment in infrastructure or equipment and do not need a significant amount of sample. In conclusion, in this work we demonstrate that bioactive peptides have great promise in particular in the identification of novel compounds to develop new drugs. If we generate an alliance in Peru between the scientific community, the industry and public financing entities, our country’s biodiversity could be responsibly exploited to proceed, in a big scale, with the massive isolation and identification of bioactive compounds of great interest worldwide / Trabajo de investigación

O arroz (Oryza sativa L.) é consumido principalmente na forma polida, porém uma alternativa nutricionalmente melhor seria o arroz integral, que contém ao redor de 10% de farelo, rico em micronutrientes e compostos bioativos. Dentre esses destacam-se a vitamina E e &#947;-orizanol, aos quais se atribuem diversos efeitos benéficos à saúde. A literatura descreve vários estudos relacionados à presença desses compostos no arroz integral, porém pouco se sabe a respeito da sua estabilidade com o armazenamento, a parboilização e a cocção caseira. Pelo fato do arroz integral apresentar curta vida de prateleira, uma alternativa para aumentar o prazo para o consumo é o emprego da parboilização. A parboilização, que consiste em um processo hidrotérmico, provoca alterações estruturais no amido do grão e a literatura é controversa no que diz respeito à diminuição da digestibilidade do amido e consequentemente à redução do índice glicêmico (IG). Este trabalho teve como objetivos: avaliar os efeitos da parboilização sobre a disponibilidade do amido por meio da cinética de hidrólise do amido estimando o índice glicêmico (IG) em cultivares de arroz integral com diferentes teores de amilose; analisar o efeito da parboilização, do armazenamento por seis meses e da cocção caseira do arroz integral sobre a estabilidade dos compostos bioativos, e correlacionar os compostos bioativos com a atividade antioxidante do arroz, antes e após o processamento. Foram analisadas 36 amostras pertencentes a diferentes cultivares selecionadas de arroz integral e parboilizado integral das safras de 2007 e 2008 e outras três amostras de arroz integral e parboilizado integral, adquiridas no comércio local da cidade de São Paulo. A partir do índice de hidrólise (IH) in vitro, foi estimado o IG. A vitamina E e o &#947;-orizanol foram extraídos com metanol, separados por CLAE-fase reversa e quantificados por curvas padrão correspondentes. A atividade antioxidante foi avaliada pelos métodos de DPPH&#8226; e ORAC. Foi verificado que a disponibilidade do amido de todas as amostras de arroz foi pouco afetada pela parboilização nas condições empregadas de modo que os IH permaneceram similares aos das amostras não parboilizadas. As amostras com teor intermediário de amilose apresentaram os menores IH (p<0,05). O IG estimado nas amostras cruas e parboilizadas foi cerca de 80%, tanto nas cultivares de arroz selecionadas quanto nas amostras comerciais e não foi observada alteração com o processamento. Com relação aos compostos bioativos, os teores de vitamina E e &#947;-orizanol no arroz integral cru foram, em média de 25 e 188mg/kg, respectivamente. A vitamina E foi sensível ao processo de parboilização, ao armazenamento e à cocção, com perdas de 50% ou mais, em relação à concentração no grão integral in natura. Já o &#947;-orizanol mostrou-se mais estável frente a qualquer tratamento, com perda de no máximo 20% da sua concentração inicial. A maior correlação entre a concentração de compostos bioativos e atividade antioxidante foi encontrada para a vitamina E. / Rice (Oryza sativa L.) is consumed mostly in the milled form. However, due to its content of bran, vitamin E and &#947;-oryzanol, the intake of brown rice has been recommended. Although previous studies ascribed some important health benefits related to these brown rice\'s compounds, information regarding the effect of cooking, storage and parboiling on these substances remains to be elucidated. Rice parboiling is a hydrothermal process consisting of soaking, heating and drying that provide technological and nutritional benefits to the consumer. Until now, if parboiling process decreases the starch digestibility and consequently reduces the glycemic index (GI) is unclear. The objectives of the present study were: to evaluate the effect of the parboiling process on starch digestibility through a GI estimation by the kinetics of starch hydrolysis in rice cultivars with different levels of amylose; to analyze the effect of traditional parboiling, cooking and storage for six months on the stability of bioactive components as well as to correlate the content of bioactive components of rice with its antioxidant activity before and after parboiling. Thirty-six samples of brown rice and parboiled brown rice harvested in 2007 and 2008 were analyzed. In addition, three commercial cultivars of both brown and parboiled brown samples from local market that were obtained in São Paulo were also studied. The glycemic index (IG) was estimated from the hydrolysis index (IH). Vitamin E homologues and &#947;-oryzanol were extracted simultaneously with methanol and analyzed by HPLC-RP/UV and fluorescence detection in a single run. Peak areas were converted to the corresponding by the standards of &#945;- and &#947;-tocopherol, &#945;- and &#947;-tocotrienol and &#947;-oryzanol. The antioxidant capacity was evaluated by both, DPPH&#8226; and ORAC methods. The results indicated that starch digestibility from all rice samples was little affected by parboiling under the conditions employed in the present study. Samples with intermediate amylose content had the lowest HI (p <0.05). The estimated GI in raw and parboiled samples was 80% in all rice cultivars selected and in commercial samples as well. The parboiling showed no effect on GI. The total tocols and &#947;-orizanol contents in raw rice were 25 and 188 mg/kg, respectively. The vitamin E homologues were the most affected by parboiling, storage and cooking than &#947;-oryzanol with a decrease of 50% or more when compared to the raw grain. However, &#947;-oryzanol was more stable during either treatment with 20% loss of when compared to raw rice. The most significant correlation between the concentration of bioactive compounds and antioxidant activity was found for vitamin E.

by Clifford Sanders. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web. / Ethylene is the simplest alkene. The carbon-carbon double bond is ubiquitous in the field of chemistry. Ethylene serves as the basis for understanding these molecules. Thus, the assignment of the electronic transitions in ethylene is an important endeavor that many scientists have undertaken, but are yet to decipher theoretically or experimentally. Synchrotron Radiation in the vacuum ultraviolet region allows for magnetic circular dichroism (MCD) measurements of ethylene and other simple alkenes. Studies of ethylene and propylene revealed that the páap* (AgáaB1u ethylene notation) transition is not the lowest energy transition. The páa3s(R) (AgáaB3u ethylene notation) is the lowest energy transition. To further this investigation, MCD and absorption measurement were carried out on isobutene. The isobutene spectra clearly showed four electronic transitions in the 156 to 212 nm wavelength region. These four isobutene transitions have been assigned as páa3s, páap*, páa3p(Sv (Band páa3px proceeding from lower energy to higher energy. The present results support the assignments in ethylene and propylene.