Rozprawy doktorskie na temat „Bioactive”

Alginates are polysaccharides extracted from the cell walls of brown seaweed or from certain bacteria such as Azotobacter vinelandii. Alginates have been shown to reduce the activity of pepsin and initial data suggest that it may affect the activity of pancreatic lipase. Pancreatic lipase plays an important role in the breakdown of triacylglycerol, if the activity of pancreatic lipase can be reduced then the breakdown of triacylglycerol would be reduce which in turn would lower the amount absorbed by the body. A pharmaceutical treatment for obesity called orlistat inhibits lipase and accounts for 66% of all prescriptions for treatments for obesity in 2008 in the UK. However the side effects of orlistat (uncontrolled diarrhoea and steatorrhea) can reduce the compliance with the treatment. If orlistat is taken with a high fibre product it has been shown that this can greatly reduce or eliminate the side effects. Since alginate is a dietary fibre it is believed that this could be an effect treatment for obesity. Colourimetric and turbidimetric assays were used to determine the effect of a wide range of alginates and other dietary fibres on pancreatic lipase. Alginates have the ability to inhibit pancreatic lipase, with a maximum inhibition of 72.2% (±9.9). Pectin could also inhibit lipase by a maximum of 71.8% (±22.3), however, specific enzymactically modified alginates could activate the enzyme by up to 22.0% (±10.1). Carrageenan and Hydroxypropyl methylcellulose could also activate the enzyme by a maximum of 37.3% (±24.5). The structure of the alginate is key to the inhibition or activation of the enzyme. Consecutive guluronate residues are important for inhibition whereas alternating mannuronate and guluronate blocks are detrimental to inhibition. The alginate that activated the enzyme was almost entirely consisting of poly-alternating uronate residues. The level of esterification was important for pectins inhibition of lipase with higher levels causing little inhibition. Alginate causes sustained inhibition at low concentrations of biopolymer compared to pectin. Therefore alginate could play a potential role in the management of obesity. ii

Montbretins A-E were isolated from the corms of Crocosmia sp., an invasive perennial plant. The montbretins are inhibitors of human pancreatic α-amylase (HPA). Montbretin A (2- 30) is a competitive inhibitor of HPA with a K₁ of 1.3 nM. The activity of the other family members varied significantly and provided structure-activity information. Saturation Transfer Difference (STD) NMR spectroscopy was used to determine that the caffeic acid region of the montbretins is important for binding. HPA is involved in the breakdown of complex carbohydrates; inhibition of this enzyme could help with regulation of blood sugar levels after a meal. In the lungs of cystic fibrosis patients, the activation of Toll-Like Receptor 5 (TLR5) in the presence of flagellin leads to inflammation and obstruction. Girolline (3-1), a known alkaloid, was isolated from a Phonpeian sponge following potent inhibition of the flagellin initiated TLR5 activation. No activity was observed in any synthetic analogues of girolline. The massacreones are a new family of ecdysteroids isolated from an unidentified Dominican cnidarian. The extract of the cnidarian had good TLR5 activity, but the massacreones – namely massacreone A (3-25) and massacreone B (3-26) have only moderate activity and a small window of activity before they are toxic. The algal pigment caulerpin (4-29) was isolated from Caulerpa sp. as a compound showing good activity in a yeast growth restoration assay designed to identify inhibitors ofhuman indoleamine-2,3-dioxygenase (IDO). Caulerpin did not show any activity in a free enzyme IDO assay. IDO is involved in immune escape, which prevents the immunological rejection of tumors.

The chemical exploration of extracts from cultures of the marine bacterial isolate PNG-276 yielded the novel antibiotic tauramamide (2.13), a non-ribosomal peptide active against cultures of Enterococcus sp. and methicillin-resistant Staphylococcus aureus (MRSA). A study of extracts of the marine sponge Spirastrella coccinea yielded the novel macrolide methylspirastrellolide C (3.14), which is active against protein phosphatase 2A (PP2A). A third study examined sponge extracts active in a cannabinoid receptor assay, yielding two known compounds, an A- nor -steroid derivative (4.10) and bengamide A (4.11). Neither purified compound was active in the cannabinoid receptor assay, although in both cases this is the first report of these compounds being isolated from Stylissa massa and Hemiasterella aff. affinis sponges, respectively. [See Thesis for Diagrams]
Science, Faculty of
Chemistry, Department of
Graduate

An artificial liposome membrane system has been employed for in vitro screening of the human absorption of biologically active molecules for applications in nutrition and drug treatments. Initial work with molecules having small permeabilities demonstrated that they could not be measured using the technique since they were absorbed by the membrane. A critical innovation was to pre-treat the membrane by equilibrating it with the molecule of interest since this avoids the absorption problem but required more complex data analysis. Bioactive molecules with strong antioxidant and anti-cancer activity extracted from green tea showed a strong affinity to the membrane, which suggests that this significantly limits bioavailability. Ethanol but not dimethyl sulfoxide (DMSO) was found to enhance the diffusion of paracetamol, theophylline, acyclovir, nadolol and amphotericin B. The potential synergistic effect on the diffusion of paracetamol in the presence of caffeine was investigated but it was shown to have a detrimental effect. Finally, an effective protection of Epigallocatechin-3-gallate (EGCG) from the environment was achieved by the preparation of beeswax microspheres as a carrier.

For thousands of years, Nature has' been considered to be essential for human beings for the treatment of their diseases. Amphibians are an important component of the Animal Kingdom and have played a significant role in sourcing many active compounds and some therapeutics. In the course of this research, genomic and proteomic techniques have been used to investigate the bioactive peptides from the skin secretions of four American amphibian species: the Central American red-eyed leaf frog, Agalychnis callidryas~ the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, Rohde's leaf frog, Phyllomedusa rohdei and the Giant Mexican leaf frog Pachymedusa dacnicolor. The synthetic peptides all exhibited significant biological activities. Medusins were a novel family of antimicrobial peptides found in all species investigated and all possessed a broad-spectrum of antibiotic activity. Phylloseptins were found to be active against both planktonic bacteria and those within biofilms. Bradykinin-like peptides (BRPs) exhibited significant and selective activities on a range of mammalian smooth muscle preparations and one was identified as a B2 receptor antagonist using the rat tail artery. These discoveries of novel peptides from amphibian skin secretions have enriched our knowledge of bioactive peptides from this source and may provide the basis for several drug development programmes.

Peptidomimetics are synthetic foldamers that expected more resistant to proteolytic degradation and enormous chemodiversity when compared with peptides. To date, the functional peptidomimetics such as β-peptides, peptoids, oligoureas, etc have been developed in many science fields. In order to explore the unnatural foldameric architectures, it’s necessary to discover the novel frameworks and molecular scaffolds. γ-AApeptides were reported to be a new class of peptidomimetics that showed its potential applications in drug discovery and chemical biology. However, a wide function and property of γ-AApeptides need to be further explored. To expand the potential application of γ-AApeptides in biochemistry, I have been focusing on the development of bioactive peptidomimetics, such as exploring the antibacterial activity of helical 1:1 α-sulfono-γ-AA heterogeneous peptides, developing the helical peptidomimetic as the inhibitor of the protein Ras_Raf interaction, identifying the protein/peptide ligands by the novel one-bead-two compound macrocyclic γ-AApeptide screening library, and elucidating the de novo dragon-boat-shaped synthetic foldamers.

Tot i que poques vegades es fa servir en els aliments, l'ordi està rebent un interès creixent com un gra saludable, per les seves propietats nutricionals. Es van estudiar a camp genotips d'ordi sense cobertes i de gra acolorit que es diferenciaven en una varietat de compostos bioactius valuosos i saludables, en diferents condicions ambientals i de temperatura. L'estrès per alta temperatura durant l'ompliment del gra va augmentar la concentració de compostos bioactius claus en el gra. S'ha de considerar la collita primerenca de grans immadurs per maximitzar la capacitat antioxidant. El 30% més extern de les fraccions perlades semblen la millor opció per explotar la capacitat antioxidant i l'alta composició fenòlica, mentre que els grans perlats poden proporcionar farines enriquides amb β-glucans. Les cobertes també són un valuós material saludable, especialment per a l'alimentació de remugants. L'ordi sense coberta i de gra morat ofereix noves vies per satisfer la creixent demanda de productes saludables, proporcionant un major contingut de fibra dietètica i compostos bioactius, una major capacitat antioxidant i un índex glucèmic estimat més baix que les farines comercials refinades i integrals de blat.
Aunque rara vez se usa en los alimentos, la cebada está recibiendo un interés creciente como un grano saludable, debido a sus propiedades nutricionales. Se estudiaron en campo genotipos de cebada sin cubiertas y de grano coloreado que se diferenciaban en una variedad de compuestos bioactivos valiosos y saludables, en diferentes condiciones ambientales y de temperatura. El estrés por alta temperatura durante el llenado del grano aumentó la concentración de compuestos bioactivos claves en el grano. Se debe considerar la cosecha temprana de granos inmaduros para maximizar la capacidad antioxidante. El 30% más externo de las fracciones perladas parecen la mejor opción para explotar la capacidad antioxidante y la alta composición fenólica, mientras que los granos perlados pueden proporcionar harinas enriquecidas con β-glucanos. Las cubiertas también son un valioso material saludable, especialmente para la alimentación de rumiantes. La cebada desnuda y de grano morado ofrece nuevas vías para satisfacer la creciente demanda de productos saludables, proporcionando un mayor contenido de fibra dietética y compuestos bioactivos, una mayor capacidad antioxidante y un índice glucémico estimado más bajo que las harinas comerciales refinadas e integrales de trigo.
Although rarely used in food, barley is receiving increasing interest as a healthy grain, due to its nutritional properties. Hull-less and coloured-grain barley genotypes differing in an array of valued and healthy bioactive compounds, were studied in the field under different temperature and environmental conditions. High temperature stress throughout grain filling increased concentration of key bioactive compounds in the grain. Early harvesting of non-mature grain should be considered as a way to maximize antioxidant capacity. The 30% outermost of the pearling fractions seem the best option to exploit antioxidant capacity and high phenolic composition, while pearled grains may provide β-glucan-enriched flours. Husks are also a valuable healthy material, particularly for ruminant feed. Hull-less and purple-grain barley offers new avenues to fulfil the increasing demand for healthy products, providing higher content of dietary fibre and bioactive compounds, greater antioxidant capacity and lower estimated glycemic index than commercial refined and whole wheat flours.

The excess of fat deposits are originated by a prolonged imbalance between the energy intake and the energy expenditure. The current food pattern based on a high consumption of saturated fats, simple sugars and processed foods, together with the sedentary lifestyle of the population, favor the development of obesity, which is responsible for 3.4 million deaths per year and is the main risk factor for the development of associated comorbidities, such as type 2 diabetes mellitus, non-alcoholic fatty liver and cardiovascular diseases. Given the current pandemic scope of obesity, finding therapeutic targets and strategies for their control and treatment is of great importance. The Mediterranean Diet, characterized by a high consumption of fruits and vegetables, provides a high levels of bioactive compounds mainly carotenoids and polyphenols, which have shown antiobesogenic properties. The main aim of this thesis is to define the molecular mechanisms involved in the metabolic impact of foods rich in bioactive compounds in animal models of obesity. We worked with sofrito and maqui to be two food products rich in bioactive compounds. Sofrito is as a typical Mediterranean preparation with a high nutritional interest due to the high content of bioactive compounds, mainly carotenoids and because beneficial effects in the primary prevention of cardiovascular and metabolic diseases such as type 2 diabetes has been attributed to it. On the other hand, the berry known as maqui (Aristotelia chilensis), characteristic of southern Chile, with a high polyphenols contents, especially from the anthocyanin group, has shown cardioprotective and hypoglycemic effects. The results show that both sofrito and maqui have beneficial effects on insulin sensitivity and glucose tolerance, respectively. As well, they are also able to increase the expression and signaling of the fibroblast growth factor pathway 21 and increase the expression of uncoupling protein 1 and browning in white adipose tissue. In addition, in the liver, the supplementation with maqui improves the hepatic steatosis caused by a high-fat diet by the expression of small heterodimer partner– interacting leucine zipper protein. The data presented allow us to point out that both dietary supplementation with sofrito and with maqui, could be good strategies in the prevention and / or treatment of obesity and its associated comorbidities.

Mestrado em Materiais e Dispositivos Biomédicos
Bone is an extremely important connective tissue in the human body, as it provides support and protection of internal organs, being also metabolically relevant as the main mineral reservoir and assuring haematopoiesis through the bone marrow. Due to the current ageing of the population, an increase in bone tissue related diseases is noticeable. Thus, more efficient therapies for treating bone diseases is crucial. Tissue Engineering appears as a promising technology for treating several of those problems, such as bone loss and joint problems. In the present work, composite biomaterials composed of a polymeric hydrogel matrix reinforced with bioactive glass particles were prepared. Individually, these materials have a high water content, which enhances their diffusive transport properties, and display osteogenic properties, respectively. The selected polymer was RGD functionalized pectin, due to its interesting properties, such as biocompatibility, cell-adhesive characteristics and adequacy for cell entrapment, and the bioactive glass selected was a novel alkali-free formulation of 70% diopside and 30% tricalcium phosphate (Di-70), composed of SiO2, CaO, MgO and P2O5. Several different composite formulations were tested, in which pectin concentration, bioactive glass content and glass particle size were varied. The biocomposite’s viscoelastic properties were assessed, as well as their biological behaviour through cytotoxicity assays, and osteogenic character by incubating mesenchymal stem cell (MSC)-laden composites into both basal and osteogenic media for up to 21 days. The results obtained demonstrated that a composite biomaterial with tuneable mechanical properties was successfully prepared, with in situ crosslinking ability within therapeutically relevant timeframes, and not requiring additional crosslinking strategies besides its own composition. Furthermore, its intrinsic osteogenic properties due to the glass composition provided the adequate conditions for promoting the differentiation of MSCs without osteogenic stimulation. The combined properties achieved indicate that the biocomposites prepared are suitable candidate cellularized biomaterials for bone tissue engineering applications.
O osso é um tecido conjuntivo de extrema importância no organismo humano, tendo funções como suporte ou proteção de órgãos internos, sendo também metabolicamente relevante como o principal reservatório de minerais e assegurando a hematopoiese com a medula óssea. Dado o envelhecimento da população, tem-se verificado um aumento da incidência de doenças degenerativas deste tecido, sendo assim essencial aplicar terapias altamente eficientes para o tratamento dessas patologias. A Engenharia de Tecidos surge como uma tecnologia promissora no tratamento destes problemas, como a perda de massa óssea e problemas nas articulações. Neste trabalho, foram produzidos biomateriais compósitos, baseados numa matriz polimérica sob a forma de hidrogel reforçada com partículas de vidro bioativo. Individualmente, estes materiais apresentam um elevado teor em água favorável ao transporte de nutrientes, e propriedades osteogénicas, respetivamente. O polímero selecionado foi a pectina funcionalizada com RGD, dadas as suas propriedades interessantes como a biocompatibilidade, capacidade de promover a adesão celular e adequabilidade para o encapsulamento de células, e o vidro bioativo apresenta uma composição de 70% de diópsido e 30% de fosfato tricálcico (Di-70) isento de alcalinos e sendo composto por SiO2, CaO, MgO e P2O5. Diferentes formulações de hidrogéis compósitos foram testadas, em que se variou a concentração de polímero, a concentração de biovidro e o seu tamanho de partícula. Analisaram-se as propriedades viscoelásticas dos biocompósitos, bem como o seu comportamento biológico, com ensaios de citotoxicidade, e ainda as propriedades osteogénicas do material, pela incubação de hidrogéis contendo células estaminais mesenquimais (MSCs) em meio basal e osteogénico durante 21 dias. Os resultados deste trabalho indicam que foi possível preparar um biomaterial compósito de propriedades mecânicas ajustáveis, com capacidade de reticular in situ em tempos clinicamente desejáveis sem necessitar agentes reticulantes externos. Para além disso, as propriedades osteogénicas intrínsecas do biovidro forneceram as condições adequadas para a promoção da diferenciação de MSCs sem estimulação osteogénica adicional. As propriedades combinadas alcançadas indicam que os biocompósitos preparados têm potencial para ser aplicados em engenharia de tecido ósseo.

Crude extracts of the rare macrofungus Serpula sp. collected from a wooded area in Sri Lanka showed antimicrobial activity. The novel fungal metabolite serpulanine (2.1) was isolated from the crude extract in very small amounts along with a number of additional secondary metabolites. In order to obtain sufficient quantities of serpulanine (2.1) for biological evaluation, a synthetic route was developed to the natural product and a small library of analogs that have been evaluated in a panel of bioassays. Serpulanine (2.1) inhibits the histone deacetylase I/II with a clear dose response curve. Halitoxins (3.1) that are frequently isolated from marine sponges have a complex macrocyclic chemical structure made of different numbers of monomeric alkylpyridinium units. An unknown halitoxin-related natural product named alotau potently inhibited the dephosphorylation activity of calcineurin. With the goal to elucidate the structure of alotau, compounds of one, two and three pyridinium rings (3.10, 3.7 and 3.8) were synthesized. Though these compounds have NMR spectra similar to the natural alotau, according to bioassay results, none of them recapitulates the activity of the unknown natural product alotau. (+)-Makassaric acid 4.1 was isolated in the Andersen Lab from the marine sponge Acanthodendrilla sp. It showed promising activity in a zebrafish screen for new drugs to treat stroke patients. The convergent synthetic scheme shown below was undertaken to conduct structure activity relationship (SAR) studies. The key intermediate 4.17 has been obtained, and further synthetic efforts will be needed to produce 4.1.
Science, Faculty of
Chemistry, Department of
Graduate

This thesis describes the investigation of the bioactivity of triterpenoids, limonoids and spiro-triterpenoids, isolated from the order of the Rutales. The focus of this research has been towards the development of botanical insecticides with non-neurotoxic modes of action in insects. It follows the recent development of Neem (Azadirachta indica, Meliaceae) as a botanical insecticide containing a limonoid azadirachtin as its main active compound. Because so few Meliaceae members have been examined for insecticidal activity the project was initiated by the screening of over sixty ethanol extracts of various parts of twenty species (collected from Costa Rica and the Tropical Museum in Miami, Florida) for bioactivity against the European corn borer (Ostrinia nubilalis) and the Variegated cutworm (Peridroma saucia). The data suggest that a majority of the extracts studied inhibited growth significantly; some are more active than neem leaf extracts. Swietenia mahogani bark, Trichilia glabra bark, T. hirta leaves, T. americana bark, T. trifolia wood, T. pleana and Azadirachta indica wood showed potent activity against the cutworm. Ruptiliocarpon caracolito bark, Cedrela odorata leaves, Aphanamixis polystachys wood, T. glabra wood and T. pleana bark showed good activity against the corn borer larvae. Bioassay guided fractionation of the most active crude extract, the bark of Ruptiliocarpon caracolito, has resulted in the isolation of six very novel spiro-CD-triterpenoids, the spirocaracolitones. The spirocaracolitones, at 100 ppm, were screened for antifungal activity against Fusarium using a hyphal growth bioassay. After 48 h a high degree of inhibition of growth (59-79%) was observed. These novel triterpenoids exhibited no antimalarial activity when screened against Plasmodium falciparum. A study of the effects of these compounds, incorporated into artificial diet, on the neonate life cycle of the European corn borer was conducted. All the spirocaracolitones effected the growth and the development of the insects but spirocaracolitone B and spirocaracolitone C were substancially more active than the other spirocaracolitones. Two postulates dealing with the biosynthesis of these unique spiro-compounds are also presented. Both commence with a friedelin derivative because canophyllol was isolated in large quantities from the hexane extract. They envisage generation of a carbocation at C12 followed by migration of the C23 methyl group this results in the formation of a carbocation at C13. The spirosystem results from migration of the C8-C14 bond. An investigation of the structure/activity relationships of gedunin was conducted to determine the moieties responsible for its antimalarial and/or antifeedant properties. Ten derivatives of gedunin were prepared (1,2-dihydrogedunin, 1,2-epoxygedunin, 1,2-dihydro-3$\beta$-gedunol, 3$\beta$-acetoxy-1,2-dihydrogedunin, 7-deacetylgedunin, 7-ketogdunin, hexahydrogedunin, tetrahydrogedunin, 21-acetylgedunin, 23-acetylgedunin) along with five other limonoids (limonin, epilimonol, nomilin, obacunone, hirtin) closely related to gedunin were evaluated for antifeedant activity (Ostrinia nubilalis) and antimalarial activity (Plasmodium falciparum). Evaluation of these compounds for antimalarial activity presented no increase in activity however it was clearly determined that alterations to the enone in ring A and at C7 of gedunin resulted in large losses of activity. Evaluation of the effects of these limonoids on the neonate life cycle of the European corn borer at 5 and 50 ppm incorporation into the artificial diet did lot produced dramatic effects on the growth and development of the larvae. At these concentrations structure/activity relationships could not be proposed.$\sp*$ ftn$\sp*$Please refer to the dissertation for diagrams.

Bioassay guided fractionation of a crude extract of the marine sponge Neopetrosia exigua resulted in the first reported isolation of exiguamines A and B. These pyrroloquinone alkaloids have an unprecedented hexacyclic skeleton that has not been previously encountered in natural products. Biological studies have identified exiguamine A as a potent in vitro inhibitor of the enzyme indoleamine-2,3-dioxygenase (IDO). IDO is an enzyme expressed by tumor cells to evade the immune system. Inhibitors against this enzyme may allow the immune system to attack cancer cells, making this enzyme a potential drug target for anti-cancer agents. Investigation of the crude extract of a Bacillus sp. collected in Dominica led to the isolation of the known diketopiperazine cyclo(S-Val-S-Phe) (3.9). In vitro biological studies revealed that cyclo(S-Val-S-Phe) is able to promote neurite outgrowth, even in the presence of physiological inhibitors. In vivo studies have shown that cyclo(S-VaI-S-Phe) is able promote sprouting in serotonergic and adrenergic axons. Synthesis of the other three diastereomers led to the discovery that cyclo(R-Val-R-Phe) is also an in vitro activator of axonal outgrowth. Inhibitors of the G2 checkpoint are able to increase the cytotoxicity of DNA damaging chemotherapeutics. Bioassay guided fractionation of an extract of the South American plant Duguetia odorata led to the isolation of the G2 checkpoint abrogator, oliveroline. This investigation also led to the isolation of the previously unreported alkaloid N-methylguatterine, and the known alkaloids dehydrodiscretine and pseudopalmatine. Chemical investigation of the marine sponge Myrmekioderma granulatum led to the isolation of the new compounds abolenone and myrmekioside C, as well as the known compounds curcudiol, curcuphenol, abolene and sesquiterpenoid. Biological studies of these compounds revealed that curcudiol is a ligand of the sex hormone-binding globulin. This protein is involved in transporting and regulating the concentration of steroids such as testosterone and estradiol. Many pathological conditions have a lower plasma concentration of these steroids. Ligands to SHBG can release steroids into the blood, so this protein is a potential drug target to treat conditions where a hormone insufficiency is present.

Macronutrient digestion is a major factor in health and metabolic diseases such as obesity and diabetes and presents a huge global challenge. Modulating macronutrient digestion with food additives and pharmaceuticals has been shown to be a fruitful approach to the treatment of obesity (Orlistat) and diabetes (Acarbose). Previous work has shown that bioactive agents have novel modulatory effects on the major enzymes of digestion, and work in this lab has shown that specific alginates can inhibit pancreatic lipase up to 70%. Alginates are now being investigated as a potential anti-obesity agent. The purpose of this thesis was to develop in vitro methodologies and an analytical approach for investigating the effects of exogenous compounds on the major digestive enzymes; -amylase, pepsin, trypsin, and lipase. A 3-step process was developed consisting of; higher-throughput single enzyme analysis, selected enzyme kinetics and model gut analysis. Alginates were shown to inhibit the action of pepsin, but have no effect on trypsin activity in vitro. The structure of alginate is key to the inhibition of pepsin, and rheological and viscometric data suggested that this effect was due to a pH dependent interaction between alginate and protein substrate as well as direct enzyme-inhibitor interactions. A similar effect was observed with Fucoidan and sulphated carrageenans. In the model gut analysis, these effects manifested as inhibition of proteolysis in the simulated gastric phase, but not in the small-intestinal phase. Alginates were shown to increase the activity of α-amylase during in vitro single enzyme analysis, but have no significant affect on carbohydrate digestion in a model gut simulation. Fat digestion in the model gut simulation was inhibited by specific alginates, adding further weight to the potential use of alginates as a therapeutic treatment of obesity.

Bioactive glasses are of great importance for orthopaedic and dental applications. The macroscopic properties of bioactive glasses, and in particular the rate of dissolution, can be tailored for specific applications by altering the compositions. It is therefore important to understand a compositional-structural-relationship as this will help when predicting glass properties and for optimising the design of future glasses for clinical applications. This thesis focused on the structural-characterisation of a wide range of bioactive glasses that are important for orthopaedic and dental applications. Experimental probes including neutron diffraction, high energy X-ray diffraction, solid-state NMR, supported by complimentary techniques such EXAFS, FTIR, XPS and XRD were used to characterise these glasses. A series of halide containing (CaF2 and CaCl2) bioactive glasses were successfully synthesised and characterised. Despite conflicting reports in the literature, the present study strongly suggests that minimal Si-Cl or Si-F bonds are present in these glasses. Instead halide ions were found to surround calcium ions. The silica network is therefore unaffected by the addition of halides ions. A series of novel zinc silicate-germanate glasses, with potential applications for bone cements, were investigated structurally. Studies revealed both Zn and Ge cations are in a tetrahedra coordination with no evidence of 5, 6-fold coordinations. An increase in the network connectivity of the glass will significant reduce its dissolution. The impact of incorporating Mg and Zn into bioglass was studied since both these ions can act as network intermediates. Whilst Zn was found to be solely tetrahedral Mg had significant fractions of 5 and 6-fold coordinations. The structure of Mg-BMG were investigated using neutron diffraction and molecular dynamics to help deconvolved overlapping partial structure factors. Isotopic neutron diffraction was further used to simplify the individual partial structure factors to further understand the structure of the glasses which is important to optimise its properties.

Modern tissue engineering (TE) scaffolds are expected to actively promote tissue repair as well as meeting the traditional requirements of non-toxicity, degradability and structural integrity. This thesis presents two novel bioactive hydrogel systems for bone and cartilage TE. A series of alginate hydrogels were developed in which all or a fraction of the calcium normally used for crosslinking alginate was replaced by bioactive strontium and/or zinc ions. Strontium was chosen for its ability to stimulate bone formation, while zinc is essential for alkaline phosphatase activity. Due to an interaction between the crosslinking ion and alginate type, the hydrogel properties could be tailored independently of the crosslinking ion used – meaning that varying biological and materials requirements can be accommodated. Strontium release from alginate gels was of a physiologically relevant magnitude, and alkaline phosphatase protein activity in Saos-2 cells was highest in strontium gels. Secondly, a biomimetic strategy for transforming growth factor beta (TGF-β) presentation and release was evaluated. TGF-β in vivo is secreted as part of an inactive latent complex, which is sequestered in a stable form within extracellular matrix until released by cells. TGF-β was therefore incorporated into poly(ethylene glycol)-hyaluronic acid hydrogels in its latent form. When compared to free TGF-β, advantages were demonstrated in terms of lower protein adsorption to tissue culture plastic and relative biological inactivity. The latter implies that high doses may be loaded into TE scaffolds without exposing cells to excessive quantities of active growth factor, with TGF-β bioavailability then being controlled by gradual activation by cells. Increased metabolic activity and ECM deposition by bovine chondrocytes were seen after almost five weeks in culture with a single initial loading of LTGF-β. These innovations correspond to current TE trends, which seek to use biomimetic principles to evoke regenerative responses from transplanted or host cells, but to do so using technically and commercially feasible means.

Melt-quenched glasses containing SiO2, CaO, Na2O and P2O5, and sol-gel derived glasses containing SiO2 and CaO are known to have bioactive properties. Foaming of binary sol-gel derived bioactive glasses containing SiO2 and CaO can be used to produce 3D porous scaffolds which mimic the structure of trabecular bone, increasing the potential for these glasses to be used as bioactive bone-regenerative materials. A range of experimental techniques have been used to investigate the atomic scale structure of these materials, and also to observe the reaction mechanisms which occur when these materials are immersed in a simulated physiological solution (simulated body fluid, SBF) and a standard cell culture medium (tris buffer solution, TBS). A robust structural model of the most bioactive of the melt-quenched glasses, namely Bioglass®, has been produced by combining high energy X-ray and neutron diffraction data, magic angle spinning nuclear magnetic resonance (MAS NMR) and reverse Monte Carlo (RMC) modelling. It has been shown that Ca clustering occurs in the glass, which is of direct relevance to the understanding of the facile nature of calcium within such glasses giving rise to its relatively rapid diffusion from the solid into solution. Hydroxyapatite has been confirmed as the calcium phosphate phase which grows on the surface of Bioglass® when immersed in the standard cell culture medium, TBS. A new method which can be used for in-situ time resolved high-energy X-ray diffraction studies of reaction mechanisms, such as those involved when a bioactive glass is immersed in a simulated physiological solution, is decribed in this thesis. Small-angle X-ray scattering has enabled the growth of mesopores to be observed during the foamed sol-gel stabilisation process. In-situ simultaneous small and wide angle X-ray scattering measurements of a foam in SBF have shown that the mesoporous network facilitates the rapid growth of relatively high-density HCA, which will therefore eventually replace the initial silicate glass as the material bounding the macropores. The data presented herein reveal the structure of highly important materials in the field of biomaterials and enable a link to be made between the atomic scale structure of the materials and their bioactive properties.

La melatonina i l’hidroxitirosol són molècules bioactives importants en diversos processos biològics en mamífers, principalment pel seu rol antioxidant. Són sintetitzades pels llevats durant la fermentació alcohòlica i la melatonina té efectes protectors contra l’estrès oxidatiu i la radiació UV en llevats. L’objectiu d’aquesta tesi ha estat determinar si la melatonina i l’hidroxitirosol protegeixen les cèl·lules de Saccharomyces cerevisiae contra estressos relacionats amb la fermentació. Per aprofundir en els mecanismes antioxidants de la melatonina, hem analitzat el seu efecte a nivell transcripcional. La melatonina exògena creua membranes cel·lulars i modula l’expressió de molts gens: en cèl·lules no estressades, activa una resposta semblant a l’hipòxia, mentre que en cèl·lules estressades provoca una resposta antioxidant. El component cel·lular amb més gens afectats és el mitocondri, per tant hem avaluat l’efecte de la melatonina en el funcionament d’aquest orgànul. Encara que l’exposició a estrès oxidatiu indueix la disfunció mitocondrial (reduint el consum d’oxigen, el potencial de membrana i les activitats de complexes de la cadena de transport d’electrons i promovent la fissió mitocondrial), la melatonina no afecta la funció mitocondrial.
La melatonina y el hidroxitirosol son moléculas bioactivas importantes en diferentes procesos biológicos en mamíferos, principalmente por su rol antioxidante. Son sintetizadas por las levaduras durante la fermentación alcohólica, y la melatonina protege contra el estrés oxidativo y la radiación UV en levaduras. El objetivo de esta tesis ha sido determinar si estos compuestos bioactivos protegen a Saccharomyces cerevisiae contra estreses relacionados con la fermentación. Para profundizar en los mecanismos antioxidantes de la melatonina analizamos su efecto a nivel transcripcional. La melatonina exógena cruza membranas celulares y modula la expresión de muchos genes: en células no estresadas, activa una respuesta parecida a la hipoxia y en células estresadas, provoca una respuesta antioxidante
Melatonin and hydroxytyrosol are bioactive molecules that play important roles in various biological processes in mammals, mainly relying on their antioxidant properties. Those molecules are synthesized by yeast during alcoholic fermentation, and melatonin has protective effect against oxidative and UV stresses in yeast cells. The aim of the present thesis was to determine if melatonin and hydroxytyrosol play a protective role against fermentation-related stresses in Saccharomyces cerevisiae cells. First, to deepen into the antioxidant mechanisms of melatonin, we analyzed its effect at the transcriptional level. Exogenous melatonin crossed cell membranes and modulated the expression of many genes: in nonstressed cells, it triggered a hypoxic-like response, while in stressed cells, it provoked an antioxidant response. As the compartment with more genes affected was the mitochondrion, we evaluated the effect of melatonin on mitochondria functionality.

In spite of their widespread application, metallic orthopaedic prosthesis failure still occur due to lack of sufficient bone-bonding and the incidence of post-surgery microbial infections. The goal of this research was to develop multifunctional composite polymer/bioactive glass coatings as a potential strategy to improve surface properties of metallic implants. Using this approach, the bioactive glass improves osseointegration and the polymer plays a dual role: firstly to improve mechanical properties and secondly as a carrier for the release of therapeutics at the implantation site. Electrophoretic deposition (EPD) was chosen as the fabrication method, as it is a room temperature technique in which deposition properties can be effectively tuned and complex architectures can be coated homogenously. 45S5 Bioglass® (BG) powder (~10 μm) and chitosan (CS); a natural polysaccharaide (85% deacetylated); were utilised for coating AISI 316L stainless steel substrate. In aqueous EPD of CS, the electrophoretic mobility and deposition rate were shown to increase with increasing pH from 2.9 to 4.1. Aqueous EPD from BG and composite CS/BG suspensions were optimised by the Taguchi design of experiments approach. For BG suspensions, the pH and the electric field had the most and the least effects on deposition rate, respectively, and a high deposition rate of BG was attained at pH=7. For CS/BG suspensions, co-deposition was very sensitive to the concentration of BG due to its effect on suspension pH, conductivity and particles mobility. Composites with smoother surface morphology and more uniform distribution of BG particles in the CS matrix were obtained at lower glass concentrations. Structural and physical evaluations of CS and different CS/BG coatings showed that they were amorphous and also confirmed the formation of hydrogen-bonding between BG and CS in the EPD suspension. Dissolution profiles and bioactivity study of the coatings confirmed hydroxycarbonate apatite (HCA) formation in simulated body fluid (SBF) for composites in the higher range of BG loading. The bioactivity response of coatings was elucidated considering the EPD suspension preparation step and the role of surface charge on HCA formation. Tape-testing showed improvement of coating adhesion with addition of BG (for up to~ 60 wt% BG) and Vickers micro-hardness testing revealed that the composite hardness increased with the amount of BG in the films. Two types of antibacterial agents were incorporated in the composites via EPD: nano-particulate silver (Ag-np) and gentamicin (GS) antibiotic. For the first time, single step processing, in-situ formation and incorporation of Ag-np during EPD of chitosan-based composite films was shown. The release of Ag ion and GS in SBF was measured showing an initial burst release followed by a reduced release rate. Although about 40% of GS was released in 5 days, less than 7% of the loaded silver was released within 28 days. Disk diffusion tests demonstrated inhibition of S. aureus growth up to 10 and 2 days for Ag-np and GS samples, respectively. A preliminary 7 day culture study of MG-63 osteoblast like-cells on coatings indicated cellular attachment and proliferation for all coatings, except for Ag-np-containing films. The high amount of silver release was identified as the reason for cytotoxicity. Overall, the results presented in this thesis demonstrate the potential of EPD as a suitable fabrication technology for preparing multifunctional CS/BG composite bioactive coatings for orthopaedic applications.

Thesis (Ph. D.)--University of California, San Diego, 2006.
Title from first page of PDF file (viewed Mar. 15, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

The colonization of immersed surfaces by a myriad of marine organisms is a complex, multi-stage, species-specific process giving rise to economic and environmental costs. This unwanted accumulation of organisms in the marine environment, called biofouling, has been attacked from different fronts, going from the ‘problem-elimination-as-problem-solving’ strategy (essentially through the use of biocides) to more elaborated and environmentally-friendly options based on the principle of ‘non-stick’ or ‘easy foul-release’ surfaces, which do not jeopardize marine life viability. Several marine organisms rely on proteinaceous adhesives to secure a holdfast to surfaces. Proteolytic enzymes have been demonstrated to be effective agents against settlement and settlement consolidation onto surfaces of marine bacteria, algae, and invertebrates, their proposed mode-of-action being the enzymatic degradation of the proteinaceous components of the adhesives. So far, however, the evidence remains inconclusive since most of the published investigations refer to commercial preparations where the enzyme is mixed with other components, like additives, which obviously act as additional experimental variables. This work aims at providing clear, conclusive evidence about the potential of serine proteases to target the adhesives produced by a group of model marine biofoulers. The strategy towards the goal consisted in the preparation and characterization of maleic anhydride copolymer nanocoatings modified by a surface-bound enzyme, Subtilisin A, the active constituent of the commercial preparations reported as effective against biofouling. The enzyme-containing maleic anhydride copolymer films were characterized (enzyme surface concentration, activity, stability, roughness and wettability) and thereafter tested in biological assays with three major biofoulers: spores of the green alga Ulva linza, cells of the pennate diatom Navicula perminuta, and cyprid larvae of the barnacle Balanus amphitrite. The purpose of the biological assays was to elucidate the efficacy of the immobilized catalyst to discourage settlement and/or to facilitate removal of these organisms from the bioactive layers. Results confirmed the initial hypotheses related to the enzymatic degradation of the biological adhesives: the immobilized protease was effective at reducing the adhesion strength of Ulva spores and Navicula diatoms in a manner that correlated with the enzyme activity and surface concentration, and deterred settlement of Balanus amphitrite barnacle cyprids even at the lowest surface activity tested. By facilitating the removal of biofilm-forming diatoms and of spores of the troublesome alga Ulva linza, as well as by interfering with the consolidation of adhesion of the calcareous Balanus amphitrite macrofouler, the enzyme-containing coatings here disclosed are considered to constitute an appealing and promising alternative to control marine biofouling without jeopardizing marine life.

This thesis is divided into two chapters detailing research on applying microwave methodology to access aminated nucleosides in significantly reduced time frames, and applying the Belluš-Claisen reaction to produce non-proteogenic dipeptides. 1. Amination of Nucleosides Using Microwave Methodology 2,2’-Anhydrouridine undergoes a ring opening reaction with aliphatic amines to give the corresponding aminated product. Under conventional heating reaction times are extremely lengthy, taking at least 3 to 4 days and up to a month in the case of very hindered amines. A modified procedure using microwave irradiation has proven to drastically reduce reaction time and has allowed access to novel nucleosides on gram scale. 2. Functionalised Amino Acids via the Belluš-Claisen Rearrangement The Belluš-Claisen reaction is a [3,3] sigmatropic rearrangement of allylic amines, ethers and thioethers to give the corresponding amide, ester of thioester. A modified procedure of the Belluš-Claisen rearrangement was used to prepare functionalised dipeptides by reaction of a ketene prepared from N-phthaloylglycyl chloride in situ with allylic amino acid derivatives in the presence of a Lewis Acid and diisopropylethylamine. Rearrangments were successfully carried out using N,N-diallyl alanine and N-allyl proline. A range of N-allyl proline derivatives are demonstrated. However, attempts to repeat the reaction with structurally more complex amino acids did not result in successful reactions.

In this thesis four projects related to bioactive carbohydrates are described. The first project is about the extraction of iminosugars from Hyacinthoides non-scripta. This is the first time that extraction from English bluebell seeds has been described. Efficient extraction and isolation methods are reported. Another project discusses the development of a total synthetic carbohydrate conjugate vaccine candidate against Streptococcus pneumoniae type 14 using Gold nanoparticles as carrier. The synthetic pathway of the introduction of a linker for conjugation, and the deprotection of the tetrasaccharide corresponding to the repeating unit of the Streptococcus pneumoniae type 14 capsular polysaccharide is described. The biological results of the developed vaccine candidate are briefly discussed. In the third project, attempts to synthesise regioselectively sulfated disaccharides to be used in binding studies with FedF adhesin of E. coli are described. In this section, an improved high-yielding method based on the Heyns rearrangement for the synthesis of N-acetyl lactosamine (LacNAc) is also reported. In the fourth part, conjugation of the Lewis b hexasaccharide to be used for studies of Lewis b blood group antigen binding adhesin is reported.

Influenza virus infection and the shikimic acid pathway are two of many examples of microbe-host interactions and microbial biosynthetic pathways that are interesting for investigation by means of small molecules. A particularly interesting structural motif common to both is the cyclohexenecarboxylic acid. In the former, this structural motif has been employed as a mimetic of the sialyl cation intermediate and forms the scaffold of the anti-influenza drug and neuraminidase inhibitor Oseltamivir (or TamifluTM). In the latter pathway, crucial modifications towards aromatic amino acids are carried out via shikimic acid, a cyclohexenecarboxylic acid, as a substrate. A straightforward method to replace the carboxylate moiety in such structures with a phosphonate would provide access to a wide variety of mimetics, for instance monoesters, that still retain a negative charge under physiological conditions usually required for bioactivity. The aim of this research project was to develop an efficient synthesis of the cyclohexenephosphonate scaffold from chiral pool precursors via two key steps, a Hunsdiecker-Barton iododecarboxylation followed by a palladiummediated coupling step to introduce the phosphonate moiety, thus giving a convenient access to interesting bioactive molecules. This approach has successfully been applied to the shikimic acid to afford ‘phospha'-shikimic acids and 3-dehydro-‘phospha'-shikimic acids, and further development of this strategy has led to the synthesis of ‘phospha'-Tamiflu and its derivatives from an Oseltamivir precursor.

Hydrogels are of great interest due to their ability to encapsulate and deliver bioactive molecules, mimic the extracellular matrix (ECM) and act as an artificial 3D scaffold. Here we report multi-component hydrogels based on low-molecular-weight gelators (LMWGs) and polymer gelators (PGs) incorporating heparin that can bind to self-assembled molecules, and their potential for controlled release and to mimic the ECM. These multi- component systems were characterised and the orthogonality of each individual component investigated. Firstly, three cationic surfactants were synthesised and their ability to self-assemble and bind to polyanionic heparin was investigated. The systems consisted of an amine-based head group connected via an amide linkage to different saturated fatty acids. Self- assembled C14-DAPMA and C16-DAPMA formed highly organised polycrystalline assemblies with heparin, proving that the micelles remain intact during the hierarchical assembly process. C16-DAPMA proved to be the most charge-efficient heparin binder, also with the lowest critical aggregation concentration, with high stability when free and solution and when electrostatically interacting with heparin. Two dibenzylidene-D-sorbitol (DBS) derivatives capable of forming hydrogels are then introduced: a pH-activated LMWG (DBS-COOH) and a thermally-activated LWMG (DBS- CONHNH2). The incorporation and release of heparin from the LMWGs hydrogels in the presence and absence of C16-DAPMA, and from hybrid hydrogels consisting of one of the LMWGs and a PG - agarose is reported. The rate of heparin release can be controlled through network density and composition, and control of the release surface area to volume ratio, while the presence of C16-DAPMA inhibits heparin release. Characterisation of this multi-component complexes (LMWG + Heparin + C16-DAPMA) showed the orthogonal self-assembly of each individual component within one single system. Cytocompatilibity of the multi-component hydrogels is reported. Heparin was then incorporated and released from three different hydrogels based on triamide cyclohexane derivatives. From these, a positively charged LMWG able to directly interact with heparin, resulted in the triggered release of heparin by hydrogel disruption through enzymatic cleavage.

In this thesis we consider several approaches to modelling interactions between fluid flow and cell proliferation in a bioactive porous medium. This is motivated by models of cell growth within tissue engineering scaffolds placed in perfusion bioreactors. These scaffolds are porous materials used to facilitate nutrient transport to cells placed within them. Recent modelling efforts have sought to understand the influence that cells have on the effective permeability of these tissue scaffolds, and hence on the capacity of the porous medium to facilitate fluid and nutrient transport, which enhances the overall growth of tissue within a scaffold. Contemporary experimental and theoretical studies have emphasized the importance of mechanical forcing on cells due to their environment. We therefore consider simple models of cell growth that assume cells are affected by the fluid itself, either via shear stress, hydrostatic pressure, or local flow rate, and that cell growth influences the local permeability of the scaffold at higher cell densities via pore-blocking. Hence, we are interested in the feedback between cell growth and fluid flow within a bioactive porous medium. It is within this simplified context of fluid-growth interaction that we explore different approaches to modelling the spatial structure of the pore network, and the influence that this has on the cell density distribution throughout the scaffold. Models in the literature are often spatially homogeneous (ODE) or spatially continuous (PDE), only implicitly accounting for the discrete pore network of the medium. The pore and scaffold length scales in typical experiments with perfusion bioreactors can lead to scaffolds with relatively few pores, and it is unclear that macroscopic spatially averaged (homogenized) continuous models will capture features present in small pore networks. We propose a suite of spatially continuous models, modified from existing PDE approaches in the literature, and compare these to discrete lattice (ODE) systems in order to elucidate differences between these modelling paradigms. We also use the structure of the lattice model to explore stochastic analogues that are computationally and theoretically amenable to analysis. We explore behaviours of these models via numerical simulations, bifurcation analyses, and asymptotic reductions to simpler systems. Our lattice modelling approach provides a 'mesoscopic' perspective, where the effective equations governing the cell growth and fluid flow are prescribed as ODEs at each pore. These capture the microscale dynamics at the pore scale, as opposed to homogenization approaches where the microscale is explicitly related to macroscale equations. This also allows us to use tools from dynamical systems theory which are substantially more tractable in the finite dimensional setting of ODEs (with algebraic conditions for the fluid flow), as opposed to the infinite dimensional setting of PDEs (consisting of coupled elliptic and parabolic equations). Additionally, we do not have to worry about issues of numerical convergence or existence of solutions that are important in the spatially continuous setting. Our emphasis on mesoscopic governing equations makes this network-based approach somewhat unique in the tissue engineering community. We demonstrate qualitative and quantitative differences between these continuum and network paradigms, and in particular show behaviours captured by explicitly accounting for the discrete pore network that are not captured in spatially continuous models. For each kind of fluid-cell interaction, we classify behaviours depending on nondimensional model parameters in order to elucidate in what regimes discrete models may provide useful insights into bioactive porous materials. We also discuss computational considerations for analyzing these kinds of models, and in particular suggest that stochastic and discrete models may be easier to simulate in some parameter regimes compared to typical spatially continuous models. Our results suggest several novel approaches to pursue in accounting for the finite discrete nature of bioactive porous media, and we highlight several useful further directions.

There is great therapeutic potential for bioactive glass nanoparticles, as they can be internalised within cells and achieve sustained intracellular delivery of active ions. Bioactive glass nanoparticles are also used in nano-composites. The Stӧber process is commonly used for synthesising spherical silica particles. This thesis reports a comprehensive study of how the process variables can be systematically altered to obtain monodispersed particles of specific sizes in the range 20 – 500 nm. Modification of the Stӧber process so that the particles can contain cations such as calcium, which can impart bioactivity, while maintaining monodispersity, is therefore highly desirable. Here, both zinc and calcium incorporation is achieved, with a homogenous distribution and without affecting the size, dispersity or morphology of the particles. However, careful characterisation shows that much of the cations were not incorporated and a washing step was essential, which suggests that previous reports are likely to have overestimated the amount of calcium incorporated. A maximum of 8.9 ± 1.1 mol % CaO and 22.7 ± 0.8 mol % ZnO was incorporated into the particles. Monodispersed mesoporous silica particles were also synthesised in this thesis, containing up to 18.5 ± 2.7 mol % Ca and 21.5 ± 1.3 mol % Zn. The differences in incorporation between the two cations were attributed to the different roles that they play within the silicate network. Whilst calcium acts purely as a network modifier, zinc can also act as a network former. These differences are seen to affect % cation incorporation, surface area, network connectivity and subsequent degradation. Nanoparticles of 80 - 100 nm were readily internalised by cells, where they were located both within vesicles and also as individual particles within the cytoplasm (having escaped from the endosomal pathway). SiO2-ZnO mesoporous particles were also seen to subsequently degrade rapidly inside cells, demonstrating preferential toxicity to cancer cells due to pH sensitive intracellular release of toxic Zn2+ ions. SiO2-CaO particles were successfully functionalised and show great potential for incorporation into nano-composites for bone regeneration.

Natural products have been a fundamental source of medicinal scaffolds for decades; with sixty percent of marketed drugs. Many synthetic chemists are focused on synthesizing potent and nontoxic compounds for pharmaceutical targets, however, nature is still proving to be a source of new bioactive compounds. Produced by the host organism for defense, reproduction and communication, secondary metabolites also demonstrate promising bioactivity against human pathogens. Hence, natural product chemists continue their quest for new leads. As a continuation of these efforts, this thesis attempts to explore fungi and sponges for new chemistry, and ultimately, new drug candidates. Antarctica is largely untapped; hence herein two Antarctic sponges were chemically investigated. This resulted in isolation and characterization of two metabolites. Concurrently, chemical investigation of fungus, from Floridian mangrove species, resulted in the isolation of two structurally diverse metabolites. Further, a dereplication process was applied to MPLC fractions, which lead to the identification of known metabolites and mycotoxins. This enabled prioritization of fractions for future studies.

Protein-Protein Interactions (PPIs) play a very important role in biological functions and therefore the inhibition of specific Protein-Protein Interactions has a huge therapeutic value. The most successful small molecular PPIs inhibitors do not fit with the prevalent `Rule of Five' drug profile. To overcome the disadvantages of small molecular PPIs inhibitors, peptide based PPIs inhibitors were developed. Herein we describe the development of a new class of peptidomimetics AA-peptides. The AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides were discussed in this dissertation "α-AApeptides" and "γ-AApeptides". The AApeptides were shown to disrupt p53/MDM2 protein-protein interaction and tomimic fMLF tripeptide to target G protein-coupled formyl peptide receptors (FPRs). Moreover, the lipidated α-AApeptides can mimic the structure and function of natural antimicrobial lipopeptides and show broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Lastly I have designed and synthesized a serials of phosphopeptides to disrupt cancer related STAT3-STAT3 dimerization.

The problem of the development of new pesticides is very urgent, mainly because of the appearance of pest forms resistant to permitted pesticides and for the strict requirements in terms of their safety for people and the environment. Small molecules produced in biological contexts have been, and still are, a large reservoir of new biologically active substances, which can become scaffolds for the discovery of new agrochemicals. The aim of this PhD work was to synthesize naturally occurring potentially antifungal and herbicidal compounds and to test their biological activity. Efforts mainly focused towards the total synthesis of Bulgarein, a fungal metabolite produced by the fungus Bulgaria inquinans. Bulgarein possesses a benzo[j]fluoranthene skeleton, that can be found in a number of polyketide-derived fungal metabolites endowed with significant biological activity, in particular inhibition of Topoisomerase I. As no attempt to synthesize any of these compounds has been reported in literature so far, a synthetic sequence to the benzo[j]fluoranthene nucleus has been developed. Crucial steps for our strategy include a Suzuki coupling followed by a McMurry ring closure. The approach is modular and rapid and can be utilized to synthesize natural products, biologically active analogues or building blocks for the preparation of materials. Following this strategy, the first total synthesis of the recently isolated natural product benzo[j]fluoranthene-4,9-diol was carried out. Efforts were made to adapt the sequence for the synthesis of Bulgarein and other variously substituted compounds with this skeleton. As a second topic in this research, attention was dedicated to another natural compound, Farinomalein A, a structurally rather simple maleimide isolated in 2009 from the entomopathogenic fungus Paecilomyces farinosus. Farinomalein shows potent inhibition of Phytophthora sojae, a plant pathogen that every year causes enormous damage to soybean crops. Recently, three new farinomalein derivatives (Farinomalein C, D & E) were isolated from an endophyte from the mangrove plant Avicennia marina, growing in Oman. Due to the interesting antifungal activity of this class of compounds, a practical synthesis of farinomalein A was developed, which may have value in the large-scale preparation of the natural compound. Starting from farinomalein A, all the three derivatives were successfully synthesized . The antifungal activity of the derivatives was evaluated against Cladosporium cladosporioides and other pathogenic fungi. An approach to the synthesis of Ascaulitoxin, a phytotoxic metabolite produced by the fungus Ascochyta caulina, was also developed.

L'objectiu principal de la present tesi doctoral va ser la caracterització de bolets cultivats a la Rioja, especialment pel que fa al seu contingut de compostos bioactius, i explorar l'efecte que podria tenir l'aplicació de Polsos Elèctrics de Moderada Intensitat de Camp (PEMIC) sobre aquests continguts aplicats tant en els substrats de cultiu com en els cossos fructífers. Per això, es va realitzar una caracterització inicial dels bolets cultivats a les instal·lacions del Centro Tecnolígico del Champiñón (CTICH) mitjançant anàlisi de composició centesimal i energia, així com del contingut en fibra dietètica, β-glucans, quitosan i l'activitat antioxidant dels bolets seleccionats. Com que els PEMIC són una tecnologia no tèrmica que permeabilitza la paret cel·lular vegetal i fúngica, això podria influir en la productivitat i facilitar un augment de concentració de compostos bioactius en els bolets. Per això, es varen aplicar PEMIC sobre el substrat utilitzat per al cultiu de bolets comestibles i sobre els cossos fructífers un cop recol·lectats, corresponents als bolets més comercialitzats i de major interès per al sector. L'estudi es va realitzar en quatre fases en què es va demostrar la qualitat nutricional dels bolets, mostrant un alt valor en proteïnes i baix en greixos, i la potencialitat dels PEMIC per augmentar la concentració de nutrients, fibra dietètica, i β-glucans, així com la seva activitat antioxidant. Els valors obtinguts es varen comparar amb substrats o cossos fructífers manejats convencionalment sense aquest tractament. Els resultats obtinguts després de l'aplicació de PEMIC són molt prometedors ja que va permetre un augment de compostos bioactius, especialment fibra dietètica i activitat antioxidant, quan es va aplicar sobre els cossos fructífers de xampinyó, Ganoderma lucidum, Lentinula edodes, Pleurotus eryngii i Pleurotus ostreatus. En aplicar els PEMIC sobre el substrat de Pleurotus eryngii es va obtenir un major rendiment (30%) del cultiu però no es varen apreciar millores en la composició centesimal ni en els compostos bioactius. No obstant això, en els paquets de Lentinula edodes, encara que els PEMIC no varen produir un augment en la producció, varen aportar als bolets una major quantitat de proteïnes, fibra i β-glucans (15,7%), i es va disminuir el contingut en greixos (14,8%). A més, l'activitat antioxidant es va veure afavorida mostrant una positiva i alta correlació afirmant que la presència de Fenols Totals influeix en l'activitat antioxidant que es va demostrar tant en la captació de radicals lliures com en la transferència d'electrons. El tractament de 2 kV/cm i 30 polsos va resultar ser excessiu en tots els paràmetres estudiats en produir un estrès a la cèl·lula danyant-la a causa d'una permeabilització irreversible pels PEMIC i no es varen trobar diferències entre aplicar 2 o 5 polsos.
El objetivo principal de la presente Tesis Doctoral fue la caracterización de setas cultivadas en La Rioja especialmente en lo relativo a su contenido de compuestos bioactivos y explorar el efecto que podría tener la aplicación de Pulsos Eléctricos de Moderada Intensidad de Campo (PEMIC) sobre dichos contenidos tanto aplicados en los sustratos de cultivo como en los cuerpos fructíferos. Para ello, se realizó una caracterización inicial de las setas cultivadas en las instalaciones del Centro Tecnológico del Champiñón en La Rioja (CTICH) mediante análisis de composición centesimal y energía, así como del contenido en fibra dietética, β-glucanos, quitosano y la actividad antioxidante de las setas seleccionadas. Como los PEMIC son una tecnología no térmica que permeabiliza la pared celular vegetal y fúngica, ello podría influir en la productividad y facilitar un aumento de concentración de compuestos bioactivos en las setas. Por ello, se aplicaron PEMIC sobre el sustrato utilizado para el cultivo de setas comestibles y sobre los cuerpos fructíferos una vez recolectados, correspondientes a las setas más comercializadas y de mayor interés para el sector. El estudio se realizó en 4 fases en las que se demostró la calidad nutricional de las setas, mostrando un alto valor en proteínas y bajo en grasas, y la potencialidad de los PEMIC para aumentar la concentración de nutrientes, fibra dietética, y β-glucanos, así como su actividad antioxidante. Los valores obtenidos se compararon con sustratos o cuerpos fructíferos manejados convencionalmente sin ese tratamiento. Los resultados obtenidos tras la aplicación de PEMIC son muy prometedores ya que permitió un aumento de compuestos bioactivos, especialmente fibra dietética y actividad antioxidante, cuando se aplicó sobre los cuerpos fructíferos de Agaricus bisporus, Ganoderma lucidum, Lentinula edodes, Pleurotus eryngii y Pleurotus ostreatus. Al aplicar los PEMIC sobre el sustrato de Pleurotus eryngii se obtuvo un mayor rendimiento (30%) del cultivo pero no se apreciaron mejoras en la composición centesimal ni en los compuestos bioactivos. Sin embargo, en los paquetes de Lentinula edodes, aunque los PEMIC no produjeron un aumento en la producción, aportaron a las setas una mayor cantidad de proteínas, fibra y β-glucanos (15,7%), y se disminuyó el contenido en grasas (14,8%). Además la actividad antioxidante se vio favorecida mostrando una positiva y alta correlación afirmando que la presencia de Fenoles Totales influye en la actividad antioxidante que se demostró tanto en la captación de radicales libres como en la transferencia de electrones. El tratamiento 2kV/cm y 30 pulsos resultó ser excesivo en todos los parámetros estudiados al producirse un estrés en la célula dañándola debido a una permeabilización irreversible por los PEMIC y no se encontraron diferencias entre aplicar 2 o 5 pulsos. Estos primeros estudios deben ser ampliados y los mecanismos implicados estudiados en profundidad de cara a poder implementar industrialmente esta tecnología en las setas de forma que pueda aumentarse tanto su potencial saludable como los rendimientos de su cultivo.
The main objective of this Doctoral Thesis was the characterization of mushrooms cultivated in La Rioja, especially in relation to their bioactive compounds content, and the exploration of the effect that applying Moderate Intensity Pulse Electric Field (MIPEF) to these bioactive compounds might have, both applied to the growing substrates as well as to the fruiting bodies. For this purpose, an initial characterization of the cultivated mushrooms was made in the facilities of the Mushroom Technological Center in La Rioja (CTICH) by analysing centesimal composition and energy, as well as the dietary fiber, β-glucans and chitosan content, and also the antioxidant activity of the selected mushrooms. As MIPEF is a non-thermal technology that permeabilizes the vegetal and fungal cell wall, it could have an impact on the productivity and facilitate an increase in the bioactive compounds concentration in mushrooms. Therefore, MIPEF treatment was applied on the substrate used for the cultivation of edible mushrooms and on the fruiting bodies once collected, being those fruiting bodies the most commercialized mushrooms and those the mushroom’s sector has a major interest in. The study was carried out in four phases, demonstrating the nutritional quality of the mushrooms, showing a high protein and low fat value, and the potential of MIPEF to increase the nutrients concentration, dietary fiber, and β-glucans, as well as its antioxidant activity. The values obtained were compared with conventionally handled substrates or fruiting bodies without any applied treatment. The results obtained after application of MIPEF are very promising since it allowed an increase in bioactive compounds, especially dietary fiber and antioxidant activity, when applied on the fruiting bodies of Agaricus bisporus, Ganoderma lucidum, Lentinula edodes, Pleurotus eryngii and Pleurotus ostreatus. When the MIPEF treatment was applied to the substrate of Pleurotus eryngii, an increase in crop yield (30 %) was obtained but no improvements were observed neither in the centesimal composition nor in the bioactive compounds. However, in the Lentinula edodes bags, although the MIPEF did not produce an increase in production, the treatment contributed to increasing the protein, fiber and β-glucans contents in the mushrooms (15,7%), and the fat content was reduced (14,8%). In addition, the antioxidant activity was favored by showing a positive and high correlation: the presence of Total Phenols has an impact on the antioxidant activity. This was demonstrated both in free radical uptake and in electron transfer. The treatment 2kV/cm and 30 pulses proved to be excessive in all the parameters studied. Stress occurred in the cell and the cell was damaged due to an irreversible permeability by the PEMIC. No differences were found between applying 2 or 5 pulses. These first studies should be expanded and the mechanisms involved studied in depth in order to be able to industrially implement this technology in the mushrooms in a way that can increase both their healthy potential and the yields of their cultivation.

Part I: In the first study of pyrethroids, twenty-one novel pyrethroid esters bearing strong electron-withdrawing groups (e.g., halomethylketo and nitro groups) in the double bond side chain of the cyclopropane acid moiety have been synthesized and evaluated for insect toxicity. Rather than the usually employed Wittig reaction for these syntheses, the novel pyrethroid acid moieties were prepared by amino acidcatalyzed Knoevenagel condensations under mild conditions. In the second study of pyrethroids, fourteen pyrethroid-like carbonates were synthesized by condensation of a variety of alcohols and the chloroformates of the corresponding known pyrethroid alcohols.

Esta Tesis Doctoral se presenta como Compendio de publicaciones. Los capítulos en los que se ha dividido la presente Tesis son los siguientes: Capítulo 1 – parte A: Enantioselective, Protecting Group-Free Synthesis of 1S-Ethyl-4-Substituted Quinolizidines Se ha descrito una síntesis enantioselectiva que no implica grupos protectores para acceder a la quinolizidina clave 6 a partir de la lactama bicíclica derivada de fenilglicinol 1. La adición de un reactivo organometálico a 6 ocurre de manera estereoselectiva para conducir a las quinolizidinas 1S-etil-4-sustituidas 4-epi-207I y 7-9. Siguiendo una secuencia sintética análoga, se preparó el compuesto 9a-epi-6. Sin embargo, la adición de reactivos de Grignard al compuesto 9a-epi-6 no ocurre de manera estereoselectiva. Capítulo 1 – parte B: A practical procedure for the removal of the phenylethanol moiety from phenylglycinol-derived lactams Las lactamas bicíclicas no racémicas derivadas de fenilglicinol se han relevado como intermedios clave en la preparación de compuestos nitrogenados enantiopuros. En este capítulo se describe la eliminación del inductor quiral de piperidonas sustituidas utilizando aire u oxígeno en medio básico. Capítulo 2: Synthesis of triheptanoin and formulation as a solid diet for rodents En el presente estudio se describe la síntesis eficaz de triheptanoina de elevada pureza a partir de glicerol y ácido heptanoico, en presencia de carbono sulfonado como catalizador. La triheptanoina se formula como un sólido estable para que constituya la base de una dieta cetogénica mediante la combinación de custro gentes de formulación comerciales; dos tipos de sílica, celulosa microcristalina y talco. La adecuación de la dieta se prueba en ratones C57BI/6 en un periodo de 15 días, comparando el estado general y el cambio de peso corporal. Capítulo 3: Toluene dioxygenase (TDO) mediated oxidation of halogen-substituted benzoate esters Una serie de esteres benzoicos metílicos sustituidos en o-, m- y p- se han sometido a hidroxilación enzimática via fermetación con E. coli JM109 8pDTG601A). Solo se metabolizaron los benzoatos sustituidos en orto. El 2-fluorobenzoato de metilo produjo regioselectivamente solo un diol mientras que el 2-cloro, 2-bromo y 2-iodobenzoato de metilo proporciona una mezcla de regioisómeros. Capítulo 4: Dauben–Michno oxidative transposition of allylic cyanohydrins Enantiomeric switch of (–)-carvone to (+)-carvone Cuando cianohidrinas alílicas se someten a la reacción de oxidación de Dauben-Michno a bajas temperaturas se accede a β-cianoenonas en buenos a excelentes rendimientos. El potencial de esta trasposición oxidativa se pone de manifiesto en enonas que contienen un plano latente de simetría como por ejemplo la conversión de la (-)-carvona en su enantiomero.
A dissertation submitted by Vladislav SEMAK to obtain a doctoral degree from University of Barcelona. This thesis was developed under the supervision of Dr. Carmen Escolano Mirón from Faculty of Pharmacy, University of Barcelona. This doctoral thesis is presented as a compendium of publications. The thesis is divided in four chapters: CHAPTER 1 – PART A: Enantioselective, Protecting Group-Free Synthesis of 1S-Ethyl-4-Substituted Quinolizidines (Amat, M.; Semak, V.; Escolano, C.; Molins, E.; Bosch, J. Org. Biomol. Chem. 2012, 10, 6866-6875.) A practical enantioselective protecting group-free four-step route to the key quinolizidinone 6 from phenylglycinol-derived bicyclic lactam 1 is reported. The organometallic addition reaction upon 6 takes place stereoselectively to give 1-ethyl-4-substituted quinolizidines 4-epi-207I and 7-9. Following a similar synthetic sequence, 9a-epi-6 is also accessed. However, the addition of Grignard reagents upon 9a-epi-6 proceeds in a non-stereoselective manner. In order to gain insight into the different stereochemical outcome in the two series, theoretical calculations on the iminium salts A and B have been performed. The study concludes that the addition of the hydride, which is the step that determines the configuration of the final products, occurs in a stereoelectronic controlled manner. CHAPTER 1 – PART B: A practical procedure for the removal of the phenylethanol moiety from phenylglycinol-derived lactams (V. Semak; C, Escolano; C. Arróniz; J. Bosch; M. Amat Tetrahedron: Asymmetry 2010, 21, 2542-2549.) Chiral non-racemic bicyclic lactams derived from phenylglycinol have been appointed as key building blocks for the preparation of enantiopure nitrogen compounds. The removal of the chiral inductor leading to substituted piperidones by using air or oxygen in basic media is presented in this chapter. CHAPTER 2: Synthesis of triheptanoin and formulation as a solid diet for rodents (Semak, V.; Semakova, J.; Halbaut, L.; Asó, E.; Ferrer, I.; Calpena, A.; Escolano, C.; Perales, J. C. Eur. J. Lipid Sci. Technol. 2012, 114, 889-895.) In the present study, we successfully synthesized triheptanoin to the highest standards of purity from glycerol and heptanoic acid, using sulfonated charcoal as a catalyst. Triheptanoin oil was then formulated as a solid, stable and palatable preparation using a ketogenic base and a combination of four commercially available formulation agents: hydrophilic fumed silica, hydrophobic fumed silica, microcrystalline cellulose, and talc. Diet compliance and safety was tested on C57Bl/6 mice over a 15-week period, comparing overall status and body weight change. CHAPTER 3: Toluene dioxygenase mediated oxidation of halogen-substituted benzoate esters (Semak, V.; Metcalf, T. A.; Endoma-Arias, M. A. A.; Mach, P.; Hudlicky, T. Org. Biomol. Chem. 2012, 10, 4407-4416.) A series of ortho-, meta-, and para-halogen-substituted methyl benzoate esters was subjected to enzymatic dihydroxylation via the whole-cell fermentation with E. coli JM109 (pDTG601A). Only ortho-substituted benzoates were metabolized. Methyl 2-fluorobenzoate yielded one diol regioselectively whereas methyl 2-chloro-, methyl 2-bromo- and methyl 2-iodobenzoates each yielded a mixture of regioisomers. Absolute stereochemistry was determined for all new metabolites. Computational analysis of these results and a possible rationale for the regioselectivity of the enzymatic dihydroxylation is advanced. CHAPTER 4: Dauben–Michno oxidative transposition of allylic cyanohydrins. Enantiomeric switch of (–)-carvone to (+)-carvone. (Hudlicky, J. R.; Werner, L.; Semak, V.; Simionescu, R.; Hudlicky, T. Can. J. Chem. 2011, 89, 535-543.) Allylic cyanohydrins were subjected to Dauben–Michno oxidation at low temperatures to provide β-cyanoenones in good to excellent yields. The potential of this oxidative transposition as a means of an enantiomeric switch of enones containing a latent plane of symmetry was tested by conversion of (–)-carvone to its enantiomer.