Tesis por compendio / [ES] La presente tesis doctoral abarcó el desarrollo de un ingrediente a partir del subproducto de la industria citrícola. Se buscó obtener un ingrediente en polvo, con alto contenido de fibra soluble, que pudiera utilizarse en la formulación de productos. El trabajo se dividió en cuatro etapas. Tras la obtención del ingrediente por diferentes metodologías, en las primeras etapas se analizó su composición química y propiedades tecno funcionales. Posteriormente se realizó un ensayo de estabilidad del ingrediente tras su almacenamiento a diferentes actividades de agua. Finalmente, se evaluó la incorporación del ingrediente, como fuente de fibra, en la formulación de un flan; se estudió el efecto de la información sobre el origen de la fibra agregada en la respuesta del consumidor y el efecto de la incorporación de dicha fibra sobre las propiedades sensoriales del producto. Se ensayaron cuatro tecnologías no contaminantes para la extracción de fibra soluble: agua caliente, extrusión + agua caliente, jet cooker y jet cooker + agua caliente. El sobrenadante de la extracción se secó en "spray dryer" obteniendo un ingrediente en polvo. El contenido de fibra alimentaria de alto y bajo peso molecular fue analizado, junto con el contenido de azúcares simples y de compuestos bioactivos. El contenido de fibra total varió entre 10 y 20 gramos por 100 gramos de producto entre los tratamientos ensayados, siendo el proceso de extrusión + agua caliente el que mayor contenido de fibra presentó. Además, este tratamiento fue el que presentó menor contenido de azúcares y mayor contenido de compuestos bioactivos. En la segunda etapa se analizaron la capacidad espumante y estabilidad de la espuma, retención de aceite, solubilidad en agua, higroscopicidad, microestructura, temperatura de transición vítrea, color y reología de las soluciones. Se observaron diferentes grados de aglomeración en los polvos, que se relacionaron con la baja temperatura de transición vítrea de los ingredientes, fruto de sus componentes de bajo peso molecular. Aunque todas las soluciones formaron espumas, la del ingrediente obtenido por extrusión + agua caliente fue la más estable, lo que pudo deberse a su mayor viscosidad. En la tercera etapa se almacenaron los ingredientes obtenidos a diferentes actividades de agua durante 15 semanas, y una vez transcurrido el tiempo se modeló su isoterma de sorción. Además, se estudió la transición vítrea, junto con el contenido de compuestos bioactivos, color y textura. Los polvos no fueron estables en su estado inicial, debido al alto contenido de humedad que presentaban. Se observó pardeamiento a medida que aumenta la actividad de agua En la última etapa se evaluó la incorporación del ingrediente en la formulación de un flan. Se estudió el efecto de la información en la respuesta del consumidor y el efecto de su incorporación en las propiedades sensoriales. Se realizó una encuesta en España y Uruguay tomando como variables de estudio la intención de compra, y las percepciones de saludable y amigable con el medio ambiente. Se evaluaron tres categorías de producto diferentes (un producto listo para consumo, un polvo para preparar el postre en casa y un flan casero), variando la información ofrecida (origen de la fibra y un logo haciendo referencia a la sostenibilidad). El comportamiento de ambas poblaciones fue diferente en las tres variables de estudio, Para evaluar la incorporación del ingrediente en la formulación de un flan "fuente de fibra" (3% de contenido de fibra), se ensayaron diferentes porcentajes de sustitución de inulina (muestra control) del postre por el ingrediente de naranja obtenido a partir de la extrusión + agua caliente. La sustitución de un 30% del contenido total de fibra por el nuevo ingrediente logró un producto similar al control, sin descriptores de sabor no agradables y con una mejora en el brillo y la textura. / [CA] La investigació de la present tesi doctoral va abastar el desenvolupament d'un ingredient a partir del subproducte de la indústria citrícola. Es va buscar obtindre un ingredient en pols, amb alt contingut de fibra soluble, que poguera utilitzar-se en la formulació d'altres productes. Per a això el treball es va dividir en quatre etapes. Després de l'obtenció de l'ingredient per les diferents metodologies, en les primeres etapes es van analitzar la seua composició química i propietats tecno funcionals. Posteriorment es va feir un assaig d'estabilitat de l'ingredient després del seu emmagatzematge a diferents activitats d'aigua. Finalment, es va avaluar la incorporació de l'ingredient, com a font de fibra, en la formulació d'un flam; es va presentar l'efecte de la informació sobre l'origen de la fibra agregada en la resposta del consumidor i l'efecte de la incorporació d'aquesta fibra sobre les propietats sensorials del producte. Es van assajar quatre tecnologies no contaminants per a l'extracció de fibra soluble: aigua calenta, extrusió+aigua calenta, jet cooker i jet cooker+aigua calenta. El sobrenedant de l'extracció es va assecar en "spray dryer" obtenint un ingredient en pols. El contingut de fibra alimentària d'alt i baix pes molecular va ser analitzat juntament amb el contingut de sucres simples i de compostos bioactius. El contingut de fibra total va variar entre 10 i 20 grams per 100 grams de producte entre els tractaments assajats, sent el procés d'extrusió+aigua calenta el que major contingut de fibra va presentar. A més, aquest tractament va ser el que va presentar menor contingut de sucres i major contingut de compostos bioactius. En la segona etapa es van analitzar la capacitat espumant i estabilitat de l'espuma, retenció d'oli, solubilitat en aigua, higroscopicitat, microestructura, temperatura de transició vítria, color i reologia de les solucions. Es van observar diferents graus d'aglomeració en les pólvores, que es van relacionar amb la baixa temperatura de transició vítria dels ingredients, fruit dels seus components de baix pes molecular. Tot i que totes les solucions van a formar espumes, la de l'ingredient obtingut per extrusió+aigua calenta va ser la més estable, la qual cosa va poder deure's a la seua major viscositat. En la tercera etapa es van emmagatzemar els ingredients obtinguts a diferents activitats d'aigua durant 15 setmanes, i una vegada transcorregut el temps es va modelar la seua isoterma de sorció. A més, es va estudiar la transició vítria, juntament amb el contingut de compostos bioactius, color i textura. Les pólvores no van ser estables en el seu estat inicial, a causa de l'alt contingut d'humitat que presentaven. Es va observar pardejament a mesura que augmenta l'activitat d'aigua. En l'última etapa es va avaluar la incorporació de l'ingredient en la formulació d'un flam. Es va estudiar l'efecte d'informació sobre l'ingredient en la resposta del consumidor i l'efecte de la seua incorporació en les propietats sensorials. Es va fer una enquesta a Espanya i l'Uruguai prenent com a variables d'estudi la intenció de compra, i la percepció de saludable i amigable amb el medi ambient. Es van avaluar tres categories de producte diferents (un producte llest per a consum, una pols per a preparar les postres a casa i un flam casolà), variant la informació oferida (origen de la fibra i un logo fent referència a la sostenibilitat). El comportament de totes dues poblacions va ser diferent en les tres variables d'estudi. Per a avaluar la incorporació de l'ingredient en la formulació d'un flam "font de fibra" (3% de contingut de fibra), es van assajar diferents percentatges de substitució d'inulina (mostra control) de les postres per l'ingredient de taronja obtingut a partir de l'extrusió + aigua calenta. La substitució d'un 30% del contingut total de fibra pel nou ingredient va aconseguir un producte similar al control, sense descriptors de sabor no agradables, amb una millora en la lluentor i la textura / [EN] This thesis covered the development of an ingredient from the by-product of the citrus industry. The aim was to obtain a powdered ingredient, with high soluble fiber content, that could be used in the formulation of other products. The work was divided into four stages. After obtaining the ingredient by the different methodologies, in the first stages its chemical composition and techno-functional properties were analyzed. Afterwards, a stability test of the ingredient was carried out after storage at different water activities. Finally, theincorporation of the ingredient as a source of fiber, in the formulation of a flan was evaluated; the effect of information on the origin of the added fiber on consumer response and the effect of incorporating said fiber on the sensory properties of the product were studied. Four non-contaminant technologies for the extraction of soluble fiber were tested. These were hot water, extrusion + hot water, jet cooker, and jet cooker + hot water. The extraction supernatant was dried in a spray dryer to obtain a powdered ingredient. The content of high and low molecular weight dietary fiber and the content of simple sugars and bioactive compounds were analyzed. The total fiber content varied between 10 and 20 grams per 100 grams of product among the tested treatments, being the extrusion + hot water process the one with the highest fiber content. In addition, this treatment was the one with the lowest sugar content and the highest content of bioactive compounds. In the second stage, the foaming capacity and stability, oil holding capacity, water solubility, hygroscopicity, microstructure, glass transition temperature, color and rheology of the solutions were analyzed. Different degrees of caking were observed in the powders, which were related to the low glass transition temperature of the ingredients, due to their low molecular weight components. Although all the solutions formed foams, the foam of the ingredient obtained by extrusion + hot water was the most stable, which could be due to its higher viscosity. In the third stage, the ingredients obtained were stored at different water activities for 15 weeks, and once the time had elapsed, their sorption isotherm was modeled. In addition, the glass transition was studied, along with the content of bioactive compounds, color, and texture. The powders were not stable in their initial state, due to their high moisture content. Browning was observed as the water activity increased. In the last stage, the incorporation of the ingredient in the formulation of a flan was evaluated. The effect of information about the ingredient on consumer response and the effect of its incorporation on the sensory properties of the dessert were studied. To evaluate the effect of information on consumer response, a survey was carried out in Spain and Uruguay, taking purchase intention and healthiness and environmental friendliness perceptions as study variables. Three different product categories were evaluated (a ready-to-eat product, a powder to prepare dessert at home and a homemade flan), varying the information offered (origin of the fiber and a logo referring to sustainability). The behavior of both populations was different in the three study variables. To evaluate the incorporation of the ingredient in the formulation of a "fiber source" (total fibre content 3%) flan, different percentages of substitution of inulin content (control sample) the total fiber content of the dessert by the orange ingredient obtained from extrusion + hot water were tested. The substitution of 30% of the total fiber content with the new ingredient achieved a product similar to the control, without unpleasant flavor descriptors and with an improvement in brightness and texture. / This work was supported by the National Innovation and Investigation Agency (ANII), Uruguay, under code POS_EXT_2018_1_154449. / Pérez Pirotto, C. (2022). Obtención de fibra alimentaria a partir del subproducto de la industria citrícola, a través de la aplicación de diferentes tecnologías de extracción [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191431 / Compendio

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: This study concerns the synthesis, properties and quantitative analysis of polydiallyldimethylammonium chloride (polyDADMAC), a water-soluble polymer used world-wide for potable water purification. The special interest in this polymer is the result of its widespread use and the current lack of adequate analytical methods for it. This is especially important for water treatment organisations. A novel gel permeation chromatography (GPC) method was developed and evaluated for polymer analysis. The scope was extended to determine the presence of unreacted monomer (DADMAC) as well as the percentage active polymer. polyDADMAC was first prepared using a known synthesis method. The product was purified and characterized by GPC and 13C-NMR spectroscopy. New and conclusive evidence of the existence of a five-member pyrrolidine ring system was obtained. A proposed mechanism of polymerization was determined. The activity of the synthesized polyDADMAC was evaluated and it was found to perform effectively as a coagulant. The physical and chemical properties of polyDADMAC were then studied under simulated water treatment conditions. The polymer reaction with chlorine revealed the formation of trihalogenated methane compounds (THMs). Gas chromatography–mass spectrometry (GC–MS) was used to conclusively identify the formation of chloroform. The polymer stability under different conditions of heat exposure, UV radiation and pH variations was studied. GPC results indicated that polyDADMAC is a very stable polymer and undergoes structural change only when subjected to extremes of pH, temperature and UV conditions. Results of a short study on microbial degradation indicated growth of the cultures, and subsequent polymer degradation. Reactions of polyDADMAC were concluded with a study of the impact of ozone on polyDADMAC. GPC results indicated a significant change in the ozonated polymer peak profile. Analytical methods to determine polyDADMAC residues in water were reviewed and critically evaluated. Methods based on complex formation/spectroscopy suffered from severe limitations and produced no meaningful results, contrary to claims made by previous researchers. Colloid titration based on an established method was promising but required extensive modification for quantitative analysis. Finally four novel methods were developed, including: solid phase extraction, membrane filtration-GPC, the HACH complexation method, and a GPC method with indirect UV detection. The study is concluded with a chemical risk assessment that indicated minimal human health risks associated with the production and use of polyDADMAC. / AFRIKAANSE OPSOMMING:Hierdie studie behels die sintese, eienskappe en kwantitatiewe analise van polidiallielmetielammoniumchloried (polyDADMAC), 'n wateroplosbare polimeer wat wêreldwyd vir drinkwatersuiwering gebruik word. Die belangstelling in hierdie spesifieke polimeer is as gevolg van die wydverspreide gebruik daarvan en die feit dat daar tans onvoldoende eenvoudige analitiese metodes daarvoor bestaan. Dit is veral belangrik vir waterbehandelingsorganisasies. 'n Nuwe gelpermeasiechromatografie (GPC) metode is ontwikkel en geevalueer vir die analise van hierdie polimeer. Die omvang van die studie is later uitgebrei om die teenwoordigheid van ongereageerde monomeer (DADMAC) asook die persentasie aktiewe polimeer te bepaal. polyDADMAC is eers volgens 'n bekende sintesemetode berei. Die produk is gesuiwer en gekarakteriseer m.b.v. GPC en 13C-KMR. Nuwe bewyse vir die bestaan van 'n vyflid pirollidoonringsisteem is verkry. 'n Meganisme vir hierdie polimerisasie metode is vasgestel. Die aktiwiteit van die bereide polyDADMAC is geevalueer en daar is bevind dat dit effektief as koaguleermiddel optree. Daarna is die chemiese en fisiese eienskappe van polyDADMAC onder gesimuleerde waterbehandelingskondisies bepaal. polyDADMAC het met chloor gerageer om trihalogeneerde metaanverbindings (THMs) te vorm. Gaschromatografie–massa-spektrometrie (GC–MS) is gebruik om die ontstaan van chloroform te bevestig. Daarna is die stabiliteit van die polimeer onder verskei reaksiekondisies bepaal: hitte, UV-bestraling, en pH. GPC-resultate het aangeduiui dat polyDADMAC baie stabiel is en ondergaan strukturele veranderings slegs onder uiterste kondisies van pH, temperatuur en UV. 'n Kort studie van die effek van mikro-organismes op polyDADMAC het egtermikrobiese kultuurgroei met die gevolglike afbreek van die polimeer getoon. Resultate van 'n studie van die impak van osoon op polyDADMAC het getoon dat daar 'n groot verandering in die GPC-profiel van die ge-osoneerde vorm van die polimeer was. Verdere analitiese metodes wat al gebruik is om polyDADMAC residue in water te bepaal, is uitgevoer en krities geevalueer. Metodes gebasseer op kompleksvorming/ spektroskopie het erge beperkings gehad en het nie betekenisvolle resultate gelewer nie. Dit was in teenstelling met wat voorheen deur ander navorsers bevind is. 'n Kolloiedtitrasie gebasseer op 'n bestaande metode het goeie resultate gelewer maar het omvattende veranderings benodig om kwantitatiewe resultate te lewer. Ten slotte is vier nuwe metodes ontwikkel: soliede fase-ekstraksie, membraanfiltrasie-GPC, die HACH-komplekseringsmetode, en 'n GPC-metode met indirekte UV-waarneming.. Die studie is afgesluit met 'n bepaling van die chemiese risiko wat poly DADMAC vir die gesondheid van die mens inhou. Daar is tot die gevolgtrekking gekom dat die produksie en gebruik van poly DADMAC slegs‘n minimum gesondheidsrisiko inhou.

Human alteration of the global nitrogen (N) cycle has had major impacts on naturally N-limited ecosystems worldwide. Peatlands, dominated by peat mosses, Sphagnum species, represent one such sensitive ecosystem. I have studied how this ecosystem is affected by increased N availability, using a small-scale N fertilization experiment in combination with a gradient study of three peatlands with varying N deposition. I found both in the experiment and in the gradient a similar pattern of Sphagnum decline accompanied by an increase of vascular plants associated with enhanced N supply. For one common Sphagnum species - both in the experiment and in the gradient study - I also found an identical, linear increase in soluble amino acid N (NAA) accumulation. As soluble amino acids function as N storage compounds among Sphagna, NAA is a suitable measure for Sphagnum N status, and indicates accumulation of excess N not used for growth. My results show that NAA can be used as a sensitive indicator to signal N pollution before the slow, and gradual, regime shift from Sphagnum to vascular plant dominance is visible. In an N-uptake experiment using Sphagnum specimens from the three peatlands varying in N deposition, I found a reduced N-uptake by both investigated Sphagnum species from a high N deposition site, in south-western Sweden. This potential of Sphagna to adjust to high N loads through N uptake regulation will, however, not prevent tissue N accumulation, and as a result a shift from Sphagnum to vascular plant dominance. In general I found similar patterns of N induced changes both in Sphagnum tissue chemistry and vegetation structure in the experiment and along the gradient study. Thus, I conclude that long-term, small-scale field experiments seem to offer reliable estimates of both the direction and strength of key vegetation responses in Sphagnum dominated peatlands. This is likely related to the key role of Sphagna as ecosystem engineers. In the experiment I found a marked time lag in vegetation response to N application treatments. The closed Sphagnum carpet did not collapse until after eight years of continuous treatments. Another result was that dwarf shrubs, e.g. cranberry Vaccinium oxycoccos, first increased, but later declined due to severe attacks by fungal diseases. One important conclusion is that long-term, manipulative field experiments are necessary for our ability to understand how ecosystems will respond to environmental change.

Poor aqueous solubilities of drug candidates limit the biopharmaceutical usefulness in either oral or parenteral dosage forms. Lipid assemblies, such as micelles, may provide a means of enhancing solubility. Despite their usefulness, little is known about the means by which micelles accomplish this result. The goal of the current dissertation is to provide the molecular level understanding of the mechanism by which simple micelle systems solubilize drugs. Specifically, the location, orientation and amount of the drug molecules in micelle systems are the focuses of the work. Three series of model drugs, steroids, benzodiazepines and parabens, in three surfactant systems with anionic, cationic and neutral hydrophilic headgroups were studied. Solubilization power of each micelle system for each model drug was determined by equilibrium solubility. The observed strong surface activities of model drug at hydrocarbon/water interface and the ability of the drugs to compete with surfactants for the model oil/water interface lend support to the hypothesis that drug molecules are mainly solubilized in the interfacial region of the micelles. A surface-localized thermodynamic model that considered the surfactant-drug competition at micelle surface was successfully applied to predict the micelle/water partitioning coefficients. The predictions were made without the use of adjustable parameters in the case of both dilute and concentrated solutions. The orientation of drug at micelle surface was determined by matching calculated occupied areas by solutes at oil/water interface using molecular modeling method to the experimental values. To look into the micro-structure of micelles, twodimensional and diffusion (or PGSE) NMR techniques were employed to detect the specific drug-surfactant interactions and the micelle sizes influenced by model drugs and electrolytes.

The majority of pharmacologically active chemotherapeutics are poorly water soluble. Solubilization enhancement by the utilization of organic solvents often leads to adverse side effects. Nanoparticle-based cancer therapy, which is passively targeted to the tumor tissue via the enhanced permeation and retention effect, has been vastly developed in recent years. Nanocrystals, which exist as crystalline and carry nearly 100% drug loading, has been explored for delivering antineoplastic agents. Additionally, the hybrid nanocrystal concept offers a novel and simple way to integrate imaging agents into the drug crystals, enabling the achievement of theranostics. The overall objective of this dissertation is to formulate both pure and hybrid nanocrystals, evaluate their performance in vitro and in vivo, and investigate the extent of tissue distribution and tumor accumulation in a murine model. Pure and hybrid nanocrystals of several model drugs, including paclitaxel (PTX), camptothecin, and ZSTK474, were precipitated by the antisolvent method in the absence of stabilizer, and their size was further minimized by homogenization. The nanocrystals of PTX, which is the focus of the study, had particle size of approximately 200 nm and close-to-neutral surface charge. Depending on the cell type, PTX nanocrystals exerted different level of cytotoxicity. In human colon and breast cancer xenograft models, nanocrystals yielded similar efficacy as the conventional formulation, Taxol, at a dose of 20 mg/kg, yet induced a reduced toxicity. Biodistribution study revealed that 3H-PTX nanocrystals were sequestered rapidly by the macrophages upon intravenous injection. Yet, apparent toxicity was not observed even after four weekly injections. The sequestered nanocrystals were postulated to be released slowly into the blood circulation and reached the tumor. Tritium-labeled-taxol, in contrast, was distributed extensively to all the major organs, inducing systemic toxicity as observed in significant body weight loss. The biodistribution results obtained from radioactive analysis and whole-body optical imaging was compared. To some degree, the correlation was present, but divergence in the quantitative result, due to nanocrystal integrity and limitations associated with the optical modality, existed. Despite their promising properties, nanocrystal suspensions must be securely stabilized by stealth polymers in order to minimize opsonization, extend blood-circulation time, and efficiently target the tumor.

Soluble organic matter represent less than 1% of total soil organic matter (SOM) - but it contributes to many terrestrial ecosystem processes, due to its high mobility and reactivity in soil. Although it has been suggested that soluble organic matter (OM) may serve as an early indicator of soil quality changes as a result of shifts in land-use and management practices, only a few studies have addressed the dynamics of soluble OM in relation to land-use and specifically soil depth. This study focuses on two aspects of soluble OM. In the first part, I hypothesized that extractable OM obtained by aqueous solutions is a continuum of substances that depending on the extraction method can be separated into two operationally different fractions. The size and properties of these fractions may consistently differ among different land uses and at different soil depths. The objective of this part of the study was then to assess dynamics (size and properties, biodegradability and seasonality) of water extractable organic matter (WEOM) and salt extractable organic matter (SEOM) in a sequence of human dominated land-uses at topsoil and subsoil. At the second part of the study, I tested the regulatory gate hypothesis –abiotic solubilization of OM- as a primary controlling factor in soluble OM production. The objective of this study was to evaluate the impact of the microbial activity on the net production of dissolved organic matter (DOM) from the native SOM in the presence of added DOM and plant residue. For the first part of the experiment, the soil samples were collected from four land-uses under bog pine (Halocarpus bidwillii) woodland, tussock grassland (Festuca novae-zelandiae and Heiracium pilosella), cropland (Medicago sativa) and plantation forest (Pinus nigra). The selected land uses were located in the Mackenzie Basin, Canterbury, New Zealand and occurring on the same soils, topography and experienced similar climates. Soil samples were obtained from topsoil (0-20 cm) and subsoil (60-80 cm) at the end of each season (November, February, May and August) during 2007-2008. The sampled soils were adjusted to the same water status prior to extraction. While WEOM was obtained during a mild extraction procedure and using 0.01M CaCl2, SEOM was extracted with 0.5M K2SO4 at high temperature (75οC for 90 min). Both extracts were filtered through a 0.45 μm filter size. In the first part of the study, I assessed the biodegradation dynamics of WEOM and SEOM (spring samples), using a double-exponential decay model. The WEOM and SEOM were inoculated and incubated at 22°C for 90d under aerobic conditions. Subsamples were removed on days 1, 3, 7, 12, 16, 30, 42, 60, 75, and 90, filtered (0.22 μm), and analyzed for organic C and N content, UV absorption, and 13C natural abundance (δ13C). The results of the biodegradation experiment indicated a similar pattern for both C and N of SEOM and WEOM as that of previously shown for soil DOM. However, C and N mineralization rate were considerably larger in the WEOM than SEOM. The parameters of the double-exponential model suggested that regardless of the land-use and soil depth, both the WEOM and SEOM can be modeled in two biological pools, with a largely similar “fast decomposable” but different “slowly decomposable” pools. However, since the extraction was not sequentially followed, a very small portion of the SEOM was comprised of the WEOM and given the greater observed biodegradability of the WEOM, the overall biodegradable portion of the SEOM would be lower than the observed. Despite a greater biodegradability of the organic N than C of both WEOM and SEOM; mainly due to a longer HL of the slowly biodegradable pool of C; the C/N ratio of the samples did not change very much during the biodegradation. This led us to conclude that the biodegradation of soluble OM may occur as a function of N availability. Parallel to C and N loss, a considerable increase in SUVA254 of SEOM, and particularly WEOM occurred during the incubation period. The greater increase in the proportion of aromatic compounds (assessed by SUVA) in the WEOM than SEOM, implied consumption of simple compounds (vs. very humified) during decomposition and further supported the observed faster biodegradation rate of the WEOM. The data indicated a relatively strong correlation (R2=0.66 and 0.74 for the WEOM and SEOM, respectively) between the amount of biodegraded C and the increase in SUVA254. This suggested that SUVA254 can be used as a simple, low-cost but reliable approach for describing the biodegradability of soluble OM, as previously suggested by others. At the end of the bioassay, the 13C natural abundance of the WEOM was significantly depleted, and showed a clear relationship with the proportion of the biodegraded C. This confirmed the previously suggested preferential biodegradation of simple organic constituents (13C enriched), resulting in the accumulation of more depleted 13C compounds (often recalcitrant compounds). Moreover, the results of the δ13C technique revealed that the relatively greater 13C enrichment of the WEOM obtained from subsoil, seems to be due to the presence of root exudates (often highly 13C enriched). In contrast, a proportionally greater 13C depletion observed in the SEOM particularly at subsoil samples, suggests that there is a close relationship between the SEOM and the typically 13C depleted humified SOM. The results of the biodegradation model (half-life of both C and N), in addition to dynamics of SUVA254 and δ13C of the WEOM and SEOM were very comparable between top and subsoil samples. This implied that the potential biodegradability of soluble OM under laboratory conditions does not necessary reflect the reported lower in situ biodegradability at soil depth, in agreement with recent evidence suggested by others. Instead, this may be largely due to the lack of optimum conditions (oxygen, nutrients, and moisture) for the decomposer community at soil depth. Although there was a tendency for a generally greater biodegradability of the samples from the soils under the crop land (both WEOC and SEOC), along with relatively greater increase in SUVA, there was not a consistent trend of the effect of land use on the biodegradation of either WEOM or SEOM. The lower C/N ratio of the soils under the crop land seemed to be related with the observed proportionally greater biodegradability of these soils. During the second part of the study, I assessed seasonal variations of the size and properties of the previously defined WEOM and SEOM, collected from top-and subsoil from the land-uses. I observed that 10-year after conversion of the degraded tussock grassland to cropland or plantation, the total C stock of topsoil (0-20 cm) when above- and below-ground plant biomass is excluded; has remained unchanged. This was attributed to the limited biomass production of the region, more likely as a result of low productivity of the soil, but also harsh climatic conditions. Not only soil depth, but land-use affected both C concentration and C/N ratio of soil organic matter (SOM), with the greatest C concentration of soils under grassland and plantation in topsoil and subsoil, respectively. Despite the WEOM, the size of SEOM was largely unaffected by land-use and soil depth; instead, the properties of SEOM was more consistent with the effect of soil depth. Given the observed large temporal and spatial variability of the WEOM, the study suggests that the SEOM more consistently reflects the influence of land use and soil depth. No consistent effect of seasonality was observed in terms of size or properties of the SOM and the WEOM and SEOM. Overall comparison of the size and properties of the WEOM and SEOM indicated that OM extraction efficiency may vary largely, depending on extraction conditions. Using more concentrated salt solutions consistently yielded greater amount of OM (N, and especially C) release from soil with properties resembling more those of total soil OM (more humified) compared to the WEOM. The SEOM was also less variable by time and space. The last part of the study was aimed to assess biotic vs. non-biotic solubilization of OM in the presence of added plant residue. Given the need to recognize the source of the solubilized OM during the experiment, I used enriched (13C) plant residue as the source of fresh OM. The above-ground part of ryegrass was added to soil either as plant residue or residue extract (extracted with CaCl2 followed by 0.45μm filtration) -termed DOM. These two forms of added OM (residue/DOM) were conceived to represent two levels of bioavailability for the decomposer community for further assessing possible biotic solubilization of OM. Two soils similar in their OM content and other properties, but different in mineralogy were selected for the experiment. Soils were incubated for 90d under sterilized vs. non-sterile conditions and leached regularly with a dilute aqueous solution (0.05M CaCl2). Plant residue was added to soil (1:100, residue: soil, w/w) prior to the start of the incubation, but DOM was frequently applied to the soils along with each leaching experiment. The greater C and N concentration in the leachates of both sterilized residue-amended and DOM-amended soils compared to that of living soils, indicated a high microbial activity, as determined by CO2 loss, in the living soils. However, the proportion of the solubilized C (determined by 13C) from sterilized soils was largely comparable to that of living soils. This supports the recently suggested “regulatory gate” hypothesis, stating that solubilisation of OM largely occurs independent of the size or community structure of microorganisms. In addition, I observed that even with the presence of adequate amount of added fresh OM (ryegrass residue), about 70% of the solubilized C consistently originated from the humified soil OM, highlighting the role of native soil OM as the source of soluble OM in soil. In addition, in the DOM-amended soils, there was strong evidence, indicating that in the sterilized soils, the added DOM was exchanged with the humified soil OM as observed by an increase in SUVA, and humification index (HI) of the leached OM. Although the results of the study did not show a considerable difference in the solubilisation rate of added OM as a function of biological activity (either in the residue- or DOM-amended soils), there was clear evidence that the presence of microbial activity has resulted in further decomposition of the solubilised OM through biological transformations. Together, the results suggested that the proposed fractionation method can be used to separate two operationally defined pools of soluble OM with consistent differences in their size (C and N), properties (δ13C, SUVA254, and C/N ratio) and biodegradability across the land-uses and soil depth. The second part of the study supported the primary role of abiotic factors on the production of soluble OM from native soil OM. Although the abiotic mechanisms involved in the solubilization remain to be addressed by future studies. Cons and pros of the methods with some suggestions for further works have been mentioned in the last chapter.

Soluble guanylyl/guanylate cyclase (sGC), the primary receptor for nitric oxide (NO), is a heme containing heterodimeric enzyme involved in numerous physiological events in animals. The small molecule YC-1 stimulates sGC, but the mechanism behind and the location of binding are unknown. I have developed a prokaryotic expression system for insect ( <italic>Manduca sexta</italic>) sGC. The recombinant holoenzyme, like its mammalian counterpart, is responsive to NO, CO and YC-1, displaying a 175-fold increase in activity on binding. Truncated constructs containing the N-terminal two-thirds of both subunits (msGC-NT) were designed to facilitate expression. With the highly pure material, we investigated NO and CO binding, reaction kinetics and regulation. Binding of NO to msGC-NT heme forms a six-coordinate intermediate followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex. The conversion rate is insensitive to nucleotides, YC-1 and changes in NO concentration up to ~30 micromolar. In contrast, NO release from msGC-NT is biphasic in the absence of YC-1, while binding of YC-1 eliminates the fast phase but has little effect on the slower phase. CO binding to msGC-NT is also regulated by YC-1. The CO release rate is reduced by YC-1 while the on rate remains unchanged, which leads to an ~50-fold increase in binding affinity. YC-1 binding leads to a substantial geminate recombination of CO to msGC-NT upon photolysis. Our data are consistent with a model for allosteric activation in which (1) YC-1 binds away from the catalytic site and (2) sGC undergoes a conformational switch between two states of an open and a closed heme pocket. The final catalysis results from the integration of the influence of numerous allosteric effectors on the equilibrium between these two states.<italic>S </italic>-nitrosoglutathione (GSNO) exists <italic>in vivo </italic> and plays important roles in NO signaling. We have developed a model cell line, in which inducible NO synthase and human sGC genes were included. GSNO stimulation of sGC has been investigated using recombinant insect and human enzymes. GSNO can activate sGC as efficiently as gaseous NO, but apparently with a distinct mechanism. GSNO or endogenous NO could <italic>S </italic>-nitrosylate sGC, which might regulate the enzyme function.

La libération de l'acide arachidonique (AA) de la membrane des cellules par les phospholipases activées, le rend accessible pour être métabolisé par plusieurs enzymes impliquées dans la biosynthèse des eicosanoïdes. Ceux-ci incluent les cyclooxygénases (COX), les lipoxygénases (LOX) et plusieurs isoformes des cytochromes P450 (CYP450) qui produisent les acides époxy-eicosatriénoïques (EET) et les acides hydroxy-eicosatétraénoïques (HETE). Tandis qu'une attention considérable a été accordée aux rôles des eicosanoïdes dérivés des COX et des LOX, relativement peu de données sont disponibles sur les rôles potentiels des eicosanoïdes produits par les CYP450 sur la réactivité des muscles lisses des voies respiratoires (MLVR). L'objectif général de ce projet était de déterminer les modes d'action de ces eicosanoïdes bioactifs sur les tissus pulmonaires humains. L' idée étant de mieux cerner les mécanismes cellulaires et moléculaires activés lorsque ces tissus sont traités avec différents médiateurs lipidiques (eicosanoïdes) et inflammatoires (cytokines). Pour cela les propriétés électrophysiologiques, pharmacomécaniques et biochimiques susceptibles d'être modulées par ces eicosanoïdes sur les MLVR ont été analysées. Premièrement, nous avons démontré que l'acide 14,15-époxy-eicosatriénoïques (14,15-EET) hyperpolarise et relaxe les MLVR humains via l'activation des canaux potassiques de grande conductance activé par le Ca[exposant]2+ (BK[indice]Ca ). De plus, nous avons démontré que cet époxy-eicosanoïde diminue la sensibilité au Ca[indice]2+ des bronchioles perméabilisées à la [bêta]-escine, ce qui est corrélé avec une baisse du niveau de phosphorylation et d'expression de la protéine CPI-17 (protein kinase C-potentiated myosin phosphatase inhibitor). Par la suite, nous avons mis au point un modèle d'hyperréactivité bronchique (HRB) induit par un prétraitement des tissus au tumor necrosis factor [alpha] (TNF[alpha]). Ce modèle nous a permis d'évaluer les effets anti-inflammatoires des époxy-eicosanoïdes. Nous avons démontré que ces eicosanoïdes interfèrent avec l'activation du Nuclear Factor [kappa] B (NF[kappa]B) via leur interaction avec le peroxisome proliferator-activated receptors [gamma] (PPAR[gamma]). De plus, le 14,15-EET et l'acide 17,18-époxy-eicosatetraénoïque (17,18-EpETE) diminuent l'hypersensibilité au Ca[exposant]2+ des bronchioles prétraitées au TNF[alpha], en interférant avec la voie de signalisation de p38 mitogen-activated protein kinase (p38-MAPK), ce qui entraîne une réduction de la phosphorylation et de l'expression de la protéine CPI-17. L'introduction de siRNA dirigés contre les transcrits de la CPI-17 dans les bronchioles humaines traitées au TNF[alpha] a permis de mettre en évidence le rôle crucial de cette protéine dans l'HRB. Cette étude a aussi permis de démontrer que l'époxyde hydrolase soluble (sEH) est surexprimée dans les bronchioles humaines traitées au TNF[alpha] et dans les biopsies de patients asthmatiques. L'utilisation d'un inhibiteur pharmacologique de la sEH, le 12-(3-adamantyl-ureido)-dodecanoic acid (AUDA), a permis d'augmenter la biodisponibilité des époxy-eicosanoïdes et leurs effets bénéfiques sur les tissus bronchiques traités au TNF[alpha]. Les stratégies expérimentales établies ont permis de définir les modes d'action de ces eicosanoïdes et de démontrer leurs rôles potentiels contre l'HRB.

Nicotinamide cofactors in their reduced and oxidised forms are important redox agents in biology. Of about 3000 dehydrogenases available to date, many require these cofactors for their activity. Dehydrogenases are of interest to chemists as they offer asymmetric catalysis to yield chiral products. The requirement of dehydrogenases for nicotinamide cofactors necessitates research into finding the best way of recycling the oxidised or reduced forms of these cofactors. Electrocatalytic NAD(P)H oxidation and NAD(P)⁺ reduction on standard electrodes is problematic due to unwanted side reactions and high overpotential requirements, but in Nature efficient enzyme catalysts are available to facilitate these reactions. The focus of this Thesis, the Soluble Hydrogenase of R. eutropha (SH) is a multimeric bidirectional hydrogenase that couples H2 oxidation to the reduction of NAD⁺ to NADH. Protein Film Electrochemistry (PFE) has been employed to study NAD⁺-reducing catalytic moieties of the SH for the first time. It is shown that SH subunits on an electrode are able to catalyse NADH oxidation and NAD⁺ reduction efficiently with minimal overpotential, which is significant because in vivo, NAD(H) cycling is coupled to 2H⁺/H₂ cycling and these reactions are closely spaced in potential. Substrate affinities and inhibition constants for the SH, determined using PFE are discussed in the context of the SH function and the related catalytic domains of respiratory Complex I. A range of molecules that are known to inhibit the related Complex I have been investigated for their ability to inhibit the SH moieties: the similarity between inhibition constants is consistent with structural and functional similarity between the SH and Complex I. The ability of the SH moieties to sustain NAD(H) catalysis in the presence of O₂ is also demonstrated and is consistent with the requirement for the SH to function under aerobic conditions and to reactivate the inactivated hydrogenase moiety by supplying low potential electrons from NADH. Engineered variants of the SH, designed to enhance the affinity towards NADP⁺, were investigated for the first time, using PFE. Electrochemical characterisation of the variants is presented and results are discussed alongside findings on the wild type SH. The variants are shown to exhibit NADP⁺ reduction, and to have higher affinity towards NADP⁺ than the wild type SH. The first efficient NADP⁺ reduction and NADPH oxidation is observed for one of the variants on a graphite electrode and the best variant showed a K_M of 1.7 mM for NADP⁺. This Thesis also provides evidence for the ability of moieties of the SH to be used in cofactor regeneration systems. Two novel systems are demonstrated. The first involves H₂ driven NADH recycling based on the NAD⁺-reducing moiety of the SH immobilised on graphite particles together with a hydrogenase or platinum, with electrons from H₂ passed from the hydrogenase through the graphite to the NAD⁺-reducing moiety. The second involves an electrode modified with the NAD⁺-reducing moiety of the SH, and is demonstrated as an electrochemical NADH recycling system coupled with NADH-dependent pyruvate reduction to lactate by lactate dehydrogenase. The ability of variants of the SH to catalyse NADP⁺ reduction suggests that it may also be possible to use these systems for recycling NADPH for catalysis of important biotransformation reactions by NADPH-dependent dehydrogenases.

Monodispersed microgels composed of poly-acrylic acid (PAAc) and poly(N-isopropylacrylamide) (PNIPAM) interpenetrating networks were synthesized by 2-step method with first preparing PNIPAM microgel and then polymerizing acrylic acid that interpenetrates into the PNIPAM network. The semi-dilute aqueous solutions of the PNIPAM-PAAc IPN microgels exhibit an inverse thermo-reversible gelation. Furthermore, IPN microgels undergo the reversible volume phase transitions in response to both pH and temperature changes associated to PAAc and PNIPAM, respectively. Three applications based on this novel hydrogel system are presented: a rich phase diagram that opens a door for fundamental study of phase behavior of colloidal systems, a thermally induced viscosity change, and in situ hydrogel formation for controlled drug release. Clay-polymer hydrogel composites have been synthesized based on PNIPAM gels containing 0.25 to 4 wt% of the expandable smectic clay Na-montmorillonite layered silicates (Na-MLS). For Na-MLS concentrations ranging from 2.0 to 3.2 wt%, the composite gels have larger swelling ratio and stronger mechanical strength than those for a pure PNIPAM. The presence of Na-MLS does not affect the value of the lower critical solution temperature (LCST) of the PNIPAM. Surfactant-free hydroxypropyl cellulose (HPC) microgels have been synthesized in salt solution. In a narrow sodium chloride concentration range from 1.3 to 1.4 M, HPC chains can self-associate into colloidal particles at room temperature. The microgel particles were then obtained in situ by bonding self-associated HPC chains at 23 0C using divinyl sulfone as a cross-linker. The volume phase transition of the resultant HPC microgels has been studied as a function of temperature at various salt concentrations. A theoretical model based on Flory-Huggins free energy consideration has been used to explain the experimental results. Self-association behavior and conformation variation of long chain branched (LCB) poly (2-ethyloxazoline) (PEOx) with a CH3-(CH2)17 (C18) modified surface are investigated using light scattering techniques in various solvents. The polymer critical aggregation concentration (cac) strongly depends on solvent polarity, decreasing as the solvent becomes more hydrophobic.