Tesis por compendio / [ES] La presente tesis doctoral, titulada "Diseño y desarrollo de nuevos nanodispositivos con puertas biomoleculares para la liberación dirigida y controlada de fármacos", está centrada en el diseño, síntesis, caracterización y evaluación in vitro de nuevos nanosistemas híbridos orgánicos-inorgánicos como estrategias innovadoras para la administración dirigida y controlada de moléculas terapéuticas. El primer capítulo de este trabajo es una introducción general que define el contexto en el que se sitúan los proyectos llevados a cabo a lo largo de esta tesis. En particular, se describe el concepto de nanomedicina, así como las principales estrategias para el desarrollo de dispositivos nanofarmacéuticos eficientes y los desafíos que esto conlleva. Además, se presentan brevemente los materiales mesoporosos de sílice. Sucesivamente, se presentan los objetivos generales que se abordan en los siguientes capítulos experimentales. El tercer capítulo se centra en el desarrollo de un sistema de administración dirigida al Toll-like receptor 3 (TLR3), basado en nanopartículas mesoporosas de sílice funcionalizadas con el RNA sintético de doble cadena ácido polinosínico-policitidílico (poly(I:C)). El poly(I:C) ha mostrado efectos citotóxicos en diferentes tipos de cáncer, y los resultados obtenidos en este trabajo demuestran su capacidad de desencadenar respuestas apoptóticas en células de cáncer de mama, gracias a su interacción con TLR3. Asimismo, la carga del antibiótico antraciclínico doxorubicina en los mesoporos, ha permitido conseguir un efecto terapéutico aún más intenso. De hecho, se ha observado una mayor disminución de la viabilidad en la línea celular SK-BR3. El capítulo cuatro muestra el diseño de una estrategia de cooperación entre nanopartículas. El objetivo de este trabajo es el de mejorar la eficacia de la aproximación terapéutica para cáncer de mama desarrollada precedentemente, a través de la combinación de dos tipos de nanopartículas mesoporosas de sílice con puertas moleculares capaces de cooperar con el fin de conseguir un objetivo terapéutico. El primer tipo, de hecho, induce cambios que incrementan la interacción del segundo con la célula tumoral diana. Para ello, se han sintetizado nanopartículas cargadas con ácido 9-cis retinoico y funcionalizadas en su superficie con interferón-¿, y nanopartículas cargadas con el colorante sulforodamina B y con poly(I:C) anclado en superficie. Aprovechando la habilidad del interferón-¿ del ácido 9-cis retinoico de aumentar la expresión del TLR3, nuestra intención era la de mejorar la interacción de las nanopartículas funcionalizadas con poly(I:C) con las células diana. Los resultados obtenidos demuestran que la estrategia de combinación propuesta realmente ha producido un aumento de los niveles de internalización de los nanodispositivos funcionalizados con poly(I:C) en el modelo celular seleccionado. En el quinto capítulo se presenta un sistema basado en nanopartículas mesoporosas de sílice dendriméricas. El volumen de poro más amplio de este tipo de materiales hace de ellos unos dispositivos adecuados para lograr el objetivo principal de este proyecto: la administración local y la liberación controlada de moléculas de RNA pequeño de interferencia (siRNA) anti-factor de crecimiento vascular endotelial (VEGF) en células del epitelio pigmentado retinal. Los nanodispositivos se han sintetizado, cargado con siRNA y finalmente funcionalizado con cadenas de polietilenimina, que actúan de puerta molecular para la liberación controlada de las moléculas de siRNA y como agentes de escape endosomal para su liberación en el citosol. Los resultados obtenidos en el silenciamiento de VEGF en las células ARPE-19 resaltan el considerable potencial del sistema diseñado como transportador de siRNA. Las conclusiones generales en relación con los trabajos recopilados en esta tesis se resumen en el capítulo sei / [CA] La present tesi doctoral, titulada "Disseny i desenvolupament de nous nanodispositius amb portes biomoleculars per a l'alliberament dirigit i controlat de fàrmacs", està centrada en el disseny, síntesi, caracterització i avaluació in vitro de nous nanosistemes híbrids orgànics-inorgànics com a estratègies innovadores per a l'administració dirigida i controlada de molècules terapèutiques. El primer capítol d'aquest treball és una introducció general que defineix el context en el qual es situen els projectes duts a terme al llarg d'aquesta tesi. En particular, es descriu el concepte de nanomedicina, així com les principals estratègies per al desenvolupament de dispositius nanofarmacèutics eficients i els desafiaments que això comporta. A més, es presenten breument els materials mesoporosos de sílice. Successivament, es presenten els objectius generals que s'aborden en els següents capítols experimentals. El tercer capítol es centra en el desenvolupament d'un sistema d'administració dirigida al Toll-like receptor 3 (TLR3), basat en nanopartícules mesoporoses de sílice funcionalitzades amb l'RNA sintètic de doble cadena àcid polinosínic-policitidílic (poly(I:C)). El poly(I:C) ha mostrat efectes citotòxics en diferents tipus de càncer, i els resultats obtinguts en aquest treball demostren la seua capacitat de desencadenar respostes apoptòtiques en cèl·lules de càncer de mama, gràcies a la seua interacció amb TLR3. Així mateix, la càrrega de l'antibiòtic antraciclínic doxorubicina en els mesopors, ha permès aconseguir un efecte terapèutic encara més intens. De fet, s'ha observat una major disminució de la viabilitat en la línia cel·lular SK-BR3. El capítol quatre mostra el disseny d'una estratègia de cooperació entre nanopartícules. L'objectiu d'aquest treball és el de millorar l'eficàcia de l'aproximació terapèutica per a càncer de mama desenvolupada precedentment, mitjançant la combinació de dos tipus de nanopartícules mesoporoses de sílice amb portes moleculars capaces de cooperar amb la fi d'aconseguir un objectiu terapèutic. El primer tipus, de fet, indueix canvis que incrementen la interacció del segon amb la cèl·lula tumoral diana. Per a això, s'han sintetitzat nanopartícules carregades amb àcid 9-cis retinoic i funcionalitzades en la seua superfície amb interferó-¿, i nanopartícules carregades amb el colorant sulforodamina B i amb poly(I:C) ancorat en superfície. Aprofitant l'habilitat de l'interferó-¿ de l'àcid 9-cis retinoic d'augmentar l'expressió del TLR3, la nostra intenció era la de millorar la interacció de les nanopartícules funcionalitzades amb poly(I:C) amb les cèl·lules diana. Els resultats obtinguts demostren que l'estratègia de combinació proposada realment ha produït un augment dels nivells d'internalització dels nanodispositius funcionalitzats amb poly(I:C) en el model cel·lular seleccionat. El cinquè capítol presenta un sistema basat en nanopartícules mesoporoses de sílice dendrimèriques. El volum de porus més ampli d'aquest tipus de materials fa d'ells uns dispositius adequats per a aconseguir l'objectiu principal d'aquest projecte: l'administració local i l'alliberament controlat de molècules d'RNA xicotet d'interferència (siRNA) anti-factor de creixement vascular endotelial (VEGF) en cèl·lules de l'epiteli pigmentat retinal. Els nanodispositius s'han sintetitzat, carregats amb siRNA i finalment funcionalizats amb cadenes de polietilenimina, que actuen de porta molecular per a l'alliberament controlat de les molècules de siRNA i com a agents d'escap endosomal per al seu alliberament en el citosol. Els resultats obtinguts en el silenciament de VEGF en les cèl·lules ARPE-19 ressalten el considerable potencial del sistema dissenyat com a transportador de siRNA. Les conclusions generals en relació amb els treballs recopilats en aquesta tesi es resumeixen en el capítol sis. / [EN] The present PhD thesis, entitled "Design and development of new bio-gated nanodevices for targeted controlled drug release" is focused on the design, synthesis, characterization and in vitro evaluation of new hybrid organic-inorganic nanosystems as innovative strategies for the targeted and controlled delivery of therapeutic molecules. The first chapter of this work is a general introduction that defines the context in which the projects carried out during this thesis are placed. In particular, the concept of nanomedicine is described, as well as the main strategies for the development of efficient nanopharmaceutical devices and the related challenges. Furthermore, a brief presentation of mesoporous silica materials is given. Next, the general objectives that are addressed in the following experimental chapters are introduced. The third chapter is focused on the development of a targeting delivery system directed to Toll-like receptor 3 (TLR3) and based on mesoporous silica nanoparticles capped with the synthetic double stranded RNA (dsRNA) polyinosinic-polycytidylic acid (poly(I:C)). Poly(I:C) has shown cytotoxic effects in different types of cancer, and the results obtained in this work demonstrate its ability to trigger apoptotic pathways in breast cancer cells, thanks to its interaction with TLR3. Furthermore, loading the mesopores with the anthracyclinic antibiotic doxorubicin, a commonly used chemotherapeutic agent, allowed to achieve an enhanced therapeutic effect. In fact, a higher decrease of the cellular viability in SK-BR-3 cell line was observed. Chapter four shows the design of a nanoparticles cooperation strategy. The aim of this work is to improve the efficacy of the previously developed therapeutic approach for breast cancer through the combination of two gated mesoporous silica nanoparticles sets able to cooperate to achieve a medical goal. The first type, in fact, induces changes that enhance the interaction of the second one with the target cancer cell. In order to do that, nanoparticles loaded with 9-cis-retinoic acid and capped with interferon-¿, and nanoparticles loaded with sulforhodamine B dye and gated with poly(I:C) were synthesized. Taking advantage of the ability of both interferon-¿ and 9-cis-retinoic to increase TLR3 expression, we intended to improve the interaction of poly(I:C) functionalized nanoparticles with target cells. The obtained results show that the proposed combination strategy actually increased the uptake levels of poly(I:C) gated nanodevices in the cellular model selected. In the fifth chapter a system based on dendrimer-like mesoporous silica nanoparticles is presented. The higher pore volume of such materials makes them suitable for the achievement of the main aim of this project: the topical administration and controlled delivery of anti-vascular endothelial growth factor (VEGF) small interfering-RNA (siRNA) molecules to retinal pigmented epithelial cells. The nanodevices were synthesized, loaded with siRNA and finally functionalized with polyethylenimine chains, that act as molecular gate for the controlled release of the siRNA molecules and endosomal escape agent for cytosolic delivery. The obtained results in VEGF silencing in ARPE-19 cells highlight the noteworthy potential of the designed system as siRNA carrier. General conclusions regarding the works collected in this thesis are summarized in chapter six. / We thank the Spanish Government (projects SAF2010-21195 and MAT2012-38429-C04-01) and the Generalitat Valenciana (project PROMETEOII/2014/047) for support. A.U. and C.G. are grateful to the Ministry of Education, Culture and Sport for their doctoral fellowships. / Ultimo, A. (2019). Design and development of new bio-gated nanodevices for targeted controlled drug release [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/129875 / Compendio

Yes / Carbon monoxide (CO) is both a substance hazardous to health and a side product of a number of industrial processes, such as methanol steam reforming and large-scale oxidation reactions. The separation of CO from nitrogen (N2) in industrial processes is considered to be difficult because of the similarities of their electronic structures, sizes, and physicochemical properties (e.g., boiling points). Carbon monoxide is also a major poison in fuel cells because of its adsorption onto the active sites of the catalysts. It is therefore of the utmost economic importance to discover new materials that enable effective CO capture and release under mild conditions. However, methods to specifically absorb and easily release CO in the presence of contaminants, such as water, nitrogen, carbon dioxide, and oxygen, at ambient temperature are not available. Here, we report the simple and versatile fabrication of a new class of hybrid materials that allows capture and release of carbon monoxide under mild conditions. We found that carborane-containing metal complexes encapsulated in networks made of poly(dimethylsiloxane) react with CO, even when immersed in water, leading to dramatic color and infrared signature changes. Furthermore, we found that the CO can be easily released from the materials by simply dipping the networks into an organic solvent for less than 1 min, at ambient temperature and pressure, which not only offers a straightforward recycling method, but also a new method for the “on-demand” release of carbon monoxide. We illustrated the utilization of the on-demand release of CO from the networks by carrying out a carbonylation reaction on an electron-deficient metal complex that led to the formation of the CO-adduct, with concomitant recycling of the gel. We anticipate that our sponge-like materials and scalable methodology will open up new avenues for the storage, transport, and controlled release of CO, the silent killer and a major industrial poison. / The Royal Society, The Romanian Ministry of Education and Research, The University of Bradford, European Regional Development Fund of the European Union / Research Development Fund Publication Prize Award winner.

Yes / Understanding structure-property relationships is critical for the development of new drug delivery systems. This study investigates the properties of Pluronic smart hydrogel formulations for future use as injectable controlled drug carriers. The smart hydrogels promise to enhance patient compliance, decrease side effects and reduce dose and frequency. Pharmaceutically, these systems are attractive due to their unique sol-gel phase transition in the body, biocompatibility, safety and injectability as solutions before transforming into gel matrices at body temperature. We quantify the structural changes of F127 systems under controlled temperature after flow, as experienced during real bodily injection. Empirical formulae combining the coupled thermal and shear dependency are produced to aid future application of these systems. Induced structural transitions measured in-situ by small angle x-ray and neutron scattering reveal mixed oriented structures that can be exploited to tailor the drug release profile.

Yes / The Molecular Stress Function (MSF) constitutive model with convective constraint release mechanism has been shown to accurately fit a large range of viscometric data, and also shown to give strong vortex growth in flows of LDPE through planar and axisymmetric contractions. This work compares simulation and experimental results for 3-D flows of Lupolen 1840H LDPE through a contraction slit; 3-D effects are introduced by using a slit with a low upstream aspect ratio of 5:3. Comparisons are made with vortex opening angles obtained from streak photography, and also with stress birefringence measurements. The comparisons are made with two versions of the convective constraint release (CCR) mechanism. The simulated vortex angles for one version of the CCR mechanism are found to approach what is seen experimentally. The best-fit value for the stress optical coefficient was found to vary between CCRs and to decrease with flow rate. This is partially explained by different centreline elongational rates with the two CCRs, which in turn is related to different opening angles. A 3-D simulation is compared to the corresponding 2-D simulation. It is shown that both velocity vectors and birefringence show only small changes to around 60% of the distance to the side wall.

The available literature indicates that to date, few research has been performed on oil powders for topical delivery. The aim of this project was to investigate the release characteristics of oil powder formulations, as well as their dermal and transdermal delivery properties. Whey protein-stabilised emulsions were used to develop oil powders. Whey protein was used alone, or in combination with chitosan or carrageenan. Nine oil powders, with salicylic acid as the active ingredient, were formulated by using the layer-by-layer method. Three different pH values (pH 4, 5 and 6) were used to prepare the formulations, because of the different charges that polymeric emulsifiers may have. The characteristics of the prepared oil powders were determined, including their droplet sizes, particle size distributions, loss on drying, encapsulation efficiencies, oil leakage and water dispersibility. Release studies (membrane diffusion studies) were conducted by utilising cellulose acetate membranes (0.2 μm pore size) and Franz-type diffusion cells over a period of eight hours. The release of the active ingredient was determined for all nine powders, their respective template emulsions, as well as their respective oil powders redispersed in water. The release of salicylic acid from the respective redispersed oil powders was then further compared to its release from the template emulsions and from the oil powders. The effect of pH and different polymer types used in preparing the oil powders, their respective redispersed oil powders and the template emulsions were determined with regards to the release of the active ingredient from all these preparations. The effect of pH and different polymers used was furthermore determined on the oil powders and their respective redispersed oil powders, with regards to their dermal and transdermal deliveries. Transdermal delivery and skin uptake were investigated on specifically selected powders only, based on the outcomes of the oil powder characterisation and release data. The qualifying formulations were chitosan pH 4, 5 and 6, whey and carrageenan pH 6 oil powders, together with their respective redispersed oil powders in water. Human abdominal skin was dermatomed (thickness 400 μm) for use in the diffusion studies. Franz-type diffusion cells were used over a period of 24 hours. The results of the membrane release studies indicated that the oil powders had achieved a significantly higher release than their respective redispersed oil powders. The release of salicylic acid from the redispersed oil powders and from their respective emulsions was similar. The transdermal delivery test outcomes showed that the effect of pH could have been influenced by the degree of ionisation, resulting in a decrease in permeation with increasing ionisation of salicylic acid, in accordance with the pH partition hypothesis. Furthermore, biopolymers, such as chitosan had demonstrated a penetration enhancing effect, which had led to the enhanced dermal and transdermal delivery of salicylic acid. A correlation was also found between the powder particle size and transdermal delivery. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

The available literature indicates that to date, few research has been performed on oil powders for topical delivery. The aim of this project was to investigate the release characteristics of oil powder formulations, as well as their dermal and transdermal delivery properties. Whey protein-stabilised emulsions were used to develop oil powders. Whey protein was used alone, or in combination with chitosan or carrageenan. Nine oil powders, with salicylic acid as the active ingredient, were formulated by using the layer-by-layer method. Three different pH values (pH 4, 5 and 6) were used to prepare the formulations, because of the different charges that polymeric emulsifiers may have. The characteristics of the prepared oil powders were determined, including their droplet sizes, particle size distributions, loss on drying, encapsulation efficiencies, oil leakage and water dispersibility. Release studies (membrane diffusion studies) were conducted by utilising cellulose acetate membranes (0.2 μm pore size) and Franz-type diffusion cells over a period of eight hours. The release of the active ingredient was determined for all nine powders, their respective template emulsions, as well as their respective oil powders redispersed in water. The release of salicylic acid from the respective redispersed oil powders was then further compared to its release from the template emulsions and from the oil powders. The effect of pH and different polymer types used in preparing the oil powders, their respective redispersed oil powders and the template emulsions were determined with regards to the release of the active ingredient from all these preparations. The effect of pH and different polymers used was furthermore determined on the oil powders and their respective redispersed oil powders, with regards to their dermal and transdermal deliveries. Transdermal delivery and skin uptake were investigated on specifically selected powders only, based on the outcomes of the oil powder characterisation and release data. The qualifying formulations were chitosan pH 4, 5 and 6, whey and carrageenan pH 6 oil powders, together with their respective redispersed oil powders in water. Human abdominal skin was dermatomed (thickness 400 μm) for use in the diffusion studies. Franz-type diffusion cells were used over a period of 24 hours. The results of the membrane release studies indicated that the oil powders had achieved a significantly higher release than their respective redispersed oil powders. The release of salicylic acid from the redispersed oil powders and from their respective emulsions was similar. The transdermal delivery test outcomes showed that the effect of pH could have been influenced by the degree of ionisation, resulting in a decrease in permeation with increasing ionisation of salicylic acid, in accordance with the pH partition hypothesis. Furthermore, biopolymers, such as chitosan had demonstrated a penetration enhancing effect, which had led to the enhanced dermal and transdermal delivery of salicylic acid. A correlation was also found between the powder particle size and transdermal delivery. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Thesis (LLM) -- Stellenbosch University, 2004. / Stellenbosch University. Faculty of Law. Dept. of Public Law. / ENGLISH ABSTRACT: When evaluating a bail application, the court must take into consideration the interests of the accused against those of the community. The main goal of this assessment is to find a balance between said interests. While an application for bail is made at a stage where guilt has not been proven, it is of extreme importance that the court must not infringe on the fundamental rights of the accused which include the right to personal freedom and the presumption of innocence. In order to protect the rights of the bail applicant within the proper functioning of the legal system, it is important to determine the nature of bail proceedings. As shown in this thesis, bail proceedings are sui gelleris in nature, which means that a separate set of rules of the law of evidence is applicable to these proceedings. The object of this thesis is to identify the rules of law of evidence applicable to bail proceedings as well as to clarify the deviation from the normal rules of evidence which apply to the trial of the accused. In chapter one the purpose and nature of bail proceedings as well as the characteristics of accusatorial and inquisitorial systems, are discussed. Problem areas within the South African legal system with regards to bail applications are also highlighted in this chapter. In chapter two the application of the primary rules of the law of evidence with regards to bail proceedings are investigated as well as the admissibility of evidence pertaining to prior convictions of the applicant, opinion evidence and character evidence. In chapter three the admissibility of hearsay evidence at bail proceedings is discussed. The constitutionality of the privilege pertaining to the police docket is dealt with in chapter four. Chapter five deals with the infom1er's privilege. The requirements that have to be met in order to qualify for protection under the said privilege, are examined. Chapter six focuses on the privilege against self-incrimination and the manner In which it is applied in bail proceedings. The provisions of s 60(11B)(c) of the Criminal Procedure Act and the role of the presiding officer are also discussed in this chapter. Chapter seven focuses on the burden of proof in bail applications. Chapter eight contains a summary and recommendations. / AFRIKAANSE OPSOMMING: By die beoordeling van 'n borgaansoek moet die hof die be lange van die beskuldigde en die belange van die samelewing teen mekaar opweeg. Die doel van die betrokke verrigtinge is om 'n balans tussen hierdie belange te vind. Omdat borgtog ter sprake kom op 'n tydstip waar daar nog geen skuldigbevinding is nie, is dit van kardinale belang dat die hof ten aile tye moet waak teen die onregverdige inbreukmaking op die beskuldigde se fundamentele regte wat onder andere die reg op individuele vryheid en die vem10ede van onskuld insluit. Ten einde die regte van die borgaansoeker na behore te beskem1 sonder om die behoorlike funksionering van die regstelsel te belemmer, is dit belangrik om vas te stel wat die aard van borgverrigtinge is. Soos in hierdie tesis aangetoon word, is borgverrigtinge sui generis van aard. Dit het tot gevolg dat daar 'n aparte stel reels van die bewysreg bestaan wat op hierdie verrigtinge van toepassing is. In hierdie tesis word daar gepoog om die reels van die bewysreg wat op borgverrigtinge van toepassing is, te identifiseer en om die afwykings van die gewone bewysregreels wat op die verhoor van toepassing is, te verklaar. In hoofstuk een word die doel en aard van borgverrigtinge bespreek en word die kenmerke van die akkusatoriale en inkwisitoriale stelsels teen mekaar gestel. Die onduidelikhede oor die aard van borgverrigtinge in die Suid-Afrikaanse reg word ook aangeraak. In hoofstuk twee word die toepassing van die relevantheidsgrondreel by borgverrigtinge ondersoek, asook die toelaatbaarheid van getuienis oor die vorige veroordelings van die beskuldigde, opiniegetuienis en karaktergetuienis. Hoofstuk drie het betrekking op die toelaatbaarheid van hoorsegetuienis by borgverrigtinge. In hoofstuk vier word kwessies rakende dossierprivilegie behandel en die grondwetlikheid van sodanige privilegie, asook die toepassing daarvan, word van naderby beskou. In hoofstuk vyf word daar gefokus op die aanbrengersprivilegie. Die aard en toepassing van die privilegie asook die vereistes waaraan voldoen moet word alvorens daar op die betrokke privilegie gesteun kan word, word aangeraak. Hoofstuk ses fokus op die borgapplikant se privilegie teen selfinkriminasie. Die bepalings van a 60(11 B)(c) asook die rol van die voorsittende beampte word ook in hierdie hoofstuk aangespreek. Die sewende kwessie wat in verband met borgverrigtinge in die stu die ondersoek word, is die ligging van die bewyslas by sodanige verrigtinge. Dit word In hoofstuk sewe gedoen. Hoofstuk agt bevat 'n opsomming van sowel bevindings as aanbevelings.

Abstract : The major objective of our study is to investigate DNA damage induced by soft X-rays (1.5 keV) and low-energy electrons (˂ 30 eV) using a novel irradiation system created by Prof. Sanche’s group. Thin films of double-stranded DNA are deposited on either glass and tantalum substrates and irradiated under standard temperature and pressure surrounded by a N[subscript 2] environment. Base release (cytosine, thymine, adenine and guanine) and base modifications (8-oxo-7,8-dihydro -2’-deoxyguanosine, 5-hydroxymethyl-2’-deoxyuridine, 5-formyl-2’-deoxyuridine, 5,6-dihydrothymidine and 5,6-dihydro-2’-deoxy uridine) are analyzed and quantified by LC-MS/MS. Our results reveal larger damage yields in the sample deposited on tantalum than those on glass. This can be explained by an enhancement of damage due to low-energy electrons, which are emitted from the metal substrate. From a comparison of the yield of products, base release is the major type of damage especially for purine bases, which are 3-fold greater than base modifications. A proposed pathway leading to base release involves the formation of a transient negative ion (TNI) followed by dissociative electron attachment (DEA) at the N-g lycosidic bond. On the other hand, base modification products consist of two major types of chemical modifications, which include thymine methyl oxidation products that likely arises from DEA from the methyl group of thymine, and 5,6-dihydropyrimidine that can involve the initial addition of electrons, H atoms, or hydride ions to the 5,6-pyrimidine double bond. / Résumé: L'objectif majeur de ce projet étude est d'étudier les lésions d'ADN induites par les rayons X mous (1,5 keV) et des électrons de faible énergie (˂ 30 eV) à partir d'un nouveau système d'irradiation créé par le groupe du Pr. Sanche. De minces couches d'ADN double brin sont déposées soit sur du verre ou sur les substrats de tantale. Celles-ci sont irradiées sous une température et pression environnante, mais dans une atmosphère de N[indice inférieur 2]. Les bases relâchées (cytosine, la thymine, l'adénine et la guanine) et les produits de modification de base (8-oxo-7,8-dihydro-2'-désoxyguanosine, 5-hydroxyméthyl-2'-désoxyuridine, 5-formyl-2'-désoxyuridine, 5,6-dihydrothymine et 5,6-dihydrouridine) sont analysés et quantifiés par LC-MS/MS. Nos résultats révèlent un plus grand rendement de dommages dans les échantillons déposés sur le tantale que celles sur le verre. Cette différence peut être expliquée par l’interaction des électrons de faible énergie qui sont photo émis des substrats métalliques. D'après les résultats obtenus, la libération de bases est un produit majeur en comparaison avec la modification de bases. Ceci provient, en particulier, surtout des purines qui libèrent la base a un niveau trois fois plus grand que la modification de la base. Une voie proposée, conduisant à la libération de base, implique la formation d'ions négatifs transitoires (TNI), suivie par l'attachement d'électrons dissociatifs (DEA) à la liaison N-glycosidique. En outre, les produits de modification de base sont composés en deux grands types de modifications chimiques. L’un des produits est l’oxydation du groupe méthyle de la thymine, qui probablement consiste de en d'hydrure (-H[indice supérieur -]) par l'intermédiaire de DEA. Alors que l’autre modification chimique est la formation de 5,6-dihydropyrimidine qui implique l'addition d'hydrure à la double liaison du 5,6-pyrimidine.

Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The growing global epidemic of human tuberculosis (TB) results in 8 million new cases of this disease and 2 million deaths annually. Control thereof will require greater insight into the biology of the causative organism, Mycobacterium tuberculosis, and into the pathogenesis of the disease. This will benefit the design of new vaccines and diagnostic assays which may reduce the degree of both disease transmission and progression. Animal models have played a vital role in the understanding of the aetiology, pathogenesis, and treatment of TB. Much of such insight has been obtained from experimental infection models, and the development of new vaccines, for example, is dependant on these. Nonetheless, studies utilising naturally occurring TB in animals, such as those which have investigated the use of interferon-gamma release assays (IGRA) for its diagnosis, have contributed substantially to the body of knowledge in this field. However, there are few such examples, and this study sought to identify and investigate naturally occuring animal TB in South Africa as an opportunity to gain further insight into this disease. During the course of this study, the dassie bacillus, a distinctly less virulent variant of M. tuberculosis, was isolated from a rock hyrax from the Western Cape Province of South Africa. This has provided new insight into the widespread occurrence of this organism in rock hyrax populations, and has given impetus to further exploring the nature of the difference in virulence between these pathogens. Also investigated was M. tuberculosis infection in dogs in contact with human TB patients. In so doing, the first reported case of canine TB in South Africa was described, v a novel canine IGRA was developed, and a high level of M. tuberculosis infection in these animals was identified. This supports human data reflecting high levels of transmission of this pathogen during the course of human disease. Additionally, the fact that infected companion animals may progress to disease and potentially act as a source of human infection was highlighted. However, an attempt to adapt a flow cytometric assay to study cell-mediated immune responses during canine TB revealed the limitations of such studies in species in which the immune system remains poorly characterised. The use of IGRAs to diagnose TB was further explored by adapting a human assay, the QuantiFERON-TB Gold (In-Tube Method), for use in non-human primates. These studies have shown that such an adaption allows for the sensitive detection of TB in baboons (Papio ursinus) and rhesus macaques (Macaca mulatta) and may be suitable for adaption for use in other species. However, they have also evidenced the limitation of this assay to specifically detect infection by M. tuberculosis. Finally, to contextualise the occurrence of the mycobacterial infections described above, and other similar examples, these have been reviewed as an opinion piece. Together, these investigations confirm that animal models will continue to make important contributions to the study of TB. More specifically, they highlight the opportunities that naturally occuring animal TB provides for the discovery of novel insights into this disease. / AFRIKAANSE OPSOMMING: Wêreldwye tuberkulose (TB) epidemie veroorsaak agt miljoen nuwe gevalle en twee miljoen sterftes jaarliks. Ingryping by die beheer hiervan vereis begrip van die biologie van die mikroörganisme Mycobacterium tuberculosis, die oorsaak van TB, asook van die patogenese van die siekte self. Hierdie kennis kan lei tot ontwerp van nuwe entstowwe en diagnostiese toetse wat gevolglik beide die oordrag- en vordering van die siekte mag bekamp. Dieremodelle speel lankal 'n rol in ons begrip van die etiologie-, patogenese- en behandeling van TB. Insig is grotendeels verkry vanaf eksperimentele infeksiemodelle, en ontwikkeling van entstowwe, onder andere, is afhanklik van soortgelyke modelle. Desnieteenstaande, studies wat natuurlike TB voorkoms in diere ondersoek, byvoorbeeld dié wat op die ontwikkeling van interferon-gamma vrystellingstoetse (IGVT) fokus, het merkwaardige bydrae gemaak tot kennis en begrip in hierdie studieveld. Daar is slegs enkele soortgelyke voorbeelde. Om hierdie rede is die huidige studie uitgevoer waarbinne natuulike diere-TB geïdentifiseer en ondersoek is in Suid-Afrika om verdere kennis en insig te win aangaande TB. Die "dassie bacillus", bekend om beduidend minder virulent te wees as M. tuberculosis, is tydens hierdie studie geïsoleer vanuit 'n klipdassie (Procavia capensis) in die Wes-Kaapse provinsie, Suid-Afrika. Insig in die wydverspreide voorkoms van hierdie organisme in klipdassie bevolkings is gevolglik verkry en verskaf momentum om die aard van verskil in virulensie tussen dié patogene te bestudeer. vii Voorts is M. tuberculosis infeksie bestudeer in honde wat in kontak is met menslike TB pasiënte en word die eerste geval van honde TB dus in Suid-Afrika beskryf. In hierdie groep diere, is 'n hoë vlak van M. tuberculosis infeksie geïdentifiseer deur gebruik te maak van 'n nuut ontwikkelde IGVT vir die diagnose van honde TB. Gevolglik ondersteun dié studie bevindinge van menslike studies wat toon dat besondere hoë vlakke van M. tuberculosis oordrag voorkom gedurende die verloop van die siekte. Verder toon die studie dat geïnfekteerde troeteldiere 'n bron van menslike infeksie kan wees. 'n Poging om 'n vloeisitometriese toets te ontwikkel om die aard van selgefundeerde immuunreaksies te bestudeer in honde met TB toon die beperkings van dergelike studies in spesies waarin die immuunsisteem gebrekkig gekarakteriseer is. Die gebruik van IGVT'e in die diagnose van TB is verder ondersoek deur 'n menslike toets (QuantiFERON-TB Gold, In-Tube Method) aan te pas vir die gebruik van nie-menslike primaat gevalle. Hierdie studies toon gevolglik dat so 'n aanpassing toepaslik is vir hoogs sensitiewe deteksie van TB in chacma bobbejane (Papio ursinus) en rhesus ape (Macaca mulatta), en mag ook aangepas word vir gebruik in ander spesies. Tog word die beperkings van hierdie toets om infeksie wat spesifiek deur M. tuberculosis veroorsaak uitgelig. Ter afsluiting word hierdie studie in konteks geplaas deur 'n oorsig te gee van bogenoemde- en soortgelyke gevalle van dierlike infeksie deur mikobakterieë in Suid-Afrika. Hierdie studies bevestig dat dieremodelle steeds belangrike toevoegings maak tydens die bestudering van TB en lig veral die moontlikhede uit dat bestudering van natuulike TB in diere kan lei tot die ontdekking van nuwe insigte ten opsigte van die siekte self.

Σκοπός της παρούσας διδακτορικής διατριβής είναι η σύνθεση, ο χαρακτηρισμός και η βιολογική αξιολόγηση τροποποιημένων πολυλειτουργικών νανοπεριεκτών (ΠΝΠ) ως συστημάτων μεταφοράς φαρμάκων (ΣΜΦ), με άμεση εφαρμογή στη θεραπεία του καρκίνου του μαστού και του προστάτη. Με τον όρο νανοπεριέκτες (ΝΠ) εννοούμε τα σωματίδια τα οποία βρίσκονται στην κλίμακα του νανομέτρου και κυμαίνονται σε διαστάσεις από περίπου 1nm έως και 100 nm. Ο όρος πολυλειτουργικά αναφέρεται στις ιδιότητες αυτών των νανοδομών και πιο συγκεκριμένα στο πως μεταβάλλονται οι ιδιότητες αυτών όταν υπάρχει επίδραση εξωτερικών παραγόντων όπως θερμοκρασία, pH, όξειδο-αναγωγικό περιβάλλον και μεταβαλλόμενο μαγνητικό πεδίο. Για την παρασκευή των πολυμερικών νανοπεριεκτών χρησιμοποιήθηκαν διάφορα είδη πολυμερισμών όπως, πολυμερισμός γαλακτώματος μέσω ριζών, πολυμερισμός σπόρου (seed), πολυμερισμός μεταφοράς ατόμου με ρίζες, πολυμερισμός διασποράς και πολυμερισμός μέσω απόσταξης-καταβύθισης. Οι διάφορες ευαισθησίες στα ΝΣ προστέθηκαν μέσω συμπολυμερισμού διαφόρων μονομερών τα οποία παρουσιάζουν τις προαναφερθείσες ιδιότητες. Κάποια από τα μονομερή τα οποία χρησιμοποιήθηκαν, όπως το υδρόξυ προπυλικό μεθακρυλαμίδιο (HPMA) το οποίο παρουσιάζει ευαισθησία στη μεταβολή της θερμοκρασίας και το 3-Μεθυλ-N-(2-((2-(3-οξοβουταναμιδο)εθυλ) δισουλαφανυλ)εθυλ)βουτ-3-εναμίδιο (Disulfide) το οποίο παρουσιάζει ευαισθησία στις μεταβολές του όξειδο-αναγωγικού περιβάλλοντος συνετέθησαν στο εργαστήριο, ενώ τα υπόλοιπα ήταν εμπορικά διαθέσιμα. Το κύριο μονομερές το οποίο χρησιμοποιήθηκε στους πολυμερισμούς είναι ο Μεθακρυλικός Μεθυλεστέρας (MMA) το οποίο είναι μη τοξικό και κατά τον πολυμερισμό του δημιουργεί σφαιρικές δομές συγκεκριμένου μεγέθους (νανοσφαίρες). Με τη χρήση του συγκεκριμένου μονομερούς συνετέθησαν συμπολυμερή, τα οποία είναι ευαίσθητα στη θερμοκρασία, στο pH και στο όξειδο-αναγωγικό περιβάλλον ή σε συνδυασμό των παραγόντων αυτών. Οι πολυμερικές νανοσφαίρες-νανοπεριέκτες οι οποίες συνετέθησαν είναι κενές στο εσωτερικό τους, ή είναι της μορφής πυρήνας-κέλυφος, όπου το κέλυφος περιέχει τα μονομερή με τις επιθυμητές ευαισθησίες. Η κοιλότητα η οποία δημιουργείται στο εσωτερικό των νανοσφαιρών, σε ορισμένες περιπτώσεις, έχει ως σκοπό τον εγκλωβισμό φαρμακευτικών ουσιών. Τα φαρμακευτικά μόρια τα οποία χρησιμοποιούνται στην παρούσα διδακτορική διατριβή, είτε στο εσωτερικό των νανοσφαιρών είτε προσδεδεμένα στην επιφάνεια αυτών, είναι η Δοξορουβικίνη (Doxorubicin) και η Δαουνοροβικίνη (Daunorubicin). Η επαγωγή των μαγνητικών ιδιοτήτων στους νανοπεριέκτες πραγματοποιήθηκε με τη σύνθεση μαγνητικών νανοσωματιδίων (ΜΝΣ) τα οποία παρασκευάστηκαν πάνω στην επιφάνειά τους. Η χρήση ενός εναλλασσόμενου μαγνητικού πεδίου αυξάνει τοπικά τη θερμοκρασία με αποτέλεσμα, αφενός να διευκολύνεται η τοπική απελευθέρωση της εγκλωβισμένης φαρμακευτικής ουσίας και αφετέρου, εξαιτίας της υψηλής θερμοκρασίας που αναπτύσσεται τοπικά, να οδηγείται το καρκινικό κύτταρο σε απόπτωση (προγραμματισμένος κυτταρικός θάνατος). Για το χαρακτηρισμό των νανοπεριεκτών χρησιμοποιήθηκε μία πληθώρα τεχνικών. Για το μορφολογικό χαρακτηρισμό χρησιμοποιήθηκε η ηλεκτρονική μικροσκοπία σάρωσης και διέλευσης, Scanning Electron Microscopy (SEM) και Transmission Electron Microscopy (ΤΕΜ) αντίστοιχα, για το δομικό, η φασματοσκοπία υπερύθρου (FT-IR), η φασματοσκοπία Raman, η φασματοσκοπία πυρηνικού μαγνητικού συντονισμού (Nuclear Magnetic Resonance, NMR) και η τεχνική περίθλασης ακτίνων-Χ, ενώ για τη μελέτη των μαγνητικών ιδιοτήτων χρησιμοποιήθηκε η φασματοσκοπία δονούμενου δείγματος Vibrating Sample Magnetometry, VSM). Επίσης χρησιμοποιήθηκαν η τεχνική της δυναμικής σκέδασης φωτός (Dynamic Light Scattering, DLS) η οποία έδωσε πληροφορίες για τις ιδιότητες και τη συμπεριφορά των νανοπεριεκτών μέσα στο διάλυμα, καθώς επίσης και η φασματοσκοπία ορατού-υπεριώδους (Ultra Violet-Visible, UV-VIS), μέσω της οποίας έγινε μελέτη του εγκλωβισμού και της απελευθέρωσης των χρησιμοποιούμενων φαρμάκων από τις νανoσφαίρες. Τέλος, πραγματοποιήθηκαν μελέτες υπερθερμίας με τη βοήθεια ενός εξωτερικού εναλλασσόμενου μαγνητικού πεδίου. / The aim of this thesis is the synthesis, characterization and biological evaluation of modified multifunctional nanoparticles (MMNs) as drug delivery systems (DDS, with immediate effect in the treatment of breast and prostate cancer. The term nanoparticles (NPs) refer to the particles which are in the nanometer scale and range in size, from about 1nm to and 100 nm. The term multifunctional refers to the properties of these nanostructures and more particularly to the way that change their properties when external factors such as temperature, pH, redox environment and alternating magnetic field are applied. For the preparation of polymeric nanoparticles, different types of polymerizations were used such as, emulsion polymerization, seed polymerization, atom transfer radical polymerization, dispersion polymerization and polymerization through distillation-precipitation process. The different sensitivities were added via copolymerization of different monomers which exhibit the aforementioned properties. Some of the monomers used like, N-(-2-HydroxyPropyl) Methacrylamide (HPMA), which is sensitive to temperature changes and N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2-methylacrylamide) (Disulfide) which is sensitive to changes in the redox environment, were synthesized in the laboratory, while the rest, were commercially available. The primary monomer, used in the polymerizations, was Methyl Methacrylate (MMA) which is non-toxic and in through to its polymerization creates spherical structures of certain size (nanospheres). By using the specific monomer, copolymers, which are sensitive to temperature, pH and redox environment or a combination of these factors, were synthesized. The synthesized polymeric nanospheres-nanocontainers, are either hollow inside, or form core-shell structures, where the shell contains the monomers with the desired sensitivity. The cavity which is created inside the nanospheres, in some cases, is intended to entrap pharmaceutical substances. The drug molecules used in this thesis, either within or tethered to the surface of the nanospheres are, Doxorubicin (DOX) and Daunorubicin (DNR). The induction of the magnetic properties in nanocontainers was performed by the synthesis of magnetic nanoparticles (MNPs), prepared on their surface. The use of an alternating magnetic field increases the temperature locally resulting on one hand to facilitate local release of the entrapped drug, and on the other hand, because of the high temperatures developed locally, to lead the tumor cell to apoptosis (programmed cell death). For the characterization of nanoparticles a variety of techniques were used. For the morphological characterization, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) were used while for the structural characterization, Fourier Transform Infrared Spectroscopy (FT-IR), Raman Spectroscopy, Nuclear Magnetic Resonance (NMR) and X-ray diffraction Spectroscopy were used. Magnetic properties were studied by Vibrating Sample Magnetometry (VSM). The Dynamic Light Scattering technique (DLS) was also used in order to get information about the properties and behavior of nanoparticles in solution and Ultra Violet-Visible Spectroscopy (UV-VIS), gave information about Loading and Release of the drugs used in nanocontainers. Hyperthermia measurements were carried out by using an external alternating magnetic field.