<p>Biotherapeutics, such as monoclonal antibodies (mAbs), represent a primary mechanism for treatment of human disease, and there has been a steady increase in Food and Drug Administration approvals since the first one in 1982. Subcutaneous (SC) injection of protein-based therapeutics is a convenient and clinically established drug delivery method that increases the convenience and reduces cost compared to other delivery methods. However, progress is needed to optimize bioavailability via this route. This dissertation describes the methods for evaluation of mass transport of high molecular weight proteins, such as mAbs, following SC injection using <em>in vitro</em> and <em>ex vivo</em> modeling developed to describe the factors relevant for optimal distribution prior to uptake into systemic circulation. The first chapter describes a novel collagen and hyaluronic acid (HA) based hydrogel for investigation of macromolecule transport based on the physiochemical properties of the diffusing molecule and the tissue matrix. This initial study demonstrated that, in collagen alone, collagen combined with HA, and HA alone, the molecules demonstrated different transport paradigms dependent primarily on molecule size, matrix viscosity, and electrostatic charge, respectively. This showed that the local tissue heterogeneity and therapeutic properties could be determining factors for molecule transport and bioavailability. The second, third, and fourth chapters describe two novel platforms for the investigation of injection plume formation in SC tissue utilizing three-dimensional X-ray tomography. Injection plume analysis has been studied comprehensively in the context of insulin transport using co-injection of radiopaque dyes to track the protein distribution. However high molecular weight therapeutics have vastly different physiochemical properties than insulin and are injected under different rates, concentrations, volumes, and viscosities due to dosing considerations. To address the gap mAb distribution, we first developed a novel protein conjugated to an x-ray contrast agent to directly track injection plume formation and investigated the effects of injection rate and tissue location through injections into ex vivo porcine tissue, described in chapters three and four. Ex vivo tissue analysis showed that the rate did not influence the distribution, however, plume volume was lower in porcine belly compared to neck tissue. Whereas porcine tissue is an excellent model to represent the structural features of human injection, the large heterogeneity between animal subjects and collected samples is a disadvantage. Therefore, the fourth chapter describes the fabrication of a gelatin hydrogel-based injection platform representing the dermal and subcutaneous tissue layers for controlled injection plume analysis. In summary, all three models represent useful platforms for the assessment of macromolecular mass transport, pharmaceutical autoinjector performance, as well as the potential impact of tissue properties and intersubject heterogeneity on plume formation. Overall, the findings in these studies might better inform drug designers and clinicians on how to most optimally engineer an injection to deliver the most efficient patient outcomes through better dosing and increased cost savings. </p>

<p>Radiotherapy is used in many clinics to deliver a sufficient and uniform dose to the cancerous tumours while the dose to normal tissues is minimized. However, there is a possibility of missing the target volume due to patient set up/motion errors, or any fluctuation in treatment delivery. Therefore, accurate dose verification tools are essential to evaluate the delivered dose distribution of the designed treatment plan under realistic treatment conditions.</p> <p>Current research is focused on developing 3D dose verification tools to record the complex dose distributions for quality assurance purposes and the evaluation of new treatment techniques. New and novel materials and read-out techniques suitable for use in hospitals are desirable. The objective of this research is to fabricate a transparent radiochromic gel dosimeter that may be used as quality assurance tool. Also, the fabricated gel must be analyzed using a simple optical read-out technique.</p> <p>Gel dosimeters are gels that undergo some chemical changes upon irradiation as a function of absorbed dose. The absorbed dose may be recorded in three dimensions depending on the type of gel dosimeter. Radiochromic gels are dosimeters that change colour upon irradiation. A radiosensitive dye, leucomalachite green (LMG) is dissolved in a matrix material to record the dose distribution in 3D. LMG changes its colour upon irradiation, and has an absorbance band of 629nm.</p> <p>In this research two different matrix materials were investigated: poly (vinyl alcohol) and gelatin. PVA was studied as the primary agent due to its adjustable mechanical strength and high transparency. PVA has also been studied to have a low diffusion rate when it was used as the matrix material in Fricke gel dosimeters [41]. Even though PVA had all the desired characteristics, fabricating a PVA based radiochromic dosimeter was not successful. Consequently, gelatin was used as the matrix material to fabricate a gelatin-based radiochromic dosimeter.</p> <p>Using gelatin, highly transparent radiosensitive gels were successfully fabricated. The absorbencies of the irradiated gels were measured as a function of absorbed dose, using a 1D set up. After, the gels were formed into 5mm thick films and used as two-dimensional dose verification tools. The relationship between absorbance and absorbed dose for 1D measurement was obtained to be 0.00241± 0.00004 , and 0.0022 ± 0.00007 for 2D gels scaled to a thickness of 1 cm.</p> <p>In all of the experiments the absorbance-dose relationships were similar in slopes, but there was an offset between different batches. The offset was 20% between the different experiments. Moreover, there was less than 5% error associated with the physical set up; the major source of error was due to the production and handling of the mixture, possibly due to the effects of inconsistent heating and UV light exposure.</p> <p>The 2D gels were used to verify the dose distribution for the purpose of quality assurance. Six different complicated beams were delivered to the gels and their dose distributions were compared to their respective Pinnacle Calculated Planar (PCP) dose maps. The difference was found to be about 35% at worst; however, this error may be reduced by utilizing more sophisticated data processing methods. Nevertheless, the images were quite similar above 20Gy. Furthermore, the dose distributions recorded by the gels are qualitatively and quantitatively similar to the (PCP) dose map. Although the fabricated gel dosimeters show some promise as future tools for quality assurance purposes, they must go through many more stages of research to be used clinically.</p> / Master of Science (MSc)

Tesis por compendio / [ES] El cartílago hialino es un tejido conectivo denso con poca capacidad de auto regeneración cuando es afectado por patologías degenerativas. Por lo tanto, la estimulación eléctrica se ha propuesto como una terapia alternativa no invasiva para mejorar la reparación del cartílago hialino. De acuerdo con esto, este trabajo presenta un enfoque computacional y experimental combinado para entender mejor la biología del cartílago hialino y su respuesta a la estimulación eléctrica usando diferentes modelos in vitro. En primer lugar, se ha desarrollado un modelo mecanobiológico para simular el proceso de osificación endocondral. Por otro lado, se ha evaluado el efecto de la estimulación eléctrica sobre el cartílago hialino en tres escenarios diferentes. Inicialmente se ha analizado la proliferación celular y la síntesis de glicosaminoglicanos de condrocitos cultivados en monocapa y estimulados con campos eléctricos. Luego, se ha realizado un análisis histomorfométrico a explantes de condroepífisis que fueron estimulados eléctricamente. Por último, se ha evaluado el efecto de los campos eléctricos sobre la diferenciación condrogénica de células madre mesenquimales cultivadas en hidrogeles. Los resultados indican que la estimulación eléctrica es un estímulo biofísico prometedor, ya que este tipo de estimulación mejora la viabilidad y la proliferación celular, induce cambios morfológicos en los condrocitos, y estimula la síntesis de las principales moléculas que componen el cartílago hialino, tales como SOX-9, glicosaminoglicanos y agrecan. Además, este proyecto es el primer paso hacia la implementación de un estímulo biofísico alternativo que modifica la dinámica celular de los condrocitos de la placa de crecimiento en condiciones ex vivo. Adicionalmente, este estudio resalta el efecto potencial de los campos eléctricos para inducir el proceso de condrogénesis de células madre mesenquimales cultivadas en condiciones basales. En general, la evaluación de la estimulación eléctrica sobre condrocitos, tejidos y andamios es una herramienta útil que puede contribuir al conocimiento actual de las terapias regenerativas enfocadas en la regeneración del cartílago hialino. / [CA] El cartílag hialí és un teixit connectiu dens amb poca capacitat d'auto regeneració quan es veu afectat per patologies degeneratives. Per tant, l'estimulació elèctrica s'ha proposat com una teràpia alternativa no invasiva per millorar la reparació del cartílag articular. D'acord amb això, aquest treball presenta un enfoc computacional i experimental combinat per entendre millor la biologia del cartílag hialí i la seva resposta a l'estimulació elèctrica usant diferents models in vitro. En primer lloc, s'ha desenvolupat un model mecanobiològic per simular el procés d'ossificació endocondral. D'altra banda, s'ha avaluat l'efecte de l'estimulació elèctrica sobre el cartílag hialí en tres escenaris diferents. Inicialment s'ha analitzat la proliferació cel·lular i la síntesi de glicosaminoglicans de condròcits cultivats en monocapa i estimulats amb camps elèctrics. Després, s'ha realitzat una anàlisi histomorfomètrica a explants de condroepífisis que van ser estimulats elèctricament. Finalment, s'ha avaluat l'efecte dels camps elèctrics sobre la diferenciació condrogénica de cèl·lules mare mesenquimals cultivades en hidrogels. Els resultats indiquen que l'estimulació elèctrica és un estímul biofîsic prometedor, ja que aquest tipus d'estimulació millora la viabilitat i la proliferació cel·lular, indueix canvis morfològics en els condròcits, i estimula la síntesi de les principals molècules que componen el cartílag hialí, com ara SOX-9, glicosaminoglicans i agrecan. A més, aquest projecte és el primer pas cap a la implementació d'un estímul biofísic alternatiu que modifica la dinàmica cel·lular dels condròcits de la placa de creixement en condicions ex vivo. Addicionalment, aquest estudi ressalta l'efecte potencial dels camps elèctrics per induir el procés de condrogènesi de cèl·lules mare mesenquimals cultivades en condicions basals. En general, l'avaluació de l'estimulació elèctrica sobre condròcits, teixits i scaffolds és una eina útil que pot contribuir al coneixement actual de les teràpies regeneratives enfocades a la regeneració del cartílag hialí. / [EN] Hyaline cartilage is a dense connective tissue with low self-healing capacity when is affected by degenerative pathologies. Therefore, electrical stimulation has been proposed as a possible non-invasive alternative therapy to enhance the restoration of the cartilaginous tissue. Accordingly, this work presents a combined computational and experimental approach to understand better the hyaline cartilage biology and its response to electrical stimulation using different in vitro models. On the one hand, a mechanobiological model was developed to simulate the endochondral ossification process. On the other hand, the electrical stimulation on hyaline cartilage was evaluated in three different scenarios. Initially, cell proliferation and glycosaminoglycans synthesis of chondrocytes, cultured in monolayer and stimulated with electric fields, was analyzed. Then, a histomorphometric analysis was performed to chondroepiphysis explants that were electrically stimulated. Finally, the effects of the electric fields on chondrogenic differentiation of mesenchymal stem cells cultured in hydrogels was assessed. The results indicated that electrical stimulation is a promising biophysical stimulus, due to the fact that this type of stimulation enhances the viability and the proliferation of cells, induces morphological changes in the chondrocytes, and stimulates the synthesis of the main molecules that compose the hyaline cartilage, such as SOX-9, glycosaminoglycans and aggrecan. Moreover, this project is the first step towards the implementation of an alternative biophysical stimulus that modifies the cellular dynamics of growth plate chondrocytes in ex vivo conditions. Additionally, this study highlights the potential effect of electric fields to induce the chondrogenesis process of mesenchymal stem cells cultured in basal conditions. Overall, the assessment of electrical stimulation on chondrocytes, tissues and scaffolds is a useful tool that may contribute to the current knowledge of regenerative therapies focused on hyaline cartilage healing. / To the financial support from COLCIENCIAS – COLFUTURO through the fellowship No. 647 for national doctorates. To the financial support from COLCIENCIAS through the research grant 712-2015 No. 50457. To the financial support from the Spanish Ministry of Economy and Competitiveness through the MAT2016-76039-C4-1-R project. / Vaca González, JJ. (2019). The effect of electric fields on hyaline cartilage: an in vitro and in silico study [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/120023 / Compendio

[EN] Articular cartilage is a tissue with low capacity for self-restoration due to its avascularity and low cell population. It is located on the surface of the subchondral bone covering the diarthrodial joints. Degeneration of articular cartilage can appear in athletes, in people with genetic degenerative processes (osteoarthritis or rheumatoid arthritis) or due to a trauma; what produces pain, difficulties in mobility and progressive degeneration that finally leads to joint failure. Self-restoration is only produced when the defect reaches the subchondral bone and bone marrow mesenchymal stem cells (MSCs) invade the defect. However, this new formed tissue is a fibrocartilaginous type cartilage and no a hyaline cartilage, which finally leads to degeneration. Transplantation of autologous chondrocytes has been proposed to regenerate articular cartilage but this therapy fails mainly to the absence of a material support (scaffold) for the adequate stimulation of cells. Matrix-induced autologous chondrocyte implantation uses a collagen hydrogel as scaffold for chondrocytes; however, it does not have the adequate mechanical properties, does not provide the biological cues for cells and regenerated tissue is not articular cartilage but fibrocartilage. Different approaches have been done until now in order to obtain a scaffold that mimics better articular cartilage properties and composition. Hydrogels are a good option as they retain high amounts of water, in a similar way to the natural tissue, and can closely mimic the composition of natural tissue by the combination of natural derived hydrogels. Their three-dimensionality plays a critical role in articular cartilage tissue engineering to maintain chondrocyte function, since monolayer culture of chondrocytes makes them dedifferentiate towards a fibroblast-like phenotype secreting fibrocartilage. Recently, injectable hydrogels have attracted attention for the tissue engineering of articular cartilage due to their ability to encapsulate cells, injectability in the injury with minimal invasive surgeries and adaptability to the shape of the defect. Following this new approach we aimed at synthesizing two new families of injectable hydrogels based on the natural protein gelatin for the tissue engineering of articular cartilage. The first series of materials consisted on the combination of injectable gelatin with loose reinforcing polymeric microfibers to obtain injectable composites with improved mechanical properties. Our results demonstrate that there is an influence of the shape and distribution of the fibers in the mechanical properties of the composite. More importantly bad fiber-matrix interaction is not able to reinforce the hydrogel. Due to this, our composites were optimized by improving matrix-fiber interaction through a hydrophilic grafting onto the microfibers, with very successful results. The second series of materials were inspired in the extracellular matrix of articular cartilage and consisted of injectable mixtures of gelatin and hyaluronic acid. Gelatin molecules in the mixtures provided integrin adhesion sites to cells, and hyaluronic acid increased the mechanical properties of gelatin. This combination demonstrated ability for the differentiation of MSCs towards the chondrocytic lineage and makes these materials very good candidates for the regeneration of articular cartilage. The last part of this thesis is dedicated to the synthesis of a non-biodegradable material with mechanical properties, swelling and permeability similar to cartilage. This material intends to be used as a platform in a bioreactor in which the typical loads of the joint are simulated, so that the hydrogels or scaffolds would fit in the recesses in the platform. The function of the platform is to simulate the effect of the surrounding tissue on the scaffold after implantation and could reduce animal experimentation by simulating in vivo conditions. / [ES] El cartílago articular es un tejido con baja capacidad de auto-reparación debida a su avascularidad y baja población celular. Se encuentra en la superficie del hueso subcondral cubriendo las articulaciones. La degeneración del cartílago articular puede aparecer en atletas, en personas con procesos genéticos degenerativos o debido a un trauma; lo que produce dolor, dificultades en la movilidad y degeneración progresiva que lleva al fallo de la articulación. La auto-reparación sólo se produce cuando el defecto alcanza el hueso subcondral y las células madre (MSCs) de la médula ósea invaden el defecto. Sin embargo, este nuevo tejido es un cartílago de tipo fibrocartilaginoso y no un cartílago hialino, el cual finalmente lleva a la degeneración. El trasplante de condrocitos autólogos ha sido propuesto para regenerar el cartílago articular pero esta terapia falla principalmente por la ausencia de un material soporte (scaffold) que estimule adecuadamente a las células. El implante de condrocitos autólogos mediante un hidrogel de colágeno no tiene las propiedades mecánicas apropiadas, no proporciona las señales biológicas a las células y el tejido regenerado no es cartílago articular sino fibrocartílago. Se han realizado diferentes enfoques para obtener un scaffold que mimetice mejor las propiedades y la composición del cartílago articular. Los hidrogeles son una buena opción ya que retienen elevadas cantidades de agua, de forma similar al tejido natural, y pueden imitar de cerca la composición del tejido natural mediante la combinación de derivados de hidrogeles naturales. Su tridimensionalidad juega un papel crítico para mantener la función de los condrocitos, ya que el cultivo en monocapa de los condrocitos hace que desdiferencien hacia un fenotipo similar al fibroblasto secretando fibrocartílago. Los hidrogeles inyectables han acaparado la atención en la ingeniería tisular de cartílago articular debido a su capacidad para encapsular células, su inyectabilidad en el daño con cirugías mínimamente invasivas y su adaptabilidad a la forma del defecto. Siguiendo este nuevo enfoque hemos sintetizado dos nuevas familias de hidrogeles inyectables basados en la proteína natural gelatina para la ingeniería tisular del cartílago articular. La primera serie de materiales combina una gelatina inyectable con microfibras poliméricas sueltas de refuerzo para obtener composites inyectables con propiedades mecánicas mejoradas. Nuestros resultados demuestran que hay una influencia de la forma y la distribución de las fibras en las propiedades mecánicas del composite. Además, la mala interacción entre las fibras y la matriz no es capaz de reforzar el hidrogel. Debido a esto, nuestros composites han sido optimizados mediante la mejora de la interacción fibra-matriz a través de un injerto hidrófilo sobre las microfibras, con resultados muy exitosos. La segunda serie de materiales se ha inspirado en la matriz extracelular del cartílago articular y ha consistido en mezclas inyectables de gelatina y ácido hialurónico. Las moléculas de gelatina proporcionan los dominios de adhesión mediante integrinas a las células, y el ácido hialurónico aumenta las propiedades mecánicas de la gelatina. Esta combinación ha demostrado la habilidad para la diferenciación de MSCs hacia el linaje condrocítico y convierte a estos materiales en buenos candidatos para la regeneración del cartílago articular. La última parte de esta tesis se dedica a la síntesis de un material no biodegradable con propiedades mecánicas, hinchado y permeabilidad similar al cartílago. Este material pretende ser empleado como plataforma en un biorreactor en el que se simulan las cargas típicas de las articulaciones, de forma que los scaffolds encajarían en los huecos de la plataforma. Su función es simular el efecto del tejido circundante en el scaffold después de su implantación y podría reducir la experimentación anim / [CA] El cartílag articular es un teixit amb baixa capacitat d'auto-reparació deguda a la seua avascularitat i baixa població cel·lular. Es troba en la superfície de l'ós subcondral cobrint les articulacions. La degeneració del cartílag articular pot aparèixer en atletes, en persones amb processos genètics degeneratius o degut a un trauma; produeix dolor, dificultats a la mobilitat i degeneració progressiva que finalment porta a la fallida de l'articulació. L'auto-reparació es produeix quan el defecte arriba fins a l'ós subcondral i les cèl·lules mare (MSCs) de la medul·la òssia envaeixen el defecte. No obstant això, aquest nou teixit format es un cartílag de tipus fibrocartilaginós i no un cartílag hialí, el qual finalment porta a la degeneració. El transplantament de condròcits autòlegs ha sigut proposat per a regenerar el cartílag articular però aquesta teràpia falla principalment per la absència d'un material de suport (scaffold) que estimuli adequadament a les cèl·lules. L'implant de condròcits autòlegs en un hidrogel de col·lagen per als condròcits no té les propietats mecàniques apropiades, no proporciona les senyals biològiques a les cèl·lules i el teixit regenerat no és cartílag articular sinó fibrocartílag. Diferents enfocs han sigut realitzats fins ara per a obtenir un scaffold que mimetitzi millor les propietats i la composició del cartílag articular. Els hidrogels son una bona opció ja que retenen elevades quantitats d'aigua, de forma similar al teixit natural, i poden imitar acuradament la composició del teixit natural mitjançant la combinació d'hidrogels naturals. La seua tridimensionalitat juga un paper crític per a mantenir la funció dels condròcits, ja que el cultiu en monocapa dels condròcits fa que aquests desdiferencien cap a un fenotip similar al fibroblàstic secretant fibrocartílag. Recentment, els hidrogels injectables han acaparat l'atenció en l' enginyeria tissular de cartílag articular degut a la seua capacitat per a encapsular cèl·lules, la seua injectabilitat en el dany amb cirurgies mínimament invasives i la seua adaptabilitat a la forma del defecte. Seguint aquesta nova aproximació hem sintetitzat dues noves famílies d'hidrogels injectables basats en la proteïna natural gelatina per a l'enginyeria tissular del cartílag articular. La primera sèrie de materials combina una gelatina injectable amb microfibres polimèriques soltes de reforç per a obtenir compòsits injectables amb propietats mecàniques millorades. Els nostres resultats demostren que hi ha una influència de la forma i la distribució de les fibres en les propietats mecàniques del compòsit. Més importantment, la mala interacció entre les fibres i la matriu no és capaç de reforçar l'hidrogel. Degut a això, els nostres compòsits han segut optimitzats mitjançant la millora de la interacció fibra-matriu a traves d'un empelt hidròfil sobre les fibres, amb resultats molt exitosos. La segona sèrie de materials està inspirada en la matriu extracel·lular del cartílag articular i ha consistit en mescles injectables de gelatina i àcid hialurònic. Les molècules de gelatina proporcionen els dominis d'adhesió mitjançant integrines a les cèl·lules, i l'àcid hialurònic augmenta les propietats mecàniques de la gelatina. Esta combinació ha demostrat l'habilitat per a la diferenciació de MSCs cap al llinatge condrocític i converteix a aquests materials en bons candidats per a la regeneració del cartílag articular. L'última part d'aquesta tesi és dedicada a la síntesi d'un material no biodegradable amb propietats mecàniques, inflat i permeabilitat similar al cartílag. Aquest material pretén ser utilitzat com a plataforma a un bioreactor que simula les cargues típiques de les articulacions, de manera que els hidrogels o scaffolds encaixarien als buits de la plataforma. La seua funció es simular l'efecte del teixit circumdant al scaffold després d / Poveda Reyes, S. (2016). Protein-based injectable hydrogels towards the regeneration of articular cartilage [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61392 / Premios Extraordinarios de tesis doctorales

In vitro liver models allow the investigation of the cell behavior in disease conditions or in response to changes in the microenvironment. A major challenge in liver tissue engineering is to mimic the tissue-level complexity: besides the selection of suitable biomaterial(s) replacing the extracellular matrix (ECM) and cell sources, the three-dimensional (3D) microarchitecture defined by the fabrication method is a critical factor to achieve functional constructs. In this study, coaxial extrusion-based 3D bioprinting has been applied to develop a liver sinusoid-like model that consists of a core compartment containing pre-vascular structures and a shell compartment containing hepatocytes. The shell ink was composed of alginate and methylcellulose (algMC), dissolved in human fresh frozen plasma. The algMC blend conferred high printing fidelity and stability to the core–shell constructs and the plasma as biologically active component enhanced viability and supported cluster formation and biomarker expression of HepG2 embedded in the shell. For the core, a natural ECM-like ink based on angiogenesis-supporting collagen-fibrin (CF) matrices was developed; the addition of gelatin (G) enabled 3D printing in combination with the plasma-algMC shell ink. Human endothelial cells, laden in the CFG core ink together with human fibroblasts as supportive cells, formed a pre-vascular network in the core in the absence and presence of HepG2 in the shell. The cellular interactions occurring in the triple culture model enhanced the albumin secretion. In conclusion, core–shell bioprinting was shown to be a valuable tool to study cell–cell-interactions and to develop complex tissue-like models.

No / The latest advances in mesocrystal formation and non-classical crystallization of pre-synthesised nanoparticles have been reviewed with the focus on providing a fuller description of a number of complex systems and their properties and applications through examination of the crystallisation mechanisms at work. Two main crystallization principles have been identified; classical crystallization and particle based aggregation modes of non-classical pathways. To understand the non-classical pathways classical crystallization and its basics are introduced before non-classical pathways, such as oriented attachment and mesocrystal formation, are examined. In particular, the various destabilization mechanisms as applied to the pre-synthesized building blocks in order to form mesocrystalline materials as well as the interparticular influences providing the driving forces are analyzed and compared to the mechanisms at work within classical crystallization. Furthermore, the new properties of the mesocrystalline materials that derive from the collective properties of the nanoparticular building units, and their applications potential are presented. It is shown that this new class of materials has the potential to impact in a number of important areas such as sensor applications, energy conversion, photonic crystals as well as for energy storage, optoelectronics and heterogeneous catalysis or photocatalysis.

A Doença de Chagas atualmente atinge a vida de aproximadamente 18 milhões de portadores do parasita. Assim, este trabalho, parte de um estudo mais amplo, teve como objetivo avaliar a atividade biológica de compostos nitro-heterocíclicos com estrutura análoga à nifuroxazida, 5-nitro-2-furfurilideno 4-hidroxibenzidrazida, frente à cepa Y de Trypanosoma cruzi antes e após a sua microencapsulação por coacervação complexa de gelatina e goma-arábica, que, em geral, se mostram pouco solúveis nos meios e concentração empregados em testes farmacológicos, indicando a necessidade do desenvolvimento de um sistema vetorial que poderá melhorar o perfil farmacológico destes compostos. Assim, padronizou-se técnica de microencapsulação, a partir de resultados de ensaios preliminares e dados da literatura, e empregou-se dois métodos de secagem das microcápsulas, a saber: álcoois e atomização. Exame microscópico dos produtos secos confirmou a formação das microcápsulas possibilitando visualizar a diferença entre os produtos obtidos pelos dois métodos de secagem, para os quais, obteve-se rendimento de microcápsulas de 76±3,6 % e 61±5, 1 % e porcentagem de microencapsulação de 66±12,0 e 24,5±5,3 respectivamente. Obteve-se curva de absorção e de calibração dos compostos analisados, em DMSO, observando-se que não há interferência do complexo gelatina-goma arábica sobre a absorção UV dos compostos possibilitando determinação confiável da concentração destes nas microcápsulas. Ensaios in vitro demonstraram a não interferência do material de encapsulação e do solvente utilizado (DMSO 5%) sobre o desenvolvimento parasitário. Os compostos puros (50 &#181;g/mL) se mostraram bastante ativos eliminando 100% das formas parasitárias em 48 horas, enquanto que, com benznidazol (100 &#181;g/ml) no 12º dia de experimento ainda haviam formas evolutivas viáveis. Os produtos secos por atomização tiveram um comportamento antichagásico semelhante aos compostos puros reduzindo a população parasitária em 63±10% em 24 horas (64±13% compostos puros) não sendo um método adequado para liberação prolongada. Os produtos secos por álcoois permitiram este tipo de liberação devido à integridade das microcápsulas, observada ao microscópio, e em 24 horas houve redução de apenas 20±9% da população, necessitando de 96 horas para eliminação total dos parasitas. Assim, a microencapsulação e a secagem por álcoois utilizados resultaram em liberação lenta dos nitro compostos indicando a possibilidade de utilização deste sistema vetor em estudos mais aprofundados com vistas a melhorar a eficácia antichagásica desses compostos. / Chagas\' Disease reaches whole Latin America exposing 100 million people to infection risk, harming seriously 18 million patients\' life. Thus, the aim of this work was evaluate biological activity of nitro-heterocyclics compounds with similar structure of nifuroxazide, 5-nitro-2-furaldehyde p-hydroxy-benzoylhydrazone, against Trypanosoma cruzi, strain Y, before and after microencapsulation by complex coacervation of gelatin and gum arabic. These compounds, in general, are poorly soluble in concentration used in pharmacological tests, thus the development of a vectorial system will improve pharmacological profile of them. Microencapsulation method was defined based on literature data and preliminary assays and it was used two drying methods: isopropanol-ethanol addition, or spray-drying. Microscopic examination of dried products shown morphological difference between alcohols and spray-dried microcapsules. Microencapsulation percentage average yields were 66.0±12.0% and 24.5±5.3% and coacervate yields were 76.0±3.6% and 61.0±5.1% respectively. After determination of &#955;MAX and standard curves, with DMSO, it was observed no interference of gelatin-acacia complex in UV absorption, making possible reliable dosage of nitro-compounds in microcapsules. ln vitro studies have not shown interference of unloaded microcapsules or DMSO 5% on parasites growth. Solutions used of free nitro-heterocyclics (50 &#181;g/mL) were plenty active against T. cruzi, have shown population reduction of 100% in 48 hours, however, benznidazol (100 &#181g/mL) in 12th day experiment there were still viable parasitic forms. Dried products by spray-drying had antichagasic behavior similar to pure compounds, with parasitic population reduction of 63±10% in 24 hours (64±13% pure compounds) not being appropriate method for extended release. Dried products by alcohols allowed this liberation type due to integrity of microcapsules, observed by microscope, and in 24 hours there was reduction of only 20±9% of the population, needing 96 hours for total parasites\' elimination. Thus, microencapsulation and drying method useing alcohols resulted in slow liberation rates of nitro compounds indicating the possibility of use this vectorial system in studies with views to improve the antichagasic effect of those compounds.

A Doença de Chagas atualmente atinge a vida de aproximadamente 18 milhões de portadores do parasita. Assim, este trabalho, parte de um estudo mais amplo, teve como objetivo avaliar a atividade biológica de compostos nitro-heterocíclicos com estrutura análoga à nifuroxazida, 5-nitro-2-furfurilideno 4-hidroxibenzidrazida, frente à cepa Y de Trypanosoma cruzi antes e após a sua microencapsulação por coacervação complexa de gelatina e goma-arábica, que, em geral, se mostram pouco solúveis nos meios e concentração empregados em testes farmacológicos, indicando a necessidade do desenvolvimento de um sistema vetorial que poderá melhorar o perfil farmacológico destes compostos. Assim, padronizou-se técnica de microencapsulação, a partir de resultados de ensaios preliminares e dados da literatura, e empregou-se dois métodos de secagem das microcápsulas, a saber: álcoois e atomização. Exame microscópico dos produtos secos confirmou a formação das microcápsulas possibilitando visualizar a diferença entre os produtos obtidos pelos dois métodos de secagem, para os quais, obteve-se rendimento de microcápsulas de 76±3,6 % e 61±5, 1 % e porcentagem de microencapsulação de 66±12,0 e 24,5±5,3 respectivamente. Obteve-se curva de absorção e de calibração dos compostos analisados, em DMSO, observando-se que não há interferência do complexo gelatina-goma arábica sobre a absorção UV dos compostos possibilitando determinação confiável da concentração destes nas microcápsulas. Ensaios in vitro demonstraram a não interferência do material de encapsulação e do solvente utilizado (DMSO 5%) sobre o desenvolvimento parasitário. Os compostos puros (50 &#181;g/mL) se mostraram bastante ativos eliminando 100% das formas parasitárias em 48 horas, enquanto que, com benznidazol (100 &#181;g/ml) no 12º dia de experimento ainda haviam formas evolutivas viáveis. Os produtos secos por atomização tiveram um comportamento antichagásico semelhante aos compostos puros reduzindo a população parasitária em 63±10% em 24 horas (64±13% compostos puros) não sendo um método adequado para liberação prolongada. Os produtos secos por álcoois permitiram este tipo de liberação devido à integridade das microcápsulas, observada ao microscópio, e em 24 horas houve redução de apenas 20±9% da população, necessitando de 96 horas para eliminação total dos parasitas. Assim, a microencapsulação e a secagem por álcoois utilizados resultaram em liberação lenta dos nitro compostos indicando a possibilidade de utilização deste sistema vetor em estudos mais aprofundados com vistas a melhorar a eficácia antichagásica desses compostos. / Chagas\' Disease reaches whole Latin America exposing 100 million people to infection risk, harming seriously 18 million patients\' life. Thus, the aim of this work was evaluate biological activity of nitro-heterocyclics compounds with similar structure of nifuroxazide, 5-nitro-2-furaldehyde p-hydroxy-benzoylhydrazone, against Trypanosoma cruzi, strain Y, before and after microencapsulation by complex coacervation of gelatin and gum arabic. These compounds, in general, are poorly soluble in concentration used in pharmacological tests, thus the development of a vectorial system will improve pharmacological profile of them. Microencapsulation method was defined based on literature data and preliminary assays and it was used two drying methods: isopropanol-ethanol addition, or spray-drying. Microscopic examination of dried products shown morphological difference between alcohols and spray-dried microcapsules. Microencapsulation percentage average yields were 66.0±12.0% and 24.5±5.3% and coacervate yields were 76.0±3.6% and 61.0±5.1% respectively. After determination of &#955;MAX and standard curves, with DMSO, it was observed no interference of gelatin-acacia complex in UV absorption, making possible reliable dosage of nitro-compounds in microcapsules. ln vitro studies have not shown interference of unloaded microcapsules or DMSO 5% on parasites growth. Solutions used of free nitro-heterocyclics (50 &#181;g/mL) were plenty active against T. cruzi, have shown population reduction of 100% in 48 hours, however, benznidazol (100 &#181g/mL) in 12th day experiment there were still viable parasitic forms. Dried products by spray-drying had antichagasic behavior similar to pure compounds, with parasitic population reduction of 63±10% in 24 hours (64±13% pure compounds) not being appropriate method for extended release. Dried products by alcohols allowed this liberation type due to integrity of microcapsules, observed by microscope, and in 24 hours there was reduction of only 20±9% of the population, needing 96 hours for total parasites\' elimination. Thus, microencapsulation and drying method useing alcohols resulted in slow liberation rates of nitro compounds indicating the possibility of use this vectorial system in studies with views to improve the antichagasic effect of those compounds.

Abstract The salt print is an important part of photography, both in its historic value and in the tonal range it can provide. This tonal range is greater than any other photographic printing process available to date attributed to the inherent masking ability of the metallic silver. However the intrinsic production problems have made it a 'forgotten' process. There are five key problems. 1. The difficulties in achieving the potential extensive tonal range. 2. The varying colour of the print. 3. Staining that appears in the print, during and after processing. 4. Instability and longevity of the salt print. 5. Contradictory and inaccurate information in material published on the salt print. Although the emphasis of the research is on exploring and controlling the colour and tonal range, the staining problems and stability of the print are also addressed. The materials used for contact negatives today vary in both capture and output, from analogue film processed in the traditional wet darkroom to a variety of transparent film printed from digital files. Inadequate density and tonal range can affect all types of negatives. To provide sufficient exposure time for the salt prints extended tonal range adjustments to the negative were necessary. These long exposures then converted sufficient silver salts to the image making metallic silver, utilising the intrinsic self-masking process. Ultimately this research has uncovered ways to control colour and tonal range and certain aesthetic qualities of the salt print, while simultaneously resolving some of the conflicts in published information. Accurate and consistent methods of processing eliminate staining, providing some stability to the print. The activities and steps carried out to make a salt print are manual; precise duplication is therefore almost unattainable. Nevertheless, although tests on a densitometer may display numeric differences, visual differences are barely noticeable.

In recent years, electrochemical capacitors have emerged as devices with the potential to enable major advances in electrical energy storage. Electrochemical capacitors (ECs) are akin to conventional capacitors but employ higher surface-area electrodes and thinner dielectrics to achieve larger capacitances. This helps ECs to attain energy densities greater than those of conventional capacitors and power densities greater than those of batteries. Akin to conventional capacitors, ECs also have high cycle-lives and can be charged and discharged rapidly. But ECs are yet to match the energy densities of mid to high-end batteries and fuel cells. On the basis of mechanism involved in the charge-storage process, ECs are classified as electrical double-layer capacitors (EDLCs) or pseudocapacitors. Charge storage in EDLCs and pseudocapacitors is brought about by non-faradaic and faradaic processes, respectively. Faradaic process, such as an oxidation-reduction reaction, involves the transfer of charge between electrode and electrolyte. By contrast, a non-faradaic process does not use a chemical mechanism and charges are distributed on surfaces by physical processes that do not involve any chemical reaction. ECs employ both aqueous and non-aqueous electrolytes in either liquid or solid form, the latter providing the advantages of freedom from leakage of any liquid component, compactness, reliability and large operating potential-window. In the literature, polymer electrolytes are the most widely studied solid electrolytes. Complexation of functional-groups of certain polymers with cations results in the formation of polymer-cation complexes commonly referred to as solid-polymer electrolytes (SPEs). Mixing a polymer with an alkali metal salt dissolved in an organic solvent result in the formation of a polymer gel electrolyte. Organic solvents with low molecular-weights, such as ethylene carbonate and propylene carbonate, employed in polymer gel electrolytes are commonly referred to as plasticizers. When water is used as a plasticizer, the polymer electrolyte is called a polymer hydrogel electrolyte. Part I of the thesis is directed to studies pertaining to Polymer Hydrogel Electrolytes for Electrochemical Capacitors and comprises four sections. After a brief survey of literature on polymer hydrogel electrolytes employed in ECs in Section I.1, Section I.2 of Part I describes the studies on electrochemical capacitors employing cross-linked poly (vinyl alcohol) hydrogel membrane electrolytes with varying perchloric acid dopant concentration. Acidic poly (vinyl alcohol) hydrogel membrane electrolytes (PHMEs) with different perchloric acid concentrations are prepared by cross-linking poly (vinyl alcohol) with glutaraldehyde in the presence of a protonic acid acting as a catalyst under ambient conditions. PHMEs are characterized by scanning electron microscopy and temperature-modulated differential scanning calorimetry in conjunction with relevant electrochemical techniques. An optimised electrochemical capacitor assembled employing PHME in conjunction with black pearl carbon (BPC) electrodes yields a maximum specific capacitance value of about 96 F g-1, phase angle value of about 79o and a discharge capacitance value of about 88 F g-1. Section I.3 of Part I describes the studies on cross-linked poly (vinyl alcohol)/ploy (acrylic acid) blend hydrogel electrolytes for electrochemical capacitors. Acidic poly (vinyl alcohol)/poly (acrylic acid) blend hydrogel electrolytes (BHEs) have been prepared by cross-linking poly (vinyl alcohol)/poly (acrylic acid) blend with glutaraldehyde in presence of perchloric acid. These acidic BHEs have been treated suitably to realize alkaline and neutral BHEs. Thermal characteristics and glass-transition behavior of BHEs have been followed by differential scanning calorimetry. Ionic conduction in acidic BHEs has been found to take place by Grötthus-type mechanism while polymer segmental motion mechanism is predominantly responsible for ion motion in alkaline and neutral BHEs. Ionic conductivity of BHEs has been found to range between 10-3 and 10-2 S cm-1 at 298 K. Electrochemical capacitors assembled with acidic PVA hydrogel electrolyte yield a maximum specific capacitance of about 60 and 1000 F g-1 with BPC and RuOx.xH2O/C electrodes, respectively. Section I.4 of Part I describes the studies on gelatin hydrogel electrolytes and their application to electrochemical capacitors. Gelatin hydrogel electrolytes (GHEs) with varying NaCl concentrations have been prepared by cross-linking an aqueous solution of gelatin with aqueous glutaraldehyde under ambient conditions, and characterized by scanning electron microscopy, temperature-modulated differential scanning calorimetry, cyclic voltammetry, electrochemical impedance spectroscopy and galvanostatic chronopotentiometry. Glass transition temperatures for GHEs range between 340 and 377 K depending on the dopant concentration. Ionic conductivity behavior of GHEs is studied with varying concentrations of gelatin, glutaraldehyde and NaCl, and conductivity values are found to vary between 10-3 and 10-1 S cm-1 under ambient conditions. GHEs have a potential window of about 1 V with BPC electrodes. The ionic conductivity of pristine and 0.25 N NaCl-doped GHEs follows Arrhenius behavior with activation energy values of 1.9×10-4 and 1.8×10-4 eV, respectively. Electrochemical capacitors employing GHEs in conjunction with black pearl carbon electrodes are assembled and studied. Optimal values for capacitance, phase angle, and relaxation time constant of about 81 F g-1, 75o, and 0.03 s are obtained for 3 M NaCl-doped GHE, respectively. EC with pristine GHE exhibits continuous cycle life for about 4.3 h as against 4.7 h for the electrochemical capacitor with 3 M NaCl-doped GHE. Unlike electrochemical capacitors, fuel cells do not store the charge internally but instead use a continuous supply of fuel from an external storage tank. Thus, fuel cells have the potential to solve the most challenging problem associated with the electrochemical capacitors, namely their limited energy-density. A fuel cell is an electrochemical power source with advantages of both the combustion engine and the battery. Like a combustion engine, a fuel cell will run as long as it is provided with fuel; and like a battery, fuel cells convert chemical energy directly to electrical energy. As an electrochemical power source, fuel cells are not subjected to the Carnot limitations of combustion (heat) engines. A fuel cell operates quietly and efficiently and, when hydrogen is used as a fuel, it generates only power and potable water. Thus, a fuel cell is a so called ‘zero-emission engine’. In the past, several fuel cell concepts have been tested in various laboratories but the systems that are being potentially considered for commercial developments are: (i) Alkaline Fuel Cells (AFCs), (ii) Phosphoric Acid Fuel Cells (PAFCs), (iii) Polymer Electrolyte Fuel Cells (PEFCs), (iv) Solid-Polymer-Electrolyte-Direct Methanol Fuel Cells (SPE-DMFCs), (v) Molten Carbonate Fuel Cells (MCFCs) and (vi) Solid Oxide Fuel Cells (SOFCs). Among the aforesaid systems, PEFCs that employ hydrogen as fuel are considered attractive power systems for quick start-up and ambient-temperature operations. Ironically, however, hydrogen as fuel is not available freely in the nature. Accordingly, it has to be generated from a readily available hydrogen carrying fuel such as natural gas, which needs to be reformed. But, such a process leads to generation of hydrogen with some content of carbon monoxide, which even at minuscule level is detrimental to the fuel cell performance. Pure hydrogen can be generated through water electrolysis but hydrogen thus generated needs to be stored as compressed / liquefied gas, which is cost-intensive. Therefore, certain hydrogen carrying organic fuels such as methanol, ethanol, propanol, ethylene glycol, and diethyl ether have been considered for fuelling PEFCs directly. Among these, methanol with a hydrogen content of about 13 wt. % (specific energy = 6.1 kWh kg-1) is the most attractive organic liquid. PEFCs using methanol directly as fuel are referred to as SPE-DMFCs. But SPE-DMFCs suffer from methanol crossover across the polymer electrolyte membrane, which affects the cathode performance and hence the cell performance during its operation. SPE-DMFCs also have inherent limitations of low open-circuit-potential and low electrochemical-activity. An obvious solution to the aforesaid problems is to explore other promising hydrogen carrying fuels such as sodium borohydride, which has a hydrogen content of about 11 wt. %. Such fuel cells are called direct borohydride fuel cells (DBFCs). Part II of the thesis includes studies on direct borohydride fuel cells and comprises three sections. After a brief introduction to DBFCs in section II.1, Section II.2 describes studies on an alkaline direct borohydride fuel cell with hydrogen peroxide as oxidant. A peak power density of about 150 mW cm-2 at a cell voltage of 540 mV could be achieved from the optimized DBFC operating at 70oC. Section II.3 describes studies on poly (vinyl alcohol) hydrogel membrane as electrolyte for direct borohydride fuel cells. This DBFC employs a poly (vinyl alcohol) hydrogel membrane as electrolyte, an AB5 Misch metal alloy as anode, and a gold-plated stainless steel mesh as cathode in conjunction with aqueous alkaline solution of sodium borohydride as fuel and aqueous acidified solution of hydrogen peroxide as oxidant. The performance of the PHME-based DBFC in respect of peak power outputs, ex-situ cross-over of oxidant, fuel, anolyte and catholyte across the membrane electrolytes, utilization efficiencies of fuel and oxidant as also cell performance durability under ambient conditions are compared with a similar DBFC employing a Nafion®-117 membrane electrolyte (NME). Peak power densities of about 30 and 40 mW cm-2 are observed for the DBFCs with PHME and NME, respectively. The PHME and NME-based DBFCs exhibit cell potentials of about 1.2 and 1.4 V, respectively, at a load current density of 10 mA cm-2 for 100 h. Publications of Nurul Alam Choudhury 1. Gelatin hydrogel electrolytes and their application to electrochemical supercapacitors, N. A. Choudhury, S. Sampath, and A. K. Shukla, J. Electrochem. Soc., 155 (2008) A74. 2. Cross-linked polymer hydrogel electrolytes for electrochemical capacitors, N. A. Choudhury, A. K. Shukla, S. Sampath, and S. Pitchumani, J. Electrochem. Soc., 153 (2006) A614. 3. Hydrogel-polymer electrolytes for electrochemical capacitors: an overview, N. A. Choudhury, S. Sampath, and A. K. Shukla, Energy and Environmental Science (In Press). 4. Cross-linked poly (vinyl alcohol) hydrogel membrane electrolytes with varying perchloric acid dopant concentration and their application to electrochemical capacitors, N. A. Choudhury, S. Sampath, and A. K. Shukla, J. Chem. Sc. (Submitted) 5. An alkaline direct borohydride fuel cell with hydrogen peroxide as oxidant, N. A. Choudhury, R. K. Raman, S. Sampath, and A. K. Shukla, J. Power Sources, 143 (2005) 1. 6. Poly (vinyl alcohol) hydrogel membrane as electrolyte for direct borohydride fuel cells, N. A. Choudhury, S. K. Prashant, S. Pitchumani, P. Sridhar, and A. K. Shukla, J. Chem. Sc. (Submitted). 7. A phenyl-sulfonic acid anchored carbon-supported platinum catalyst for polymer electrolyte fuel cell electrodes, G. Selvarani, A. K. Sahu, N. A. Choudhury, P. Sridhar, S. Pitchumani, and A. K. Shukla, Electrochim. Acta, 52 (2007) 4871. 8. A high-output voltage direct borohydride fuel cell, R. K. Raman, N. A. Choudhury, and A. K. Shukla, Electrochem. Solid-State Lett., 7 (2004) A 488. 9. Carbon-supported Pt-Fe alloy as a methanol-resistant oxygen-reduction catalyst for direct methanol fuel cells, A. K. Shukla, R. K. Raman, N. A. Choudhury, K. R. Priolkar, P. R. Sarode, S. Emura, and R. Kumashiro, J. Electroanal. Chem., 563 (2004) 181.