The unsafe use of needles propagates cross infections with bloodborne pathogens and reduces the positive impact of vaccinations on global health. While a plethora of needle-free injection devices exist, the reformulation of protein-based vaccines is largely empirical and costly, which presents a barrier to their widespread clinical application. This thesis contributes to the identification of approaches that facilitate rapid vaccine reformulation and enhance the immunogenicity of needle-free dry-powder vaccines with the help of novel antigen delivery platforms. We hypothesised that the thermodynamic stabilisation of diphtheria toxin mutant 197 (CRM197), a glycoconjugate vaccine carrier protein, may enhance its structural preservation during spray-freeze-drying (SFD), and that its formulation in either soluble, surface-adsorbed, or nanoparticle form impacts the elicited immune response. Differential scanning fluorimetry was used to study the effect of excipients on the thermal stability of CRM197. Dry-powder formulation of CRM197 used i) encapsulation into a thermodynamically stabilising excipient matrix by SFD, ii) surface-immobilisation via physisorption onto a novel potassium-doped hydroxyapatite (kHA) carrier microparticle formed by molten salt synthesis, and iii) chemical conjugation and surface presentation on amphiphilic block copolymer nanoparticles that were incorporated into SFD-powders (SFD-NP). The structural integrity of CRM197 was assessed by size separation in addition to various spectral and thermal analysis methods. The immunogenicity of dry-powder CRM197 formulations was subsequently tested in vivo. The results suggest that the thermodynamic stability of CRM197 in solution does not ensure its structural stability during SFD. While needle-free dermal vaccination with kHA-adsorbed CRM197 induced comparable antibody titres to conventional IM injection of alum-adjuvanted CRM197, needle-free SFD and SFD-NP powders were less immunogenic. The highest mean IgG titre and most balanced Th1/Th2 response was achieved with nanoparticle-conjugated CRM197 by IM, which outperformed the current clinical standard. Therefore, future vaccine design should combine thermodynamic and kinetic stability screening, and place special emphasis on the delivery and structural presentation of the antigen to the immune system.

Investigations concerning the cellular response of tumor tissue to treatment with dimethyl sulfoxide and hematoxylin have not been reported. To establish the response of neoplastic tissue and cells to this combination of agents, this study was undertaken to determine the effects of dimethyl sulfoxide and hematoxylin on a transplantable lymphosarcoma in mice.

Developmental cortical malformations are a major cause of intractable seizures. Determining the location and timing of susceptibility for epileptiform activity is critical to identifying what mechanisms contribute to epileptogenesis in any model. Using the freeze lesion rat model of polymicrogyria, we have identified, in lesioned cortex, these two aspects of epileptogenesis. Previous studies have demonstrated that epileptiform activity cannot be evoked prior to postnatal day (P) 12, but the malformed cortex is more susceptible to seizures as early as P10. An increase in excitatory afferents to the epileptogenic zone occurs before the onset of network epileptiform activity. Whether or not these afferents are a major contributor to the hyperexcitability of the malformed cortex can be investigated by determining if they specifically create a susceptibility for epileptiform activity. We have examined that here by measuring whether that timing coincides with an increased susceptibility for evoked and spontaneous epileptiform activity. We report that the malformed cortex is more susceptible to evoked epileptiform activity than control cortex as earlier as P7 and as late as P36. Further, we also find that the form of spontaneous epileptiform activity in malformed cortex is altered as early as P7. The timing of these early disruptions of cortical function found here suggests additional epileptogenic mechanisms exist prior to the reported increase in excitatory afferents at P10. Determining the location of the seizure initiation is an essential part of epilepsy research. Some patients with developmental cortical malformations have seizures initiated within the malformation, while others have seizures generated by the surrounding cortex. Previous data in the freeze lesion model of microgyria suggests that the timing of freeze lesion (from P0 to P1) can shift the epileptogenic focus from the malformation to the paramicrogyrial region (PMR). We report that both the timing of the freeze lesion and the survival age of the animal can alter the epileptogenic circuitry of the malformation and surrounding tissue. These findings provide new insight to the timeline of hyperexcitability in malformed cortex and will possibly lead to greater surgical success for patients with intractable epilepsy.

L’objectif principal de ce travail était l’étude et l’optimisation du procédé de lyophilisation en milieu aqueux et organique de l’ibuprofène. Une étude comparative approfondie a été réalisée à différents stades de la fabrication de l’ibuprofène lyophilisé pour les deux formulations étudiées. Cette étude comparative a été menée sur l’étape de formulation, sur le procédé de lyophilisation (congélation, sublimation, désorption) et sur les propriétés finales du produit (humidité, réhydratabilité, aspect…). On a observé, que l’utilisation comme solvant du mélange eutectique A, correspondant à une composition de 20% en TBA + 80% en eau (% massique), permettait une forte augmentation de la solubilité de l’ibuprofène et conduisait pour les mêmes conditions opératoires (Tshelf et Pc), à une augmentation des cinétiques de séchage d’un facteur 1,8, par rapport aux résultats observés avec la formulation d’ibuprofène à base aqueuse. Par ailleurs, la détermination expérimentale des valeurs des pressions de vapeur à l’équilibre solide-vapeur et de l’enthalpie de sublimation a été effectuée par deux méthodes différentes : la méthode thermogravimétrique et la méthode statique. Ces déterminations nous ont permis de conclure que l’augmentation des cinétiques de sublimation résultait essentiellement des valeurs plus élevées de la pression de vapeur à l’équilibre solide-vapeur et d’une valeur plus faible de l’enthalpie de sublimation de l’eutectique A par rapport à la glace. Ces données thermodynamiques ont aussi été utilisées comme paramètres clefs pour la modélisation de l’étape de sublimation sous Comsol Multiphysics. In fine, l’optimisation des paramètres opératoires de l’étape de sublimation par la méthodologie du Design Space a conduit à un lyophilisat final d’ibuprofène formulé à base organique qui satisfait entièrement aux critères standard de qualités recherchés (couleur, aspect, résidus de solvants etc). Un développement industriel de la formulation à base de co-solvant organique apparait plus avantageux qu’avec la formulation à base aqueuse. / The main objective of this work was the study and the optimization of the freeze-drying process of ibuprofen with aqueous and/or organic based formulations. A detailed comparative study was realized at different steps of the ibuprofen freeze-drying process for both types of investigated formulations. This comparative study was realized at the succesives stages of freezing, sublimation and desorption and also concerned some properties of the final freeze-dried product (humidity, rehydratability, aspect…). We observed that the use of eutectic A mixture, corresponding to 20 % TBA + 80 % water (mass %) as co-solvent, allowed a strong increase of the solubility of ibuprofen and for the same operating conditions (Tshelf and Pc), led to an increase of drying kinetics values by a factor 1.8 by comparison to aqueous based formulations of ibuprofen. Besides, the experimental determination of equilibrium solid-vapour pressure and of sublimation enthaly values was carried out by using two different methods: the thermogravimetic method and the static method.These determinations allowed to conclude that the increase of sublimation kinetics resulted essentially from the higher equilibrium solid-vapour pressure values and from the lower sublimation enthalpy value of eutectic A by camparison to pure ice values. Next, these thermodynamical data were used as key parameters in the modelling of the sublimation stage under Comsol Multiphysics. In fine, the overall optimization of the operating parameters of sublimation stage was achieved by using the Design Space aproach. It proved that the organic based formulation led to final freeze-died cakes which fulfilled largely the main quality criteria (color, aspect, solvents residues, etc.). Thus, the development of an ibuprofen freeze-drying process with organic based formulation seems more advantageous than an aqueous based formulation process.

6 ABSTRACT Thermal and moisture regime of patterned ground is closely associated with the presence of the regelation phenomenon. Generally, the regelation includes all processes leading to the water freeze-thaw alternations in soil or bedrock. As a result of cyclic freezing and thawing of soil water under specific conditions, processes, such a moisture migration, ice segregation or frost heaving, operate. Termal and moisture regime is the main factor of these processes and consequentely, of the patterned ground formation. The submited diploma thesis is concerned with thermal and moisture regime of patterned ground in the High Tatras. The main aims of the thesis was to characterize thermal and moisture regime of patterned ground, to evaluate and confront the patterned ground regelation activity depending on the diverse soil depths and different types of the patterned ground and to evaluate the soil temperature relationship to the air temperatures, soil moisture content and the water level regime of the nearest lakes. The partial purpose of the thesis was to assess the applicability of different methods of the regelation cycle determination based on the soil temperature measurements. During the study period 2007 - 2012 the regelation activity of sorted paterned ground (Hincove oká, Lúčne sedlo and Skalnaté...

Orientador: Luis Vitor Silva do Sacramento / Banca: Marcia Luzia Rizzatto / Banca: Juliana Cristina Bassan / Resumo: Objetivo: quantificar teores de compostos fenólicos totais, carotenoides totais, ácido ascórbico e flavonoides totais, bem como avaliar a atividade antioxidante, pelos métodos de sequestro do radical DPPH e ABTS+, de duas espécies de pimentas: Dedo-de-Moça (Capsicum baccatum) e Biquinho (Capsicum chinense) em função de dois processos de manufatura (pasteurização e cozimento). Métodos: foram obtidas amostras de pimentas biquinho e dedo-de-moça em mercados de Matão-SP e Araraquara-SP, formando uma amostra composta de cada espécie, cuja homogeneidade foi obtida após quarteamento das amostras menores. Foram realizados os processos térmicos de cozimento (100ºC/5min) e pasteurização (65ºC/30min). Após resfriamento, as amostras foram liofilizadas (48h/-60ºC), realizando-se as extrações de acordo com as metodologias utilizadas. Realizaram-se análises de quantificação dos compostos fenólicos totais, flavonoides totais, vitamina C, carotenoides totais e capacidade antioxidante por DPPH˙ e ABTS⁺. Resultados: os processamentos térmicos não afetaram os teores de fenólicos totais em Capsicum chinense (pimenta biquinho); no entanto, para Capsicum baccatum (pimenta dedo-de-moça) houve diminuição no teor de fenólicos totais frente a pasteurização. Para o teor de flavonoides totais em ambas as variedades o processo de pasteurização demonstrou perdas significativas. A capacidade antioxidante em ambas as espécies foi favorecida devido ao cozimento, e o processo de pasteurização contrariamente de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Objective: to quantify total phenolic compounds, total carotenoids, ascorbic acid and total flavonoids, as well as to evaluate the antioxidant activity, by the methods of scavenging of the DPPH˙ and Abts˙⁺ radical, of two species of peppers: finger-of-the-girl (Capsicum baccatum) and Biquinho (Capsicum chinense) as a function of two manufacturing processes (pasteurization and baking). Methods: samples of pout and finger-of-the-girl peppers were collected in the cities of Matão-SP and Araraquara-SP, composing a homogeneous sample of each pepper, the method of Quarteamente was performed in each of the varieties studied, so as to obtain a Best sampling. The thermal cooking processes (100ºC/5min) and pasteurization (65ºC/30min) were performed after the samples were freeze-dried (48h/ -20 º C). Aqueous and ethanolic extractions were performed to meet the methodologies used. Analysis of quantification of total phenolic compounds, total flavonoids, vitamin C, total carotenoids and antioxidant capacity by DPPH˙ and ABTS˙⁺ were performed. Results: the thermal processes did not affect the total phenolic contents in Capsicum chinense; however, for Capsicum baccatum (finger pepper) there was a decrease in the total phenolics content against pasteurization. For the total flavonoid content in both varieties the pasteurization process is the only one that has had significant losses. For the antioxidant capacity for both varieties the cooking process causes an increase of this capacity to oc... (Complete abstract click electronic access below) / Mestre

Freeze-drying of protein pharmaceuticals is a procedure frequently used to obtain stability of the active pharmaceutical ingredientduring distribution and storage. It can be performed in pre-filled syringes, with a lubricous coating of silicone on the inside, to enable the piston moving. The coating changes the environment potentially affecting the features of the freeze-dried cake since the wetting behavior and adhesion to the inner wall is affected.This project aimed to investigate the effect of the siliconization of the cakes. Three different formulations were freeze-dried in nonsiliconized (NS) and siliconized vials using different siliconization protocols. Analysis was done using differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA),scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and an embedding method, intended to give information about the cake’s shrinkage, cracking and pore-structure. The water content in the bottom of the cakes was consistently higher than in the top, a difference decreasing over time. Increased surface hydrophobicity lead to increased shrinkage of the cake’s volume and a decrease in fogging. The bottom of the protein cake in the vial siliconized with a commercial silicone emulsion consisted of pores with regularly equal pore size and thick pore walls, a structure not seen in any other cake. All cakes in the silicone emulsion siliconized vials had lower water content than the cakes in the vials using the other siliconization method. The XPS-analysis showed that the cakes in the emulsion siliconized vials contained silicon, indicating an excess of silicone when siliconizing and/or an unstable coating. / NextBioForm

β-sitosterol/poly(ethylene glycol)-block-poly(lactic acid) (PLA-b-PEG) complexes were prepared by solution blending in purified water and ethanol. The mixture of water and ethanol is a suitable solvent system for the two components. The complex was studied by using Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DSC). β-sitosterol is a drug that may reduce the swelling of benign prostatic hyperplasia (BPH) and diminishing inflammation. However, it is hydrophobic and difficult to deliver in aqueous solution. Since PLA-b-PEG has amphiphilic properties, the complex described here may enhance delivery of this drug for treatment of BPH. Proton NMR (1HNMR) of the complexes shows that the methylene (CH2) protons of the PEG, the (-O-CH-) of PLA, and (CH3) of PLA are slightly shifted because of its non-covalent interaction with β-sitosterol. The complex formation was supported by 2-D NMR (NOESY) spectroscopy. NOESY spectra show cross peaks, indicating the interaction between the two components. DSC of the complexes shows thermal characteristics that are different from the individual components. In particular, the PEG in the complex shows a lower melting point and decreased crystallinity compared to the pure PEG. The melting point is lowered from 57°C to 55.3 °C for the PEG-b-PLA/β-sitosterol (5%) complex. Under the same condition, the melting point of PLA dropped from 170 °C to 130 °C. Atomic force microscopy shows changes in the surface morphology of the copolymer from crystalline to amorphous when incorporated with the drug. NMR, DSC, AFM, and MTT assay studies suggest the formation of a relatively stable β-sitosterol/poly(lactic acid)-block-poly(ethylene glycol) complex. The cell proliferation assay (MTT assay) suggests significant inhibition of the stimulation of growth of prostate cancer cells upon addition the complex.

Considerando as diferentes aplicações clínicas dos enxertos ósseos nas reconstruções alveolares e a dificuldade de se obter ganhos ósseos em altura, o presente estudo avaliou do ponto de vista histológico a integração do enxerto autógeno (AU), do aloenxerto ósseo liofilizado desmineralizado (ALD), do aloenxerto ósseo congelado mineralizado (ACM) e do coágulo sanguíneo (CO) em um modelo de regeneração óssea vertical. Foram utilizados nove coelhos, sendo um animal doador primário de enxertos ósseos e oito animais submetidos a um modelo de regeneração óssea guiada (ROG), onde 32 cilindros de titânio foram fixados na calota craniana e preenchidos aleatoriamente com AU, ALD, ACM e CO. Após 13 semanas, os animais sofreram eutanásia e o conteúdo dos cilindros submetido à avaliação histológica e histomorfometrica para quantificar a área total de tecido neoformado (AT), o osso neoformado (ON) e o remanescente do material enxertado (MR). Os dados foram submetidos aos testes t-Student e Mann-Whitney com nível de significância de 5%. Os resultados mostraram que em relação à AT os valores médios foram significantes para ACM e ALD e seguiram a seguinte relação: ACM = ALD > AU > CO. Para a variável neoformação óssea as intervenções ALD e ACM mostraram maior quantidade de tecido ósseo formado do que as que empregaram osso autógeno ou coágulo. Já em relação à MR, a média da variável obedeceu à relação: ACM > ALD = AU = CO (valores-p < 5%). Todas as intervenções apresentaram médias mais significativas de crescimento tecidual nas regiões mais próximas ao leito receptor. Foi possível concluir que os aloenxertos podem ser considerados soluções adequadas para o crescimento ósseo vertical. / Regarding different clinical applications for bone grafts in alveolar reconstructions and difficulties on achieving vertical osseous increase the present study performed a comparative histological evaluation of demineralized freeze-dried bone allograft (DFDBA), of fresh frozen bone allograft (FFBA), autogenous graft (AU) and blood clot (CO) on vertical guided bone regeneration (GBR) in rabbit calvarium. Nine rabbits were used, with one as the primary bone graft donor and eight that were subjected to a model of GBR, whereby 32 titanium cylinders were fixed to the calvaria and randomly filled with DFDBA, FFBA, AU, or CO. The animals were sacrificed 13 weeks later, and the content of the cylinders was subjected to hitomorphological and histomorphometric analysis to quantify the total area of neoformed tissue (AT), the new bone tissue (NB) and residual graft particles (RG). The results showed that mean values for AT were significant to DFDBA and FFBA and followed the relation DFDBA = FFBA > AU > CO. Considering new bone formation DFDBA and FFBA showed better results than the AU and CO. The amount of residual bone particles was larger in the DFDBA and followed the relation FFBA > DFDBA = AU = CO (pvalues < 5%). All interventions showed greater new tissue formation nearby the receptor site. It was possible to conclude that allografts DFDBA and FFBA can be considered good strategies for new bone formation in vertical increasing bone.

A busca de fontes alternativas de pigmentos naturais tem estimulado o desenvolvimento de pesquisas com diferentes frutos e resíduos tropicais. No presente trabalho, optou-se por estudar o aproveitamento do bagaço gerado na produção de suco de jabuticaba. A jabuticaba é uma fruta rica em antocianinas, um pigmento natural que, além da capacidade de conferir cor, também possui atividades benéficas à saúde. No entanto, o seu uso como corante natural na indústria de alimentos como uma forma de substituir os corantes sintéticos é limitado pela sua instabilidade frente às condições de processamento e da presença de outros componentes. Uma alternativa para aumentar a estabilidade das antocianinas é através da técnica de microencapsulação no qual o ingrediente sensível é protegido dentro do material de revestimento. Assim, o presente estudo objetivou a produção, caracterização e a verificação da estabilidade dos pós obtidos por liofilização utilizando a maltodextrina, a pectina e a proteína isolada de soja como materiais de parede em diferentes proporções. Os pós foram caracterizados quanto ao teor de umidade, atividade de água, solubilidade, higroscopicidade, tamanho de partícula, morfologia, análise térmica e colorimétrica, teor de fenólicos totais, antocianinas monoméricas e atividade antioxidante com o radical ABTS. Os mesmos também foram avaliados quanto à estabilidade na presença da luz UV durante a estocagem e comparadas com o extrato de antocianinas liofilizado não microencapsulado. As antocianinas presentes no bagaço de jabuticaba foram extraídas com ultrassom utilizando a água acidificada com ácido cítrico (1 %) O extrato de antocianinas concentrado apresentou uma quantidade de antocianinas monoméricas de 510 ± 0,09 mg.100 g-1 de bagaço, um teor de fenólicos totais de 12.860 ± 1,5 mg AGE.100 g-1 bagaço e uma atividade antioxidante de 39.590 ± 1,25 μM TE.g-1 de bagaço todos expressos em base seca. Os pós produzidos por liofilização apresentaram estruturas irregulares amorfas sem estrutura cristalina com tamanho médio de partículas entre 311,66 – 419,74 μm com distribuição bimodal. Além disso, as amostras apresentaram baixos valores de umidade (2,09 ± 0,10 a 4,15 ± 0,32%), atividade de água (0,053 ± 0,003 a 0,162 ± 0,002), higroscopicidade (10,8 ± 0,1 a 14,2 ± 0,3 g/100g) e baixa solubilidade em água (10,9 ± 0,1 a 21,3 ± 0,2 %), atributos desejáveis na obtenção de alimentos em pó. Com a análise térmica foi possível verificar que houve a microencapsulação das antocianinas com os diferentes materiais de revestimento testados devido à formação de novas transições endotérmicas, e ainda foi possível observar que os pós apresentaram uma estabilidade térmica até a temperatura de 130 °C. A fotoestabilidade dos pós liofilizados bem como do extrato de antocianinas liofilizado não microencapsulado armazenados por 90 dias mostraram que a degradação desse pigmento seguiu uma cinética de primeira ordem e os parâmetros cinéticos foram calculados Para o extrato não microencapsulado os valores obtidos para a constante de degradação e tempo de meia-vida foram iguais a 0,0135 d-1 e 51 dias, respectivamente, correspondendo a uma redução de 77% de antocianinas monoméricas, 56 % de fenólicos totais e 43 % da capacidade antioxidante. Por outro lado, os materiais de parede protegeram o pigmento do efeito deletério da luz em mais de 90 % em relação ao teor de fenólicos totais, 80 % para a quantidade de antocianinas monoméricas, de modo que a atividade antioxidante foi mantida em 70 % durante o período de estocagem. Os valores da constante de degradação para todas as formulações variaram de 0,0022 a 0,0070 d-1 e os tempos de meia-vida variaram de 101 a 320 dias. Portanto, os resultados obtidos neste estudo demonstraram que a pectina e a proteína isolada de soja podem ser consideradas potenciais materiais de revestimento na microencapsulação de antocianinas a serem utilizados na indústria de alimentos. / The search for alternative sources of natural pigments has stimulated the development of research with different tropical fruits and their waste; in the present work, we chose to study the use of the bagasse generated in the production of jaboticaba juice was studied. Jaboticaba is a fruit rich in anthocyanins, a natural pigment, which besides the ability of providing color, also has beneficial health activities. However, its use as a natural colorant in the food industry, replacing the synthetic coloring agents is limited by their instability due to the process conditions and the presence of other components. An alternative to increase stability of anthocyanins is microencapsulation technique in which the sensitive ingredient is protected within the coating material. The present work aims the production, characterization and verification of the stability of the powders obtained by freeze-drying using maltodextrin, pectin and the isolate soy protein as wall materials in different proportions. The powders were analyzed for moisture content, water activity, solubility, hygroscopicity, particle size, morphology, thermal and colorimetric analysis, total phenolics content, total monomeric anthocyanin and capacity to scavenge the ABTS.+. They were also evaluated for stability in the presence of UV light during storage and compared with the lyophilized extract of anthocyanins not microencapsulated. Anthocyanins were extracted from jaboticaba pomace with ultrasound using water acidified with citric acid (1%) The concentrated extract anthocyanins showed an amount of monomeric anthocyanins of 510 ± 0.09 mg. 100g-1 a content of total phenolics of 12.860 ± 1.5 mg GAE.100g-1 and antioxidant activity of 39,590 ± 1.25 μM TE.g-1 pomace dry basis. In addition, the samples presented low moisture values (2.09 ± 0.10 to 4.15 ± 0.32%), low water activity (0.053 ± 0.003 to 0.162 ± 0.002) and hygroscopicity ranging from 10.8 ± 0.1 to 14.2 ± 0.3 g/100g as well as low solubility in water (10.86 ± 0.1 to 21.28 ± 0.2%) which are desirable attributes for food powder. Through the thermal analysis it was verified that the anthocyanins were effectively microencapsulated with the different coating materials tested due to the formation of new endothermic transitions; it was also observed that the powders showed a thermal stability up to 130 °C. The photostability the powders and extract of anthocyanins lyophilized not microencapsulated stored for 90 days showed that this pigment degradation followed a first-order kinetics. For the extract not microencapsulated the values obtained of degradation and half-life were equal to 0.0135 d-1 and 51 days, respectively, corresponding to a 77 % reduction of monomeric anthocyanins, 56% total phenolics content and 43 % of antioxidant capacity In contrast, the wall material protected the pigment against the deleterious effect of light in more than 90 % for total phenolic content and 80 % for the amount of monomeric anthocyanins, so the antioxidant activity remained at 70 % for the storage period. The rate constants according to a first-order reaction for all formulations ranged from 0.0022 to 0.0070 d-1 and the half-life times ranged from 101 to 320 days. Therefore, the results of this study showed that the pectin and the isolate soy protein can be potentially be used as coating materials in the microencapsulation of anthocyanins for use in the food industry.