Among the different pulmonary fungal infections, aspergillosis, and in particular invasive pulmonary aspergillosis (IPA), are becoming the most worrying diseases in immunocompromised patients. This is due to their high incidence and mortality. Indeed, invasive aspergillosis manifests as invasive pulmonary disease accounting for 50/60% of all cases, with a mortality of 50-90% in severely immunocompromised patients. Triazoles act by inhibiting 14-α demethylase, a fungal cytochrome P450 enzyme implicated in the synthesis of ergosterol, an essential constituent of fungal cell walls. Moreover, they interact with the same cytochrome present in large quantities in the human liver, inducing possible drug-drug interactions in IPA patients. Consequently, interactions resulting from inhibitors, inductors, or substrates of cytochromes can modify the plasmatic concentrations of triazoles or other drugs administered concomitantly. To overcome these important issues, pulmonary delivery of triazoles could be an interesting alternative to conventional routes.The aim of this work was to develop triazole-based dry powders for inhalation able to be deposited adequately in the lungs, with a release of drug and a lung retention that can optimize its pharmacological action. This work focused on two active pharmaceutical ingredients (API): itraconazole (ITZ), for which improved solubility was needed, and voriconazole (VCZ), for which slow release was required.Concerning ITZ, solid dispersions for inhalation (SDIs) comprising ITZ and mannitol were previously developed in our laboratory. The selected SDI showed interesting results in terms of improved dissolution and lung retention in vivo in mice during a pharmacokinetic study. Therefore, this SDI was tested in a murine preclinical model of IPA and showed promising results in terms of prophylaxis efficacy. One aim of this work was to continue the pharmaceutical development of this promising SDI by making a scaling-up study. These methods were intended to improve the SDI’s ecological footprint and productivity by increasing the production yield and decreasing the amount of solvents and time used in its manufacture. During the first step of this study, the obtained SDI showed interesting results obtaining similar powder characteristics (i.e. amorphous content, aerodynamic performance, and dissolution profiles) from concentrated solutions using a laboratory-scale spray-dryer B-290 (Büchi, Switzerland) before using a pilot-scale spray-dryer (GEA Niro, Denmark). Then, the upscaling was performed on the pilot spray-dryer allowing the production of SDIs with increased productivity (yield and process duration). These SDIs had similar powder characteristics than the optimized lab-scale SDIs. During the second part of this work we developed VCZ based dry powder for inhalation. The aim was to slow down the release of this highly permeable and very slightly soluble API and to prolong its lung residence. To this end, various lipidic excipients were chosen. The selection took into account the potential good pulmonary tolerance of the lipids and their hydrophobicity to evaluate their ability to slow down the VCZ release (FPFs 20-25%, slowed release up to 24h, burst effect of ± 58% of VCZ dissolved within 30min). Immediate-release SDIs were also developed to have a comparator reference for the pharmacokinetic and efficacy studies (FPFs of 40%).Then, a pharmacokinetic study in mice was performed following the pulmonary administration of one immediate-release and two sustained-release SDIs (with or without PEG excipient). With an 80-fold higher pulmonary exposure over 24 hours, the slow-release SDIs presented a real interest compared to the immediate-release SDI. Moreover, in accordance with these results, VCZ plasma exposure following the administration of the SDI with PL90-H was more than 1.5-fold higher than its pulmonary exposure (AUC0-24 of 8.70 µg.h/g in the lungs and 14.70 µg.h/mL in the plasma). The slow-release formulations presented plasma exposures at least 15 times lower than their pulmonary exposures (AUC0-24 in lung of 741.40 and 686.85 µg.h/g vs plasmatic AUC0-24 of 37.44 and 42.81 µg.h.mL, respectively with and without PEG excipient). Moreover, the presence of PEG excipient did not influence the residence time and the exposure of the VCZ within the lungs. Finally, the sustained-release SDIs administration by inhalation led to VCZ lung and plasma concentrations higher than the minimal inhibitory concentration (MIC) of VCZ against Aspergillus fumigatus (1 μg/mL) over 24 h. Finally, a murine model of IPA was developed in our lab. The immunosuppression model was fixed and performed by the intraperitoneal (IP) injection of corticosteroids to induce a neutropenia state. Then, different doses of spores (from 1.10^4 to 5.10^6 spores) were inoculated to the neutropenic mice via an endotracheal instillation and the survival rate of each group was observed. Unfortunately, the survival rate resulting from the different infections were not reproducible. Therefore, these models were not suitable to conduct the efficacy study. This underlined the link between the immunosuppressive model and the infection. Indeed, the IPA murine model should be developed according to the immune state of the animal, the Aspergillus conidia species and its concentration to be used. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Practitioners have proposed that Digital Natives prefer graphics while Digital Immigrants prefer text. While Instructional Design has been extensively studied and researched, the impact of the graphical emphasis in instructional designs as it relates to digital propensity has not been widely explored. Specifically, this study examined the performance of students when presented with text-only and graphic-only instructional formats. The purpose of this study was to test the relationship between Digital Propensity Index scores of individuals and their performance when interpreting online instruction. A sample of students from the population of a large metropolitan university received the Digital Propensity Index questionnaire, which is a measure of an individual's time spent interacting with digital media. Each student was randomly assigned varying formats of a computer-based instructional unit via a public survey. The instructional unit consisted of the DPI questionnaire and six tasks related to the Central Florida commuter rail system. Participants were asked to answer the DPI questionnaire on a website by clicking on a link in an emailed invitation. Following the DPI questionnaire, participants were randomly assigned to one of two groups. Group One saw three instructional tasks shown in text and shuffled in random order. Each task was displayed on its own webpage. By submitting an answer to the task, the group progressed through the website to the next task. Group Two saw graphic tasks first, again, shuffled in random order. After the first three tasks, the groups swapped instructional formats to view the opposing group's initial questions. Participants were timed on how many seconds they spent reviewing each task. Each task had an assessment question to evaluate the learning outcomes of the instructional unit. Finally, the DPI score of the participant was matched with the time spent viewing each presentation format. The findings indicate that DPI score had a statistically significant prediction of time spent navigating each type of instruction. Though the link between DPI score and time spent navigating instruction was statistically significant, the actual measurable time difference between navigating text and graphic formats was only a fraction of a second for each increment in DPI score. Limitations and potential future research related to the study are discussed as well.

No description available.

As advances in thin films are made there is a parallel requirement to develop equipment capable of measuring their properties accurately and consistently. In addition there is a need to understand the parameters that are measured. Typical DPI measurements allow both the refractive index (related to density) and the thickness of the adsorbed layer to be calculated with relatively few assumptions, to a very high precision in real time. This thesis presents the research undertaken to develop multiple path length dual polarisation interferometry (MPL-DPI) and absorption enhanced dual polarisation interferometry (AE-DPI). In addition research is presented that can be used to improve the interpretation of the measured parameters for inhomogeneous films and uniaxial films. The new Interferometric technique MPL-DPI allows the thickness and refractive index of in situ and ex-situ coated ultra-thin films to be measured. The procedures and the mathematics required to calculate the properties of films have been described and the technique verified. The technique was demonstrated using films of PMMA, where good agreement was found with complementary techniques. Furthermore, some key features of MPL-DPI were demonstrated using the measurements of interfacially grafted acrylic acid. The absorption enhanced DPI uses the attenuation of the light within the waveguide, due to the light absorbing properties of a film on its surface. As the composition of a film changed, it was shown that the refractive index and extinction coefficients could be used to separate the mass of the components of the film that absorbed light, from the components of the film that did not. With the use of a semi-uniaxial model, the extra data from the attenuation in two polarisations was used to fit the extraordinary and ordinary extinction coefficients. The extraordinary and ordinary extinction coefficients were used to demonstrate that molecular orientation could be implied. The influence that an inhomogeneous film has on the measured thickness, refractive index and extinction coefficient fitted by homogeneous models were investigated. Formulas are presented to explain the thickness, refractive index and extinction coefficient of the measured film. A formula for the total mass per unit area that uses the refractive index was created to account for films that contain molecules of different refractive index increments (dn/dc's). To separate the mass of the individual molecular species from the total mass per unit area, formulas that use the extinction coefficient were derived so molecules that absorb light could be separated from those that do not. The mass calculated from the refractive index and the mass calculated from the extinction coefficient were also examined for uniaxial films. For uniaxial films both measures of the adsorbed mass were found to be relatively accurate and benefited from a partial cancellation of errors. The accuracy of the measurements made by dual polarisation interferometry technology is systematically examined throughout this thesis. Improvements in the calibration routines are suggested and a procedure for the identification and reduction of errors in the phase and contrast is demonstrated.

Herewithin, two transcription factor (TF) regulatory complexes were investigated. A bHLH–MYB–WDR (BMW) DNA-binding complex from maize was the first complex to be studied. R, a maize bHLH involved in the activation of genes in the anthocyanin pathway, had been characterized to indirectly bind DNA despite the presence of a functional DNA-binding domain. Findings presented here reveal that this is only partially correct. Direct DNA-binding by R was found to be dependent upon two distinct dimerization domains that function as a switch. This switch-like mechanism allows R to be repurposed for the activation of promoters of differing cis-element structure. The second regulatory complex studied was of the Arabidopsis thaliana MIKC-MADS TF family. For many TFs, DNA-binding site recognition is relatively straightforward and very sequence specific, while others exhibit relaxed sequence specificity. MADS-domain TFs are one family of TFs with a wider range of cis-element sequences. Though consensus cis-element sequences have been determined for various MADS-domains, correctly predicting and identifying biologically functional cis-elements has been a challenge. In order to study the influence of nucleobase associations within the cis-element, a DNA-Protein Interaction (DPI)-ELISA method was modified and optimized to screen a panel of specific probes. Screening of the SEP3 homodimer against a panel of sequential, palindromic probes revealed that nucleobases in position -1:+1 of the CArG-box influence binding strength between the MADS-domain and DNA. Additionally, the specificity of AGL15 towards CT-W6-AG forms was discovered to be determined by the functional groups present in the minor groove at position -4:+4 using inosine:cytosine (I:C) base pairs. Finally, the FLC–SVP MADS-domain heterodimer, bound to a native cis-element, was modeled and binding simulated using molecular dynamics. In conjunction with simulations of AGL15 and SEP3 homodimers, a potential binding mechanism was identified for this unique heterodimer. DNA sequence recognition by the MADS-domain was found to occur asymmetrically. In the case of the FLC–SVP heterodimer, the direction of asymmetrical DNA-binding in heterodimers was found to be fixed. Furthermore, the molecular dynamics simulations provided insight towards understanding the results generated from previous DPI-ELISA experiments, which should provide an improved means for predicting biologically significant CArG-boxes around genes.

As ligas à base de níquel de alta resistência são utilizadas em uma infinidade de sistemas avançados, onde baixo peso e sistemas de transmissão mecânica de alta densidade de energia são necessários. Componentes, tais como, engrenagens, rolamentos e eixos poderiam ser consideravelmente menor e mais durável se uma grande melhoria em propriedades mecânicas de ligas à base de níquel for alcançada. Um refinamento significativo no tamanho de grão (incluindo nível nano) é um método promissor para a obtenção de melhorias fundamentais nas propriedades mecânicas. O tamanho de grão é conhecido por ter um efeito significativo sobre o comportamento mecânico dos materiais. Um dos métodos mais favoráveis de alcançar refinamento de grão extremo é submetendo os materiais à deformação plástica intensa. As principais variáveis microestruturais nas superligas são a quantidade de precipitados e sua morfologia, o tamanho e a forma do grão e a distribuição de carbonetos (Cr7C3 e Cr23C6) que poderão reduzir propriedades mecânicas da liga. Neste trabalho é apresentada análise por microscopia óptica e eletrônica de transmissão e também os dados de dureza após deformação plástica intensa (tensão de cisalhamento puro) e alguns tratamentos térmicos. / High strength nickel based alloys are used in a multitude of advanced systems where lightweight, high power density mechanical power transmission systems are required. Components such as gears, bearings and shafts could be made significantly smaller and more durable if a major improvement in nickel based alloy mechanical properties could be achieved. A significant refinement in grain size (includes nano level) is thought to be a promising method for achieving fundamental improvements in mechanical properties. Grain size is known to have a significant effect on the mechanical behavior of materials. One of the most favorable methods of achieving extreme grain refinement is by subjecting the materials to severe plastic deformation. The principal microstructural variations in superalloys are the precipitation amount and morphology, grain size and the distribution of carbide precipitation (Cr7C3 and Cr23C6) that could reduce the mechanical properties of the alloys. This work shows optical and transmission electron microscopy analysis and also hardness data after severe plastic deformation (pure shear stress) and some thermal treatments.

As ligas à base de níquel de alta resistência são utilizadas em uma infinidade de sistemas avançados, onde baixo peso e sistemas de transmissão mecânica de alta densidade de energia são necessários. Componentes, tais como, engrenagens, rolamentos e eixos poderiam ser consideravelmente menor e mais durável se uma grande melhoria em propriedades mecânicas de ligas à base de níquel for alcançada. Um refinamento significativo no tamanho de grão (incluindo nível nano) é um método promissor para a obtenção de melhorias fundamentais nas propriedades mecânicas. O tamanho de grão é conhecido por ter um efeito significativo sobre o comportamento mecânico dos materiais. Um dos métodos mais favoráveis de alcançar refinamento de grão extremo é submetendo os materiais à deformação plástica intensa. As principais variáveis microestruturais nas superligas são a quantidade de precipitados e sua morfologia, o tamanho e a forma do grão e a distribuição de carbonetos (Cr7C3 e Cr23C6) que poderão reduzir propriedades mecânicas da liga. Neste trabalho é apresentada análise por microscopia óptica e eletrônica de transmissão e também os dados de dureza após deformação plástica intensa (tensão de cisalhamento puro) e alguns tratamentos térmicos. / High strength nickel based alloys are used in a multitude of advanced systems where lightweight, high power density mechanical power transmission systems are required. Components such as gears, bearings and shafts could be made significantly smaller and more durable if a major improvement in nickel based alloy mechanical properties could be achieved. A significant refinement in grain size (includes nano level) is thought to be a promising method for achieving fundamental improvements in mechanical properties. Grain size is known to have a significant effect on the mechanical behavior of materials. One of the most favorable methods of achieving extreme grain refinement is by subjecting the materials to severe plastic deformation. The principal microstructural variations in superalloys are the precipitation amount and morphology, grain size and the distribution of carbide precipitation (Cr7C3 and Cr23C6) that could reduce the mechanical properties of the alloys. This work shows optical and transmission electron microscopy analysis and also hardness data after severe plastic deformation (pure shear stress) and some thermal treatments.

Local delivery of medication to the lung is highly desirable as the principal advantages include reduced systemic side effects and higher dose levels of the applicable medication at the site of drug action. This administration could be particularly useful for patients with specifically chronic pulmonary infections or pulmonary diseases, such as cystic fibrosis, asthma or lung cancer. In order to deliver a high dose range of medication for highly-dosed drugs such as antibiotics, “carrier-free” DPI formulations of tobramycin were developed with the aim of minimizing the use of excipients. Briefly, dry powders were prepared by spray drying various suspensions of tobramycin in isopropanol. First, as particle size is a key parameter in defining drug deposition in the lungs, the new Spraytec® laser diffraction method specifically modified for measuring the PSD of aerosolized drug was evaluated. The dispersion properties of various dry powder formulations were investigated using different laser diffraction and impaction apparatuses at different flow rates and using different inhalator devices. Different correlations between geometric and aerodynamic size data were demonstrated in this study. As a potential application, for the flow rate, the different inhalation devices and the drug formulations examined, the tobramycin fine particle fraction could be predicted from measurements obtained from the Spraytec® using linear relationships. Correlations (R² > 0.9) between the MMAD and the percentage of particles with a diameter below 5 µm could be demonstrated between the results obtained from the laser diffraction technique and the impaction method. Consequently, the Spraytec® laser diffraction technique was proved to be an important tool for initial formulation and process screening during formulation development of DPIs. In order to modify the surface properties of the raw tobramycin powder, different powder compositions were formulated with the aim of studying the influence of the concentration of tobramycin in drug suspensions used for spray-drying, the lipid film composition (cholesterol:Phospholipon ratio) and the coating level (in percentage) on the physicochemical and aerodynamic characteristics of the antibiotic. The results indicated that the application of a lipid coating around the active particles allowed an improvement in particle dispersion from the inhalator, decreasing raw powder agglomeration and thus enhancing drug deposition deep in the lungs. Moreover, these results seemed to be influenced by the amount and composition of the lipids in the formulations. The evaluation of the influence of the coating level showed that the deposition of only 5% w/w lipids (on a dry basis) was sufficient to improve particle dispersion properties during inhalation. The FPF, which is around 36% for the uncoated micronized tobramycin, was increased to up to about 68% for the most effective lipid-coated formulation. Of particular importance, these results revealed the need to add sufficient amounts of covering material in order to significantly modify the particle surface properties and reduce their tendency to agglomeration, while limiting the lipid level in the formulations in order to avoid any undesirable sticking and to allow the delivery of more of the active drug to the deep lung. Another approach used to modify the surface properties of raw tobramycin was to coat the micronized particles with nanoparticles of the drug, produced by high pressure homogenization. The evaluation of the influence of the level of nanoparticle coating of the micronized particles showed that the presence of nanoparticles in the formulations improved the particle dispersion properties during inhalation. One microparticle was completely covered with a single layer or several layers of nanoparticles, in function of the percentage of nanoparticles in the mixture. Coating the fine drug particles with particles in the nanometer range was believed to reduce Van Der Waals forces and powder agglomeration. These various layers of nanoparticles also allowed a decrease in the cohesion of the powder by improving the slip between the particles. On the other hand, suspensions containing solely nanoparticles were spray dried with various concentrations of surfactant in order to produce easily dispersible and reproducible micron-size agglomerates of nanoparticles during inhalation. The evaluation of the influence of the concentration of surfactant showed that deposition of only 2% w/w (on a dry basis) of Na glycocholate is sufficient to improve particle dispersion properties during inhalation. Consequently, the use of nanoparticles in dry powder formulations increased the FPF from 36% for the uncoated micronized tobramycin to about 61% for this latter formulation. To modify the balance between the different forces of interactions without the need for any excipient, the influence of formulation components on the aerosolization characteristics of spray-dried tobramycin through the use of various proportions of water in the solvent used to prepare initial suspensions was investigated. These results showed that it is possible to modify the surface properties of the particles by coating the particles of drug with a homogeneously distributed film of the active compound dissolved in a solvent system containing a mixture of different solvents such as isopropanol and water. During nebulization of the suspension, droplets are composed of one or more particles in solid state surrounded with solvent containing the dissolved drug. It is hypothesized that during the drying step, dissolved tobramycin forms a coating of the amorphous drug around particles in suspension. The coating of drug particles can thus be used as an alternative approach that permits the modification of the surface properties of the particles, increasing the flowability, the desagglomeration tendency and the fine particle fraction deposited in the deep lung. So, the evaluation of the influence of the water content of the suspensions and the effect of the inlet temperature during spray-drying showed that the addition of 2% water v/v is sufficient to improve particle dispersion during inhalation. Of particular interest, as tobramycin is a very hygroscopic drug, the addition of water turned out to be a critical step. It was thus important to add a small amount of water to the solvent system and to process the drying step at a high temperature to produce formulations containing solely the active drug and showing a FPF of up to 50%. Moreover, stability studies demonstrated that these optimized formulations (lipid-coated formulation, nanoparticle formulation and amorphous drug-coated formulation) were stable over a long time period at various ICH temperature and relative humidity storage conditions (25°C/60% RH, 30°C/65% RH and 40°C/75% RH). The formulations were shown to keep their crystalline state, initial PSD, redispersion characteristics and deposition results for more than twelve months. In order to confirm these encouraging results, two optimized formulations (one with a lipid coating and another with amorphous drug coating) were selected and compared to the only commercially available tobramycin formulation for inhalation, Tobi® (nebulizer solution), by performing a combined in vivo scintigraphic and pharmacokinetic evaluation of tobramycin DPIs in nine CF patients. In comparison with Tobi®, it was estimated that lung deposition, expressed as a percentage of the nominal dose, was 7.0 and 4.5 times higher for the lipid-coated and amorphous tobramycin-coated formulations, respectively. Moreover, the pharmacokinetic data, adjusted to the same drug dose as that of the Tobi® deposited in the lungs, showed that the AUC values were found to be 1.6 times higher for Tobi® than for DPI formulations. So this evaluation confirmed the superiority of dry powder formulations in terms of drug deposition and reduced systemic exposure in comparison with the conventional comparator product, Tobi®. Thus, these new and orginal tobramycin DPI formulations based on the use of very low excipient levels and presenting very high lung deposition properties, were shown to offer very good prospects for improving the delivery of drugs to the pulmonary tract and to the widest possible patient population.

The cellular response to a biomaterial, such as a dental implant, is mainly governed by the surface properties, and can thus be altered by the introduction of a surface coating. In this thesis the buildup of a biomacromolecule-based coating formed by layerby-layer (LbL) deposition of the charged polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) has been studied. In an attempt to make these coatings bioactive and useful for bone-anchored implants, an amelogenin protein mixture (EMD), has been immobilized in these thin polyelectrolyte multilayer (PEM) films. Multilayers were also built by LbL deposition of the natural biomacromolecules collagen (Col) and hyaluronic acid (HA). Multilayer films of these two extra-cellular biomacromolecules should be of interest for use as a scaffold for tissue engineering. The buildup of the multilayer films has been followed in situ, using ellipsometry, quartz crystal microbalance with dissipation (QCM-D), and dual polarization interferometry (DPI). The studied PLL/PGA multilayers were found to be highly hydrated, and to exhibit a two-regime buildup behavior, with an initial “slow-growing” regime, and a second “fast-growing” regime with a linear growth in film thickness and more than linear growth in mass. A net diffusion of polypeptides into the film during the buildup led to an increase in density of the films for each layer adsorbed. A change in density was also observed in the Col/HA film, where HA penetrated and diffused into the porous fibrous Col network. The formed PLL/PGA films were further found to be rather stable during drying, and post-buildup changes in temperature and pH, not losing any mass as long as the temperature was not raised too rapidly. The film thickness responded to changes in the ambient media and collapsed reversibly when dried. A swelling/de-swelling behavior of the film was also observed for changes in the temperature and pH. The EMD protein adsorbed to silica surfaces as nanospheres, and could by itself form multilayers. The adsorption of EMD onto PLL/PGA multilayer films increased at lower pH (5.0), and EMD could be immobilized in several layers by alternate deposition of EMD and PGA. / QC 20101019

<p>The cellular response to a biomaterial, such as a dental implant, is mainly governed by the surface properties, and can thus be altered by the introduction of a surface coating. In this thesis the buildup of a biomacromolecule-based coating formed by layerby-layer (LbL) deposition of the charged polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) has been studied. In an attempt to make these coatings bioactive and useful for bone-anchored implants, an amelogenin protein mixture (EMD), has been immobilized in these thin polyelectrolyte multilayer (PEM) films. Multilayers were also built by LbL deposition of the natural biomacromolecules collagen (Col) and hyaluronic acid (HA). Multilayer films of these two extra-cellular biomacromolecules should be of interest for use as a scaffold for tissue engineering.</p><p>The buildup of the multilayer films has been followed in situ, using ellipsometry, quartz crystal microbalance with dissipation (QCM-D), and dual polarization interferometry (DPI). The studied PLL/PGA multilayers were found to be highly hydrated, and to exhibit a two-regime buildup behavior, with an initial “slow-growing” regime, and a second “fast-growing” regime with a linear growth in film thickness and more than linear growth in mass. A net diffusion of polypeptides into the film during the buildup led to an increase in density of the films for each layer adsorbed. A change in density was also observed in the Col/HA film, where HA penetrated and diffused into the porous fibrous Col network.</p><p>The formed PLL/PGA films were further found to be rather stable during drying, and post-buildup changes in temperature and pH, not losing any mass as long as the temperature was not raised too rapidly. The film thickness responded to changes in the ambient media and collapsed reversibly when dried. A swelling/de-swelling behavior of the film was also observed for changes in the temperature and pH.</p><p>The EMD protein adsorbed to silica surfaces as nanospheres, and could by itself form multilayers. The adsorption of EMD onto PLL/PGA multilayer films increased at lower pH (5.0), and EMD could be immobilized in several layers by alternate deposition of EMD and PGA.</p>