A lesão medular traumática é uma patologia incapacitante, ainda sem tratamento eficaz. As terapias celulares representam uma nova estratégia para o tratamento destas lesões. As células-tronco adultas são fontes potenciais para o transplante celular com o objetivo de minimizar a lesão e promover a recuperação de tecidos lesados, como a medula espinhal. O objetivo desta tese foi avaliar a eficácia do transplante de células-tronco adultas na recuperação funcional e regeneração da lesão medular traumática em modelo experimental de lesão medular contusa em ratas fêmeas Wistar. Os principais objetivos foram: a) comparar os efeitos do transplante da fração mononuclear de sangue de cordão umbilical humano e de células-tronco mesenquimais dos vasos da parede do cordão umbilical humano; b) determinar a janela terapêutica deste tipo de intervenção, comparando os implantes de células- tronco realizados 1 hora, 24 horas e 9 dias após a lesão; c) demonstrar a possível diferenciação das células-tronco implantadas, bem como sua integração no tecido lesado. Os resultados obtidos demonstraram que o transplante de células foi mais eficaz para a recuperação funcional da lesão medular em ratas Wistar quando realizado pela via de administração local 1h após a lesão, quando comparado com a administração na cisterna magna e a aplicação 9 dias a lesão. O tratamento com a fração de células mononucleares ou com as células-tronco mesenquimais do sangue do cordão umbilical 24h após a lesão, não apresentou resultado funcional significativo.Observou-se a neuroproteção do tecido medular quando foi realizado o transplante de células-tronco mesenquimais 1h após a lesão medular. As células humanas transplantadas migraram e sobreviveram no local da lesão quando administradas na cisterna magna ou quando administradas diretamente no local da lesão, porém não se diferenciaram em células gliais ou neurônios. Concluímos que o transplante de células-tronco adultas promoveu a recuperação funcional após a lesão medular contusa, principalmente quando realizado 1h após a lesão diretamente no local da lesão. Apesar das células transplantadas sobreviverem na área da lesão, não foi evidenciada diferenciação celular. / Spinal cord injury is a debilitating disease and yet no effective treatment is available. In this framework cell therapy represents a new strategy to treat this condition. Adult stem cells are potential sources for cell transplantation in order to minimize injury and promote the recovery of damaged tissues, such as the spinal cord. The purpose of this Thesis was to evaluate the action of adult stem cells in the regeneration and functional recovery of spinal cord injury in experimental contusion spinal cord injury in female Wistar rats. Main goals were: a) to compare the effects of transplantation of the mononuclear cells of human umbilical cord blood and mesenchymal stem cells of the vessel wall of human umbilical cord; b) to determine the therapeutic window of this type of intervention, comparing the stem cell implants performed 1 hour, 24 hours and 9 days after injury; c) to demonstrate the possible differentiation of cells implanted, as well as their integration into the damaged tissue. Results reported demonstrate that the transplantation of stem cells was more effective for functional recovery of spinal cord injury when performed into the site of the lesion 1 h after injury, as compared with administration in the cisterna magna 9 days after injury. Treatment with mononuclear cells and mesenchymal cells from umbilical cord blood 24 hours after injury, not showed functional outcome. Neuroprotection was observed when mesenchymal stem cells were transplanted 1 hour after spinal cord injury. The transplanted human cells survived and migrated to the site of injury either when administered in the cisterna magna or directly onto the injury site, but did not differentiated into glial cells or neurons. It is suggested that the transplantation of adult stem cells promotes functional recovery after spinal cord injury when performed 1 hour after injury directly at the injury site, however differentiation of transplanted cells was not detected.

<p>This thesis presents methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during thermal degradation of polymers such as polyurethane (PUR) or epoxy.</p><p>Isocyanates, amines and anhydrides are airway irritants known to cause occupational asthma. Some of the compounds are listed as human carcinogens. Many workers are exposed.</p><p>Isocyanates and anhydrides are reactive and needs to be immediately derivatized during sampling. Methods have been developed for determination of airborne isocyanates, aminoisocyanates and anhydrides using di-n-butylamine (DBA) as reagent to form stabile urea derivatives or amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled detection limits as low as 10 attomoles. A nitrogen-selective LC-detector enabled quantification of DBA-derivatives in reference solutions. </p><p>A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (<1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Aromatic isocyanates become associated to small particles (<1 µm). As a reference method, air-sampling was performed using an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder-impactor sampler and the reference method.</p>

This thesis presents methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during thermal degradation of polymers such as polyurethane (PUR) or epoxy. Isocyanates, amines and anhydrides are airway irritants known to cause occupational asthma. Some of the compounds are listed as human carcinogens. Many workers are exposed. Isocyanates and anhydrides are reactive and needs to be immediately derivatized during sampling. Methods have been developed for determination of airborne isocyanates, aminoisocyanates and anhydrides using di-n-butylamine (DBA) as reagent to form stabile urea derivatives or amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled detection limits as low as 10 attomoles. A nitrogen-selective LC-detector enabled quantification of DBA-derivatives in reference solutions. A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (&lt;1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Aromatic isocyanates become associated to small particles (&lt;1 µm). As a reference method, air-sampling was performed using an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder-impactor sampler and the reference method.

Advancements in pulmonary drug delivery technologies have improved the use of dry powder inhalation therapy to treat respiratory and systemic diseases. Despite remarkable improvements in the development of dry powder inhaler devices (DPIs) and formulations in the last few years, an optimized DPI system has yet to be developed. In this work, we hypothesize that Thin Film Freezing (TFF) is a suitable technology to improve inhalation therapies to treat lung and systemic malignancies due to its ability to produce brittle powder with optimal aerodynamic properties. Also, we developed a performance verification test (PVT) for the Next Generation Cascade Impactor (NGI), which is one of the most important in vitro characterization methods to test inhalation. In the first study, we used TFF technology to produce amorphous and brittle particles of rapamycin, and compared the in vivo behavior by the pharmacokinetic profiles, to its crystalline counterpart when delivered to the lungs of rats via inhalation. It was found that TFF rapamycin presented higher in vivo systemic bioavailability than the crystalline formulation. Subsequently, we investigated the use of TFF technology to produce triple fixed dose therapy using formoterol fumarate, tiotropium bromide and budesonide as therapeutic drugs. We investigated applications of this technology to powder properties and in vitro aerosol performance with respect to single and combination therapy. As a result, the brittle TFF powders presented superior properties than the physical mixture of micronized crystalline powders, such as excellent particle distribution homogeneity after in vitro aerosolization. Lastly, we developed a PVT for the NGI that may be applicable to other cascade impactors, by investigating the use of a standardized pressurized metered dose inhaler (pMDI) with the NGI. Two standardized formulations were developed. Formulations were analyzed for repeatability and robustness, and found not to demonstrate significant differences in plate deposition using a single NGI apparatus. Variable conditions were introduced to the NGI to mimic operator and equipment failure. Introduction of the variable conditions to the NGI was found to significantly adjust the deposition patterns of the standardized formulations, suggesting that their use as a PVT could be useful and that further investigation is warranted. / text

A lesão medular traumática é uma patologia incapacitante, ainda sem tratamento eficaz. As terapias celulares representam uma nova estratégia para o tratamento destas lesões. As células-tronco adultas são fontes potenciais para o transplante celular com o objetivo de minimizar a lesão e promover a recuperação de tecidos lesados, como a medula espinhal. O objetivo desta tese foi avaliar a eficácia do transplante de células-tronco adultas na recuperação funcional e regeneração da lesão medular traumática em modelo experimental de lesão medular contusa em ratas fêmeas Wistar. Os principais objetivos foram: a) comparar os efeitos do transplante da fração mononuclear de sangue de cordão umbilical humano e de células-tronco mesenquimais dos vasos da parede do cordão umbilical humano; b) determinar a janela terapêutica deste tipo de intervenção, comparando os implantes de células- tronco realizados 1 hora, 24 horas e 9 dias após a lesão; c) demonstrar a possível diferenciação das células-tronco implantadas, bem como sua integração no tecido lesado. Os resultados obtidos demonstraram que o transplante de células foi mais eficaz para a recuperação funcional da lesão medular em ratas Wistar quando realizado pela via de administração local 1h após a lesão, quando comparado com a administração na cisterna magna e a aplicação 9 dias a lesão. O tratamento com a fração de células mononucleares ou com as células-tronco mesenquimais do sangue do cordão umbilical 24h após a lesão, não apresentou resultado funcional significativo.Observou-se a neuroproteção do tecido medular quando foi realizado o transplante de células-tronco mesenquimais 1h após a lesão medular. As células humanas transplantadas migraram e sobreviveram no local da lesão quando administradas na cisterna magna ou quando administradas diretamente no local da lesão, porém não se diferenciaram em células gliais ou neurônios. Concluímos que o transplante de células-tronco adultas promoveu a recuperação funcional após a lesão medular contusa, principalmente quando realizado 1h após a lesão diretamente no local da lesão. Apesar das células transplantadas sobreviverem na área da lesão, não foi evidenciada diferenciação celular. / Spinal cord injury is a debilitating disease and yet no effective treatment is available. In this framework cell therapy represents a new strategy to treat this condition. Adult stem cells are potential sources for cell transplantation in order to minimize injury and promote the recovery of damaged tissues, such as the spinal cord. The purpose of this Thesis was to evaluate the action of adult stem cells in the regeneration and functional recovery of spinal cord injury in experimental contusion spinal cord injury in female Wistar rats. Main goals were: a) to compare the effects of transplantation of the mononuclear cells of human umbilical cord blood and mesenchymal stem cells of the vessel wall of human umbilical cord; b) to determine the therapeutic window of this type of intervention, comparing the stem cell implants performed 1 hour, 24 hours and 9 days after injury; c) to demonstrate the possible differentiation of cells implanted, as well as their integration into the damaged tissue. Results reported demonstrate that the transplantation of stem cells was more effective for functional recovery of spinal cord injury when performed into the site of the lesion 1 h after injury, as compared with administration in the cisterna magna 9 days after injury. Treatment with mononuclear cells and mesenchymal cells from umbilical cord blood 24 hours after injury, not showed functional outcome. Neuroprotection was observed when mesenchymal stem cells were transplanted 1 hour after spinal cord injury. The transplanted human cells survived and migrated to the site of injury either when administered in the cisterna magna or directly onto the injury site, but did not differentiated into glial cells or neurons. It is suggested that the transplantation of adult stem cells promotes functional recovery after spinal cord injury when performed 1 hour after injury directly at the injury site, however differentiation of transplanted cells was not detected.

A lesão medular traumática é uma patologia incapacitante, ainda sem tratamento eficaz. As terapias celulares representam uma nova estratégia para o tratamento destas lesões. As células-tronco adultas são fontes potenciais para o transplante celular com o objetivo de minimizar a lesão e promover a recuperação de tecidos lesados, como a medula espinhal. O objetivo desta tese foi avaliar a eficácia do transplante de células-tronco adultas na recuperação funcional e regeneração da lesão medular traumática em modelo experimental de lesão medular contusa em ratas fêmeas Wistar. Os principais objetivos foram: a) comparar os efeitos do transplante da fração mononuclear de sangue de cordão umbilical humano e de células-tronco mesenquimais dos vasos da parede do cordão umbilical humano; b) determinar a janela terapêutica deste tipo de intervenção, comparando os implantes de células- tronco realizados 1 hora, 24 horas e 9 dias após a lesão; c) demonstrar a possível diferenciação das células-tronco implantadas, bem como sua integração no tecido lesado. Os resultados obtidos demonstraram que o transplante de células foi mais eficaz para a recuperação funcional da lesão medular em ratas Wistar quando realizado pela via de administração local 1h após a lesão, quando comparado com a administração na cisterna magna e a aplicação 9 dias a lesão. O tratamento com a fração de células mononucleares ou com as células-tronco mesenquimais do sangue do cordão umbilical 24h após a lesão, não apresentou resultado funcional significativo.Observou-se a neuroproteção do tecido medular quando foi realizado o transplante de células-tronco mesenquimais 1h após a lesão medular. As células humanas transplantadas migraram e sobreviveram no local da lesão quando administradas na cisterna magna ou quando administradas diretamente no local da lesão, porém não se diferenciaram em células gliais ou neurônios. Concluímos que o transplante de células-tronco adultas promoveu a recuperação funcional após a lesão medular contusa, principalmente quando realizado 1h após a lesão diretamente no local da lesão. Apesar das células transplantadas sobreviverem na área da lesão, não foi evidenciada diferenciação celular. / Spinal cord injury is a debilitating disease and yet no effective treatment is available. In this framework cell therapy represents a new strategy to treat this condition. Adult stem cells are potential sources for cell transplantation in order to minimize injury and promote the recovery of damaged tissues, such as the spinal cord. The purpose of this Thesis was to evaluate the action of adult stem cells in the regeneration and functional recovery of spinal cord injury in experimental contusion spinal cord injury in female Wistar rats. Main goals were: a) to compare the effects of transplantation of the mononuclear cells of human umbilical cord blood and mesenchymal stem cells of the vessel wall of human umbilical cord; b) to determine the therapeutic window of this type of intervention, comparing the stem cell implants performed 1 hour, 24 hours and 9 days after injury; c) to demonstrate the possible differentiation of cells implanted, as well as their integration into the damaged tissue. Results reported demonstrate that the transplantation of stem cells was more effective for functional recovery of spinal cord injury when performed into the site of the lesion 1 h after injury, as compared with administration in the cisterna magna 9 days after injury. Treatment with mononuclear cells and mesenchymal cells from umbilical cord blood 24 hours after injury, not showed functional outcome. Neuroprotection was observed when mesenchymal stem cells were transplanted 1 hour after spinal cord injury. The transplanted human cells survived and migrated to the site of injury either when administered in the cisterna magna or directly onto the injury site, but did not differentiated into glial cells or neurons. It is suggested that the transplantation of adult stem cells promotes functional recovery after spinal cord injury when performed 1 hour after injury directly at the injury site, however differentiation of transplanted cells was not detected.

Cette thèse se consacre à la mise au point de techniques d’échantillonnage suivies d’analyses réalisées au laboratoire pour mettre en évidence les espèces majoritaires en air intérieur et caractériser leurs variations temporelles. Le développement analytique a été effectué pour 52 COVs et pour 16 HAPs associés à la phase particulaire de l’air. Les prélèvements des COVs ont été réalisés au moyen d’un préleveur automatique permettant d’échantillonner l’air sur des tubes extraits par désorption thermique et analysés par chromatographie gazeuse couplée à la détection FID. Pour la quantification des HAPs, le prélèvement des particules est effectué par un impacteur à cascade à trois étages permettant de fractionner l’aérosol en fonction de leur diamètre aérodynamique. Les HAPs sont quantifiés par chromatographie liquide haute performance couplée à la détection par fluorescence. Ces deux techniques ont été mises en application dans le cadre de la 1ère campagne intensive du projet MERMAID. / This work aims at developing rapid and simple air sampling techniques followed by laboratory analyses to highlight the occurrence of the main species in indoor air and to describe their temporal variations. The analytical development was focused on 52 VOCs belonging to a wide variety of chemical families to determine their concentrations. The sampling step was performed using an automatic sampler allowing the sampling of air on adsorbent tubes. These tubes were then thermally desorbed and analyzed by gas chromatography coupled to FID. Another analytical development was conducted to determine the particle bound PAHs concentrations. The sampling of particles was realized by the mean of a 3-stages cascade impactor allowing an aerosol fractionation depending on their aerodynamic diameter. Finally, quantification of PAHs was realized by liquid chromatography coupled to fluorescence detection. These two techniques were applied in a field campaign in the frame of the MERMAID program.

An issue of inhalation therapy is a complex topic, actively discussed in last decades, and its progress in various scientific fields is more than required. First part of this thesis brings a theoretical introduction into principles of aerosol therapy and into the requirements resulting from them. Commonly available technologies of inhalers and nebulisers for medical usage, parameters that determinate their effectivity are briefly described. Usage mistakes influencing the effectivity of inhalation are discussed, as well. Second part deals with experimental measurements of aerosol that selected inhalers generate. It also describes difficulties connected with the methods of these measurements, with sampling and following analyses. Gained results are compared with an available literature.

Kvůli stále značnému počtu usmrcených chodců při dopravních nechodách se výrobci automobilů snaží pomocí vhodné konstrukce automobilu zmírnit následky způsobené nehodou. Při srážce impaktoru hlavy s kapotou automobilu dochází ke zpomalení impaktoru a na toto zpomalení je aplikováno HIC kitérium. HIC kritérium vyhodnocuje míru možnosti poranění hlavy při srážce. Cílem diplomové práce je výběr nejvhodnějšího časového průběhu zpomalení dětského impaktoru hlavy při srážce s kapotou automobilu Škoda Superb II z hlediska HIC kritéria a navržení konstrukčních úprav kapoty a okolních částí, které se dostanou do kontaktu tak, aby se skutečný průběh blížil teoretické fyziologicky přípustné variantě. Pro výpočtové modelování úlohy byla použita explicitní varianta metody konečných prvků (MKP). V první části práce jsou analyzovány data ze simulací úderu impaktoru dětské hlavy na kapotu auta, zvlástě pak analýzy částí energie spotřebované kapotou při srážce s impaktorem. Tyto data byly poskytnuté firmou Škoda Auto a.s. Další část se zaobírá návrhem křivek zpomalení se sinusovým, čtvercovým a trojúhelníkým tvarem. Dále je také navržena 2 vrcholová trojúhelníhová křivka zpomalení, jež je pomocí parametrů modifikovatelná. Optimalizací aproximovaného modelu geometrie kapoty automobilu při dopadu impaktoru hlavy na navržený model geometrie kapoty je zajištěno podobné shody s dříve optimalizovanou 2 vrcholovou trojúhleníkovou křivkou zpomalení. V další části je použit originální model geometrie kapoty automobilu Škoda Superb II a další optimalizace modelu geometrie kapoty vůči optimálnímu časovému průběhu zpomalení.

The European New Car Assessment Programme (Euro NCAP) performs several different tests to evaluate vehicles and rate their safety. Some of these tests are subsystem tests made to mimic different body parts of a pedestrian in an interaction with a vehicle. However, some criticism to the test method for the upper leg has been presented, stating that there is a discrepancy between this test method and a real-life interaction. Therefore, a modified test method for the upper leg has been proposed. The aim of this thesis was to evaluate the upper leg test method used today by Euro NCAP, and compare it with the proposed modified test method as well as to computer simulations with a Human Body Model (HBM). The evaluation was performed by comparing different parameters obtained in the two test methods. These have also been compared to computer simulations using a HBM in interaction with a passenger vehicle model. Prior to the evaluation of the test methods, the HBM was positioned into different stances to mimic postures in the human walking cycle. The vehicle model was positioned at four different heights, and three different impact points along the bonnet were used. The results showed that the different methods had their own advantages for some parameters. However, no general conclusion of which method showed the closest correlation to the HBM reference simulations could be determined. / European New Car Assessment Programme (Euro NCAP) utför flera olika tester för att utvärdera fordon och betygsätta deras säkerhet. Några av dessa tester sker med delsystem skapade för att efterlikna olika kroppsdelar hos en fotgängare i en kollision med ett fordon. Viss kritik har dock riktats mot testmetoden för lårben och höft, då studier visat att det finns en skillnad mellan testmetoden och en verklig kollision. En modifierad testmetod för lårbenet och höften har därför föreslagits. Syftet med detta examensarbete har varit att utvärdera testmetoden för lårben och höft, som idag används av Euro NCAP, och jämföra den med den föreslagna modifierade testmetoden men även datorsimuleringar med en humanmodell. Utvärderingen har genomförts genom att jämföra olika parametrar som erhållits från de två testmetoderna. Dessa parametrar har även jämförts med datorsimuleringar av fotgängarkollisioner med en humanmodell och en bilmodell. Humanmodellen positionerades i olika kroppsställningar innan utvärderingen av testmetoderna genomfördes. Detta för att efterlikna verkliga positioner i en mänsklig gångcykel. Bilmodellen positionerades vid fyra olika höjder och tre träffpunkter längs motorhuven användes. Resultaten visade att båda metoderna hade fördelar gentemot varandra för vissa parametrar. Ingen generell slutsats kunde dock dras om vilken metod som visade närmast korrelation till referenssimuleringarna med humanmodellen.