No description available.

Neste trabalho foram desenvolvidas condições específicas de RP-HPLC para análise de preparações recombinantes e naturais de hLH, de hCG, e de suas subunidades. O hLH e o hCG heterodimérico e suas subunidades e migraram com tempos de retenção (tR) significativamente diferentes, na seguinte ordem de hidrofobicidade crescente: -hCG < -hLH < hCG < hLH < -hCG < -hLH. Nestas condições, onze preparações foram estudadas: o Padrão Internacional recombinante hLH-WHO 96/602, uma preparação comercial recombinante, duas preparações hipofisárias altamente purificadas de hLH, uma preparação recombinante e duas preparações urinárias de hCG e quatro produtos urinários heterogêneos, contendo hLH + hFSH. Todas as preparações de hLH mostraram um tempo de retenção similar para o pico principal (tR = 38,35 ± 0,42 min; DPR = 1,1 %; n = 4 preparações), enquanto o pico principal do hCG migrou cerca de 4% mais rápido, quando comparado a este valor médio. Picos de hLH, hFSH e hCG foram também identificados nas preparações urinárias heterogêneas. O método foi validado para as sete preparações homogêneas, sendo a exatidão, precisão e sensibilidade calculadas com base na curva dose-resposta, altamente linear (r=0,99998; p<0,0001; n=20). A quantificação de diferentes gonadotrofinas nas preparações heterogêneas foi também realizada, embora com claras limitações de exatidão. / Specific RP-HPLC conditions for the analysis of recombinant and native hLH and hCG preparations and of their subunits were set up. Heterodimeric hLH and hCG and their - and - subunits all migrated with significantly different retention times (tR) in the following order of increasing hydrophobicity: -hCG < -hLH < hCG < hLH < -hCG < -hLH. With basis on these conditions, a total of eleven preparations were studied: the International Standard of recombinant hLH-WHO 96/602, a commercial recombinant and two highly purified pituitary hLH, a recombinant and two urinary hCG preparations and four heterogeneous urinary products containing hLH + hFSH. All hLH preparations showed very similar retention times for the main peak (tR = 38.35 ± 0.42 min; RSD = 1.1 %; n = 4 preparations), while the hCG main peak ran about 4 % faster when compared to this average value. Human LH, hFSH and hCG peaks could also be identified in the heterogeneous urinary preparations. Quantitative analysis could be validated for the seven homogeneous preparations and accuracy, precision and sensitivity were calculated on the basis of a highly linear dose-response curve (r=0.99998; p<0.0001; n=20). Quantification of the differents gonadotropins in the heterogeneous urinary preparations was also carried out, though with clear accuracy limitations.

Neste trabalho foram desenvolvidas condições específicas de RP-HPLC para análise de preparações recombinantes e naturais de hLH, de hCG, e de suas subunidades. O hLH e o hCG heterodimérico e suas subunidades e migraram com tempos de retenção (tR) significativamente diferentes, na seguinte ordem de hidrofobicidade crescente: -hCG < -hLH < hCG < hLH < -hCG < -hLH. Nestas condições, onze preparações foram estudadas: o Padrão Internacional recombinante hLH-WHO 96/602, uma preparação comercial recombinante, duas preparações hipofisárias altamente purificadas de hLH, uma preparação recombinante e duas preparações urinárias de hCG e quatro produtos urinários heterogêneos, contendo hLH + hFSH. Todas as preparações de hLH mostraram um tempo de retenção similar para o pico principal (tR = 38,35 ± 0,42 min; DPR = 1,1 %; n = 4 preparações), enquanto o pico principal do hCG migrou cerca de 4% mais rápido, quando comparado a este valor médio. Picos de hLH, hFSH e hCG foram também identificados nas preparações urinárias heterogêneas. O método foi validado para as sete preparações homogêneas, sendo a exatidão, precisão e sensibilidade calculadas com base na curva dose-resposta, altamente linear (r=0,99998; p<0,0001; n=20). A quantificação de diferentes gonadotrofinas nas preparações heterogêneas foi também realizada, embora com claras limitações de exatidão. / Specific RP-HPLC conditions for the analysis of recombinant and native hLH and hCG preparations and of their subunits were set up. Heterodimeric hLH and hCG and their - and - subunits all migrated with significantly different retention times (tR) in the following order of increasing hydrophobicity: -hCG < -hLH < hCG < hLH < -hCG < -hLH. With basis on these conditions, a total of eleven preparations were studied: the International Standard of recombinant hLH-WHO 96/602, a commercial recombinant and two highly purified pituitary hLH, a recombinant and two urinary hCG preparations and four heterogeneous urinary products containing hLH + hFSH. All hLH preparations showed very similar retention times for the main peak (tR = 38.35 ± 0.42 min; RSD = 1.1 %; n = 4 preparations), while the hCG main peak ran about 4 % faster when compared to this average value. Human LH, hFSH and hCG peaks could also be identified in the heterogeneous urinary preparations. Quantitative analysis could be validated for the seven homogeneous preparations and accuracy, precision and sensitivity were calculated on the basis of a highly linear dose-response curve (r=0.99998; p<0.0001; n=20). Quantification of the differents gonadotropins in the heterogeneous urinary preparations was also carried out, though with clear accuracy limitations.

No description available.

This thesis presents the development of a novel robotically effected plastic foam sculpting system for rapid prototyping and manufacturing purposes. The developed system is capable of rapidly sculpting physical objects out of expanded and extruded polystyrene using an electrically heated Nichrome sculpting tool. An overview of current conventional rapid prototyping systems indicated that the main disadvantages lie in the limited size of objects which can be built, the relatively long time involved to produce one part and the high cost of the systems and materials. An extensive literature and technology review was conducted on work which was similar to the novel system presented in this thesis. The literature provided many good ideas which could be applied. Two sections of experimental work were conducted. The first was aimed at simply proving the concept of robotically effected sculpting of plastic foams. A crude procedure was developed which proved to be rather tedious and manual, especially in terms of generating the tool paths. Qualitative observations of the cut surfaces were used to change the testing parameters to explore their effects and discover which parameters produced accurate and smooth sculpted surfaces. 12 tests were documented and proved that the sculpting of satisfactory surfaces was achievable. The second section of experimental work involved developing the aforementioned crude procedure to make it more automated, especially in terms of the tool path generation and optimisation step. An innovative five step procedure was developed which if followed can produce accurately sculpted artefacts using CAD models of the artefacts as the primary input. Two artefacts were successfully sculpted using the developed procedure. The first was a simple lofted surface; the CAD model of which was created in SolidWorks. The second artefact was a patient customised medical radiation therapy head and neck support; the CAD model of which was created by scanning the back of the author's head and neck with a 3D scanner. The sculpted support fitted the author perfectly. The implementation of the procedure in the two tests highlighted several points including the speed in which the whole process can be carried out. The time taken from the scanning of the authors head and neck with the 3D scanner through to the physical sculpted artefact, was a mere 80 minutes; of which only 13 minutes was consumed in the actual setup and sculpting step! This is extremely quick when compared to conventional rapid prototyping systems and CNC milling. Several areas of future work were outlined and included, tool and fixture design, automation and integration of the system procedure, tool pathing strategy for foam cutting and robot control system issues. The work presented in this thesis provides an excellent foundation for future development of the robotic foam sculpting system.

Com a intenção de estabelecer métodos físico-químicos como uma alternativa ao bioensaio in vivo para determinação de atividade biológica, o conteúdo de hFSH, hTSH e hLH de diferentes preparações, nativas e recombinantes, foi determinado por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) e comparado ao dado obtido pelo clássico bioensaio in vivo em camundongos ou ratos (BA). Para estes hormônios foi encontrada uma relação linear entre os dois métodos: hFSH BAUI = 0,9925 RP-HPLCUI - 1,3165, r = 0,9371, p < 0,001, n = 24; hTSH BA&mu;g = 0,9790 RP-HPLC&mu;g - 0,052, r = 0,8725 , p < 0,001, n = 14; hLH BAUI = 0,8771 RP-HPLCUI + 12,41; r = 0,9786, p < 0,01, n = 5. Para outras nove preparações de hFSH e onze preparações de hTSH foi determinada a diferença média (d) entre a bioatividade predita pela RP-HPLC através destas equações e da média das bioatividades obtidas com os dois métodos. Para o hLH não foi possível determinar esta diferença em virtude das poucas amostras disponíveis. No caso do hFSH, d ± DP = -2,11 ± 3,49 % sendo a precisão de 1,16% e no caso do hTSH, d ± DP = -2,01 ± 5,56 % com precisão de 1,68%. Amostras parcialmente alteradas apresentaram diferentes graus de atividade de hFSH, hTSH e hLH que puderam ser preditas por RP-HPLC com uma aceitável concordância com os bioensaios in vivo. Estes resultados demonstraram que o emprego de um ensaio físico-químico sem o uso de animais, tal como a RP-HPLC, é uma alternativa viável ao uso do bioensaio in vivo para a determinação da potência de hFSH e hTSH, reduzindo assim o número de animais em geral utilizados para assegurar a qualidade e eficácia de um produto farmacêutico. / With the intention of setting up physico-chemical methods as an alternative to in vivo bioassay for determining biological activity, the hFSH, hTSH and hLH content of native and recombinant preparations was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and compared with the data obtained by the classical mouse or rat in vivo bioassays (BA). A linear relationship between the two methods was found for these hormones: hFSH BAIU = 0.9925 RP-HPLCIU 1.3165, r = 0.9371, p < 0.001, n = 24; hTSH BA&mu;g = 0.9790 RP-HPLC&mu;g - 0.052, r = 0.8725, p < 0.001, n = 14; hLH BAIU = 0.8771 RP-HPLCIU + 12.41, r = 0.9786, p < 0.01, n = 5. For nine other hFSH and eleven hTSH preparations, the mean difference (d) between the bioactivity predicted from RP-HPLC data via these equations and the mean of the bioactivities obtained with the two methods was as follows. For hLH this difference could not be estimated due to lack of different samples. In the case of hFSH, d ± SD = -2.11 ± 3.49% with a precision of 1.16% and in the case of hTSH, d ± SD = -2.01 ± 5.56 %, with precision of 1.68%. Partly-degraded hFSH, hTSH and hLH samples presented different activity degrees that could be predicted by RP-HPLC, with an acceptable agreement with the in vivo bioassays. These results demonstrate that the employment of a non-animal physico-chemical assay, such as RP-HPLC, is a viable alternative to the use of an in vivo bioassay for hFSH and hTSH potency determination, thus reducing the number of animals currently used for assuring quality and efficacy of a pharmaceutical product.

Com a intenção de estabelecer métodos físico-químicos como uma alternativa ao bioensaio in vivo para determinação de atividade biológica, o conteúdo de hFSH, hTSH e hLH de diferentes preparações, nativas e recombinantes, foi determinado por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) e comparado ao dado obtido pelo clássico bioensaio in vivo em camundongos ou ratos (BA). Para estes hormônios foi encontrada uma relação linear entre os dois métodos: hFSH BAUI = 0,9925 RP-HPLCUI - 1,3165, r = 0,9371, p < 0,001, n = 24; hTSH BA&mu;g = 0,9790 RP-HPLC&mu;g - 0,052, r = 0,8725 , p < 0,001, n = 14; hLH BAUI = 0,8771 RP-HPLCUI + 12,41; r = 0,9786, p < 0,01, n = 5. Para outras nove preparações de hFSH e onze preparações de hTSH foi determinada a diferença média (d) entre a bioatividade predita pela RP-HPLC através destas equações e da média das bioatividades obtidas com os dois métodos. Para o hLH não foi possível determinar esta diferença em virtude das poucas amostras disponíveis. No caso do hFSH, d ± DP = -2,11 ± 3,49 % sendo a precisão de 1,16% e no caso do hTSH, d ± DP = -2,01 ± 5,56 % com precisão de 1,68%. Amostras parcialmente alteradas apresentaram diferentes graus de atividade de hFSH, hTSH e hLH que puderam ser preditas por RP-HPLC com uma aceitável concordância com os bioensaios in vivo. Estes resultados demonstraram que o emprego de um ensaio físico-químico sem o uso de animais, tal como a RP-HPLC, é uma alternativa viável ao uso do bioensaio in vivo para a determinação da potência de hFSH e hTSH, reduzindo assim o número de animais em geral utilizados para assegurar a qualidade e eficácia de um produto farmacêutico. / With the intention of setting up physico-chemical methods as an alternative to in vivo bioassay for determining biological activity, the hFSH, hTSH and hLH content of native and recombinant preparations was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and compared with the data obtained by the classical mouse or rat in vivo bioassays (BA). A linear relationship between the two methods was found for these hormones: hFSH BAIU = 0.9925 RP-HPLCIU 1.3165, r = 0.9371, p < 0.001, n = 24; hTSH BA&mu;g = 0.9790 RP-HPLC&mu;g - 0.052, r = 0.8725, p < 0.001, n = 14; hLH BAIU = 0.8771 RP-HPLCIU + 12.41, r = 0.9786, p < 0.01, n = 5. For nine other hFSH and eleven hTSH preparations, the mean difference (d) between the bioactivity predicted from RP-HPLC data via these equations and the mean of the bioactivities obtained with the two methods was as follows. For hLH this difference could not be estimated due to lack of different samples. In the case of hFSH, d ± SD = -2.11 ± 3.49% with a precision of 1.16% and in the case of hTSH, d ± SD = -2.01 ± 5.56 %, with precision of 1.68%. Partly-degraded hFSH, hTSH and hLH samples presented different activity degrees that could be predicted by RP-HPLC, with an acceptable agreement with the in vivo bioassays. These results demonstrate that the employment of a non-animal physico-chemical assay, such as RP-HPLC, is a viable alternative to the use of an in vivo bioassay for hFSH and hTSH potency determination, thus reducing the number of animals currently used for assuring quality and efficacy of a pharmaceutical product.

Naturschutzgebiete sind ein essentieller Bestandteil zur Wahrung natürlicher Lebensräume. Oft verfehlt die Einrichtung solcher Schutzzonen jedoch den erwarteten Effekt. Die größte Gefahr liegt hierbei neben dem Klimawandel im direkten Einfluss des Menschen. Besonders in Phasen sozioökonomischen Umschwungs und damit verbundenen Landnutzungsveränderungen oder auch illegaler Nutzung natürlicher Ressourcen, sind Naturschutzgebiete in ihrer Funktion gefährdet. Ziel dieser Arbeit ist somit, Ursachen des Landnutzungswandels sowie dessen Auswirkungen und Ausmaß am Beispiel des rumänischen Teils der Karpaten-Ökoregion abzuleiten. Das Untersuchungsgebiet ist ein wichtiges Zentrum für Biodiversität und in ihm befindet sich Osteuropas größte gemäßigte Waldregion sowie einige der letzten europäischen Urwälder. Rumänien umschließt mehr als die Hälfte der Karpaten und es ist hiernach von besonderem Interesse, Gründe und Auswirkungen des rezenten post-sozialistischen Landschaftswandels zu untersuchen. Mit Hilfe von Landsat Aufnahmen sowie einer ad hoc entwickelten Methode zur Klassifizierung großräumiger Gebiete, wurden Veränderungen in der Waldbedeckung für die post-sozialistische Zeit abgeleitet. Die Ergebnisse offenbaren großflächige Forstveränderungen, auch innerhalb von Naturschutzgebieten und Urwäldern. Institutionelle Umbrüche und eine rapide Umgestaltung in den Eigentumsverhältnissen, wurden als Hauptursachen herausgestellt. Rumänische Naturschutzgebiete erreichen nicht die gewünschte Effektivität und Urwäldern werden weiterhin dezimiert. Die Arbeit verdeutlicht den Einfluss sozioökonomischer Veränderungen auf die Entstehung von Raubbau und legt Defizite in der Effektivität von Schutzbemühungen zum Erhalt der Biodiversität und verbundener Ökosystemleistungen offen. / Protected areas are one cornerstone of conservation efforts to safeguard natural habitats from destruction and overexploitation. Still, many of these areas remain less effective than initially envisioned. Besides climate change, main threats originate from enduring human activities. Protected areas are particularly at risk during periods of rapid socio-economic changes, which can trigger widespread land-use change and illegal resource use. The main goal of this thesis is to assess the extend and underlying causes of land-use change in protected areas and forest habitats within the Carpathian Ecoregion. The Romanian Carpathians were selected as a focus area in this study, because they comprise Eastern Europe’s largest continuous temperate forest region as well as some of the last and largest tracts of European old-growth forests, and they are a major hotspot of biodiversity. Romania comprises more than half of the Carpathian Ecoregion and it is of particular interest to study the causes and effects of land-use changes, which have emerged after the collapse of socialism in 1989. Post socialist forest cover change was quantified for the last 25 years using Landsat images and an ad hoc developed large area classification technique. Results show widespread forest disturbances, even inside protected areas and old-growth forests. Drivers of these disturbances can be related to institutional change and changes in ownership. The effectiveness of Romania’s protected area network in terms of its ability to safeguard biodiversity is most likely decreasing, and intact old-growth forests continue to disappear. This thesis reveals how rapid socio-economic changes may lead to overexploitation, and highlights substantial shortcomings in the effectiveness of protection efforts to safeguard biodiversity and related ecosystem services.

L’objectif général de ma thèse concerne l’étude de la régulation de l’activité cardiaque chez Drosophila melanogaster. Différentes questions ont été abordées : i) la régulation par le pH de l’activité cardiaque à travers l’étude d’un Transporteur du Bicarbonate dépendant du Na+, NDAE1 ; ii) l’implication du canal TRP Painless dans la mécanosensibilité du cœur ; iii) l’élaboration de tests quantitatifs permettant de mesurer le vieillissement cardiaque chez la mouche adulte. Le Na+-Driven Anion Exchanger (NDAE1) constitue l’unique transporteur chez la Drosophile capable de transporter le Bicarbonate dépendant du Na+, alors que l’on trouve dans le génome des mammifères 7 de ces transporteurs appartenant à la famille SLC4. NDAE1 permet l’échange de protons et de Cl- avec le Na+ et HCO3- et agit de manière réversible. Etant donné l’importance potentielle et reconnue de ce type d’échangeur durant certaines pathologies cardiaques intervenant par exemple lors d’épisodes d’ischémie-reperfusion, j’ai analysé sa fonction dans l’activité cardiaque. De manière surprenante, l’inactivation du gène spécifiquement dans le tube cardiaque par interférence à l’ARN n’a aucun effet sur les paramètres mesurables de l’activité cardiaque dans des conditions basales d’élevage, ni sur la viabilité. En revanche, la fonction de NDAE1 peut être révélée dans des conditions de stress où l’on déséquilibre l’homéostasie des ions transportées dans l’échange dépendant de nDAE1. Ainsi, une acidose provoquée dans les individus privés de la fonction de NDAE1 génère de très fortes arythmies, qui sont moins présentes dans les animaux de type sauvage, et conduisent à des arrêts cardiaques définitifs. En outre, arythmies et arrêts cardiaques sont irréversibles quand le pH physiologique est restoré, contrairement aux contrôles qui retrouvent complètement leur activité cardiaque normale. De même l’activité de NDAE1 est requise pour mieux résister aux stress provoqués par l’absence de Na+, de Cl- et de HCO3- dans le milieu extracellulaire et d’adapter à un choc osmotique. En outre, j’ai mis en évidence une forte interaction génétique de ndae1 avec ncx, qui code pour l’échangeur Sodium-Calcium, et dont la fonction est de réguler l’homéostasie calcique et sodique. Cette étude constitue la première démonstration in vivo de la fonction cardiaque des Transporteurs du Bicarbonate dépendant du Na+. J’ai d’autre part contribué à l’étude de la réponse des cardiomyocytes aux stress mécaniques et participé à la démonstration que Painless, un canal TRPA de la Drosophile, était requis pour cette réponse. Finalement, dans le cadre du programme « Identification of genetic markers of cardiac aging in Drosophila », j’ai cherché à proposer des tests capables de mesurer le déclin des performances cardiaques avec l’âge. Parmi ceux-ci, le plus prometteur consiste en une quantification des arythmies mesurée par l’analyse in vivo détaillée des battements cardiaques / Mechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood.Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response.In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen, based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints.This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway.

No description available.