Made available in DSpace on 2016-08-18T18:19:28Z (GMT). No. of bitstreams: 1 Mauro Bogea Pereira.pdf: 2672209 bytes, checksum: 6ffa34d37be7c980ea311a6504843760 (MD5) Previous issue date: 2011-04-28 / FUNDAÇÃO SOUSÂNDRADE DE APOIO AO DESENVOLVIMENTO DA UFMA / In this work the electronic and structural properties of the hetero-nanotube bundles, composed of a CNT (6,0) encapsulated in a BNNT (14,0) (CNT (6,0)@BNNT (14,0)) and a BNNT (6,0) encapsulated in a CNT (14,0) (BNNT(6,0)@CNT (14,0)), under hydrostatic pressure. For this, we applied density functional theory and generalized gradient approximation to the exchange and correlation term, as implemented in SIESTA code. We observed that structural changes occur when pressure is applied. For the CNT(6,0)@BNNT(14,0) these changes begin when the pressure is close to 1,0 GPa for the outer tube, while for the inner tube the changes are observed around 10,0 GPa. In the case of BNNT(6,0)@CNT(14,0) the structure begins to be modified when the applied pressure is close to 3,0 GPa. The analysis of the band structure shown that the metallic character of the CNT(6,0)@BNNT(14,0) and the semiconductor character of the BNNT(6,0)@CNT(14,0) did not change when the pressure is applied. / Neste trabalho foram estudadas as propriedades eletrônicas e estruturais de bundles de hetero-nanotubos, compostos por um CNT (6,0) encapsulado em um BNNT (14,0) (CNT(6,0)@BNNT(14,0)) e por um BNNT (6,0) encapsulado em um CNT (14,0) (BNNT(6,0)@CNT(14,0)), submetidos a pressão hidrostática. Para tanto, fizemos uso da teoria do funcional da densidade e da aproximação do gradiente generalizado para tratar o termo de troca e correlação, implementados no programa computacional SIESTA. Observou-se que ocorrem alterações estruturais nos bundles a medida que a pressão é aplicada. Para o CNT(6,0)@BNNT(14,0) estas alterações têm início a pressões próximas de 1,0 GPa para o tubo externo, enquanto que para o tubo interno as alterações só começam a ser observadas próximo de 10,0 GPa. No caso do BNNT(6,0)@CNT(14,0) a estrutura só começa a ser modificada quando o pressão aplicada é próxima de 3,0 GPa. A análise da estrutura de bandas de energia das duas estruturas mostrou que o caráter eletrônico, metálico para os CNT(6,0)@BNNT(14,0) e semicondutor para os BNNT(6,0)@CNT(14,0), mantêm-se estáveis mesmo para os maiores valores de pressão aplicados.

O escoamento hidrodinâmico é descrito por meio das equações da continuidade e da quantidade de movimento em conjunto com as condições iniciais e de contorno. Neste trabalho de pesquisa, o escoamento não permanente na saída de um canal retangular curto de declividade nula, resultante da abertura rápida de uma comporta foi experimentalmente investigado. Para determinar o coeficiente de pressão e o comprimento do trecho a montante da comporta com distribuição de pressão não hidrostática, usou-se um modelo unidimensional, descrito pelas equações de Saint Venant, para representar o fenômeno. Utilizou-se o método de Lax-Wendroff para obter as curvas características do fenômeno e compará-las com os resultados obtidos experimentalmente. Várias comparações foram feitas possibilitando a avaliação de valores para os parâmetros que definem a região com distribuição de pressão não hidrostática. Os resultados calculados para os valores de parâmetros constantes, estão de acordo com os resultados experimentais. Os resultados experimentais ajustam-se melhor quando os valores dos parâmetros variam no decorrer do tempo. / The hydrodynamic flow is described by means of the equations of the continuity and momentum together with the initial conditions, the boundary conditions. In this research work, the unsteady flow at the exit of a short rectangular channel of zero slope, resulting from the fast opening of a floodgate was investigated experimentally. For determining the pressure coefficient and the length of the upstream reach of the floodgate with non-hydrostatic pressure distribution, a one-dimensional model described by Saint Venant equations is used to represent the phenomenon. The Lax-Wendroff method was used to obtain the curves characteristics of the phenomenon and to compare them with the results obtained experimentally. Several comparisons were made to facilitate the evaluation of values for the parameters that define the region with non-hydrostatic pressure distribution. The results calculated for the values of constant parameters are in agreement with the experimental results. The experimental results are better adjusted when the values of the parameters vary in elapsing of the time.

Orientadores: Carlos Francisco Sampaio Bonafé, Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T08:59:23Z (GMT). No. of bitstreams: 1 Souza_AncelmoRabelo_M.pdf: 2604011 bytes, checksum: 68977693022bd1bef29d71963a2562d5 (MD5) Previous issue date: 2012 / Resumo: Mycobacterium abscessus é um importante patógeno de origem hospitalar que contamina materiais cirúrgicos e biofarmacêuticos. Estes quando mal esterilizados tornam-se agentes infectantes gerando graves patologias nas pessoas que os utilizam. A crescente incidência desse patógeno, as dificuldades de tratamento, a gravidade clínica e, a dificuldade de esterilizar fômites contaminados com este patógeno motivou a investigação de processos alternativos de esterilização. Atualmente, utiliza-se a alta pressão hidrostática como método bastante adequado para reduzir a carga microbiana e esterilizar materiais hospitalares e biofarmacêuticos que são sensíveis à autoclave. Desta forma, investigou-se a inativação de M. abscessus induzida por pressão em diferentes condições de temperatura e de pH visando progredir em direção a um método de esterilização. De acordo com os resultados obtidos, tratamentos a 250 MPa não inativaram significativamente (5 a 8 ordens de magnitude) a bactéria em até 90 min a 20°C. Entretanto em -15°C a inativação foi completa. O tratamento a 250 MPa a 60ºC por 45 min promoveu significativa inativação bacteriana de até 9 unidades logarítmicas, incluindo o teste com PVC. Além disso, extremos de pH (4 ou 9) também diminuíram acentuadamente o número de bactérias tratados por alta pressão (250 MPa), com inativação completa após 45 min. Assim, este trabalho torna-se de suma importância, uma vez que aponta para o melhoramento de protocolos de descontaminação de fômites hospitalares / Abstract: Mycobacterium abscessus is an important hospital-acquired pathogen which induces infections from medical surgical and biopharmaceutical materials. The increasing incidence of this pathogen, the difficulties of treatment and clinical seriousness motivates the investigation of alternatives in sterilization processes. High hydrostatic pressure is a very adequate method for reducing microbial load and for sterilization of hospital materials and biopharmaceutical that are sensitive to autoclaving. We investigated the pressure-induced inactivation of M. abscessus combined with different temperatures and pH conditions aiming improvements toward a sterilization methodology. According to our findings, treatment at 250 MPa did not inactivate the bacteria significantly (5 to 8 orders of magnitude) up to 90 min at 20 oC, nevertheless at -15°C the inactivation was complete. The treatment at 250 MPa and 60ºC or above by 45 min promoted significant bacteria inactivation, at least 9 logarithmic units, including the test PVC. Extremes of pH values (4 or 9) also decrease very much the bacteria number induced by pressure (250 MPa), with complete inactivation at 45 min. The better knowledge of the effect of high hydrostatic pressure in micobacteria may contribute to improvements in the decontamination of medical materials and pharmaceuticals / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

La carencia de servicios básicos como agua potable, para zonas como Cajamarquilla, es un problema social que se debe solucionar. Ante esta situación existen proyectos para dotar de servicios de agua a la zona, que por la geografía que presentan se utilizarán reservorios apoyados en los cerros aledaños. El procedimiento de diseño de un reservorio circular de concreto para la zona, servirá como guía para futuros proyectos. Siendo el objetivo de la investigación proponer criterios y un método adecuado para diseñar la estructura de un reservorio de concreto armado que cumplan con los parámetros de la propuesta de norma E030 2014 para la zona de Cajamarquilla, dada la poca información que existe de estos a pesar de ser de uso muy común en nuestro país. El tipo de la investigación es cualitativo, correlacional y de diseño experimental puro, con el fin de obtener resultados certeros para el diseño de reservorio. Finalmente, el método a usar es el que propone la norma del ACI 350-06, basándose en masas impulsivas y convectivas, del modelo de Housner, para el análisis estático mientras que para análisis el dinámico se utilizó el software Sap2000, por el método de elementos finitos y aplicando el espectro de diseño de la norma ACI 350 adecuándose a la norma peruana, como también para el diseño estructural de los elementos del tanque. La distorsión hallada es de 0.001 y que las cortantes basales estáticas y dinámicas son similares estando en un rango de 90%, por lo que se cumplen con los parámetros de la nueva propuesta de norma E030 2014. The lack of basic services such as water, to areas like Cajamarquilla, is a social problem to be solve. In this situation there are plans to provide water services to the area, by geography reservoirs that have supported in the surrounding hills are used. The process of designing a concrete circular reservoir area will serve as a guide for future projects. As the aim of the research propose criteria and an appropriate method to design the structure of a reinforced concrete reservoir that meet the parameters of the proposed rule for the 2014 E030 Cajamarquilla area, given the little information that exists in spite of these It is very commonly used in our country. The type of research is qualitative, pure correlational and experimental design, in order to obtain accurate results reservoir design. Finally, the method used is the standard proposed by the ACI 350-06, based on impulsive and convective mass Housner model for static analysis while for the Sap2000 dynamic analysis software used by the method of and applying the finite element design spectrum ACI 350 standard adapting to the Peruvian standard, as well as for the structural design of the elements of the tank. The distortion founded that the cutting 0.001y static and dynamic baseline are like being in a range of 90%, so that meet the parameters of the new E030 2014 proposed rule.

Les bactéries sulfato-réductrices sont largement présentes dans les environnements marins profonds. Ces écosystèmes sont caractérisés par un paramètre physique très important, la pression hydrostatique. Afin de mieux comprendre l'adaptation des bactéries sulfatoréductrices à la pression hydrostatique, la technique RNAseq ainsi que des analyses métaboliques ont été utilisées pour étudier la réponse à différentes pressions de croissance chez deux bactéries piézophiles du genre Desulfovibrio, D. hydrothermalis et D. piezophilus. L’analyse transcriptomique révèle que le métabolisme et le transport des acides aminés, le métabolisme énergétique et la transduction du signal jouent un rôle important dans l'adaptation à la pression hydrostatique chez les souches étudiées. Le dosage du glutamate intracellulaire a montré son accumulation à haute pression hydrostatique, suggérant son rôle de piézolyte chez les espèces du genre Desulfovibrio. Les analyses métaboliques ont montré que la production d'énergie chez ces bactéries est optimisée pour la vie en profondeur et qu’elle est plus efficace à la pression optimale de croissance de chacune des souches. De plus, ces analyses nous ont fourni des informations sur les mécanismes adoptés par la souche D. piezophilus pour modifier son métabolisme énergétique afin de s’adapter à des conditions de haute pression hydrostatique. L’analyse de gènes co-régulés à haute pression chez la souche D. piezophilus nous a permis d'identifier un motif de régulation en amont de gènes essentiellement impliqués dans la production d’énergie et qui serait impliquée dans l'adaptation à des pressions hydrostatiques élevées chez D. piezophilus. / Sulfate-reducing bacteria are widely distributed in deep marine environments These biotopes are characterized by a very important physical parameter, the hydrostatic pressure, which increases by 1 MPa each 100 m depth. To better understand adaptation of sulfate-reducing bacteria to high pressure, RNAseq and metabolic analyses were used to study the response of the piezophilic strains of the genus Desulfovibrio, D. hydrothermalis and D. piezophilus to various hydrostatic pressure growth conditions. The transcriptomic datasets obtained revealed that amino acids metabolisms and transport, energy metabolism and signal transduction are important for adaptation to hydrostatic pressure. Glutamate quantification showed its accumulation at high hydrostatic pressure in both strains, suggesting its role as piezolyte in Desulfovibrio species. Metabolic analyses showed that energy production of those bacteria is optimized for deep-sea life conditions and more efficient at the optimum pressure growth of each strain. Moreover, these analyses provide new insights into mechanism of metabolism pathway modification in response to hight hydrostatic pressure in the strain D. piezophilus. Analysis of hight pressure co-regulated genes in the strain D. piezophilus, allowed us to identify a regulatory motif in the upstream of genes essentially involved in energy production. This motif could be a binding site for a regulatory protein involved in the adaptation to high hydrostatic pressure in D. piezophilus that need to be identified.

Orientadores: Sergio Gama, Pedro Jorge von Ranke / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-08T01:09:57Z (GMT). No. of bitstreams: 1 Carvalho_AlexandreMagnusGomes_D.pdf: 6808779 bytes, checksum: b779b54728ac489a69ae449f1e33d093 (MD5) Previous issue date: 2006 / Resumo: Este trabalho apresenta resultados de estudos estruturais, magnéticos e magnetocalóricos de alguns compostos à base de gadolínio, germânio e silício. Os estudos estruturais incluem análises por microscopia ótica e eletrônica, além de difração de raios-X. As análises magnéticas restringem-se a medidas de magnetização em função da temperatura e do campo magnético. Utilizando essas técnicas, são estudados compostos sob diferentes condições de processamento, tais como: amostras como fundidas; tratadas termicamente; pulverizadas e sinterizadas. Os compostos Gd5Ge4 e Gd5Ge2Si2 são também analisados magneticamente sob pressão hidrostática. Adicionamos hidrogênio aos compostos Gd5Si4, Gd5G e2Si2 e Gd5Ge2,1S i1,9 e substituímos Ge e Si por Sn no composto Gd5Ge2Si2, criando novas famílias de materiais, as quais também foram analisadas pelas técnicas supracitadas. Além do trabalho experimental, são apresentados resultados teóricos para o efeito magnetocalórico, utilizando o modelo de Landau-Devonshire. Apresentamos também resultados fenomenológicos para o composto Gd5Ge2Si2 sob pressão hidrostática, utilizando o Modelo de Acoplamento Spin-Rede (MASR) / Abstract: This work presents the results from structural, magnetic and magnetocaloric studies about some compounds based on Gd, Ge and Si. Structural studies include optical and electronic microscopy analyses, besides X-ray diffraction. Magnetic analyses are limited to measurements of magnetization as a function of temperature and magnetic field. Using these techniques, the compounds are investigated under different processing conditions, such as: as-cast, heat-treated, powdered and sintered samples. Gd5Ge4 and Gd5Ge2Si2 compounds are also analyzed through magnetic measurements performed under hydrostatic pressure. We have inserted hydrogen atoms into Gd5Si4, Gd5G e2Si2 and Gd5Ge2,1S i1,9 compounds and substituted Sn for Ge and Si in Gd5Ge2Si2 compound. Thus, new families of materials were developed, which are also investigated in this work using the techniques mentioned above. Besides the experimental work, theoretical results are presented for the magnetocaloric effect using the Landau-Devonshire model. We also present phenomenological results for Gd5Ge2Si2 compound under hydrostatic pressure using the Coupling Magnetic-Lattice Model / Doutorado / Física da Matéria Condensada / Doutor em Ciências

Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T02:52:00Z (GMT). No. of bitstreams: 1 LimaNeto_DanielFerreira_D.pdf: 8189026 bytes, checksum: 64631e8e2ed546b6da6a944505049080 (MD5) Previous issue date: 2012 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Summary Juliane Schneevoigt „Investigation of time- and pressure-dependent expression of different components of the extracellular matrix by chondrocytes in vitro” Institute of Anatomy, Histology and Embryology of the University of Leipzig Submitted in June 2015 98 pages, 34 figures, 41 tables, 153 references keywords: cartilage, chondrocytes, hydrostatic pressure, bioreactor, qPCR Introduction Hyaline cartilage maintains the basic function of transmitting articular pressure load within synovial joints and therefore provides the basis for the movements of an organism. Being a small coverage of the joint surface, it shows a composition designed to match this function specifically. A high amount of proteoglycans and its associated water determines the elastic formability of the hyaline cartilage which allows the absorbance of pressure and shear forces. These proteoglycans, mainly based on aggrecan as core-protein, are embedded into a meshwork of collagen fibres, primarily formed of collagen type II. This composition is not to be understood as a static construct; moreover it is exposed to biophysical forces that permanently require a dynamic adaptation. This adaptation of the extracellular matrix formed by proteoglycans and collagen type II is organised by a small number of embedded chondrocytes, the cells of the hyaline cartilage. As chondrocytes are post-mitotic cells and due to the lack of vascularisation within hyaline cartilage, there is hardly any chance for regeneration of defects in order to maintain the integrity of the tissue. The resulting replacement is formed as fibrocartilage, which has not the capability to withstand the biodynamical forces within the joint. As these defects in hyaline cartilage represent a gross of the diseases of the musculoskeletal system, there is a high medical interest in the development of innovative cell-based therapies, as autologous chondrocyte transplantation (ACT) is one. With this type of therapy in vitro cultivated chondrocytes are seeded into a cartilage defect with the aim of producing hyaline cartilage. Aims of the study In the last decades the need for a detailed understanding of the biodynamics of cartilage became obvious for further development of therapies. The aim of this study was therefore to establish a cell culture system to provide an insight into the biodynamics of chondrocytes. Aside from the examination of the differentiation of in vitro cultivated chondrocytes and their synthesis of extracellular matrix as a function of the cultivation time, another aim of this study was to determine whether the application of hydrostatic pressure might have beneficial influence on the expression of extracellular matrix components by chondrocytes in vitro, in accordance with the hyaline cartilage. Material and methods Human articular chondrocytes were cultivated in vitro without the application of hydrostatic pressure in the first place. The cells were observed phase contrast microscopically and the distribution of collagen type I and II was detected immuncytochemically. In further experiments optical confluent chondrocytes were transferred to a bioreactor system applying a hydrostatic pressure of 5 or 10 bar with variable time periods of the pressure applied. Subsequently, the expression of collagen type I, collagen type II and aggrecan was investigated and quantified using qPCR and Western Blot. Chondrocytes cultivated exclusively without the application of hydrostatic pressure served as controls. In this pilot-study the samples were analysed using arithmetic mean and standard deviation to evaluate the power statistically. In addition, similar test conditions with marginal differences were pooled and the necessary sample size to meet a power of 80 % with an alpha error of 0.05 was calculated using the maximum potential standard deviation. In cases where this statistic power was obtained, an analysis of significance (\"One Way Analysis of Variance”) was carried out meeting a significance level under 0.05. Results During the cultivation of chondrocytes in vitro without hydrostatic pressure the length of the cultivation time did neither show an effect on the phase contrast microscopical morphology nor on the immuncytochemically detected distribution of collagen typ I and II. The application of increased hydrostatic pressure for 24 hours results in a 0.2-0.8-fold decrease of the expression of collagen type I and II and a 1.7-2.2-fold increase of aggrecan expression compared to the unloaded controls. This effect was more distinct with 5 bar but was accompanied by instabilities in the cell culture. This is why further investigations concentrated on the use of 10 bar pressure with subsequently shortened time period of the applied pressure. With short times of loading (1.5 and 3 hours) a pressure load of 10 bar led to a 0.8 fold decrease of the expression of collagen type I and II and showed a 1.6-2.4 fold increase of aggrecan expression. These qPCR results were supported by the protein expression of collagen type I, II and aggrecan detected in Western Blot. Conclusions A cell culture system was established to examine the effect of hydrostatic pressure on the expression of chondrocytes on the one hand, which can further be modified for the assembly of cell transplants on the other hand. Subsequently the results of this study led to a definition of cell culture conditions, stimulating the extracellular matrix production of chondrocytes towards the composition of hyaline cartilage. This was the case using a seeding density of 104 cells/cm2 and a pre-cultivation time of 6 days of normal pressure, followed by the application of 10 bar hydrostatic pressure for 1.5-3 h. With the help of this pilot-study a cell culture system was established to gain more information on biodynamics of hyaline cartilage. Moreover it is possible that this information will provide a basis for further development of cell based therapies of cartilage defects, such as ACT. / Zusammenfassung Juliane Schneevoigt „Untersuchung der zeit- und druckabhängigen Expression verschiedener Komponenten der extrazellulären Matrix durch Chondrozyten in vitro“ Veterinär-Anatomisches Institut der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im Juni 2015 98 Seiten, 34 Abbildungen, 41 Tabellen, 153 Literaturangaben Schlüsselwörter: Knorpel, Chondrozyten, hydrostatischer Druck, Bioreaktor, qPCR Einleitung Der hyaline Knorpel gewährleistet die grundlegende Funktion der Druckübertragung innerhalb der synovialen Gelenke und stellt somit die Grundlage für die Bewegung des Organismus dar. Als schmaler Überzug der Gelenkflächen ist er in seinem Aufbau an diese Funktion spezifisch angepasst. Dabei bedingt der hohe Gehalt an Proteoglykanen und das an diese assoziierte Wasser die elastische Verformbarkeit des hyalinen Knorpels, die es ermöglicht, Druck- und Scherkräfte abzufedern. Die Proteoglykane, die hauptsächlich auf Aggrekan als Kernprotein basieren, sind in ein Maschenwerk kollagener Fasern eingelagert, welches im Wesentlichen durch Kollagen Typ II gebildet wird. Diese Zusammensetzung darf nicht als statisches Konstrukt verstanden werden. Vielmehr ist der hyaline Knorpel in vivo verschiedenen biophysikalischen Einflüssen ausgesetzt, die eine dynamische Anpassung erfordern. Solche Anpassungsvorgänge in Form einer Änderung der Zusammensetzung der aus kollagenen Fasern und Proteoglykanen bestehenden extrazellulären Matrix werden durch die wenigen eingelagerten Chondrozyten, die Zellen des hyalinen Knorpels, organisiert. Da die reifen Chondrozyten jedoch keine Zellteilungen aufweisen und dem hyalinen Knorpel eine Vaskularisierung fehlt, ist eine Defektregeneration kaum möglich, sodass eine Wiederherstellung der Integrität des Gewebes unterbleibt und stattdessen ein Ersatzknorpel, der Faserknorpel, gebildet wird, welcher den einwirkenden biodynamischen Belastungen jedoch nicht standhalten kann. Da die Defekte des hyalinen Knorpels einen Großteil der Erkrankungen des Bewegungsapparats darstellen und zudem die Arthrose als Langzeitfolge nach sich ziehen, besteht ein hohes medizinisches Interesse an der Entwicklung zellbasierter Therapieansätze, wie der Autologen Chondrozytentransplantation (ACT). Hierbei werden - bislang mit unterschiedlichen Erfolgen – in vitro kultivierte Chondrozyten mit dem Ziel, neuen hyalinen Knorpel zu bilden, in einen Knorpeldefekt eingebracht. Ziele der Untersuchungen In den letzten Jahrzehnten zeigte sich, dass die Entwicklung von Therapieansätzen zur Behandlung von Knorpeldefekten ein detaillierteres Verständnis des Knorpelgewebes und speziell dessen Biodynamik erfordert. Ziel dieser Arbeit war es daher, im Rahmen einer Pilotstudie, ein In-vitro-System zu etablieren, welches die Untersuchung der Biodynamik der Chondrozyten ermöglicht. Neben der Untersuchung der Morphologie der Chondrozyten und der durch sie synthetisierten extrazellulären Matrix in Abhängigkeit von der Kultivierungszeit der Zellen, wurde die Fragestellung bearbeitet, ob durch die Wirkung eines hydrostatischen Drucks günstige Effekte in Hinblick auf die Expression einer extrazellulären Matrix, wie sie im hyalinen Knorpel vorliegt, erzielt werden kann. Materialien und Methoden Eine Primärkultur humaner artikulärer Chondrozyten wurde zunächst unter Standardzellkulturbedingungen und atmosphärischem Druck kultiviert. Die Zellen wurden phasenkontrastmikroskopisch und hinsichtlich der Verteilung von Kollagen Typ I und II immunzytochemisch untersucht. In den weiteren Versuchen wurden optisch konfluente Chondrozyten in einen Bioreaktor überführt und weiter unter einem hydrostatischen Druck von 5 oder 10 bar kultiviert. Dabei wurde die Dauer der Druckeinwirkung auf die Chondrozyten variiert. Das Expressionsmuster der so kultivierten Chondrozyten wurde quantitativ in Hinblick auf Kollagen Typ I und II sowie Aggrekan mittels qPCR und Western Blot untersucht. Dabei dienten jeweils Chondrozyten, die ohne erhöhte Druckbedingungen kultiviert wurden, als Kontrollen. In dieser Pilotstudie wurden die Proben unter Berechnung der Mittelwerte und Standardabweichung hinsichtlich ihrer statistischen Power ausgewertet. Neben dieser Analyse der Einzelergebnisse wurden die Versuchsbedingungen, die kaum Unterschiede in den Ergebnissen aufwiesen, in Gruppen zusammengefasst und mit Hilfe der größtmöglichen vorhandenen Standardabweichung der Stichprobenumfang eines Versuchs errechnet, welcher die statistische Power der Ergebnisse bei einem Alpha-Fehler von 0,05 auf 80% erhöht. In den Fällen, in denen diese Power erreicht wurde, erfolgte eine Untersuchung der Unterschiede auf Signifikanz („One Way Analysis of Variance“) bei einem Signifikanzniveau < 0,05. Ergebnisse Während der In-vitro-Kultivierung der Chondrozyten unter atmosphärischem Druck zeigte die Länge der Kultivierungszeit weder einen Einfluss auf die phasenkontrastmikroskopisch untersuchte Morphologie der Zellen noch auf die immunzytochemisch detektierte Verteilung des Kollagen Typ I und II. Die Wirkung eines erhöhten hydrostatischen Drucks (5 bar, 10 bar) für 24 Stunden führte zu einer Abnahme der Expression von Kollagen Typ I und Typ II auf das 0,2-0,8-fache bei gleichzeitiger Zunahme der Expression des Aggrekan auf das 1,7-2,2-fache, verglichen mit der unbehandelten Kontrolle. Dieser Effekt war bei 5 bar ausgeprägter als bei 10 bar, führte jedoch gleichzeitig zu einer starken Instabilität des Zellkultursystems. Vor diesem Hintergrund wurde für den höheren Druck (10 bar) die Zeitdauer der Druckeinwirkung verkürzt. Hierbei konnten bei kurzzeitiger Druckeinwirkung von 10 bar (1,5 und 3 Stunden) bei Erhalt der Zellen ähnliche Effekte erzielt werden wie für die Bedingung 5 bar, 24 Stunden. Die Expression von Kollagen Typ I und Typ II sank auf das 0,8-fache, wohingegen ein Anstieg der Aggrekanexpression auf das 1,6-2,4-fache erreicht wurde. Diese Ergebnisse der qPCR konnten durch die im Western Blot für Kollagen Typ I, II und Aggrekan detektierte Proteinexpression gestützt werden. Schlussfolgerungen Im Rahmen dieser Arbeit wurde ein In-vitro-System etabliert, welches einerseits der Untersuchung des Einflusses von hydrostatischem Druck auf die Expression von Chondrozyten dienen und andererseits für die Herstellung von Zelltransplantaten weiter modifiziert werden kann. Die Ergebnisse der Untersuchungen führten zur Definition von Bedingungen für das In-vitro-System, unter denen die Expression der extrazellulären Matrix durch die Chondrozyten in Richtung der Zusammensetzung im hyalinen Knorpel stimuliert werden kann. Dies zeigte sich bei einer Aussaat der humanen Chondrozyten in einer Konzentration von 104 Zellen/cm2 und einer Vorkultivierungszeit von 6 Tagen unter Normaldruck, gefolgt von der Kultivierung unter hydrostatischem Druck von 10 bar für 1,5 bis 3 Stunden. Mit Hilfe dieser Pilotstudie wurde somit ein In-vitro-System etabliert, auf dessen Basis Untersuchungen durchgeführt werden können, die weiterführende Erkenntnisse zur Biodynamik des hyalinen Knorpels liefern und der zukünftigen Entwicklung zellbasierter Therapieansätze der Knorpeldefekte, wie der ACT, zu Gute kommen.

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High interstitial fluid pressure (IFP) in tumours is recognized as a barrier to drug delivery resulting in reduced efficacy. High IFP impedes the normal process of convective fluid flow (CFF) from blood vessels into the interstitium. The aim of this study was to develop an in vitro model that could be used to measure CFF and to study its effects on drug delivery. The model consists of a transwell cell culture insert which supports the growth of multicell layers (MCL) on collagen coated membranes. A graduated tube is inserted into the transwell and a pressure gradient is applied across the membrane by raising the volume of medium in the tube above that of the bottom chamber. CFF is determined by measuring the weight of medium in the bottom chamber as a function of time. CFF was inversely proportional to MCL thickness and 41.1±3.6µm thick MCL has completely stopped CFF. Using a physiologically relevant hydrostatic pressure of 28mmHg, a CFF of 21µL/min was recorded using a DLD-1 MCL that was 12.21±3.2µm thick. Under these conditions, the rates of penetration of doxorubicin, imatinib and gefitinib were respectively 42, 26 and 13 folds greater than when no CFF exists. Reversing the CFF so that it opposed the drug diffusion gradient significantly impairs drug penetration. In conclusion, a novel in vitro model for assessing the impact of CFF on drug delivery has been developed. This model could be used to evaluate strategies designed to increase drug delivery to solid tumours by modifying the CFF.