Made available in DSpace on 2014-06-11T19:25:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-10-29Bitstream added on 2014-06-13T20:53:38Z : No. of bitstreams: 1 souza_at_me_sjrp_parcial.pdf: 48305 bytes, checksum: 2ab2a37aa6e511084d3705cb0e675c4c (MD5) Bitstreams deleted on 2014-10-03T16:24:37Z: souza_at_me_sjrp_parcial.pdf,Bitstream added on 2014-10-03T16:27:36Z : No. of bitstreams: 2 souza_at_me_sjrp_parcial.pdf.txt: 18111 bytes, checksum: c5bd29ac564839820b6ca688b23abec8 (MD5) 000680523.pdf: 1511132 bytes, checksum: c20e5a6a65edf2cba90d4de2b5073a08 (MD5) Bitstreams deleted on 2014-10-03T16:33:13Z: 000680523.pdf,Bitstream added on 2014-10-03T16:43:27Z : No. of bitstreams: 2 souza_at_me_sjrp_parcial.pdf.txt: 18111 bytes, checksum: c5bd29ac564839820b6ca688b23abec8 (MD5) 000680523.pdf: 1511132 bytes, checksum: c20e5a6a65edf2cba90d4de2b5073a08 (MD5) Bitstreams deleted on 2014-10-03T16:48:53Z: 000680523.pdf,Bitstream added on 2014-10-03T16:49:45Z : No. of bitstreams: 1 000680523.pdf: 1511132 bytes, checksum: c20e5a6a65edf2cba90d4de2b5073a08 (MD5) Bitstreams deleted on 2014-10-27T11:47:06Z: 000680523.pdf,Bitstream added on 2014-10-27T11:48:03Z : No. of bitstreams: 1 000680523.pdf: 1511132 bytes, checksum: c20e5a6a65edf2cba90d4de2b5073a08 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Nanopartículas magnéticas (NPMs) têm sido alvo de inúmeras investigações por seu grande potencial de aplicação nos mais diferentes campos tecnológicos. Dentre tantos, elas destacam-se na área biomédica, seja no diagnóstico ou tratamento de diversas doenças. Neste trabalho foram sintetizadas nanopartículas magnéticas (NPMs) de óxido de ferro (magnetita) pelo método de coprecipitação de íons Fe2+ e Fe3+ em meio alcalino. O objetivo central foi estudar as características morfológicas, estruturais, magnéticas e o comportamento biológico desses compostos em células cancerígenas, visando futuras aplicações biomédicas. Inicialmente as nanopartículas magnéticas foram avaliadas em função dos parâmetros físico-químicos que influenciam diretamente as características finais do produto (pH, molaridade, temperatura e tipo de base) para se observar as melhores condições de síntese e a influência de cada um nas características do produto. As nanopartículas foram caracterizadas por difratometria de raios-X, microscopia eletrônica de varredura, potencial zeta e magnetometria. Desse primeiro estudo concluiu-se que a coprecipitação produz partículas com polidispersão de tamanhos alta e que os parâmetros de síntese influenciam drasticamente as propriedades dos materiais, no entanto, todas as amostras exibiam características magnéticas. Depois de estabelecida esta etapa, as NPMs foram submetidas à transfecção em cultura celular de carcinoma cervical humano (células HeLa) e a testes biológicos como coloração com Azul da Prússia e hematoxilina-eosina, ensaio de MTT e ensaio de apoptose para averiguação da citotoxidade. A principal observação vinda desses resultados foi que as nanopartículas magnéticas sintetizadas, salvo algumas adaptações de síntese... / Magnetic nanoparticles (MNPs) have been the subject of numerous investigations because of its great potential application in many different fields of technology. Among many, they stand out in the biomedical area, either in diagnosis or treatment of various diseases. In this work were synthesized magnetic nanoparticles (MNPs) of iron oxide (magnetite) by the coprecipitation method of Fe2+ and Fe3+ in an alkaline medium. The main objective was to study the morphological, structural, magnetic and biological behavior of these compounds in cancer cells, in order to future biomedical applications. Initially the magnetic nanoparticles were evaluated against the physical and chemical parameters that directly influence the final characteristics of product (pH, molarity, temperature and type of base) to observe the best synthesis conditions and influence of each characteristics. The nanoparticles were characterized by X-ray diffraction, sccaning electron microscopy, zeta potential and magnetometry. In this first study showed that coprecipitation produces particles with high polydispersity of sizes and that the synthesis parameters dramatically influence the properties of materials, however all samples exhibited magnetic characteristics. After you make this step, the MNP were subjected to transfection in cell culture of human cervical carcinoma (HeLa) and biological tests such as staining with Prussian blue and hematoxylin-eosin, MTT assay and apoptosis assay to investigate cytotoxicity. The main observation was that these results coming magnetic nanoparticles synthesized, except for some adjustments in short, constitute a class of nanocomposites with enormous potential for therapeutic and / or diagnosis. The work brings together information ranging from the synthesis of nanoparticles to their behavior inside the cells, emphasizing the best conditions for each procedure

Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 RuthMO_DISSERT_partes_autorizadas.pdf: 1326808 bytes, checksum: b857ffe38a32dadfb7993ebe2bf2b79c (MD5) Previous issue date: 2011-03-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The species of the genus Marsdenia, Apocynaceae, are widely used in folk medicine of several countries. In Brazil is found several species belonging to this genus. The in vitro antioxidant, anticoagulant and antiproliferative activities were evaluated to aqueous extracts of stalk, leaf and root of Marsdenia megalantha. In the total antioxidant capacity assay (expressed as ascorbic acid equivalents) the stalk extract showed 76.0 mg/g, while leaf and root extracts 141.3 mg/g and 57.0 mg/g, respectively. The stalk and leaf extracts showed chelating activity around 40% at 1.5 mg/mL, while root extract, at the same concentration showed, 17%. Only the leaf extract showed a significant ability in superoxide scavenging (80% at 0.8 mg/mL). Any extract was able in scavenge hydroxyl, as well anticoagulant activity. The antiproliferative activity of the extracts was evaluated against HeLa tumor cell line. The extracts inhibited in a dose-dependent manner the cell growth. However, the leaf extract showed 80% of inhibition at 1.0 mg/mL, while stalk and root extracts inhibited 63% and 30%, respectively. To assess the mechanism of cell death caused by the leaf extract in HeLa, was performed flow cytometry and western blot. The results show that leaf extract induces cell death by apoptosis through an activation caspase-independent pathway. These data indicate that stalk and leaf extracts obtained have potential to be used as antioxidants and anticancer drugs / As esp?cies do g?nero Marsdenia, Apocynaceae, s?o bastante utilizadas na medicina popular de v?rios pa?ses. No Brasil s?o encontradas v?rias esp?cies pertencentes a esse g?nero. As atividades antioxidante, anticoagulante e antiproliferativa foram avaliadas para os extratos de caule, folha e raiz de Marsdenia megalantha. Na capacidade antioxidante total (expressa como equivalente de ?cido asc?rbico), o extrato do caule apresentou 76,0 mg/g, enquanto os extratos da folha e raiz apresentaram, respectivamente, 14,3 mg/g e 57,0 mg/g. Os extratos do caule e da folha mostraram habilidade quelante em torno de 40% na concentra??o de 1,5 mg/mL, enquanto o extrato da raiz, na mesma concentra??o, apresentou 17%. Apenas o extrato da folha apresentou uma capacidade significante em sequestrar radicais super?xidos (80% em 0,8 mg/mL). Nenhum extrato mostrou capacidade em seq?estrar radicais hidroxila, bem como atividade anticoagulante. A atividade antiproliferativa dos extratos foi avaliada contra a linhagem tumoral HeLa. Os extratos inibiram, de forma dose-dependente, o crescimento celular. Entretanto, o extrato da folha foi capaz de inibir em 80% a prolifera??o celular na concentra??o de 1,0 mg/mL, enquanto os extratos de caule e raiz inibiram 63% e 30%, respectivamente. Para avaliar o mecanismo de morte celular causada pelo extrato da folha nas c?lulas HeLa, foi realizada citometria de fluxo e western blot. Os resultados mostraram que o extrato da folha induz morte celular por apoptose atrav?s de uma via de ativa??o independente de caspase. Estes resultados indicam que os extratos de caule e folha obtidos t?m potencial para serem futuramente utilizados como f?rmacos antioxidantes e antic?ncer

Orientador: José Geraldo Nery / Banca: Paula Rahal / Banca: Maria Cristina Nonato Costa / Resumo: Nanopartículas magnéticas (NPMs) têm sido alvo de inúmeras investigações por seu grande potencial de aplicação nos mais diferentes campos tecnológicos. Dentre tantos, elas destacam-se na área biomédica, seja no diagnóstico ou tratamento de diversas doenças. Neste trabalho foram sintetizadas nanopartículas magnéticas (NPMs) de óxido de ferro (magnetita) pelo método de coprecipitação de íons Fe2+ e Fe3+ em meio alcalino. O objetivo central foi estudar as características morfológicas, estruturais, magnéticas e o comportamento biológico desses compostos em células cancerígenas, visando futuras aplicações biomédicas. Inicialmente as nanopartículas magnéticas foram avaliadas em função dos parâmetros físico-químicos que influenciam diretamente as características finais do produto (pH, molaridade, temperatura e tipo de base) para se observar as melhores condições de síntese e a influência de cada um nas características do produto. As nanopartículas foram caracterizadas por difratometria de raios-X, microscopia eletrônica de varredura, potencial zeta e magnetometria. Desse primeiro estudo concluiu-se que a coprecipitação produz partículas com polidispersão de tamanhos alta e que os parâmetros de síntese influenciam drasticamente as propriedades dos materiais, no entanto, todas as amostras exibiam características magnéticas. Depois de estabelecida esta etapa, as NPMs foram submetidas à transfecção em cultura celular de carcinoma cervical humano (células HeLa) e a testes biológicos como coloração com Azul da Prússia e hematoxilina-eosina, ensaio de MTT e ensaio de apoptose para averiguação da citotoxidade. A principal observação vinda desses resultados foi que as nanopartículas magnéticas sintetizadas, salvo algumas adaptações de síntese... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Magnetic nanoparticles (MNPs) have been the subject of numerous investigations because of its great potential application in many different fields of technology. Among many, they stand out in the biomedical area, either in diagnosis or treatment of various diseases. In this work were synthesized magnetic nanoparticles (MNPs) of iron oxide (magnetite) by the coprecipitation method of Fe2+ and Fe3+ in an alkaline medium. The main objective was to study the morphological, structural, magnetic and biological behavior of these compounds in cancer cells, in order to future biomedical applications. Initially the magnetic nanoparticles were evaluated against the physical and chemical parameters that directly influence the final characteristics of product (pH, molarity, temperature and type of base) to observe the best synthesis conditions and influence of each characteristics. The nanoparticles were characterized by X-ray diffraction, sccaning electron microscopy, zeta potential and magnetometry. In this first study showed that coprecipitation produces particles with high polydispersity of sizes and that the synthesis parameters dramatically influence the properties of materials, however all samples exhibited magnetic characteristics. After you make this step, the MNP were subjected to transfection in cell culture of human cervical carcinoma (HeLa) and biological tests such as staining with Prussian blue and hematoxylin-eosin, MTT assay and apoptosis assay to investigate cytotoxicity. The main observation was that these results coming magnetic nanoparticles synthesized, except for some adjustments in short, constitute a class of nanocomposites with enormous potential for therapeutic and / or diagnosis. The work brings together information ranging from the synthesis of nanoparticles to their behavior inside the cells, emphasizing the best conditions for each procedure / Mestre

Doctor en Bioquímica / El retículo endoplásmico (RE) y la mitocondria son organelos celulares que entablan una continua y dinámica conversación que permite a la célula responder coordinadamente a diversas situaciones fisiopatológicas. Datos previos de nuestro Laboratorio mostraron que ambos organelos incrementan los puntos de contacto físico durante la fase temprana del estrés de RE, permitiendo una mayor transferencia de Ca2+ desde el RE hacia la mitocondria y estimulando así el metabolismo de este último organelo y favoreciendo la sobrevida celular. El presente proyecto estudió la participación de proteína quinasa A (PKA) y caveolina-1 (Cav1) como agentes reguladores de esta interacción física y funcional entre el RE y la mitocondria. Como modelo de estudio se utilizaron células HeLa tratadas con tunicamicina como agente inductor de estrés de RE por 4 h. Primero, se investigó la participación de PKA en la fase temprana del estrés de RE. Esta quinasa se activó ante condiciones de estrés, medido por la fosforilación de DRP1 en Ser637 por Western blot y concomitante elongación mitocondrial (microscopía confocal). El inhibidor específico de PKA H89 previno estos cambios, comprobando que ambos son dependientes de PKA. Posteriormente, se estudió la participación de PKA en el acoplamiento RE-mitocondria a través de la medición de proximidad (microscopía confocal), contactos físicos (microscopía electrónica), transferencia de Ca2+ (microscopía de fluorescencia) y bioenergética mitocondrial (tasa de consumo de oxígeno). El papel de PKA en estos procesos se evaluó por modulación farmacológica con H89. Los resultados mostraron que la activación de PKA es necesaria para inducir la formación de contactos RE-mitocondria, y de esta forma, gatillar la respuesta adaptativa frente a estrés de RE. Luego se investigó el papel de Cav1 sobre la interface RE-mitocondria. La expresión de Cav1 abolió completamente la respuesta adaptativa frente a estrés de RE temprano, evidenciado mediante proximidad entre organelos, transferencia de Ca2+ y respiración mitocondrial. Además de alterar esta plasticidad organelar frente a condiciones de estrés, la expresión de Cav1 disminuyó significativamente la bioenergética mitocondrial basal, junto con inducir un remodelado basal de la interface RE-mitocondria medido por purificación de las membranas del RE asociadas a mitocondria (MAM). Finalmente, se determinó que la expresión de Cav1 antagonizó la señalización de PKA, impidiendo que ella fosforile a DRP1 y promueva la elongación mitocondrial en respuesta a estrés de RE. Más aún, mediante fraccionamiento subcelular, se estableció que Cav1 afectó la localización subcelular de PKA, evitando que se relocalice a las membranas microsomales en respuesta al estrés de RE. De este modo, Cav1 actúa como un regulador negativo de PKA, previniendo su activación frente a estrés de RE, evitando así la respuesta adaptativa celular / The endoplasmic reticulum (ER) and mitochondria are two organelles that continuously communicate between one another. This organelle crosstalk allows mitochondria and ER to coordinate responses to a variety of physiological and pathophysiological situations. Data from a previous work show that during the early phase of ER stress, both organelles increase their contact sites, augmenting thereby calcium transfer from ER to mitochondria. This response boosts mitochondrial bioenergetics and ultimately promotes cell survival. Here, we sought to study the role of Protein Kinase A (PKA) and Caveolin-1 (Cav1) as regulators of such organelle crosstalk. As an experimental model, HeLa cells were treated with tunicamycin to induce ER stress. First, we observed that PKA was activated during early stages of ER stress, as assessed by increased phosphorylation of DRP1 at the inhibitory site Ser637 (Western blot), and that this event was followed by mitochondrial elongation (confocal microscopy). The specific PKA inhibitor H89 prevented these changes, thus confirming that they were due to PKA and not another kinase. Subsequently, we studied ER-mitochondria coupling by evaluating organelle proximity (confocal microscopy), physical contact sites (electron microscopy), Ca2+ transfer (fluorescence microscopy) and mitochondrial bioenergetics (oxygen consumption rate). PKA was again involved in these processes by pharmacological modulation using the inhibitor H89. Our results show that PKA activation is necessary to induce the formation of ERmitochondria contacts and to trigger the adaptive metabolic responses to ER stress. Next, we investigated the role of Cav1 as a modulator of the ER-mitochondria interface. Cav1 overexpression completely abolished the early adaptive response to ER stress, as assessed by evaluating the proximity between organelles, calcium transfer and mitochondrial respiration. In addition to altering organelle plasticity in response to stress conditions, Cav1 overexpression severely decreased baseline mitochondrial bioenergetics and induced basal remodelling of the ER-mitochondria interface, as determined by purification and analysis of mitochondria-associated ER membranes. Finally, we determined that Cav1 overexpression antagonizes PKA signalling, preventing PKA-dependent DRP1 phosphorylation and mitochondrial elongation in response to ER stress. Moreover, subcellular fractionation revealed that Cav1 affected PKA localization, prevented redistribution to microsomal membranes in response to ER stress, and altered the pattern of DRP1 phosphorylation. Thus, Cav1 functions as a negative regulator of PKA that precludes PKA activation during early ER stress and thereby prevents the adaptive cellular response / Fondecyt; Fondap;

Syftet med min undersökning var att belysa förutsättningen för studie- och yrkesvägledaren att utföra sitt uppdrag beroende på rektors strategier att implementera studie- och yrkesvägledning i hela organisationen. Detta ämnade jag belysa på en kommunal gymnasieskola ur ett ledarskaps och organisationsperspektiv. De två frågeställningarna var på vilket sätt påverkas studie- ochyrkesvägledarens förutsättningar att utföra sitt uppdrag beroende av hur implementeringen av att studie- och yrkesorienteringen skett i organisationen samt på vilket sätt ledarskapet och organisationen av studie- och yrkesvägledningen kan underlätta för studie- och yrkesvägledarenatt utföra sitt uppdrag i organisationen. Jag utförde undersökningen med hjälp av kvalitativa semistrukturerade forskningsintervjuer. I undersökningen intervjuades tre rektorer och tre studie- och yrkesvägledare. Resultatet i min undersökning visar att om inte studie- och yrkesvägledningen implementeras som hela skolans ansvar kommer inte heller studie- ochyrkesvägledarna att utföra sitt uppdrag på ett tillfredsställande sätt. Vidare visar resultatet att det fanns en form av segregering av studie- och yrkesvägledningen eftersom den ansågs vara självständig och att de hade en otydlig profession.

To the reader <p> Since I worked on two separate projects towards my doctorate thesis, the arrangement of my thesis is rather unusual. The reader will find that my thesis is divided into four parts. Part 1 is dedicated to a very general introduction about the basic knowledge needed to guide you, the reader, through the rest of the thesis. Within this part, different sections focus on different fundamental aspects of Biophysics related to my work. In Part 2, I discuss my studies of the distribution and dynamics of the nuclear protein Ran in live interphase HeLa cells. This part contains a background section specific to this project, the materials and methods used for this study, experimental results, a discussion of our findings, and it ends with conclusions. Part 3 is dedicated to the study of the dynamical mechanisms responsible for the establishment of the Bed protein concentration gradient along the anterior-posterior axis in live Drosophila melanogaster embryos. Again, a specific background section is included in this part, followed by the materials and methods used to perform this research, results, discussions and finally I will summarize my results to conclude this work. The last part, part 4, is rather short and contains the summary of the overall results of my work on both nuclear proteins with some emphasis on the similarities and differences in their dynamical behavior.</p> / Thesis / Doctor of Philosophy (PhD)

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by Lui Po Yee Pauline. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 211-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.