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Microtubular systems are an integral and essential element of eukaryotic cells. Many of these systems display linkages between the microtubules. The aim of this study was to investigate one such system, the cytostomal apparatus of Tetramitus rostratus. It was hoped that some insight could be gained into the nature of inter-microtubule linkages as well as investigating conditions for possible isolation via differential solubilization. The cytostomal apparatus consists of two sheaves of microtubules that originate at the basal bodies and extend through two-thirds of the length of the cell, apparently supporting the gullet. These microtubules are extremely stable: they are cold stable, calcium stable and griseofulvin/colchicine stable. They are highly cross-linked by fine filaments. These "linkers" have an average length of 107 nm. Linker width varies from the mid-portion (6 nm) to the ends (12 nm). In this and other respects they are similar to the highly contested microtrabecular lattice. The linkers were observed in positively stained samples and in thin-sections. Glutaraldehyde was found to have a very destructive effect on the linkers. A combination of paraformaldehyde and glutaraldehyde (2.25\/0.5\) was found not to have this effect over times that are normally used for primary fixation. Another component of the cytostomal apparatus is the system of longitudinal filaments that is especially wellrevealed with high salt (0.25M NaCl or KCl) extraction. Since this extraction solubilizes the microtubules, it may allow the longitudinal filaments to be isolated. These filaments may be related to the tektin filaments found in flagellar microtubules. In addition, a novel set of crossfibres is found to originate at the juncture of the two sheaves of microtubules and fan out across two-thirds of the width of the apparatus. These fibres were found to be especially stable in urea which may, again, allow for their isolation and characterization. The linkers have been investigated for motility. Linker length was measured after treatment with ATP (which successfully reactivated the flagellar dynein) and after treatment with ATP followed by treatment with calcium (which efficiently halted all reactivated movement of the flagella). The length of the ATP treated linkers was very close (5 nm) to the control linkers and their morphology was indistinguishable. The length of the calcium treated linkers was considerable shorter (20 nm) but the morphology suggested that this was artifactual rather than due to a physiological cause. / Thesis / Master of Science (MSc)

The focus of this thesis has been four-fold. On one hand it has been to decipher, understand and manipulate the role of microtubule-trafficking in the cell. Secondly we have concentrated on the mechanism of action of agents that target the microtubules, and thirdly we have developed a model to explain acquired drug resistance to these microtubule-targeting agents. Lastly, we tested new microtubule-targeting agents that overcome acquired and intrinsic drug resistance to microtubule-targeting agents. Microtubules (microtubules) are major dynamic structural components in cells that are essential for the development and maintenance of cell shape, cell signaling, movement, and division. We sought to understand the role microtubules play within the cellular context. Microtubules act as "active highways" within the cells and are essential for the correct operation of the cell by controlling the delivery, location, and function of a plethora of proteins. We have focused on the p53 and the HIF1-α proteins. Both these proteins are crucial players in tumor progression and angiogenesis. p53 is a tumor suppressor gene commonly referred to as the guardian of the genome and HIF1-α is a transcriptional factor that plays a key role in adaptation to hypoxia. Upon DNA damage or hypoxia, p53 or HIF1-α respectively are induced and quickly localize to the cell nucleus; whereas upon DNA repair or normoxia they must quickly concentrate in the cytoplasm for degradation. Their fast movement rate is not random and is directed by a microtubule-driven motor. Furthermore, we have shown that HIF1-α mRNA also uses the microtubule network to travel from the nucleus to the site of translation.Drugs that bind to either tubulin or microtubules form one of the most effective classes of anticancer agents. The so-called anti-mitotic drugs usually arrest cells in mitosis leading to apoptosis. We analyzed the differential effects of taxol treatment on parental and taxol-resistant cells. Survivin is a protein that senses mitotic arrest and leading to apoptotic cell death. Despite the clinical success of microtubule-targeting agents, the emergence of acquired resistance to the drug is a limiting factor for curing cancer. Acquired drug resistance is the most common reason for the failure of drug treatment in cancer patients with initially sensitive tumors, and as such, is presently responsible for the majority of deaths from cancer. We sought to understand the timeline of events that takes place during the development of drug resistance to microtubule-targeting agents. While it has been widely published that a major mechanism of resistance to anti-mitotic drugs is due to acquired β-tubulin mutations, we have shown loss of heterozygosity of the wild type allele of β-tubulin must occur to confer higher levels of resistance. To overcome drug resistance to microtubule-targeting agents we have focused on alternate drug regimens that are active in anti-mitotic drug-resistant cells. The synergistic interaction of farnesyltransferase inhibitors (FTI) and taxol has recently been introduced in the clinic and surprisingly overcomes taxol resistance. We have shown that FTIs increase the binding of taxol to β-tubulin tubulin, even in taxol-resistant cells. In an effort to dissect the molecular mechanism underlying the synergistic interaction of FTIs with taxanes, we have recently discovered that FTIs affect microtubule acetylation and stability, partly due to inhibition of the tubulin deacetylase HDAC6. The inhibition of HDAC6 by the FTI lonafarnib leads to increased tubulin acetylation and that this is the molecular basis for the synergy of FTIs with Taxol. / La tesis titulada "Anti-microtubule drugs: Mechanisms of Action and Resistance. Agents anti-microtubulars: Mecanismes d'Acció i Resistència" tiene cuatro objetivos principales. El primer objetivo ha sido descifrar, entender y manipular el papel del tráfico microtubular. El segundo objetivo se ha centrado en el mecanismo de acción de los agentes que tienen como diana los microtúbulos. En el tercer objetivo se ha desarrollado un modelo para explicar la quimioresistencia adquirida a estos agentes anti-microtubulares. En el cuarto y ultimo objetivo se han probado nuevos agentes anti-microtubulares que son activos en casos de quimioresistencia adquirida o endógena a agentes anti-microtubulares.Los microtúbulos, componentes dinámicos y estructurales de las células, son esenciales para el desarrollo y mantenimiento de la forma celular, el movimiento y división celulares. Se ha intentado entender el papel que juegan los microtúbulos en el contexto celular. Los microtúbulos actúan como "autopistas activas" dentro de las células y son esenciales para la correcta operación celular ya que controlan la entrega, localización y función de una multitud de proteínas. El primer objetivo de la tesis se ha centrado en las proteínas p53 y HIF1-α. Estas dos proteínas son principales protagonistas en la progresión tumoral y la angiogenesis. La p53 es un gen supresor de tumor comúnmente llamado el guardián del genoma y el HIF1-α es un factor de trascripción que tiene un papel crucial en la adaptación celular a la hipoxia (la baja concentración de oxigeno). En los casos de el daño al ADN o hipoxia, la p53 o el HIF1-α, respectivamente, son inducidos y se translocan rápidamente al núcleo celular, mientras que cuando el ADN ha sido reparado o el regreso al estado de normoxia, las proteínas se deben concentrar en el citoplasma para su degradación por el proteosoma. El rápido movimiento de estas proteínas no es aleatorio y esta dirigido por un motor microtubular. Además, se ha demostrado que el ARN mensajero del HIF1-α también usa la red de microtúbulos para llegar del núcleo al sitio de translación a proteína. Las drogas que se unen a la tubulina forman parte de una de las clases más efectivas de agentes anticáncer. Estas drogas comúnmente referidas como antimitóticas arrestan las células en mitosis conllevando a la apoptosis celular. Se han analizados los diferentes efectos del tratamiento del taxol en líneas celulares sensibles al taxol y en líneas celulares resistentes al taxol derivadas de las líneas sensibles. La survivina es una proteína clave en el pase del arresto mitótico en la célula a la muerte por apoptosis. A pesar del éxito clínico de las drogas antimicrotubulares, un factor que limita su aplicación universal es la apariencia de focos resistentes. La quimioresistencia adquirida es la razón más común del fracaso de la quimioterapia en pacientes con cáncer que inicialmente responden al tratamiento. En el tercer objetivo de la tesis se ha descrito un modelo temporal para explicar el desarrollo de la quimioresistencia en líneas celulares tratadas continuamente con drogas antimicrotubulares. Aunque se ha publicado extensamente que el principal mecanismo de quimioresistencia a los agentes antimicrotubulares es debido a la apariencia de mutaciones en el gen de la beta-tubulina, en este objetivo se ha demostrado que es necesario que también haya perdida de heterocigosidad en el alelo wt para que las células tengan unos altos niveles de resistencia. En el cuarto objetivo se han estudiado nuevos regimenes de quimioterapia que son activos en las células resistentes. La interacción sinergística entre los inhibidores de la farnesiltransferasa (FTI) y los taxanos se introdujo recientemente en la clínica y es muy activa contra la resistencia al taxol. Se ha demostrado que los FTIs intensifican la unión del taxol a la beta-tubulina, incluso en células resistentes al taxol. Los FTIs afectan la acetilación microtubular, a través de la inhibición de la HDAC6, la tubulina deacetilasa. La mayor acetilación de la tubulina, conlleva a una tubulina más estable y más propensa a la unión del taxol.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2015-10-19T14:46:36Z No. of bitstreams: 1 Dissertação_ICS_ Clarissa Abreu Santos Teles.pdf: 2293057 bytes, checksum: 31af060d561a4d1be1960b0acba8d71c (MD5) / Made available in DSpace on 2015-10-19T14:46:36Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Clarissa Abreu Santos Teles.pdf: 2293057 bytes, checksum: 31af060d561a4d1be1960b0acba8d71c (MD5) / CAPES;PETROBRÁS;FAPUR / A espécie Ricinus communis L. conhecida como mamona, apresenta grande importância econômica e vem se destacando no cenário das plantas oleaginosas utilizadas para produção de biodiesel e na indústria ricinoquímica, sendo uma cultura promissora para a região semiárida do Brasil. Visando conhecer o comportamento das sementes de mamona em condições normais de germinação e sob restrição hídrica, objetivou-se com este trabalho avaliar os aspectos fisiológicos de germinação, resposta ao osmocondicionamento, verificar um tratamento para qualidade fisiológica e sanitária das sementes e caracterizar os eventos do ciclo celular através de técnicas biotecnológicas. Os ensaios fisiológicos foram realizados com sementes de mamona da cultivar EBDA MPA 11. Para simular a restrição hídrica foram utilizadas soluções de PEG 8000 nos potenciais osmóticos -0,2 MPa a -0,4 MPa e para o osmocondicionamento foram testadas soluções de PEG 8000 nos potenciais osmóticos -0,3 MPa a -0,9 MPa. Sementes secas embebidas em água (0,0 MPa) foram utilizadas como controle. Para qualidade fitossanitária, foram testados tratamentos com hipoclorito de sódio e fungicidas. Para análise do ciclo celular, foram testadas as técnicas de Western blotting para verificar o acúmulo da proteína tubulina, Imunocitoquímica para visualizar configurações do citoesqueleto microtubular e Citometria de fluxo para verificar a síntese de DNA. O tratamento com hipoclorito de sódio foi o mais eficaz para desinfestação superficial das sementes, melhorando a qualidade sanitária. As sementes de mamona restringiram completamente a germinação a partir do potencial -0,4 MPa e não apresentaram o efeito de priming em resposta ao osmocondicionamento. Os resultados do ciclo celular demonstraram que o acúmulo de tubulina foi proporcional ao aumento do conteúdo de DNA 4C nos núcleos das células e com a organização do citoesqueleto microtubular durante o período de embebição em água. Houve uma inibição deste processo durante a restrição hídrica, inibindo também a reativação do ciclo celular, entretanto houve uma progressão maior do processo em resposta ao tratamento de osmocondicionamento. As divisões celulares na zona meristemática das radículas das sementes de mamona ocorreram antes da protrusão radicular, indicando que o alongamento da radícula acontece por expansão e divisões celulares.

Research and prototyping of a fuel cell stack system for micro aerial vehicles (MAVs) was conducted by Virginia Tech in collaboration with Luna Innovations, Inc, in an effort to replace the lithium battery technology currently powering these devices. Investigation of planar proton exchange membrane (PEM) and direct methanol (DM) fuel cells has shown that these sources of power are viable alternatives to batteries for electronics, computers, and automobiles. However, recent investigation about the use of microtubular fuel cells (MTFCs) suggests that, due to their geometry and active surface areas, they may be more effective as a power source where size is an issue. This research focuses on hydrogen MTFCs and how their size and construction within a stack affects the power output supplied to a MAV, a small unmanned aircraft used by the military for reconnaissance and other purposes. In order to conduct this research effectively, a prototype of a fuel cell stack was constructed given the best cell characteristics investigated, and the overall power generation system to be implemented within the MAV was modeled using a computer simulation program. The results from computer modeling indicate that the MTFC stack system and its balance of system components can eliminate the need for any batteries in the MAV while effectively supplying the power necessary for its operation. The results from the model indicate that a hydrogen storage tank, given that it uses sodium borohydride (NaBH4), can fit inside the fuselage volume of the baseline MAV considered. Results from the computer model also indicate that between 30 and 60 MTFCs are needed to power a MAV for a mission time of one hour to ninety minutes, depending on the operating conditions. In addition, the testing conducted on the MTFCs for the stack prototype has shown power densities of 1.0, an improvement of three orders of magnitude compared to the initial MTFCs fabricated for this project. Thanks to the results of MTFC testing paired with computer modeling and prototype fabrication, a MTFC stack system may be possible for implementation within an MAV in the foreseeable future. / Master of Science

Submitted by Natalie Mendes (nataliermendes@gmail.com) on 2015-08-04T01:30:32Z No. of bitstreams: 1 CRIS TESE FINAL.pdf: 3970188 bytes, checksum: 37240b01333f3a3c2c98ce2597450f53 (MD5) / Made available in DSpace on 2015-08-04T01:30:32Z (GMT). No. of bitstreams: 1 CRIS TESE FINAL.pdf: 3970188 bytes, checksum: 37240b01333f3a3c2c98ce2597450f53 (MD5) Previous issue date: 2015-03-13 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The life cycle of a seed plant involves subsequent stages of development including seed formation, germination and seedling establishment. Together these stages represent the critical phase of intersection between two generations and are characterized by deep cytological, morphological, metabolic and physiological changes. Jatropha curcas L. (Euphorbiaceae) is popularly known as physic nut and produces seeds rich in oil with properties that allow its use in various industries, including the production of biodiesel. This study aimed to advance on the understanding of morphophysiological patterns and elucidate morphoanatomical adaptations involving embryogenesis, maturation, germination and seedling growth in J. curcas. Therefore, it was initially analysed and described the morphophysiological profile based on 13 stages of development and maturation associated to color of the fruit exocarp and seed coat, and description of the structures present at each stage (Chapter 1). Analysis of microtubular cytoskeleton configurations during embryogenesis showed cell cycle activity by the presence of cortical and mitotic microtubules during histodifferentiation and organogenesis, whilst it was possible to characterize a new organogenetic profile of embryogenesis revealed by the presence of a multimeristematic radicle and stomata in embryos of J. curcas seeds (Chapter 2). The multimeristematic embryos formed by a central apical meristem and four lateral meristems interconnected by a complex vascular system have revealed a new model of root formation during seed germination and seedling development, in which there is simultaneous protrusion of a larger main root and four smaller adventitious roots, all growing at the same time during the formation of the seedling root system (Chapter 3). The stomata occurred in the radicle-hypocotyl transition area, exhibited different sizes and ontogenic phases and short lifespan by degenerating during seedling development. This demonstrates it?s functioning as restricted to the simultaneous growth stage of the five roots during germination, apparently due to high demand in gas exchange and energy metabolism, and a likely evolution onto the lenticels present in the stem of this species (Chapter 4). / O ciclo de vida de uma planta com sementes envolve est?dios subsequentes de desenvolvimento, como a forma??o da semente, a germina??o e o estabelecimento da pl?ntula. Essas etapas juntas representam a fase cr?tica de interse??o entre duas gera??es e s?o caracterizadas por profundas mudan?as citol?gicas, morfol?gicas, metab?licas e fisiol?gicas. Jatropha curcas L. (Euphorbiaceae) conhecida popularmente como pinh?o-manso, produz sementes ricas em ?leo com propriedades e aplica??es em diversos setores industriais, incluindo a produ??o de biodiesel. O presente estudo teve como objetivo caracterizar padr?es morfofisiol?gicos e elucidar adapta??es morfoanat?micas envolvendo a embriog?nese, matura??o, germina??o e o crescimento de pl?ntulas de J. curcas. Para tanto, foi inicialmente analisado e descrito o perfil morfofisiol?gico baseado em 13 est?dios de desenvolvimento e matura??o, associados ? colora??o do exocarpo do fruto e do tegumento das sementes e descri??o das estruturas presentes em cada est?dio (Cap?tulo 1). A an?lise das configura??es do citoesqueleto microtubular durante embriog?nese evidenciou atividade do ciclo celular por meio da presen?a de microt?bulos corticais e mit?ticos durante a histodiferencia??o e organog?nese. Foi poss?vel caracterizar um novo padr?o organogen?tico de embriog?nese revelado pela presen?a de rad?cula multimeristem?tica e de est?matos em embri?es de sementes de J. curcas (Cap?tulo 2). Os embri?es multimeristem?ticos, providos de um meristema apical central e quatro meristemas laterais, revelaram um novo modelo de forma??o de sistema radicular durante a germina??o de sementes e desenvolvimento de pl?ntulas, em que h? protrus?o simult?nea de uma raiz principal maior e quatro ra?zes advent?cias menores, todas crescendo ao mesmo tempo, durante a forma??o inicial do sistema radicular da pl?ntula (Cap?tulo 3). Os est?matos ocorrem na ?rea de transi??o hipoc?tilo-rad?cula e exibem diferentes tamanhos e fases ontog?nicas. Estas estruturas apresentaram um curto per?odo de vida, degenerando-se durante o desenvolvimento da pl?ntula, sugerindo seu funcionamento restrito ? etapa de crescimento simult?neo das cinco ra?zes durante a germina??o, aparentemente devido ? alta demanda em trocas gasosas e metabolismo energ?tico, e uma prov?vel evolu??o para as lenticelas presentes no caule desta esp?cie (Cap?tulo 4).

Archamoebae is a small group of anaerobic protists belonging to the eukaryotic supergroup Amoebozoa. Historically, they were regarded as primitively amitochondriate. However, a mitochondrial remnant has been found in some archamoebae. Phylogenetic analyses showed that Archamoebae are closely related to the aerobic slime moulds (Mycetozoa). Trophozoites of archamoebae are amoeboflagellates or aflagellated amoebae. The group includes both parasitic (Entamoeba, Endolimax and, possibly, Endamoeba and Iodamoeba) and free-living (Mastigamoeba, Mastigella, Pelomyxa) genera. The genus Mastigina comprises both endozoic and free-living representatives. Flagellated genera Mastigina, Mastigamoeba, Mastigella and Pelomyxa possess a single basal body associated with a microtubular cone which may or may not be associated with nucleus. The cone is a common feature for Archamoebae and mycetozoan slime moulds. The phylogeny of Archamoebae has not been fully elucidated yet and the taxonomy of free-living representatives is confusing. In the present study, we obtained 42 stable isolates of free-living Archamoebae. We sequenced and analyzed SSU rDNA of 15 of them. The Archamoebae split into five lineages. Based on TEM, we were able to recognize genera Mastigamoeba and Mastigella. The isolate IND8 probably represents a new...