O alumínio é limitante à atividade agrícola em todo o mundo. Nos solos ácidos a disponibilidade de Al aumenta. Estes solos constituem a maioria dos solos do mundo e dois terços dos solos brasileiros. O problema da acidez do solo e da toxicidade por Al é altamente significativo para as perdas na produtividade agrícola e florestal. Para se ter Al disponível, primeiramente tem que se ter condições de pH baixo. O primeiro sintoma causado pela toxicidade por Al é a inibição no alongamento do sistema radicular. Existem trabalhos vinculando a inibição a alterações nos processos de divisão e expansão celular. Embora os mecanismos de toxicidade e resistência ao Al não estejam totalmente elucidados, admite-se que em algumas plantas, a quelação do Al por ácidos orgânicos é um dos mecanismos que confere resistência das células ao Al, assim como em outras plantas a elevação do pH da rizosfera, por compostos liberados pelo sistema radicular, atua na queda da disponibilidade do Al na solução do solo. Porém, existem outras alternativas que vêm sendo propostas na literatura como possíveis mecanismos de resistência das plantas ao Al, principalmente ao nível celular e molecular. Alterações nas composições lipídica e protéica da membrana plasmática, assim como na sua estrutura física; ativação do sistema antioxidante celular; alterações na sinalização celular e de atividade dos canais de troca da membrana plasmática vêm sendo estudados como possíveis contribuintes para os mecanismos de resistência ao Al. A sensibilidade celular ao Al depende do seu estágio de desenvolvimento. As células sensíveis ao Al acumulam o metal, enquanto que as resistentes acumulam muito pouco. Foi constatado em nosso trabalho que as células sensíveis ao Al também são sensíveis ao baixo pH. As células sensíveis não conseguem recuperar seu crescimento e sua viabilidade celular após a exposição ao Al ou ao baixo pH.A sacarose ou manitol conferiram proteção às células quanto ao acúmulo de Al. Isso fez com que a viabilidade mantivesse-se em níveis próximos ao controle (pH5,6) e a cultura conseguisse recuperar seu crescimento e viabilidade após a exposição ao Al e ao baixo pH. O efeito protetor não foi devido ao caráter energético da sacarose, pois o manitol não é metabolizado pelas células BY-2 e os resultados foram semelhantes quando se usou sacarose ou manitol, nas mesmas concentrações. Sabe-se que o Al aumenta a peroxidação lipídica e a oxidação protéica da membrana plasmática, pela geração de EAO?s, desencadeando o processo de estresse oxidativo na célula. Em nosso estudo, nas células sensíveis houve peroxidação dos lipídios, ativação do sistema de enzimas antioxidantes, como SOD, GST, GR, CAT e APX, alteração nos níveis de carboidratos e alteração no perfil protéico de frações enriquecidas de membrana plasmática, obtido por eletroforese 2D. O mesmo comportamento foi verificado em células sensíveis tratadas a baixo pH. Pode-se concluir que o sistema antioxidante celular foi ativado na presença de baixo pH ou Al, pela ocorrência de peroxidação lipídica, que gera maiores concentrações de H2O2 nas células sensíveis (fase log). E que existem diferenças no perfil protéico de células tratadas com Al em relação a células mantidas sob condições de cultivo, tanto em presença de spots como em expressão diferencial. Porém estas diferenças necessitam ser melhores exploradas. A peroxidação lipídica é um bom indicador da sensibilidade celular ao Al e ao baixo pH, assim como a ativação do sistema antioxidante e a geração do peróxido de hidrogênio. Poderiam ser realizados experimentos no tempo, medindo-se o acúmulo de Al e relacionando-o aos níveis de peroxidação lipídica, atividade das enzimas antioxidantes e geração do peróxido, para que pudéssemos indicar talvez um processo que se iniciasse antes que outro, ou mesmo que decaísse antes do outro. Assim como um monitoramento das condições de oxidação protéica na presença de Al. / Aluminum limits crop production in all over the world. In acid solis the Al disponibility is larger. Acid soils compose the major part of the brazillian soils. The problem of acidity and Al toxicity results in losses of productivity in agriculture and forestry. The first symptom of Al toxicity is inhibition of root growth. There is many studies that indicate relations between the inhibition of root growth and cell division and expansion alterations. The mechanisms of Al toxicity and resistance aren?t completely understood in plants. The resistance mechanism of Al chelation by organic acid is one of the mechanisms accept, like the elevation of the rizosphere pH by substances exsudated by the root system. Other possible mechanisms that are being mentionated are the alterations in plasma membrane composition and structure, antioxidant cell system activation, alterations in cell signal and alterations in the membrane channels activity. Aluminum cell sensibility depends of the status cellular. The cells that are sensible to Al, are in the log phase of growth and accumulate the metal, whereas the resistant cells do not accumulate and were in the stationary phase of growth. In our work, we observed that the sensible cells are sensible to low pH too. The sensible cells don?t recover their growth rate and cellular viability after the treatment exposition. Sucrose or mannitol confers cellular protection against the Al. The cellular viability was high (next to the control, pH5,6) and the cell culture recovery their growth and viability after the Al or low pH exposition. The protective effect don?t occurs in response to the energetic role of sucrose, because cells treated with mannitol showed the same results and the mannitol did not metabolizated by tobacco BY-2 cells. Al induces lipid peroxidation and protein oxidation in plasma membrane, by the ROS generation promoting the oxidative stress. We found that sensible Al cells showed lipid peroxidation, H2O2 generation, antioxidant enzymes activation (SOD, le carbohydrate levels and protein profile alterations by 2D electrophoresis. The same responses were observed in the pH sensible cells, at log phase of growth. This differences should be more explored. We concluded that the lipid peroxidation is an indicator of sensitivity to Al and low pH, like the antioxidant enzymes activities and the H2O2 generation. Studies should be done with the Al accumulated in time, measuring the activities of antioxidant system and the lipid peroxidation with the objective to indicated what process could start firstly

Eine wichtige Eigenschaft der Plasmamembran eukaryotischer Zellen ist die stabile transversale Asymmetrie der Phospholipide. Sie wird energieabhängig durch die Aktivität einer Aminophospholipidtranslokase aufrechterhalten und gilt als wichtige Voraussetzung für die Homöostasis der Zellen. Die Plasmamembran einiger Säugerzellen weist zudem laterale Lipiddomänen auf, denen eine wesentliche Bedeutung bei der Signaltransduktion zugeschrieben wird. Während der Genese durchlaufen die Membranen der Säugerspermien intensive Veränderungen. Um die Bedeutung der Phospholipidasymmetrie für die Funktion der Spermien zu untersuchen, wurde die Lokalisation und Dynamik von Phosphatidylserin in der Zell- und Akrosommembran von Eberspermien im Verlauf von Kapazitation und Akrosomreaktion betrachtet. Unter Ausnutzung der selektiven, kalziumabhängigen Bindung von AnnexinV an endogenes Phosphatidylserin konnte dessen Lokalisation an morphologisch differenzierten Zellen verfolgt werden. Eine Markierung der Zellen mit NBD-markierten Phospholipidanaloga lieferte zudem Informationen zur Dynamik der Phospholipide in der Plasmamembran. Die Differenzierung der Zellen erfolgte entweder am Durchflusszytometer oder fluoreszenz- bzw. elektronenmikroskopisch. Die Ergebnisse der vorliegenden Arbeit weisen sowohl auf eine transversale als auch laterale Ungleichverteilung der Lipide in der Zell- und Akrosommembran während der Genese der Spermien hin. Neben der stabilen transversalen Phospholipidasymmetrie der Plasmamembran konnte erstmals eine zytoplasmatische Lokalisation von Phosphatidylserin auf der äußeren Akrosommembran nachgewiesen werden. Somit akkumulieren die beiden einander zugewandten zytoplasmatischen Monolayer von Plasmamembran und äußerer Akrosommembran Phosphatidylserin. Kapazitationsbedingt kommt es zu einer engen Wechselwirkung zwischen Plasmamembran und äußerer Akrosommembran. Die Ausbildung lateraler Membrandomänen, in denen Phosphatidylserin zytoplasmatisch akkumuliert, wird als Voraussetzung für diese enge Assoziation diskutiert. Weitere Hinweise auf eine funktionelle Bedeutung lateraler Membrandomänen lieferten die Arbeiten zur Isolation Triton-unlöslicher Lipiddomänen aus der Plasmamembran von Forellenspermien. / One of the essential qualities of cell membranes in Eucaryotae is a stable transverse phospholipid asymmetry. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase and is a major prerequisite for cell homeostasis. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The membranes of mammalian spermatozoa undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of boar sperm shows a stable transversal lipid asymmetry characterised by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Applying microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably essential for the strong association of plasma and outer acrosome membrane finally leading to local fusions of both membranes. An indication for the functional meaning of lateral membrane domains in sperm cells was futher deduced from the isolation of Triton-insoluble lipid domains from membranes of trout sperm cells.

Der ABC-Transporter ABCA1 ist unmittelbar in die zelluläre Lipidhomeostasie einbezogen, in dem er die Freisetzung von Cholesterol an plasmatische Rezeptoren, wie ApoA-I, vermittelt. Trotz intensiver Untersuchungen ist dieser molekulare Mechanismus nicht verstanden. Verschiedene Studien deuten daraufhin, dass durch die Aktivität von ABCA1 bedingte Veränderungen in der Lipidphase der äußeren Hälfte der Plasmamembran (PM) wichtig für die Freisetzung des Cholesterols sind. In der vorliegenden Arbeit wird die Lipidumgebung von ABCA1 in der PM lebender Säugetierzellen unter Anwendung der Fluoreszenzlebenszeitmikroskopie von fluoreszierenden Lipidsonden untersucht. Es wurde eine breite Verteilung der Fluoreszenzlebenszeiten der Sonden gefunden, die sensitiv gegenüber Veränderungen der lateralen und transversalen Organisation der Lipide ist. Im Einklang mit Studien an riesengroßen unilamellaren Vesikeln und Plasmamembranvesikeln weisen unsere Ergebnisse die Existenz einer größeren Vielfalt submikroskopischer Lipiddomänen auf. Die FLIM-Untersuchungen an ABCA1 exprimierenden HeLa-Zellen weisen eine die Lipidphase destabilisierende Funktion des Transportes aus. Dieses wurde unterstützt durch die Lipidanalyse von Fraktionen der PM. Auf der Basis unserer Untersuchungen und früheren Daten stellen wir die Hypothese auf, dass die Exponierung von Phosphatidylserin (PS) auf der Zelloberfläche ein zentrales Ereignis der ABCA1 bedingten Veränderungen ist. Allerdings zeigen vergleichende Studien an ABCA7 exprimierenden Zellen, dass dies nicht ausreicht, um die ABCA1 verursachten Veränderungen in der Lipidpackung der PM zu erklären. Unsere Ergebnisse beweisen, dass die Fähigkeit von ABCA1, den Cholesterolefflux zu vermitteln, auf durch den Transporter bedingte Veränderungen in der LP der PM zurückzuführen sind, die unabhängig von der Bindung von ApoA-1 sind und dieser vorausgehen. Diese Veränderungen sind notwendig für die Lipidierung von ApoA-1 und der Generierung von HDL-Partikeln. / The ABCA1 transporter organizes cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors such as ApoA-I. Despite intensive investigation, the molecular mechanism of such a process has not yet been clarified. In the present study we report on the analysis of the ABCA1 lipid microenvironment at the plasma membrane of living cells, by a novel approach based on fluorescence lifetime imaging microscopy (FLIM). In the plasma membrane of mammalian cells, a broad fluorescence lifetime distribution sensitive to treatments interfering with the membrane lateral and transbilayer organization was found. In agreement with investigations in giant unilamellar vesicles and giant plasma membrane vesicles, our results are consistent with the existence of a large variety of submicroscopic lipid domains. Based on that, FLIM in HeLa cells expressing ABCA1 revealed the destabilizing function of the transporter on the lipid arrangement at the membrane, indicating that lipid packing was a primary target of ABCA1 activity. This was corroborated by the analysis of plasma membrane fractions isolated by density fractionation. On the basis of our analysis and previous data, we speculate that the exposure of phosphatidylserine on the cell surface is a central event for ABCA1-dependent modifications. However, a comparative study of cells expressing ABCA7, the member of the ABCA subfamily with the highest homology to ABCA1, revealed that exposure of PS alone cannot account for the detected effects. Collectively, our data suggest that the ability of ABCA1 to promote cholesterol efflux is independent and precedes its actual binding to ApoA-I. Rather, ABCA1-induced plasma membrane modifications are necessary for the lipidation of ApoA-I and the generation of high density lipoprotein particles.

Caveolae sind charakteristische Plasmamembraneinstülpungen, die in vielen Zelltypen vorkommen und deren biologische Funktion umstritten ist. Ihre besondere Form und ihre Häu gkeit in Zellen, die stets mechanischen Belastungen ausgesetzt sind, führten zu der Annahme, dass Caveolae die Plasmamembran vor mechanischen Belastungen schützen und als Membranreservoir dienen. Dies sollte mit dieser Dissertation experimentell geprüft werden. Zunächst wurde der Ein uss der Caveolae auf die Membranspannung von Zellen im Normalzustand untersucht. Dann wurden die Zellen mechanisch belastet. Mit Fluoreszensmikroskopie wurde das Verschwinden von Caveolae nach Strecken der Zellen oder nach einem hypo-osmotischen Schock beobachtet. Messungen der Membranspannung vor und unmittelbar nach dem hypo-osmotischem Schock zeigten, dass Caveolae einen Anstieg der Membranspannung verhindern, unabhängig von ATP und dem Cytoskelett. Die Erzeugung von Membranvesikel mit Caveolae erlaubte es, diesen Effekt der Caveolae in einem vereinfachten Membransystem zu beobachten. Schliesslich wurden Muskelzellen untersucht. Zellen, die genetisch bedingt weniger Caveolae haben und mit Muskelschwundkrankheiten in Verbingung stehen, waren mechanisch weniger belastbar als gesunde Zellen. Zusammenfassend wird mit dieser Dissertation die These bestärkt, dass Caveolae einem Anstieg der Membranspannungen entgegenwirken. Dass dies in Zellen und in Vesikeln unabhängig von Energie und Cytoskelett geschieht, lässt auf einen passiven, mechanisch getriebenen Prozess schliessen. Diese Erkenntnis trägt zum Verständnis der Rolle von Caveolae in Zellen bei und kann dem besseren Verständnis von Krankheiten bedingt durch Caveolin-Mutationen, wie z.B. Muskelschwundkrankheiten, dienen. / Caveolae, the characteristic plasma membrane invaginations present in many cells, have been associated with numerous functions that still remain debated. Taking into account the particular abundance of caveolae in cells experiencing mechanical stress, it was proposed that caveolae constitute a membrane reservoir and bu er the membrane tension upon mechanical stress. The present work aimed to check this proposition experimentally. First, the in uence of caveolae on the membrane tension was studied on mouse lung endothelial cells in resting conditions using tether extraction with optically trapped beads. Second, experiments on cells upon acute mechanical stress showed that caveolae serve as a membrane reservoir bu ering surges in membrane tension in their immediate, ATP- and cytoskeleton-independent attening and disassembly. Third, caveolae incorporated in membrane vesicles also showed the tension bu ering. Finally, in a physiologically more relevant case, human muscle cells were studied, and it was shown that mutations with impaired caveolae which are described in muscular dystrophies render muscle cells less resistant to mechanical stress. In Summary the present work provides experimental evidence for the hypothesis that caveolae bu er the membrane tension upon mechanical stress. The fact that this was observed in cells and membrane vesicles in an ATP and cytoskeleton independent manner reveals a passive, mechanically driven process. This could be a leap forward in the comprehension of the role of caveolae in the cell, and in the understanding of genetic diseases like muscular dystrophies. / Cavéoles sont des invaginations caractéristiques de la membrane plas- mique présents dans beaucoup de types cellulaires. Ils sont liées à plusieurs fonctions cellulaires, ce qui sont encore débattues. Prenant compte de l importance des cavéoles dans les cellules soumises au stress mécanique, les cavéoles sont proposées de constituer un réservoir membranaire et de tamponner la tension membranaire pendant des stresses mécaniques. Cette étude a eu le but de tester cette hypothèse expérimentalement. En premier, l in uence des cavéoles sur la tension membranaire au repos a été étudiée sur des cellules endothéliales du poumon de la souris. Puis, on a montré que les cavéoles tamponnent l augmentation de la tension membranaire après l application d un stress mécanique. En suite, la réalisation des vésicules membranaires contenant des cavéoles a permit de montrer leur rôle comme réservoir membranaire dans un système simpli é. Finalement, dans un contexte physiologiquement plus relevant, l étude des cellules musculaires a montrée que les mutations du cavéolin associées aux dystrophies musculaires rendent les cellules moins résistante aux stresses mécaniques. En conclusion, cette étude supporte l\'hypothèse que les cavéoles tamponnent la tension membranaire pendant des stresses mécaniques. Le fait que cela se passe dans les cellules et les vésicules indépendamment d ATP et du cytosquelette révèlent un processus passif et mécanique. Cela pourrait servir à une meilleure compréhension du rôle des cavéoles dans la cellule et les maladies génétiques comme les dystrophies musculaires.

This thesis examined the translocation of fatty acid (FA) transport proteins to the plasma membrane (PM) in human and rat skeletal muscle during moderate intensity exercise. The responses to the post-exercise period and to acute moderate intensity exercise after 6 weeks of high intensity interval training (HIIT) were also examined in humans. The overall hypotheses were that 1) FAT/CD36 and FABPpm would translocate to the PM in human skeletal muscle during 120 min of moderate intensity exercise, 2) FAT/CD36 and FABPpm would translocate to the PM in rat skeletal muscle during 120 min of moderate intensity exercise and this would correlate to an increase in palmitate uptake, 3) FAT/CD36 and FABPpm would translocate to the PM during 120 min of moderate intensity exercise, but return to basal levels by 45 min post-exercise, 4) six weeks of HIIT would increase PM content of FABPpm but not FAT/CD36 in resting skeletal muscle, 5) six weeks of HIIT would cause a further increase in the translocation of FAT/CD36 and FABPpm to the PM during moderate intensity exercise and this would correspond to an increase in whole body fat oxidation compared to exercise pre-training, and 6) six weeks of HIIT would increase whole muscle content of FATP1 and FATP4. In human skeletal muscle, PM FAT/CD36 and FABPpm increased 75% and 20% respectively after 120 min of cycling at ~60% VO2 peak which corresponded to a 110% increase in whole body fat oxidation. In rat skeletal muscle, PM FAT/CD36 and FABPpm increased 20% and 30% respectively, which correlated to a 30% increase in palmitate uptake following 120 min of treadmill running at ~65% VO2 peak. The PM content of FAT/CD36 increased further to 120% of resting values by 45 min of post-exercise following 120 min of cycling at ~60% VO2peak, which correlated with a heavy reliance on fat as a fuel during the post-exercise period. FABPpm returned to resting levels of PM content by 15 min post-exercise. After 6 wk of HIIT, whole muscle FAT/CD36 (50%), FABPpm (21%) and FATP4 (25%) were increased in human skeletal muscle, while FATP1 remained unchanged. There were no changes in PM content of FAT/CD36 or FABPpm at rest following training. FAT/CD36 and FABPpm were also measured before and after 120 min of cycling at ~60% of pre-trainingVO2 peak following training, but no differences in the magnitude of the PM content increases were seen compared to pre-training, despite a 27% increase in fat oxidation. These studies demonstrate that FA transport proteins translocate to the PM during moderate intensity exercise, which correlates with increased FA uptake and whole body fat oxidation. This relationship does not appear to hold during the post-exercise period, as further increases in the PM content of FAT/CD36 does not correspond with the decrease in fat oxidation. The PM content of FAT/CD36 and FABPpm were not increased at rest following training, and there was no effect of training on the translocation of FAT/CD36 or FABPpm to the PM during moderate intensity exercise at the same absolute power output, however there may be a further increase at a relative power output. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research, Ontario Graduate Scholarship

Although spinal cord injury (SCI) is a debilitating condition that presents a large socioeconomic problem in the United States, there is currently no treatment that reliably reduces morbidity and mortality. Current research is aimed at identifying mechanisms involved in the pathophysiology of SCI and using this knowledge to develop rational treatments. We have observed plasma membrane compromise in the acute (within 10 minutes), sub-acute (3 days), and chronic phases (5 weeks) in a rat model of contusion SCI and postulate that it negatively affects neurological outcome. Holes/tears in the plasma membrane were assessed with a dye exclusion assay, in which a fluorescent cell-impermeant dye was injected into the cerebrospinal fluid prior to sacrifice; therefore, cellular uptake of the dye is indicative of plasma membrane compromise. As early as 10 minutes after SCI, widespread uptake of permeability markers was evident in neuronal cell bodies as well as axonal projections. The number of permeable cells and the size of the membrane breaches (measured by using permeability markers of various sizes) varied with distance from the injury site, with larger disruptions located closer to the epicenter. Greater cellular uptake was observed when the impact force was increased (200 > 150 > 100 kdyn > sham). At longer time points (3 days and 5 weeks), substantial permeability marker uptake was observed in axons but not in cell bodies. Cells with increased permeability displayed a variety of pathomorphological alterations, including swelling, blebbing, retraction bulb formation, neurofilament loss, and fragmentation, suggesting that increased plasma membrane permeability is detrimental to cell survival and function. We therefore investigated a clinically-relevant treatment strategy designed to restore plasma membrane integrity. Animals were treated with citicoline, a molecule utilized in the endogenous synthesis of phosphatidylcholine (the major membrane component in mammalian cells). Citicoline has been shown to be beneficial in numerous studies of neurological disease, improving overall outcome by increasing phospholipid synthesis and attenuating phospholipid destruction (by reducing phospholipase A2 activity). However, these mechanisms have not been explored in a model of SCI. When compared to injured animals receiving vehicle (saline) injections, citicoline treatment after SCI did not have a statistically significant effect on cytoplasmic PLA2 activity (at 24h post-injury), the density of permeable axons (at 3 days post-injury), or the lesion volume (at 3 days post-injury). Since citicoline may improve neurological outcome after SCI through mechanisms we did not directly assess, we then conducted a longer-term study to evaluate the overall efficacy of citicoline treatment in terms of longer-term functional and histological consequences. Citicoline did not have a biologically significant effect on behavioral recovery (evaluated during open field locomotion, grid walk and hyperalgesia testing weekly for up to 5 weeks post-injury) or lesion volume (at 5 weeks post-injury). The lack of citicoline-mediated effect may be attributed to experimental parameters (e.g., dosing or sensitivity of outcome measures) or biological inefficacy. Although we were not able to demonstrate that citicoline improves outcome after SCI, the finding that plasma membrane damage occurs in a persistent fashion and is associated with pathophysiological cellular alterations may provide fundamental knowledge necessary for developing treatments targeted at membrane repair. Future work examining the complex mechanisms causing prolonged membrane damage after SCI and evaluating strategies for manipulating these pathways (potentially using citicoline in combination with other pharmacological agents) may lead to a clinically effective therapy.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Orientadora: Profa. Dra. Mirian Pacheco Silva Albrecht / Dissertação (mestrado) - Universidade Federal do ABC, Programa De Pós-Graduação em Ensino, História, Filosofia das Ciências e Matemática, 2015. / O estudo da permeabilidade seletiva da Membrana Plasmática envolve conceitos microscópicos que exigem grande imaginação dos alunos e para trabalhar com esses conceitos o professor pode utilizar diversos recursos didáticos. Por isso, como estamos vivendo em uma era digital, na qual as informações são veiculadas com muita rapidez, faz-se necessário repensar os recursos didáticos visando aproximá-los do cotidiano dos alunos. Sabemos que os jogos eletrônicos se tornaram uma das principais atividades dos adolescentes, interferindo em vários aspectos da vida cotidiana, inclusive na educação. Neste contexto, surge a questão: Como elaborar um jogo eletrônico para ser disponibilizado aos professores como um recurso didático, contemplando um equilíbrio entre o conteúdo curricular de permeabilidade seletiva da membrana plasmática e a jogabilidade? O objetivo desta pesquisa foi desenvolver um jogo eletrônico e educativo como recurso didático para o ensino de conteúdos curriculares sobre a permeabilidade seletiva da membrana plasmática, contemplando um equilíbrio entre o conteúdo proposto e a jogabilidade. A fundamentação da pesquisa é composta de estudo sobre as dificuldades no ensino e aprendizagem deste conteúdo e também, sobre a utilização de jogos educativos na educação. Para a construção do jogo utilizamos o software livre Game Maker Studio, três livros didáticos aprovados pelo Programa Nacional do Livro Didático, os Parâmetros Curriculares Nacionais e as Orientações Curriculares Nacionais, resultando na construção de um jogo sobre a permeabilidade seletiva da membrana plasmática com quatro fases que correspondem aos processos de osmose, difusão simples, difusão facilitada e transporte ativo e que devido ao conteúdo curricular abordado recebeu o nome de Cell Membrane. A pesquisa foi realizada segundo os pressupostos da pesquisa qualitativa, sendo que o jogo foi aplicado em um teste piloto a quatorze alunos de Ensino Médio e, posteriormente foi avaliado por vinte alunos de graduação em Biologia. Como resultado os participantes, tanto da aplicação quanto da avaliação do jogo, o descreveram como um recurso auxiliar, desafiador e divertido, demonstrando que o jogo contempla o equilíbrio proposto entre a jogabilidade e o conteúdo curricular. / The study of the selective permeability of the Plasma Membrane involves microscopic concepts that require great imagination of students and to work with these concepts the teacher can use a variety of teaching resources. Therefore we are living in a digital age where the information is transmitted very quickly and it is necessary to rethink the teaching resources aimed at bringing them closer to the students' daily lives. We know that electronic games have become one of the main hobbies of teenagers interfering in various aspects of everyday life including education. In this context the question arises: How to design a game to be made available to teachers as a teaching tool comprising a balance between curriculum content of selective permeability of the plasma membrane and the gameplay? The objective of this research is to develop an electronic and educational game as a teaching resource for teaching content of the selective permeability of the plasma membrane comprising a balance between the proposed content and gameplay. The foundation of the research consists of study of the difficulties in teaching and learning of this content and also on the use of educational games in education. For the construction of the game we used the free software Game Maker Studio and three textbooks approved by the National Textbook Program and the National Curriculum Guidelines Program that result a game on the selective permeability of the plasma membrane with four phases that correspond to the osmosis process, simple diffusion, facilitated diffusion and active transport, because the content the game was named Cell Membrane. The research is being carried out according to the assumptions of qualitative research. The game was applied in a pilot test with fourteen high school students and also valued by twenty undergraduate students in Biology. Both participants described the game as extra teaching tool, challenging and fun, demonstrating that the game include the proposed balance between gameplay and curricular content.

Arabinogalactan proteins (AGPs) are proteoglycans heavily substituted by arabinogalactan polysaccharides. These are composed of arabinose and galactose, and minor sugars such as glucuronic acid (GlcA), fucose and xylose. The arabinogalactan polysaccharides do not decorate classical AGPs exclusively, but they can also be found decorating a wide range of proteins. Arabinogalactan proteins have been implicated in many processes of plant development. Recently, AGPs were proposed to bind and store calcium at the plasma membrane. They are extracellular, and are localised mainly at the plasma membrane via a GPI-anchor. They can also be soluble in the apoplast. Their low abundance, chemical similarity and high functional redundancy have hindered their study. My strategy to overcome these difficulties was to study knock-out Arabidopsis thaliana plants of glycosyltransferases that transfer sugars specifically onto AG-polysaccharides. Glucuronic acid makes up about 10% of the arabinogalactan polysaccharide structure in Arabidopsis thaliana cell culture AGPs. Previously, the glucuronic acid transferase A TGLCA T14A, a member of the CAZy Glycosyl Transferase 14 family, was shown to transfer GlcA specifically onto AGPs, and knock-out Arabidopsis plants showed a 30% reduction in [Me]GlcA substitution in AGP-enriched preparations. However, no clear growth phenotype was observed. The characterisation of knock-out plants of other GT14 family members and combinations thereof is described here. Based on previous studies (Lamport and Várnai, 2013), I assayed in vitro the calcium binding capacity of AGP extracts from WT and knock-out plants. The results showed that AGP extracts from knock-out plants can hold less calcium than WT plants in vitro. A wide range of plant growth phenotypes were identified. Growth phenotypes can be explained by changes in the cytoskeleton and deficiencies in calcium signaling. Our evidence suggests links between structural deficiencies of extracellular proteoglycans to extracellular calcium and cytoskeleton. This research has the potential to create a new model system for the study of molecular mechanisms dependent on calcium that drive cell expansion, division and differentiation in plants.

Os solos ácidos recobrem, cerca de 40% da área terrestre, constituindo-se em um dos principais fatores limitantes à população à produção vegetal do planeta. No Brasil, os solos ácidos abrangem cerca de dois terços do território nacional. De um modo geral, a acidez do subsolo reduz a profundidade do sistema radicular, aumentando a susceptibilidade à seca e diminuindo o uso de nutrientes pelas plantas. Além da alta atividade de íon hidrogênio (H+), os solos ácidos geralmente apresentam níveis tóxicos de alumínio, sendo que a toxicidade por AI tem sido intensivamente investigada nos últimos anos. No entanto, a toxicidade por AI ocorre apenas a pH baixo e em condições onde a toxicidade por prótons geralmente também se manifesta. Apesar disto, trabalhos envolvendo a toxicidade por prótons são escassos. A sensibilidade de células a pH baixo depende da fase de crescimento e desenvolvimento em que estas se encontram. Em raízes, as células mais sensíveis são as da região de alongamento e em suspensões celulares as células na fase log de crescimento são mais sensíveis do que as células na fase estacionária. Este trabalho faz parte de uma linha de pesquisa que procura explorar as diferenças que existem entre células quanto à sensibilidade a pH baixo para melhor entender a toxicidade e tolerância a prótons. Foram utilizadas células da cultura de tabaco cv. BY-2, um sistema modelo que apresenta diversas vantagens sobre o uso de raízes para a realização deste trabalho. Os objetivos deste estudo foram de a) determinar condições apropriadas para a exposição destas células a acidez; b) caracterizar a resposta destas células a pH baixo, sob influência de diferentes fatores ambientais, quando se encontram em estados distintos de sensibilidade ao pH baixo; e c) verificar se mudanças na sensibilidade das células a pH baixo podem ser decorrentes de alterações na composição da membrana plasmática ou na atividade das ATPases. Vários testes iniciais foram realizados com o intuito de se definir algumas condições experimentais - o tempo de exposição, a composição do meio e o tampão a ser empregado. Optou-se por lavar as células e depois incubá-las por 1h em meio simples com pH desejado e contendo apenas Ca \'Cl IND. 2\' 2mM, KCl 10mM. E o tampão MES ou biftalato (10 mM). O biftalato foi testado porque o tampão MES, usado normalmente no meio de cultura completo, não é eficiente na faixa de pH abaixo de 5,0. O biftalato (pKa = 4,1) praticamente não afetou a viabilidade celular avaliada pela permeabilidade a trypan blue, mas inibiu o crescimento celular no meio de cultura completo. Mesmo assim, os dois tampões foram utilizados em paralelo em diversos experimentos e verificou-se que os resultados foram semelhantes. A viabilidade das células na fase log (2 dias) foi reduzida quando se abaixou o pH de 5,6 a 3,8, sendo que caiu mais acentuadamente até o pH de 4,8. As células da fase estacionaria (7d) foram insensíveis ao baixo pH. A um pH fixo de 4,2 aumentando-se a concentração de Ca \'Cl IND. 2\' para cerca de 8 a 16 mM praticamente aboliu o efeito deletério do pH baixo. Para se ter o mesmo efeito com a adição de KCl, foi necessário adicionar entre 80 e 160 mM. A adição de sacarose também amenizou os efeitos do pH\'sendo praticamente revertido a uma concentração de 100 Mm. Os resultados indicam a importância da força iônica e da osmolaridade da solução, mas o efeito de Ca não parece depender apenas destas duas propriedades. A inibição de ATPases, pelo uso de DCCD, não pareceu ter qualquer relação com a sensibilidade a pH baixo. Tanto células de tabaco na fase log quanto estacionárias foram sensíveis à aplicação de ortovanadato de sódio. Em células da fase estacionária, este efeito mais acentuado a pH 4,2, sugerindo que nestas células, as H+ ATPases do tipo P da membrana plasmática podem ter algum papel na tolerância destas células ao baixo pH. Encontrou-se diferenças no perfil protéico de frações enriquecidas em membrana plasmática entre células da fase log e estacionárias e entre células tratadas ou não a pH baixo. Estas diferenças precisam ser melhor estudadas. / Acid soils account for about 40 % of the surface area of the world and are one of the major factors limiting plant productivity. In Brazil, these soils comprise roughly two-thirds of its total territory. In general, soil acidity is detrimental because it limits the depth of the root system, increasing susceptibility to drought and decreasing the use of nutrients. In addition to the high levels of hydrogen ion activity, acid soils usually have Al toxicity hazards, a topic which has been intensely studied in the past years. However, Al toxicity only occurs at low pH, under conditions in which proton toxicity is also a problem. Despite this, studies of proton toxicity are lacking. The sensitivity of cells to low pH depends on their growth and developmental status. In roots, the most sensitive cells are those of the elongation zone and in cell cultures, cells in the log phase are more sensitive than those of the stationary phase. This study is part of a larger attempt to explore the differences that exist between cells with respect to their sensitivity to low pH to further understand toxicity and tolerance to protons. Cells of tobacco BY-2, a plant cell model system which has several advantages over the use of roots, was used in this study. The objectives were a) to determine the appropriate conditions to expose these cells to low pH; b) characterize the response of these cells to low pH, when different environmental factors are varied and at different stages of cellular sensitivity to acidity; and c) examine if changes in the sensitivity of cells to low pH are due to changes in the composition of plasma membranes or in the activity of ATPases. Several preliminary tests were performed to define the experimental conditions to be used ? the duration of exposure to low pH, the composition of the medium and the buffer to be used. A simple solution containing only CaCl2 2mM, KCl 10 mM and MES or phthalate buffer (10 mM) was chosen to wash and incubate the cells at the pH of interest. Phthalate was tested because MES, the buffer normally used in the culture medium, is not effective at pH values below 5.0. Phthalate (pKa = 4,1) had very little effects on cell viability as evaluated by membrane permeability to trypan blue, but it severely inhibited the growth of the cell culture in complete medium. Nevertheless, both buffers were used in several subsequent experiments, the results of which were found to be similar between both buffers. The viability of log-phase cells (2 day-old) was reduced when the pH was lowered from pH 5,6 to 3.8, but this was sharper down to pH 4,8. Cells in stationary phase (7 day-old) were insensitive to low pH. At a fixed pH of 4,2, increases in the concentration of CaCl2 up to 8 or 16 mM abolished most of the deleterious effects of low pH. To generate the same effect, KCl had to be added at concentrations between 80 and 160 mM. The addition of sucrose also alleviated the effects of low pH. The results suggest the importance of solution ionic strength and osmolarity on sensitivity to low pH, but the effects of Ca do not appear to depend only on these two properties. The inhibition of ATPases, by DCCD, did not appear to bear any relation to cellular sensitivity to low pH. Both log-phase and stationary-phase cells were affected by the addition of sodium orthophosphate. In stationary-phase cells, this effect was more pronounced at pH 4,2, suggesting that in these cells, P-type H+-ATPases of the plasma membrane may play a role in the tolerance of these cells to lo pH. Differences were found in the protein profile of enriched plasma membrane fractions both between log-phase and stationary-phase cells and between cells treated or not with low pH. These differences, however, need to be better examined.