Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-28Bitstream added on 2014-06-13T19:26:54Z : No. of bitstreams: 1 carreira_jt_me_jabo.pdf: 1115702 bytes, checksum: f2dddb9edf2109b5192f0e4f605b5b97 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O objetivo deste trabalho foi avaliar os efeitos da gota citoplasmática proximal (GCP) no sêmen de bovinos quanto à integridade do DNA, das membranas citoplasmática, acrossomal, no potencial mitocondrial e verificar a taxa de produção de embriões in vitro. Três amostras descongeladas de cinco (Controle: espermiograma normal), e oito touros Bos indicus (Gota: GCP ≥15%) foram avaliadas. Foram realizados os seguintes testes: motilidade e vigor pós-descongelação, concentração, morfologia espermática, teste de termo-resistência lento (TTL), integridade da membrana acrossomal, plasmática e potencial mitocondrial utilizando sondas fluorescentes (PI, FITC-PSA e JC-1) e integridade da cromatina pelo método de coloração com laranja de acridina. Dois touros com índices elevados de GCP e três animais controle foram selecionados para fertilização in vitro (FIV). As análises estatísticas foram efetuadas empregando-se o programa Statistical Analysis System. O nível de significância foi de 5%. Os resultados obtidos demonstraram que altos índices de GCP não afetaram a motilidade e o vigor, antes e após o TTL, assim como não interferiram na porcentagem de acrossomas intactos. Os resultados destas avaliações mostraram que a alta incidência de GCP afetou a integridade da membrana acrossomal e plasmática bem como a presença de potencial mitocondrial. No entanto, a alta incidência de GCP não promoveu aumento na porcentagem de injúrias à cromatina após descongelação, mas os resultados sugerem que podem ser mais sensíveis à desnaturação quando incubados por três horas. No experimento II, os índices de produção de embriões in vitro podem ter sido afetados pela interação da alteração morfológica e o efeito individual do touro. / The objective of this study was to evaluate the effects of the proximal cytoplasmic droplets (PCD) in bovine semen, on the integrity of DNA, cytoplasmic membrane, acrossome, mitochondrial function and the rate of in vitro embryo production. Three batches of five (control group G1: normal sperm parameters) and eight Bos indicus bulls (G2: PCD ≥15%) were analysed. The following tests were carried out: post thaw motility and, vigor, concentration, sperm morphology, slow thermo-resistance (TRT), membrane integrity, acrossome status, mitochondrial function through fluorescent probes (FITC-PSA, PI and JC-1) and integrity of chromatin was accessed by acridine orange stain. Two bulls with high rates of PCD and three animals (control group) were selected for in vitro fertilization (IVF). Statistical analyses were performed using the Statistical Analysis System. The significance level was 5%. The results showed that high rates of PCD did not affect motility and vigor, before and after the TRT, and did not affect the percentage of intact acrossome. The results showed that the high incidence of PCD affected membrane integrity, acrossome status and mitochondrial function when compared to the G1 group due. However, the high incidence of PCD did not affect the percentage of chromatin injury after thawing, but results suggest that spermatozoa may be more susceptible to damage when incubated for three hours. In experiment II the embryo production rate may have been affected by the interaction of the morphology traits and the bull effect.

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

Au cours de ces dernières années, des études ont montré l’existence d’une compartimentation latéraledes composants de la membrane plasmique végétale, de manière analogue à ce qui avait été montréchez les animaux et les levures. L’objectif de cette thèse était d’apporter de nouveaux éléments decaractérisation de cette compartimentation (propriétés physiques de domaines particuliers, mécanismesde mise en place de ces domaines, de contrôle de leur taille, etc…) et d’étudier son rôle dans laphysiologie de la cellule végétale.Le développement d’une méthodologie de microscopie confocale spectrale couplée à l’utilisationd’une sonde environnementale a permis d’apporter la première description à l'échellesubmicrométrique de l’organisation du plasmalemme en territoires aux propriétés physiquesdifférentiées. Ces domaines coexistent au sein de la membrane plasmique de cellules en suspension,comme à celle de membranes artificielles composées de lipides modèles ou de lipides de membranescellulaires, de vésicules géantes constituées de membrane plasmique purifiée, ou de protoplastes.Cependant, les différences de l’organisation latérale observées chez ces différentes membranes ontpermis de montrer l’importance des phytostérols qui seraient, par le biais d'interactions spécifiquesavec d’autres lipides végétaux tels que les GIPCs, des composés essentiels pour la formation locale dedomaines lipidiques ordonnés. La grande diversité des lipides végétaux organiserait ainsi lacompartimentation de la membrane plasmique permettant la ségrégation dynamique des composantsmembranaires. Si les stérols augmentent de manière importante le degré de compaction de la bicouche,les protéines le diminuent. Le cytosquelette et la paroi ne semblent, quant à eux, modifier ni laprésence, ni l’organisation des domaines ordonnés de la membrane plasmique. Nous avons égalementmontré que l’organisation de ces domaines évolue transitoirement lors des étapes précoces de lacascade de signalisation induite par des réactions de défense. De fait, nous avons identifié desmodifications des propriétés physiques globales et de l’organisation fine de la membrane provoquéespar différents éliciteurs de réactions de défense, dont la cryptogéine, une protéine sécrétée parl’oomycète Phytophthora cryptogea. Nous avons montré que ces modifications sont un élémentgénérique de la signalisation de défense, sous la dépendance de phénomènes de phosphorylation, leburst oxydatif étant également une étape clé de l’augmentation du degré d’ordre observé dans lesphases précoces de cette signalisation. La cryptogéine, qui présente une aptitude singulière pour piégerles stérols, a également montré une capacité spécifique à augmenter la fluidité membranaire, ceparamètre pouvant contrôler l’intensité de la cascade de signalisation, mesurée par la production deformes actives d’oxygène.Ces résultats ouvrent de nouvelles perspectives dans la compréhension des interactions cellule-élicitineet apportent un nouvel éclairage sur le rôle des lipides végétaux dans l’organisation latérale de lamembrane plasmique végétale et positionne la dynamique membranaire comme un élément designalisation de défense des plantes. / Recent studies have shown the existence of lateral sub-compartmentalization of plant plasmamembrane similar to that of animal cells and yeasts. The aim of this thesis was to provide newelements to characterize this compartmentalization (physical properties of specific domains,mechanisms of their formation, determination of their size, etc...) and to study its role in thephysiology of plant cells.The development spectral confocal microscopy coupled with the use of an environment-sensitiveprobe enabled to obtain the first description at the submicron scale of plasma membrane organizationinto domains exhibiting various physical properties. These domains coexist at the plasma membranesurface of tobacco suspension cells as well as the membrane of vesicles composed of models lipids orcell plasma membrane lipids, purified plasma membrane vesicles, and protoplasts. However,differences in the lateral organization observed in these membranes have shown the importance ofphytosterols which are, through specific interactions with neighboring plant lipids such as GIPCs,essential for local formation of ordered domains. The huge diversity of plant lipids drives thecompartmentalization of the plasma membrane allowing the dynamic segregation of membranecomponents. Sterols greatly increase membrane order, whereas proteins tends to decrease it.Cytoskeleton and cell wall do alter neither presence nor organization of ordered domains of the plasmamembrane. We have also shown that the organization of these domains is transiently modified duringthe early stages of defense signaling cascade. In fact, we have identified changes in overall physicalproperties and fine lateral organization of the membrane caused by various elicitors of defensereactions, including cryptogein, a protein secreted by the oomycete Phytophthora cryptogea. We haveshown that these changes are a generic element of defense signaling cascade and depend onphosphorylation processes; oxidative burst being also a major actor of the control of the increase ofmembrane order observed during the very early stages of the signalling process. Cryptogein, whichexhibits the particular ability to trap sterols, also showed a specific capacity to increase membranefluidity and amplify the intensity of the signalling cascade, as measured by the production of reactiveoxygen species.These results open new perspectives in the understanding of cell-elicitin interactions and provide anew view on the central role of sterol composition in the lateral organization of plant plasmamembrane. They also identify membrane dynamics as a new player in the signalling cascade occurringduring plant defense.

Les infections bactériennes touchant la circulation sanguine conduisent à un vaste éventail de graves pathologies, comme les chocs septiques ou les infections locales (endocardites et méningites). Neisseria meningitidis colonise avec succès l’endothélium vasculaire et cause des sepsis sévères. Ces infections résultent de la colonisation des cellules endothéliales de l’hôte, étape clef de la pathophysiologie à laquelle les travaux présentés dans ce manuscrit se sont intéressés. La colonisation de l’endothélium par N. meningitidis est un processus complexe qui implique l’adhésion et la multiplication des bactéries à la surface des cellules endothéliales dans le contexte particulier de la circulation sanguine, où des forces mécaniques sont générées par le flux sanguin sur les objets circulants. Bien que de nombreuses études se soient intéressées à l’interaction entre les cellules endothéliales et N. meningitidis, plusieurs aspects demeurent incertains comme par exemple l’impact des contraintes générées par le flux sanguin et la participation relative des deux partenaires de l’interaction dans la colonisation de l’endothélium par N. meningitidis.L’adhésion de la bactérie à la surface des cellules endothéliales est dépendante de facteurs bactériens (les pili de type IV, PT4) et induit une réponse de la part de la cellule hôte, qui se traduit par un remodelage de la membrane plasmique et une réorganisation du cytosquelette d’actine sous les microcolonies. Dans un premier temps, ces travaux de thèse montrent que la réponse cellulaire induite par N. meningitidis participe activement à la colonisation. En effet, la formation de projections membranaires permet à chaque bactérie de la microcolonie d’établir des contacts avec la cellule hôte, nécessaires à la résistance des microcolonies face aux forces mécaniques générées par le flux sanguin. De plus, nous montrons que la protéine PilV, composant des PT4, est impliquée dans le remaniement de la membrane plasmique et la réorganisation du cytosquelette. Nous avons développé une méthode combinant vidéo-microscopie et analyse de fluorescence pour décrypter les événements précoces prenant place lors du contact entre les bactéries et la surface des cellules hôtes. Nous avons alors montré que le remodelage de la membrane induit par N. meningitidis ne dépend pas de la réorganisation du cytosquelette d’actine au site d’infection mais plutôt des propriétés intrinsèques de la bicouche lipidique.Dans un second temps, nous nous sommes intéressés aux étapes tardives de l’infection, c'est-à-dire à l’initiation d’un nouveau cycle de colonisation. Bien que solidement ancrées à la surface des cellules par l’intermédiaire des projections membranaires, quelques bactéries se détachent des microcolonies pour coloniser des nouveaux sites au sein de l’hôte. Nous avons démontré l’importance de modifications post-traductionnelles de la piline majeure dans cette étape de l’infection et caractérisé les mécanismes impliqués.Cette étude a permis d’affiner les mécanismes impliqués dans l’induction de la réponse cellulaire induite par N. meningitidis et son impact sur la colonisation efficace de l’endothélium par ce pathogène. / Bacterial infections targeting the bloodstream lead to a wide array of severe clinical manifestations, such as septic shock or focal infections (endocarditis and meningitis). Neisseria meningitidis colonizes successfully the vascular wall and causes severe sepsis. Such infections result from an efficient colonization of host endothelial cells, a key step in meningococcal diseases which has been the subject of the work presented here. Endothelium colonization by N. meningitidis is a complex process implying bacterial adhesion and multiplication on the endothelial cell surface in the specific context of the bloodstream, where mechanical forces generated by the blood flow are applied on circulating bacteria. Even though many studies focused on the interaction between N. meningitidis and the endothelial cell, many aspects remain elusive, such as the impact of shear stress generated drag forces and the relative contribution of the two partners involved in this interaction.Adhesion to the endothelial cell surface is dependent on bacterial factors called type IV pili (Tfp) and leads to induction of a host cell response, characterized by a local remodeling of the plasma membrane and reorganization of actin cytoskeleton underneath bacterial microcolonies. First, we have shown that the cellular response induced by N. meningitidis actively participate in the colonization process. Indeed, membrane deformation allows contact with every bacterium inside the microcolony, which is necessary for microcolony resistance to mechanical forces. Additionally, we have demonstrated that the PilV protein, a Tfp component, is involved in plasma membrane remodeling and actin cytoskeleton reorganization. We designed a method combining high resolution live-cell fluorescence video-microscopy and fluorescence quantification to decipher the early events induced on contact of bacterial aggregates with the host cell surface. Using this technique we have shown that membrane remodeling does not rely on actin cytoskeleton reorganization but rather on intrinsic properties of the lipid bilayer. Second, we focused on latter steps of the infection process when initiation of a new colonization cycle is initiated. While firmly attached to the host cell surface through the membranous projections, some bacteria can detach from the microcolony to disseminate throughout the host. We have demonstrated the importance of post-translational modification of the major piline in this step and characterized the underlying mechanisms.This work allows refinement of the molecular mechanisms involved in the induction of the cellular response induced by N. meningitidis and its impact on successful endothelium colonization by this pathogen.

Our research aims at understanding the roles of seminolipid (sulfogalactosylglycerolipid or SGG) and its associated membrane domains in male reproduction. SGG is a sulfoglycolipid present selectively and abundantly in mammalian male germ cells. Therefore, information on its properties would be relevant towards the development of male fertility biomarkers and spermicide-based contraceptives. We have shown that SGG has direct affinity for zona pellucida (ZP, egg extracellular matrix) and plays a role in the formation of sperm lipid rafts, the ZP-binding platforms on the sperm anterior head plasma membrane (APM), the initial ZP binding site. For a better understanding of mechanisms underlying sperm-ZP interaction, I performed proteomic characterization of APM vesicles (SGG-associated membrane domains with ZP affinity) isolated from sperm before and after capacitation, a process through which sperm gain maximal ZP affinity. Proteomic results revealed that capacitated APM vesicles contained high-molecular-weight protein complexes, with higher ZP affinity and levels of ZP-binding proteins as compared with those of the non-capacitated samples. ZP-binding proteins known to exist in the acrosome (i.e., zonadhesin, proacrosin/acrosin) were found in these APM protein complexes. Immunofluorescence suggested that a fraction of these proteins trafficked from the acrosome to APM during capacitation. These findings provided a new mechanism on how sperm gain full ZP-binding ability during capacitation. Since SGG is a major component of APM, proper SGG levels at this site would be important for male fertility. Levels of sperm SGG are regulated through the synthesis and degradation. In fact, lack of SGG-synthesis enzymes causes a spermatogenesis disruption, resulting in male infertility. However, significance of SGG degradation remains unknown. SGG can be desulfated in vitro by arylsulfatase A (ARSA), an enzyme existing in the acrosomes of sperm/spermatids and lysosomes of Sertoli cells, testicular somatic cells that nurture developing germ cells. Sertoli cells also phagocytose ~50% of germ cells that become apoptotic during spermatogenesis. To understand physiological importance of SGG degradation, the fertility status and SGG levels of Arsa-/- male mice were determined. We found that Arsa-/- males became subfertile when they were older than 5 months, and when they were 8-month-old (~40-year-old men) they produced sperm at 50% wild type rate. Arsa-/- sperm had minimal in vitro fertilizing ability and a number of them showed abnormal morphology. Quantitative mass spectrometry revealed that SGG levels in Sertoli cells of 8-month-old Arsa-/- mice were increased to ~250% of the wild type level; this SGG accumulation may lead to a decrease in Sertoli cell ability to support spermatogenesis. However, SGG levels in sperm of 8-month-old Arsa-/- mice were ~50% of the wild type value, a result that partly explained the decreased fertilizing ability of these sperm. The reduced SGG level of Arsa-/- sperm was likely due to a lack of SGG’s building-block lipid (palmitylpalmitoylglycerol) putatively generated in Arsa-/- Sertoli cells and recycled to the next generation of primary spermatocytes for SGG synthesis. Hence, levels of sperm SGG are a promising bioindex for male fertility. Since Sertoli cells also regulate SGG homeostasis, their functionality should be now included in male fertility/subfertility diagnosis.

Les champs électriques pulsés sont largement utilisés dans la recherche, la médecine, l'industrie alimentaire et d'autres procédés biotechnologiques. L'interaction d'une impulsion de 100 µs avec la membrane plasmique et la membrane du réticulum endoplasmique a été évaluée dans deux types cellulaires différents. La perméabilisation des organites cellulaires avec ce type d'impulsions est démontrée expérimentalement pour la première fois. L'utilisation d'une telle impulsion afin de contrôler les oscillations calciques spontanées dans les cellules souches mésenchymateuses humaines issues du tissu adipeux a été évaluée. En créant des pics calciques électro-induits d’amplitudes différentes, l'impulsion peut ou bien induire un pic calcique supplémentaire ou bien inhiber les oscillations spontanées pour quelques dizaines de minutes. Cette inhibition rend possible d’imposer à la cellule des pics d’amplitude et de fréquence désirés. Un essai d’application de l'impulsion 100 µs à des cellules souches subissant une différenciation osseuse a aussi été réalisé. Une impulsion électrique semble retarder la différenciation. Lors d'une différenciation osseuse, plusieurs couches cellulaires ont été observées. La caractérisation de ces couches a donné des résultats qui pourraient aider à obtenir des ostéoblastes matures dans un temps moindre que la normale. L'utilisation des champs électriques pulsés microsecondes, pour perméabiliser la membrane plasmique et les membranes internes des cellules, ainsi que pour moduler les concentrations du calcium intracellulaire, semble donc très intéressante pour étudier le rôle du calcium dans de nombreux processus physiologiques et pour manipuler les dynamiques calciques (oscillations, vagues, pics) dans différents types de cellules. Ainsi, cette technologie simple, facile à appliquer et disponible dans beaucoup de laboratoires serait envisageable pour la modulation et le contrôle de fonctions cellulaires basiques telles que la prolifération, la différenciation et l'apoptose. / Pulsed electric fields are widely used in research, medicine, food industry and other biotechnological processes. The interaction of one 100 µs pulse with the plasma membrane and the endoplasmic reticulum membrane was evaluated in two different cell types. Pulse amplitude ranged between 100 and 3 000 V/cm. Organelles membrane permeabilization using this kind of pulses was experimentally demonstrated for the first time. The use of such a pulse to control the spontaneous calcium oscillations in human-adipose mesenchymal stem cells was also assessed. By creating electro-induced calcium spikes of different amplitudes, the pulse can either add a supplementary spike, or, on the contrary, inhibit the spontaneous oscillations for some tens of minutes. During this inhibition period, the electric pulse-mediated addition of calcium spikes of desired amplitude and frequency is still possible. The delivery of 100 µs pulses to stem cells undergoing osteodifferentiation was also performed. The electric pulse seemed to delay the differentiation. Moreover, during osteogenic differentiation, cells cultures displayed an organization in a few cell layers. The characterization of these layers gave results that may help to obtain mature osteoblast in less time than usual one. The use of the microsecond electric pulses technology to permeabilize the plasma and the internal cell membranes as well as to modulate internal calcium concentrations is therefore interesting to study the role of calcium in many physiological processes and to manipulate the cell calcium dynamics (oscillations, waves, spikes) in different cell types. Doing so, this available, simple and easy to apply technology could be used for the modulation and the control of basic cellular functions such as proliferation, differentiation and apoptosis.

Die dynamische Verknüpfung zwischen Plasmamembran und dem unterliegenden Zytoskelett der Zelle ist fundamental für zelluläre Prozesse wie Zellmorphogenese, Zellmotilität und Zelladhäsion. Ezrin als Bestandteil der ERM (Ezrin, Radixin, Moesin) Proteinfamilie verbindet L-α-Phosphatidylinositol-4,5-bisphosphat (PIP2) der Plasmamembran mit filamentösem Aktin (F-Aktin) des Zytoskeletts. Die Ezrinbindung an F-Aktin wird reguliert über den Aktivierungsgrad des Proteins, welcher von der N-terminalen PIP2 Bindung und der Phosphorylierung des Threoninrests 567 abhängt. Aufgrund der Bindung an PIP2 und der Phosphorylierung wechselt Ezrin von einer inaktiven, N- und C-terminal assoziierten Konformation in einen aktivierten, geöffneten Zustand, welcher die C-terminale F-Aktinbindung ermöglicht. Ziel dieser Arbeit war es Aspekte der Verknüpfung zwischen Plasmamembran und Zytoskelett zu untersuchen. Basierend auf Bindung von Ezrin an PIP2-haltige artifizielle Lipidmembranen und der anschließenden F-Aktinbindung, wurden Bindungseigenschaften, die Organisation des F-Aktinnetzwerkes und die durch das Aktinnetzwerk beeinflusste Lipidmembranmechanik untersucht. Im ersten Abschnitt dieser Arbeit wurde der molekulare Aktivierungsprozess von Ezrin anhand der Charakterisierung von Bindungsaffinitäten und der Organisation von Ezrin an Lipidmembranen untersucht. Aufgrund einer reduzierten Proteinhöhe und FRET (FÖRSTER-Resonanzenergietransfer)-Effizienz im Fall der vollständigen Aktivierung (PIP2-Bindung und Phosphorylierung) wurde postuliert, dass Ezrin eine weniger dicht gepackte, geöffnete Konformation gebunden an Lipidmembranen ausbildet. Dies ermöglicht dem Protein C-terminal F-Aktin zu binden. Im zweiten Teil der Arbeit wurden Aktinnetzwerke an festkörperunterstützten Lipidmembranen (SLBs) immobilisiert und über Ezrin an PIP2- oder elektrostatisch an 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholin (DOEPC)-haltige SLBs gebunden. Die Netzwerkorganisation wurde mit Hilfe der Fluoreszenzmikroskopie untersucht und unter Berücksichtigung der Immobilisierungsstrategie in Hinblick auf den Einfluss der Anzahl an Verknüpfungspunkten und aktinbindender Proteine (Fascin und α-Actinin) analysiert. Es konnte gezeigt werden, dass beide Immobilisierungsstrategien zu Aktinnetzwerken mit ähnlichen Eigenschaften führten, bezugnehmend auf Maschengröße und Filamentsegmentlänge. Die Aktinnetzwerkdichte konnte direkt über die Anzahl an Verknüpfungspunkten und aktinbindende Proteine (ABPs) reguliert werden, dies demonstriert die physiologische Relevanz der Ergebnisse. Es ist bekannt, dass die Aktindichte in Zellen über PIP2- und ABP-Konzentration gesteuert wird. Im dritten Teil der Arbeit wurde das etablierte Modelsystem auf poröse Substrate übertragen. Unter Kenntnis der vorangegangenen Teile der Arbeit wurde der Einfluss des F-Aktinnetzwerkes auf die Lipidmembranmechanik untersucht. Mit Hilfe der Rasterkraftmikroskopie wurden Indentationsexperimente an porenüberspannenden Lipidmembranen (PSLBs) durchführt, welche zeigten, dass ein aufliegendes F-Aktinnetzwerk die PSLBs versteift. Dies ließ sich auf die reduzierte laterale Mobilität der Lipide innerhalb der PSLBs aufgrund des Aktinnetzwerkes zurückführen, vergleichbar mit dem Picket-Fence-Modell der Plasmamembran bei welchem die Mobilität der Lipide und (Membran-)Proteine, aufgrund der Kompartimentierung der Membran durch das Aktin-Zytoskelett, eingeschränkt ist.

Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.

The exact role of opioid receptors in drug addiction and modulatory mechanism of action of monovalent cations on these receptors are still not fully understood. Our results support the view that the mechanism of addiction to morphine is primarily based on desensitization of μ- and δ-opioid receptors. Desenzitization of agonist response proceeds already at the level of G protein functional activity. Long-term exposure of rats to morphine resulted in increase of number of δ-opioid receptors and change of their sensitivity to sodium ions. Analysis of the effect of different monovalent ions on agonist binding in δ-OR- Gi1α (Cys351 -Ile351 )-HEK293 cell line confirmed the preferential sensitivity of δ-opioid receptor to sodium ions. We have distinguished the high- and low-affinity Na+ sites. Biophysical analysis of interaction of lithium, sodium, potassium and cesium ions with plasma membranes isolated from HEK293 cells with the help of fluorescent probes indicated that monovalent ions interact, in low-affinity manner, with the polar, membrane-water interface of membrane bilayer. Key words: morphine, forebrain cortex, opioid receptors, G proteins, monovalent ions, plasma membrane, fluorescence spectroscopy.