O sistema colinérgico possui papel importante na modulação dos processos de aprendizagem e memória. Nesse trabalho, avaliamos a evocação da memória de ratos de 3, 6, 12, 18 e 22 meses em esquiva inibitória e, também, analisamos a densidade dos receptores nicotínicos a7 e da subunidade b2 no hipocampo, além de enzimas colinérgicas, a colina acetiltransferase e a acetilcolinesterase. Os grupos com 18 e 22 meses apresentaram uma redução na evocação da memória de longa duração (MLD) e um aumento na densidade de receptores a7 nas piramidais de CA3 e nas células de PoDG, este apenas em 22 meses. Em contrapartida, os demais marcadores colinérgicos estavam inalterados nas diferentes idades. Assim, a redução na evocação da MLD dos animais de 18 e 22 meses acompanhada do aumento na densidade de receptores a7 pode ser caracterizada como: 1) estratégia adaptativa do envelhecimento (plasticidade colinérgica em neurônios glutamatérgicos) ou 2) modulação nos interneurônios GABAérgicos desencadeando, assim, um bloqueio no processamento da informação e o declínio da memória. / The cholinergic system plays an important role in modulating learning and memory. In this work, we evaluated the memory recall of 3, 6, 12, 18 and 22 months old rats in passive avoidance apparatus and we also evaluated, in the hippocampus, the density of a7 nicotinic receptors, b2 subunit receptor, choline acetiltransferase (ChAT) and acetilcholinesterase (AChE). The 18 and 22 months old showed a deficit in long-term memory (LTM) and an increase in a7 density in CA 3 pyramidals cells (py CA3) and polymorphic cells of Dentate Gyrus (PoDG).On the other hand, the other cholinergic markers were unchanged with aging. Thus, the deficit in LTM observed in 18 and 22 months combined with the increased a7 receptors in PoDG and py CA 3 in the hippocampus could be explained by: 1) as an adaptative strategy of aging (cholinergic plasticity in glutamatergic neurons) or 2) as a modulation in GABA interneurons triggering inhibition in the memory evocation.

Ions in the soil solution traverse many layers (epidermis, exodermis, central cortex, and endodermis) within the root to reach the stele. The endodermis is present in almost all vascular plants while the exodermis is found only in majority of angiosperm roots tested. The maturation of the exodermis and the death of epidermis alter the plasma membrane surface areas (PMSA) potentially available for ion uptake. Do these changes reduce the ion uptake in proportion to the loss of absorptive surface areas? To answer this question onion (Allium cepa L cv. Wolf) adventitious root segments representing above features: Immature Exodermis Live Epidermis (IEXLEP), Mature Exodermis Live Epidermis (MEXLEP), Mature Exodermis Dead Epidermis (MEXDEP) were excised. Using a compartmental elution technique, radioactive sulphate and phosphate present in various internal compartments were quantified. Quantities of ions moved across the plasma membrane, a summation of quantities in the cytoplasm, ‘vacuole’, and ‘bound’ compartments, indicated that the maturation of the exodermis reduces the uptake of sulphate but not phosphate. In contrast, epidermal death reduced the movement of both ions across the plasma membranes. Although there is a reduction in the available PMSA with the maturation of the exodermis and death of the epidermis, these events do not necessarily reduce the ion movement into the plasma symplast. The endodermal cells of onion roots deposit suberin lamellae as secondary walls. As seen in cross-sections some cells remain without these lamellae and are known as ‘passage cells’. What is the pattern of suberin lamella deposition along the root? Is the suberin lamella a continuous layer? To answer these questions, endodermal layers isolated from onion adventitious roots were used in the present study. These layers were observed using four stains (Sudan Red 7B, Fluorol yellow 088 [Fy], berberine, and Nile red) and three microscopes (compound-white light, compound-epifluorescence and confocal scanning). In differentiating cells with and without suberin lamellae in endodermal layers Sudan Red 7B served the best results for compound-white light microscope, Fy for compound-epifluorescence microscope and Nile for confocal laser scanning microscope (CLSM). Suberin lamellae deposition initiated almost in a random manner; they continued to be deposited resulting in the production of longitudinal files alternating with files with passage cells, and were ultimately deposited in almost all cells at a distance of 255 mm from the tip. The suberin lamellae are perforated with pores, a consistent feature even as far as 285 mm from the tip. These pores may serve as portals for water, ions, and pathogen movement.

Ions in the soil solution traverse many layers (epidermis, exodermis, central cortex, and endodermis) within the root to reach the stele. The endodermis is present in almost all vascular plants while the exodermis is found only in majority of angiosperm roots tested. The maturation of the exodermis and the death of epidermis alter the plasma membrane surface areas (PMSA) potentially available for ion uptake. Do these changes reduce the ion uptake in proportion to the loss of absorptive surface areas? To answer this question onion (Allium cepa L cv. Wolf) adventitious root segments representing above features: Immature Exodermis Live Epidermis (IEXLEP), Mature Exodermis Live Epidermis (MEXLEP), Mature Exodermis Dead Epidermis (MEXDEP) were excised. Using a compartmental elution technique, radioactive sulphate and phosphate present in various internal compartments were quantified. Quantities of ions moved across the plasma membrane, a summation of quantities in the cytoplasm, ‘vacuole’, and ‘bound’ compartments, indicated that the maturation of the exodermis reduces the uptake of sulphate but not phosphate. In contrast, epidermal death reduced the movement of both ions across the plasma membranes. Although there is a reduction in the available PMSA with the maturation of the exodermis and death of the epidermis, these events do not necessarily reduce the ion movement into the plasma symplast. The endodermal cells of onion roots deposit suberin lamellae as secondary walls. As seen in cross-sections some cells remain without these lamellae and are known as ‘passage cells’. What is the pattern of suberin lamella deposition along the root? Is the suberin lamella a continuous layer? To answer these questions, endodermal layers isolated from onion adventitious roots were used in the present study. These layers were observed using four stains (Sudan Red 7B, Fluorol yellow 088 [Fy], berberine, and Nile red) and three microscopes (compound-white light, compound-epifluorescence and confocal scanning). In differentiating cells with and without suberin lamellae in endodermal layers Sudan Red 7B served the best results for compound-white light microscope, Fy for compound-epifluorescence microscope and Nile for confocal laser scanning microscope (CLSM). Suberin lamellae deposition initiated almost in a random manner; they continued to be deposited resulting in the production of longitudinal files alternating with files with passage cells, and were ultimately deposited in almost all cells at a distance of 255 mm from the tip. The suberin lamellae are perforated with pores, a consistent feature even as far as 285 mm from the tip. These pores may serve as portals for water, ions, and pathogen movement.

<p>The spectrin-ankyrin network assembles diverse plasma membrane domains including axon initial segments and nodes of Ranvier, cardiomyocyte T-tubules and intercalated discs, epithelial lateral membranes, costameres and photoreceptor inner and outer segments. However the mechanism that targets the spectrin-ankyrin network to those plasma membrane domains is unknown. This thesis identifies two lipid inputs from protein palmitoylation and phosphoinositides that together control the precise localization of the spectrin-ankyrin network. In Chapter 2, we identify a linker peptide encoded by a single divergent exon that distinguishes the subcellular localization of ankyrin-B and -G by selectively suppressing protein binding through autoinhibition. In Chapter 3, we demonstrate that ankyrin-G is S-palmitoylated at a conserved C70 residue which is required to assemble epithelial lateral membranes and neuronal axon initial segments. We continue to interrogate how palmitoylation regulates ankyrin-G activities in Chapter 4, and identify DHHC5 and DHHC8 as the palmitoyltransferases in MDCK cells. We showed that palmitoylated ankyrin-G, in concert with phosphoinositide lipids, determines the polarized localization of beta II spectrin though a coincidence detection mechanism. This palmitoyltransferases/ ankyrin-G/beta II spectrin pathway determines the cell height of columnar epithelial cells. In Chapter 5, we elucidated the molecular mechanism through which the spectrin-ankyrin network assembles epithelial lateral membranes. We demonstrated that ankyrin-G and beta II spectrin function by opposing clathrin-mediated endocytosis to build the lateral membrane in MDCK cells. Together, this thesis dissects the mechanisms of how the spectrin-ankyrin network achieves precise membrane targeting and how it assembles lateral membranes to determine the morphogenesis of columnar epithelial cells, and provides the first molecular insight to understand how cells control the assembly of diverse plasma membrane domains.</p> / Dissertation

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.

La connaissance et la compréhension des propriétés électrocinétiques des cellulesapportent de multiples applications en recherche biomédicale, comme le diagnostic et le suivi del'évolution d'un cancer. L'application de champs électriques alternatifs non-uniformes dans desmicrosystèmes, et plus particulièrement la force de diélectrophorèse, permet de caractériser despopulations de cellules sans marqueur spécifique.Nous avons tout d'abord mené une étude fondamentale pour décrire de la réponse des celluleshumaines dans telles conditions. La compétition entre les forces diélectrophorétique et électro-hydrodynamiques (effets électrothermiques, électro-osmose) a été modélisée. La confrontation dumodèle à l'observation expérimentale du mouvement de telles cellules dans des canaux micro-fluidiques comportant des électrodes micro structurées a été effectuée. À partir de cette étude,une nouvelle méthode de détermination de la fréquence de coupure de cellules humaines sur unnombre statistique de cellules représentant une population a été élaborée.Ensuite, nous avons étudié les fréquences de coupure entre des lignées issues de différents tissusépithéliaux (rein et prostate) ou de cellules circulantes. Il a été démontré que les fréquences decoupure sont statistiquement différentes entre les lignées. Les méthodes développées ont ainsipermis de mesurer les différentes signatures électriques de cellules cancéreuses de prostates àchaque stade d'évolution du cancer.Enfin, nous nous sommes intéressés à comprendre les mécanismes de polarisation des cellulessous champ électrique alternatif. Nous avons modifié la membrane des cellules chimiquement oubiologiquement pour comprendre l'origine moléculaire de la fréquence de coupure. Il a été mis enévidence que la concentration en protéines et l'activité de certains canaux ioniques augmententsignificativement la fréquence de coupure des cellules.En exploitant les effets de la diélectrophorèse sur les cellules, il devient possible de caractériserfinement leurs propriétés diélectriques, et de proposer à plus long terme de nouvelles technologiesde détection et de diagnostic / Measuring and understanding cells' electrokinetic properties bring several appli-cations in the biomedical field, like the diagnosis and the monitoring of cancer diseases. Theapplication of alternative non uniform electric fields in microsystems, in particular the dielec-trophoretic force, allows a label-free characterization of cell populations.In this Thesis, a comprehensive study has been established to describe the response of humancells in non-uniform AC fields. We have modeled the competition between dielectrophoresisand electro-hydrodynamical forces (electrothermal effects, AC electro-osmosis). This model wascompared to the observation of cell motions in microfluidic channels with structured electrodes.We have established a new method to determine the crossover frequencies of human cells on astatistically relevant number of cells, which represents a population.Then, the Clausius-Mossotti factor of cell lines has been measured, from different epithelial tis-sues (kidney, prostate) or circulating cells. We have demonstrated that the crossover frequenciesare statistically different between these lines. This method has been used to monitor the differentelectrical signatures of prostate cancer cells at each grade of cancer.Finally, we have focused on the polarization process of cells regarding the electric field. We havemodified chemically and biologically cell membranes to understand the molecular origin of thecrossover frequency. The membrane proteins depletion and the activity of some ion channelssignificantly increase the cell crossover frequency.By taking advantage of the dielectrophoretic response of cells, it becomes possible to characte-rize their dielectric properties and to develop new technologies for cancer detection and diagnosis

Abstract Chronic overload of the heart is the major cause of left ventricular hypertrophy (LVH) and eventually heart failure. It is generally accepted that autocrine/paracrine factors, such as angiotensin II (Ang II) and endothelin-1 (ET-1) contribute to the development of LVH. Cardiac hypertrophy and failure are characterized by attenuated responsiveness to β- adrenergic stimulation and accumulation of collagenous material to the left ventricular wall. The present study aimed to characterize the roles of ET-1 and Ang II in the regulation of cardiac function. The role of the plasmamembrane Ca2+-ATPase (PMCA) in ET-1 induced cardiac responses and the role of type XV collagen in cardiac function were also studied. Both ET-1 infusion and mechanical loading were able to induce positive inotropic effect and induction of early response genes in isolated perfused hearts. ET-1 also induced strong vasoconstriction. Cardiomyocyte-specific PMCA overexpression inhibited the ET-1 induced hypertrophic response, while inotropic response remained unaltered. ET-1 was found to induce release of adrenomedullin (AM), a potent vasorelaxing and inotropic peptide. Infusion of AM antagonized the vasoconstrictive effect of ET-1 independently of nitric oxide. In hypertrophied rat hearts ET-1 was found to contribute significantly to the Frank-Starling response, a fundamental mechanism regulating contractile performance of the heart. In mice hearts, ET-1 was found to play a dual role in load induced elevation of contractile strength: ETA receptors mediated an increase, while ETB receptors mediated an inhibitory effect on contrcatile force. Ang II was not contributing to the contractile response to load in either rat or mice hearts. Blunted response to β-adrenergic stimulus and increased vulnerability as a result of exercise was observed in mice lacking collagen XV. In conclusion, the present results underscore the importance of the local factors, especially ET-1, in regulation of cardiac function, not only in terms of hypertrophic but also in terms of contractile response to load. The results also suggest a role for PMCA in regulation of cardiac function. Lack of type XV collagen was found to result in cardiac dysfunction with many features similar to those of early heart failure.

A proteína de matriz do Virus Respiratório Sincicial humano foi o foco deste trabalho. Verificamos que o gene de matriz possui sítios internos de poliadenilação, sinais de instabilidade de RNA, baixo índice de adaptação de codons (CAI) e conteúdo GC, que podem impedir a expressão gênica vitro. Quando clonado sob controle do promotor de CMVie, o gene selvagem não apresenta expressão detectável, enquanto um gene sintético com a sequência do gene de matriz otimizada apresenta altos níveis de expressão em células transfectadas. Esse alto nível de expressão permitiu a confirmação da presença da proteína M no núcleo no início de sua expressão, por análise em microscopio confocal de varredura a laser, além de sua associação a membranas em regiões conhecidas como lipid rafts. Também foi observado que a proteína M é capaz de associar à proteína tropomiosina. Ainda foram analisados os possíveis domínios funcionais através de expressão de variantes da proteína M com deleções de trechos da proteína. Finalmente foi analisada a capacidade de indução de resposta imune. / The Human Respiratory Syncytial Vírus was the focus of this work. We found that matrix gene has internal polyadenilation sites, RNA instability motifs, low codon adaptation index (CAI) and GC content, that may impair its expression in vitro. When cloned under control of the ieCMV promoter, the wild-type M gene expression was not detectable, whereas a synthetic optimized matrix gene was highly expressed in transfected cells. This high level of expression made possible to follow M nuclear localization in the beginning of its expression by confocal laser scanning microscopy, and its association with membranes in regions known as lipid rafts. It has also been found that the matrix protein associates with tropomyosin. It was further analyzed the possible functional through expression of deviations of the M protein that lack portions of the protein. Finally it was analyzed its capacity to induce an immune response.

Made available in DSpace on 2016-01-26T18:55:30Z (GMT). No. of bitstreams: 1 DISSERTACAO_HUBERSON_FINAL.pdf: 232987 bytes, checksum: 845a7b338d4388c6a546aefabcc59018 (MD5) Previous issue date: 2010-06-21 / For the economy, the Brazilian industry of cattle is a very important activity because it is one area that gives the possibility of having a very big potential in terms of growth. Having this in mind, it can conclude that the artificial insemination is probably one of the most important way for contributing with the advancement of the modern animal techniques used for the production, however, it requires the use of the frozen semen for this realization. During the cryopreservation process, a decrease in the number of sperm viability happens because of the osmotic effects, temperature, and morphological changes that occur because of the organization changes, fluidity, permeability and the lipid composition of sperm membranes. The interaction presents between sperm cells and the thinner represents a crucial factor to preserve the integrity and the fertilizing ability. The thinner has as main objective to protect the sperm cells during the cold shock, and also in the freezing and thawing. Conventional parameters used in the sperm evaluation have showed that they are limited in their ability of predicting the semen potential fertility, and then tests that have different characteristics of the sperm and evaluation of many attributes can provide a better fertility estimate. To evaluate the integrity of the plasmatic membrane, it was successfully utilized one combination of fluorescent probe (IP that means propidium iodide and CFDA that means carboxyfluorescein diacetate). The system CASA (one computer program that analyzes the semen) provides real information about the individual cell movement and also the subpopulations of sperm cells realizing draw for each sperm that has as objective to represent the real path. This review has the objective of presenting the relevant information to the effect of thinners in the frozen ox semen (Bos taurus indicus). / A pecuária brasileira é uma atividade de grande importância para a economia do país por se destacar pelo seu elevado potencial e possibilidade de crescimento. Neste sentido, a inseminação artificial é um dos instrumentos mais importantes para contribuir no avanço das modernas técnicas de produção animal, entretanto, para sua realização é necessário o uso do sêmen congelado. Durante o processo de criopreservação, ocorre diminuição da viabilidade espermática devido aos efeitos osmóticos, temperatura e alterações morfológicas que ocorrem por mudanças na organização, fluidez, permeabilidade e composição lipídica das membranas espermáticas. A interação entre as células espermáticas e o meio diluidor representa um fator crucial para a preservação da integridade espermática e habilidade de fecundação. O meio diluente tem como finalidade proteger a célula espermática durante o choque térmico, na congelação e descongelação. Parâmetros convencionais utilizados na avaliação espermática têm se mostrado limitados quanto à capacidade de predizer o potencial de fertilidade do sêmen, assim testes que mensuram diferentes características do espermatozóide e avaliação de vários atributos podem fornecer uma melhor estimativa da fertilidade. Para avaliar a integridade da membrana plasmática foi utilizada, em diversos trabalhos com sucesso a associação de sondas fluorescentes como Iodeto de Propídeo (IP) e Diacetato de Carboxifluoresceína (CFDA). O CASA (sistema informatizado para análise de sêmen) oferece informações precisas do movimento individual de cada célula bem como as subpopulações de células espermáticas realizando um desenho para cada espermatozóide com a finalidade de representar a sua trajetória real. A presente revisão de literatura tem o objetivo de apresentar informações relativas ao efeito de meios diluentes sobre a viabilidade de sêmen congelado bovino (Bos taurus indicus).

Made available in DSpace on 2016-07-18T17:53:07Z (GMT). No. of bitstreams: 1 DISSERTACAO_HUBERSON_FINAL.pdf: 232987 bytes, checksum: 845a7b338d4388c6a546aefabcc59018 (MD5) Previous issue date: 2010-06-21 / For the economy, the Brazilian industry of cattle is a very important activity because it is one area that gives the possibility of having a very big potential in terms of growth. Having this in mind, it can conclude that the artificial insemination is probably one of the most important way for contributing with the advancement of the modern animal techniques used for the production, however, it requires the use of the frozen semen for this realization. During the cryopreservation process, a decrease in the number of sperm viability happens because of the osmotic effects, temperature, and morphological changes that occur because of the organization changes, fluidity, permeability and the lipid composition of sperm membranes. The interaction presents between sperm cells and the thinner represents a crucial factor to preserve the integrity and the fertilizing ability. The thinner has as main objective to protect the sperm cells during the cold shock, and also in the freezing and thawing. Conventional parameters used in the sperm evaluation have showed that they are limited in their ability of predicting the semen potential fertility, and then tests that have different characteristics of the sperm and evaluation of many attributes can provide a better fertility estimate. To evaluate the integrity of the plasmatic membrane, it was successfully utilized one combination of fluorescent probe (IP that means propidium iodide and CFDA that means carboxyfluorescein diacetate). The system CASA (one computer program that analyzes the semen) provides real information about the individual cell movement and also the subpopulations of sperm cells realizing draw for each sperm that has as objective to represent the real path. This review has the objective of presenting the relevant information to the effect of thinners in the frozen ox semen (Bos taurus indicus). / A pecuária brasileira é uma atividade de grande importância para a economia do país por se destacar pelo seu elevado potencial e possibilidade de crescimento. Neste sentido, a inseminação artificial é um dos instrumentos mais importantes para contribuir no avanço das modernas técnicas de produção animal, entretanto, para sua realização é necessário o uso do sêmen congelado. Durante o processo de criopreservação, ocorre diminuição da viabilidade espermática devido aos efeitos osmóticos, temperatura e alterações morfológicas que ocorrem por mudanças na organização, fluidez, permeabilidade e composição lipídica das membranas espermáticas. A interação entre as células espermáticas e o meio diluidor representa um fator crucial para a preservação da integridade espermática e habilidade de fecundação. O meio diluente tem como finalidade proteger a célula espermática durante o choque térmico, na congelação e descongelação. Parâmetros convencionais utilizados na avaliação espermática têm se mostrado limitados quanto à capacidade de predizer o potencial de fertilidade do sêmen, assim testes que mensuram diferentes características do espermatozóide e avaliação de vários atributos podem fornecer uma melhor estimativa da fertilidade. Para avaliar a integridade da membrana plasmática foi utilizada, em diversos trabalhos com sucesso a associação de sondas fluorescentes como Iodeto de Propídeo (IP) e Diacetato de Carboxifluoresceína (CFDA). O CASA (sistema informatizado para análise de sêmen) oferece informações precisas do movimento individual de cada célula bem como as subpopulações de células espermáticas realizando um desenho para cada espermatozóide com a finalidade de representar a sua trajetória real. A presente revisão de literatura tem o objetivo de apresentar informações relativas ao efeito de meios diluentes sobre a viabilidade de sêmen congelado bovino (Bos taurus indicus).