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‣ Isolamento e caracterização de células progenitoras endoteliais de medula óssea de camundongos para utilização em terapia celular; Isolation and characterization of endothelial progenitor cells derived from bone marrow of mice for use in cell therapy

Giane Daniela Carneiro
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 13/04/2011 Português
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As células progenitoras endoteliais (CPEs) foram descritas por Asahara et al. (1997), isoladas a partir do sangue periférico e são células originadas da medula óssea. Estas células podem ser reconhecidas pela expressão de marcadores como o CD34, CD133, VEGFR2 e o CD31, pela capacidade de internalização de acLDL e de formar estruturas semelhantes a vasos quando cultivas em matrizes tridimensionais. Quando ocorrem lesões endoteliais, elas podem ser mobilizadas da medula migrando para o sangue periférico e atuar no local da lesão arterial, podendo se diferenciar em células endoteliais maduras e promover a re-endotelização do vaso lesionado. Contudo, estas células estão presentes na medula óssea e no sangue periférico em quantidade muito pequena, 0,1 e 0,01% do total de células respectivamente, o que leva diversos grupos de pesquisa buscarem selecionar e cultivar estas CPEs visando aumentar seu número para melhor caracterizá-las in vitro e in vivo. Em nosso trabalho, buscamos estabelecer um meio de cultura capaz de sustentar o crescimento, proliferação e diferenciação das células progenitoras endoteliais isoladas a partir de medula óssea de camundongos C57bl/6, e de caracterizar seus vários estágios de diferenciação destas células em diferentes tempos de cultura. Para tal...

‣ Numerical and experimental investigation of hollow sphere structures in sandwich panels; Métodos experimentais e numericos no estudo de estruturas de esferas ocas em painéis "sandwich"

Fiedler, Thomas
Fonte: Universidade de Aveiro Publicador: Universidade de Aveiro
Tipo: Tese de Doutorado
Português
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O presente trabalho propõe estudar o comportamento de estruturas compostas por esferas ocas metálicas em painéis sanduíche. Estudos numéricos utilizando o método de Elementos Finitos foram feitos para caracterizar o comportamento de provetos representativos. A primeira parte do estudo envolveu o estudo de diferentes tipos de estruturas metálicas compostas por esferas ocas metálicas, adesivamente fundidas, MHSS (“Metallic Hollow Sphere Structures”). A influência da morfologia, topologia e da técnica de fusão das esferas nas propriedades materiais foi avaliada numericamente. Testes de compressão uni-axial com provetes MHSS confirmaram os resultados numéricos. A condutividade térmica dos provetes MHSS foi também avaliada. A segunda parte do trabalho concentra-se na deformação utilizando 3 pontos de aplicação de força em painéis sanduíche compostos por chapas de alumínio envolvendo vários materiais celulares como espumas M-Pore® e Alporas® em alumínio, estruturas em forma de colméia e MHSS adhesivamente fundidas. A resistência à deformação, a capacidade de carga do material e o modo de falha foram verificados através de testes de flexão utilizando 3 pontos de aplicação de força. A comparação dos resultados experimentais possibilita a comparação entre os vários materiais celulares no que diz respeito às propredades testadas.; This thesis addresses the performance of novel metallic hollow sphere structures (MHSS) in sandwich panels. Numerical finite element analyses and experimental tests are conducted. The first part of this thesis focuses on different types of metallic hollow sphere structures. The influence of the morphology...

‣ Inhibition of hepatitis C virus IRES-mediated translation by small RNAs analogous to stem–loop structures of the 5′-untranslated region

Ray, Partho Sarothi; Das, Saumitra
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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Translation of the hepatitis C virus (HCV) RNA is mediated by the interaction of ribosomes and cellular proteins with an internal ribosome entry site (IRES) located within the 5′-untranslated region (5′-UTR). We have investigated whether small RNA molecules corresponding to the different stem–loop (SL) domains of the HCV IRES, when introduced in trans, can bind to the cellular proteins and antagonize their binding to the viral IRES, thereby inhibiting HCV IRES-mediated translation. We have found that a RNA molecule corresponding to SL III could efficiently inhibit HCV IRES-mediated translation in a dose-dependent manner without affecting cap-dependent translation. The SL III RNA was found to bind to most of the cellular proteins which interacted with the HCV 5′-UTR. A smaller RNA corresponding to SL e+f of domain III also strongly and selectively inhibited HCV IRES-mediated translation. This RNA molecule interacted with the ribosomal S5 protein and prevented the recruitment of the 40S ribosomal subunit. This study reveals valuable insights into the role of the SL structures of the HCV IRES in mediating ribosome entry. Finally, these results provide a basis for developing anti-HCV therapy using small RNA molecules mimicking the SL structures of the 5′-UTR to specifically block viral RNA translation.

‣ The catalytic activity of Src is dispensable for translocation to focal adhesions but controls the turnover of these structures during cell motility.

Fincham, V J; Frame, M C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 02/01/1998 Português
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The Src family of protein tyrosine kinases is involved in transducing signals at sites of cellular adhesion. In particular, the v-Src oncoprotein resides in cellular focal adhesions, where it induces tyrosine phosphorylation of pp125FAK and focal adhesion loss during transformation. v-Src is translocated to cellular focal adhesions by an actin-dependent process. Here we have used mutant v-Src proteins that are temperature-dependent for translocation, but with secondary mutations that render them constitutively kinase-inactive or myristylation-defective, to show that neither v-Src kinase activity nor a myristyl group are required to induce association of v-Src with actin stress fibres and redistribution to sites of focal adhesions at the stress fibre termini. Moreover, switching the constitutively kinase-inactive or myristylation-defective temperature-sensitive v-Src proteins to the permissive temperature resulted in concomitant association with tyrosine-phosphorylated focal adhesion kinase (pp125FAK) and redistribution of both to focal adhesions. However, both catalytic activity and myristylation-mediated membrane association are required to induce dissociation of pp125FAK from v-Src, later degradation of pp125FAK and focal adhesion turnover during transformation and cell motility. These observations provide strong evidence that the role of the tyrosine kinase activity of the Src family at sites of cellular focal adhesions is to regulate the turnover of these structures during cell motility.

‣ Ataxin-2 Interacts with the DEAD/H-Box RNA Helicase DDX6 and Interferes with P-Bodies and Stress Granules

Nonhoff, Ute; Ralser, Markus; Welzel, Franziska; Piccini, Ilaria; Balzereit, Daniela; Yaspo, Marie-Laure; Lehrach, Hans; Krobitsch, Sylvia
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /04/2007 Português
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Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.

‣ Regulation of Glycan Structures in Animal Tissues: TRANSCRIPT PROFILING OF GLYCAN-RELATED GENES*S⃞

Nairn, Alison V.; York, William S.; Harris, Kyle; Hall, Erica M.; Pierce, J. Michael; Moremen, Kelley W.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 20/06/2008 Português
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Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes...

‣ Formalin-Induced Fluorescence Reveals Cell Shape and Morphology in Biological Tissue Samples

Leischner, Ulrich; Schierloh, Anja; Zieglgänsberger, Walter; Dodt, Hans-Ulrich
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 28/04/2010 Português
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Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques.

‣ Comparison of the Multiple Oligomeric Structures Observed for the Rvb1 and Rvb2 Proteins

Cheung, Kevin L. Y.; Huen, Jennifer; Houry, Walid A.; Ortega, Joaquin
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/2010 Português
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The Rvb1 and Rvb2 proteins are two members of the AAA+ family that are involved in roles as diverse as chromatin remodeling, transcription, small nucleolar RNA maturation, cellular transformation, signaling of apoptosis and mitosis. These proteins are capable of playing a role in such diverse cellular processes because they are components of different macromolecular assemblies. In the last few years, there has been a number of groups reporting on the structure of purified Rvbs. The reported results have been rather controversial because there are significant differences observed among the published structures in spite of the high degree of homology among these proteins. Surprisingly, contradictions are observed not only between structures representing the Rvb proteins from different species, but also between protein structures from the same species. This review describes the available Rvb structures from the different species and makes also a comparative analysis of them. Finally, we identify some aspects of these structural studies worth pursuing additional investigations to ensure that the reported structures reflect physiologically relevant conformations of the Rvb1/Rvb2 complex.

‣ Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

Zhu, Jianyu; Korostelev, Andrei; Costantino, David A.; Donohue, John P.; Noller, Harry F.; Kieft, Jeffrey S.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA•mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.

‣ Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures▿†

Snyder, Jamie C.; Brumfield, Susan K.; Peng, Nan; She, Qunxin; Young, Mark J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2011 Português
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Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

‣ Cellular prion protein conformation and function

Damberger, Fred F.; Christen, Barbara; Pérez, Daniel R.; Hornemann, Simone; Wüthrich, Kurt
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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In the otherwise highly conserved NMR structures of cellular prion proteins (PrPC) from different mammals, species variations in a surface epitope that includes a loop linking a β-strand, β2, with a helix, α2, are associated with NMR manifestations of a dynamic equilibrium between locally different conformations. Here, it is shown that this local dynamic conformational polymorphism in mouse PrPC is eliminated through exchange of Tyr169 by Ala or Gly, but is preserved after exchange of Tyr 169 with Phe. NMR structure determinations of designed variants of mouse PrP(121–231) at 20 °C and of wild-type mPrP(121–231) at 37 °C together with analysis of exchange effects on NMR signals then resulted in the identification of the two limiting structures involved in this local conformational exchange in wild-type mouse PrPC, and showed that the two exchanging structures present characteristically different solvent-exposed epitopes near the β2–α2 loop. The structural data presented in this paper provided a platform for currently ongoing, rationally designed experiments with transgenic laboratory animals for renewed attempts to unravel the so far elusive physiological function of the cellular prion protein.

‣ Molecular and Cellular Aspects of Rhabdovirus Entry

Albertini, Aurélie A. V.; Baquero, Eduard; Ferlin, Anna; Gaudin, Yves
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 18/01/2012 Português
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Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.

‣ Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures

Saibil, Helen R.; Seybert, Anja; Habermann, Anja; Winkler, Juliane; Eltsov, Mikhail; Perkovic, Mario; Castaño-Diez, Daniel; Scheffer, Margot P.; Haselmann, Uta; Chlanda, Petr; Lindquist, Susan; Tyedmers, Jens; Frangakis, Achilleas S.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Yeast prions constitute a “protein-only” mechanism of inheritance that is widely deployed by wild yeast to create diverse phenotypes. One of the best-characterized prions, [PSI+], is governed by a conformational change in the prion domain of Sup35, a translation-termination factor. When this domain switches from its normal soluble form to an insoluble amyloid, the ensuing change in protein synthesis creates new traits. Two factors make these traits heritable: (i) the amyloid conformation is self-templating; and (ii) the protein-remodeling factor heat-shock protein (Hsp)104 (acting together with Hsp70 chaperones) partitions the template to daughter cells with high fidelity. Prions formed by several other yeast proteins create their own phenotypes but share the same mechanistic basis of inheritance. Except for the amyloid fibril itself, the cellular architecture underlying these protein-based elements of inheritance is unknown. To study the 3D arrangement of prion assemblies in their cellular context, we examined yeast [PSI+] prions in the native, hydrated state in situ, taking advantage of recently developed methods for cryosectioning of vitrified cells. Cryo–electron tomography of the vitrified sections revealed the prion assemblies as aligned bundles of regularly spaced fibrils in the cytoplasm with no bounding structures. Although the fibers were widely spaced...

‣ Atrial fibrillation in the elderly: the potential contribution of reactive oxygen species

Schillinger, Kurt J.; Patel, Vickas V.
Fonte: Science Press Publicador: Science Press
Tipo: Artigo de Revista Científica
Publicado em /12/2012 Português
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Atrial fibrillation (AF) is the most commonly encountered cardiac arrhythmia, and is a significant source of healthcare expenditures throughout the world. It is an arrhythmia with a very clearly defined predisposition for individuals of advanced age, and this fact has led to intense study of the mechanistic links between aging and AF. By promoting oxidative damage to multiple subcellular and cellular structures, reactive oxygen species (ROS) have been shown to induce the intra- and extra-cellular changes necessary to promote the pathogenesis of AF. In addition, the generation and accumulation of ROS have been intimately linked to the cellular processes which underlie aging. This review begins with an overview of AF pathophysiology, and introduces the critical structures which, when damaged, predispose an otherwise healthy atrium to AF. The available evidence that ROS can lead to damage of these critical structures is then reviewed. Finally, the evidence linking the process of aging to the pathogenesis of AF is discussed.

‣ Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

Holt, Brian D.; Shams, Hengameh; Horst, Travis A.; Basu, Saurav; Rape, Andrew D.; Wang, Yu-Li; Rohde, Gustavo K.; Mofrad, Mohammad R. K.; Islam, Mohammad F.; Dahl, Kris Noel
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 23/05/2012 Português
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With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs) are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

‣ Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/05/1984 Português
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Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton...

‣ Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes

Luitweiler, Eric M.; Henson, Brandon W.; Pryce, Erin N.; Patel, Varun; Coombs, Gavin; McCaffery, J. Michael; Desai, Prashant J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2013 Português
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All herpesviruses encode a complex of two proteins, referred to as the nuclear egress complex (NEC), which together facilitate the exit of assembled capsids from the nucleus. Previously, we showed that the Kaposi's sarcoma-associated herpesvirus (KSHV) NEC specified by the ORF67 and ORF69 genes when expressed in insect cells using baculoviruses for protein expression forms a complex at the nuclear membrane and remodels these membranes to generate nuclear membrane-derived vesicles. In this study, we have analyzed the functional domains of the KSHV NEC proteins and their interactions. Site-directed mutagenesis of gammaherpesvirus conserved residues revealed functional domains of these two proteins, which in many cases abolish the formation of the NEC and remodeling of nuclear membranes. Small in-frame deletions within ORF67 in all cases result in loss of the ability of the mutant protein to induce cellular membrane proliferation as well as to interact with ORF69. Truncation of the C terminus of ORF67 that resides in the perinuclear space does not impair the functions of ORF67; however, deletion of the transmembrane domain of ORF67 produces a protein that cannot induce membrane proliferation but can still interact with ORF69 in the nucleus and can be tethered to the nuclear membrane by virtue of its interaction with the wild-type-membrane-anchored ORF67. In-frame deletions in ORF69 have varied effects on NEC formation...

‣ Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells

Neupane, Bhanu; Jin, Tao; Mellor, Liliana F.; Loboa, Elizabeth G.; Ligler, Frances S.; Wang, Gufeng
Fonte: MDPI Publicador: MDPI
Tipo: Artigo de Revista Científica
Publicado em 18/09/2015 Português
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Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

‣ Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

Pereira, D. R.; Canadas, Raphael Faustino; Silva-Correia, J.; Marques, A. P.; Reis, R. L.; Oliveira, J. M.
Fonte: Universidade do Minho Publicador: Universidade do Minho
Tipo: Conferência ou Objeto de Conferência
Publicado em /09/2015 Português
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 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth)...

‣ Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Staufenbiel, Matthias; Deppert, Wolfgang
Fonte: Rockefeller University Press Publicador: Rockefeller University Press
Tipo: Article; PeerReviewed Formato: application/pdf
Publicado em 01/05/1984 Português
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Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton...