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Morgan, Rachel E. "Is the Cytoskeleton Necessary for Viral Replication?" Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_theses/38.
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The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.
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McDermott, Joshua D. "The ovine lens cytoskeleton." Lincoln University, 2007. http://hdl.handle.net/10182/700.
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The lens of the eye is a vital tissue in the visual system, responsible for the collection and focusing of light on to the retina. Comprised of epithelial cells at differing stages of differentiation, the transparency of the lens is dependent on the highly ordered crystalline structure of lens proteins. The lens consists of several proteins including crystallins (α, β, γ) that make up 90% of the soluble protein, and the lens cytoskeletal proteins. Cytoskeletal proteins contribute only a fraction of the total lens protein, but are thought to play an important role in the establishment and maintenance of transparency. Calpain-induced degradation of these proteins may be involved in the development of cataracts. This has been an area of research at Lincoln University where a flock of sheep genetically predisposed to cataract maintained as a cataract development model. The aim of this research was to investigate the distribution of cytoskeletal proteins in the lens, and to examine the effects of calpain proteolysis on these proteins, with the goal of establishing the role of the lens cytoskeletal proteins in the ovine cataract model. A combination of techniques was used including immunohistochemistry, which required the development of a specific protocol for ovine lenses. Cytoskeletal proteins were identified using immunohistochemistry in lens tissue sections and exhibited characteristic distributions. Actin displayed preferential distribution in the short sides of the fibre cells in the cortex of the lens but was absent in the lens nucleus, while spectrin in the cortex and nucleus was associated with the fibre cell membrane. Filensin was observed in the outer cortex of lens sections associated with the fibre cell membrane and cytoplasm, although the pattern of localisation was indistinct due to the abundance of filensin breakdown products. Vimentin displayed membrane and cytoplasmic association in the outer cortex that diminished toward the lens nucleus, with membrane associated vimentin only persisting in the deeper regions of the cortex and nucleus. Additionally, the effect of novel calpain inhibitors (Cat0059 and Cat811) in preventing proteolysis of lens cytoskeletal protein was investigated and compared with calpain inhibitors developed elsewhere (SJA6017). The inhibitors were tested at between 10 and 0.1 μM (100 nM). All inhibitors were effective at 10 μM. SJA6017 provided significant protection to vimentin at 1 μM. Cat0059 was found to protect spectrin and filensin at 1 μM, but not vimentin, while inhibitor Cat811 was found to protect spectrin only. SJA6017 added to assays at 100 nM offered significant protection to spectrin, and Cat0059 was found to protect filensin and spectrin to a significant degree at 100 nM, indicating the novel inhibitors were comparable to those developed elsewhere in terms of their effectiveness. Taken together, the evidence presented in this thesis shows the cytoskeletal proteins as crucial elements in the lens. Their pervasive presence coupled with evidence that lens cytoskeletal proteins are sensitive to calpain-induced proteolysis that is inhibited with novel calpain inhibitors suggests that the lens cytoskeletal proteins may be useful targets in cataract prevention for future research.
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Snyder, Heidi Ghent. "Fiber type-specific desmin content in human single muscle fibers /." Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1253.pdf.
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Fawell, E. H. "Studies on the microvillus cytoskeleton." Thesis, University of Leeds, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355700.
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Schneider, André. "The cytoskeleton of Trypanosoma brucei /." [S.l.] : [s.n.], 1988. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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McCarthy, David James. "Analysis of the novel Lyn-associated cytoskeletal modular protein, LACM." University of Western Australia. School of Medicine and Pharmacology, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0180.
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A yeast-two hybrid screen with Lyn identified a novel 130 kDa multidomain protein with a 36% identity to Actin Filament Associated Protein (AFAP) 110 and similar domains, including PH domains, potential sites of tyrosine and serine/threonine phosphorylation, a leucine-zipper domain, a potential actin binding site and multimerization site. AFAP110 has been shown to have a role in modulating actin filament integrity and induce lamellipodia formation, and is known to interact with Src family kinases. The aim of this thesis was to characterize this novel protein named Lyn-Associated Cytoskeletal Modulator (LACM) and determine any molecular interactions in order to attempt to elucidate a role for the protein in cell signaling through Lyn. LACM is encoded by a gene consisting of 18 exons and is located on human chromosome 5q33.1 and mouse chromosome 18 E1. LACM protein is expressed through a number of cell types including the R11 erythroid cell line, and mouse tissues including brain, lung, heart and embryos. LACM was shown to multimerize, and subcellular localization of the protein was observed to concentrate around the cell membrane at sites of filamentous actin in filopodia, lamellipodia and stress fibres. The carboxy-terminus of LACM was observed to localize the protein to sites at the cell membrane and through the cytoplasm. Removal of this terminal region resulted in all LACM protein localizing to the nucleus in punctuate spots. LACM protein was observed in heart muscle and potentially has a role at sites of nerve junctions on cardiac myocytes. LACM was shown to interact with the SH3 domain of Lyn at a polyproline motif on LACM. LACM was observed to co-localize and co-immunoprecipitate with Lyn and was tyrosine phosphorylated by the kinase domain of Lyn. Interestingly, the consititutively active Lyn and LACM caused transfected cells to
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Tharmann, Rainer. "Mechanical properties of complex cytoskeleton networks." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=97998002X.
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Brown, Jennifer. "Investigating the actin cytoskeleton in cancer." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7266/.
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Dynamic alterations in the actin cytoskeleton, under the regulation of the Rho/ROCK pathway, permit cell motility, cell-to-cell and cell-to-matrix adhesion, and have also been shown to participate in apoptosis and cell proliferation. These facets of cellular behaviour all have the capacity to become dysregulated in cancer; components of the Rho/ROCK pathway are known to play varying roles in these processes, both within primary tumours and within the tumour microenvironment. The LIM kinases are phosphorylated and activated by ROCK, leading to inactivation of cofilin and subsequent stabilisation of actin filaments. In addition, LIM kinase 2 serves as a p53 target and is upregulated in response to DNA damage. In some solid tumours (e.g. breast and prostate), LIM kinase levels are elevated. However, we found that LIM kinase 2 expression is downregulated in colon cancer, with a progressive reduction noted with advancing tumour stage. I found that LIMK2 expression in colon cancer is under epigenetic regulation, with hypermethylation of the promoters leading to transcriptional silencing; this implicates LIMK2 as a tumour suppressor gene in this context. This has potential translational implications as loss of LIMK2 could be utilised as a biomarker to stratify patients in the future. Elevated mechanical tension within the tumour microenvironment is known to be an adverse prognostic indicator due to its association with desmoplasia. ROCK activation has previously been shown to increase epidermal tissue stiffness and thickness, but little was known about the mechanisms by which this occurs. I found that ROCK activation leads to the deposition of extracellular matrix components, with a presumed consequent further increase in stromal stiffness. This indicates that a positive feedback cycle is established in the tumour microenvironment, maintaining a fibrotic stromal reaction that permits tumour progression. These results highlight the disparate roles that the actin cytoskeleton and constituents of the Rho/ROCK pathway play in tumour initiation and propagation, indicating the need for further research.
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Balanda, Matthew L. "The use of cytoskeletal inhibitors to determine the role of the cytoskeleton in the activation of hypertonicity-induced currents in xenopus oocytes /." View abstract, 1999. http://library.ctstateu.edu/ccsu%5Ftheses/1561.html.
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Thesis (M.A.)--Central Connecticut State University, 1999.<br>Thesis advisor: Kathy Martin. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaves 42-44).
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Huber, Florian. "Emergent structure formation of the actin cytoskeleton." Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-86666.
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Anders als menschengemachte Maschinen verfügen Zellen über keinen festgeschriebenen Bauplan und die Positionen einzelner Elemente sind häufig nicht genau festgelegt, da die Moleküle diffusiven Zufallsbewegungen unterworfen sind. Darüber hinaus sind einzelne Bauteile auch nicht auf eine einzelne Funktion festgelegt, sondern können parallel in verschiedene Prozesse einbezogen sein. Basierend auf Selbstorganisation und Selbstassemblierung muß die Organisation von Anordnung und Funktion einer lebenden Zelle also bereits in ihren einzelnen Komponenten inhärent enthalten sein.
Die intrazelluläre Organisation wird zum großen Teil durch ein internes Biopolymergerüst reguliert, das Zytoskelett. Biopolymer-Netzwerke und –Fasern durchdringen die gesamte Zelle und sind verantworlich für mechanische Integrität und die funktionale Architektur. Unzählige essentielle biologische Prozesse hängen direkt von einem funktionierenden Zytoskelett ab.
Die vorliegende Arbeit zielt auf ein besser Verständnis und den Nachbau zweier verschiedener funktionaler Module lebender Zellen anhand stark reduzierter Modellsysteme. Als zentrales Element wurde Aktin gewählt, da dieses Biopolymer eine herausragende Rolle in nahezu allen eukaryotischen Zellen spielt.
Mit dem ersten Modellsystem wird der bewegliche Aktin-Polymerfilm an der Vorderkante migrierender Zellen betrachtet. Die wichtigsten Elemente dieser hochdynamischen Netzwerke sind bereits bekannt und wurden in dieser Arbeit benutzt um ein experimentelles Modellsystem zu etablieren. Vor allem aber lieferten detailierte Computersimulationen und ein mathematisches Modell neue Erkenntnisse über grundlegende Organisationsprinzipien dieser Aktinnetzwerke. Damit war es nicht nur möglich, experimentelle Daten erfolgreich zu reproduzieren, sondern das Entstehen von Substrukturen und deren Charakteristika auf proteinunabhängige, generelle Mechanismen zurückzuführen.
Das zweite studierte System betrachtet die Selbstassemblierung von Aktinnetzwerken durch entropische Kräfte. Aktinfilamente aggregieren hierbei durch Kondensation multivalenter Ionen oder durch Volumenausschluss hochkonzentrierter inerter Polymere. Ein neu entwickelter Experimentalaufbau bietet die Möglichkeit in gut definierten zellähnlichen Volumina, Konvektionseinflüsse zu umgehen und Aggregationseffekte gezielt einzuschalten. Hierbei wurden neuartige, regelmäßige Netzwerkstrukturen entdeckt, die bislang nur im Zusammenhang mit molekularen Motoren bekannt waren. Es konnte ferner gezeigt werden, dass die Physik der Flüssigkristalle entscheidend zu weiteren Variationen dieser Netzwerke beiträgt. Dabei wird ersichtlich, dass entstehende Netzwerke in ihrer Architektur direkt die zuvor herrschenden Anisotropien der Filamentlösung widerspiegeln.
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Boey, Seng Kee. "Computer simulation of the human erythrocyte cytoskeleton." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq24296.pdf.
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Nuydens, Rony Maria. "Dynamics of the neuronal cytoskeleton during apoptosis." Maastricht : Maastricht : Universiteit Maastricht ; University Library, Maastricht University [Host], 1999. http://arno.unimaas.nl/show.cgi?fid=6860.
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Förster, Florian. "Targeting the actin cytoskeleton with natural compounds." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-168914.
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Targeting the cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. Whereas drugs which address microtubule CSK such as vinca alkaloids or taxanes are well established in the clinic, compounds binding to the actin CSK are still far away from their therapeutical application. One reason might be the lacking knowledge on their mode of cytotoxicity and moreover their tumor specific mechanism of action.
We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity by actin targeting in different breast cancer cells, namely MCF7 and MDA-MB-231. Chondramide inhibits actin filament assembly and dynamics shown by a fluorescence-based analysis (FRAP) in whole cells and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases (-9 and -3). Detailed analysis revealed, that Chondramide induces apoptosis by enhancing the occurrence of mitochondrial permeability transition (MPT). Known MPT-modulators were found to be affected by Chondramide: Hexokinase II (HkII) bound to the voltage dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad was recruited to the mitochondria. Importantly, PKCε, a prosurvival serine/threonine kinase possessing an actin-binding site and known to regulate the HkII/VDAC interaction as well as Bad phosphoylation was identified as the link between actin CSK and apoptosis induction. PKCε which was found overexpressed in breast cancer cells accumulated in actin bundles induced by Chondramide and lost its activity.
The second goal of our work was to inform on a potential tumor specific action of actin binding agents such as Chondramide. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide induced apoptosis and notably express very low level of PKCε we claim that trapping PKCε via Chondramide induced actin hyperpolymerization displays tumor cell specificity.
Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCε, thus setting the stage for actin-stabilizing agents as innovative cancer drugs. This is moreover supported by the in vivo efficacy of Chondramide triggered by abrogation of PKCε signaling shown in a xenograft breast cancer model.
For the actin targeting compound Doliculide we could show that Doliculide impairs the dynamics of the actin CSK similar to Chondramide. Moreover, it reduces the proliferation rate and migration of cancer cells and also leads to the induction of apoptosis, thus Doliculide is also an interesting lead structure for further preclinical investigations.
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Kim, Taeyoon Ph D. Massachusetts Institute of Technology. "Simulation of actin cytoskeleton structure and rheology." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39875.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.<br>Includes bibliographical references (p. 81-87).<br>Structures consisting of G-actin or other filament-forming monomers show a variety of morphologies with widely different properties in regard to pore size, degree of isotropy, and extent of cross-linking. These characteristics are primarily determined by the concentration and feature of proteins which cross-link filaments, but little is known how the filament-forming monomers and cross-linking proteins are organized in order to produce various network morphologies. In addition, it's generally known that mechanical force plays an important role in the physiology of eukaryote cells whose major structural component in cortex is actin cytoskeleton. Thus, understanding the origin of viscoelasticity of cross-linked networks should be crucial to figure out the exact role of cytoskeletal behaviors in many cellular functions. Here, we introduce a Brownian dynamics (BD) simulation model in three dimensions in which actin monomers polymerize into a filament and become cross-linked by two types of cross-linking molecules that constitute either perpendicular or parallel cross-links. We evaluate the influences of system parameters on the morphology of resultant networks. Some scaling behaviors that are independent of the specific choice of most parameters appear.<br>(cont.) Additionally, the modified model is employed to investigate the viscoelastic property of actin-like network by tracking the trajectories of filaments. This method is theoretically more direct and more precise than micro-bead rheology used in experiments. The viscoelastic property appears to be highly affected by characteristics of cross-linking molecules, average filament length, and concentration of actin monomers. Our model has the high potential as a BD model that can be applied for investigating a variety of actin-related phenomena after further refinement and modification.<br>by Taeyoon Kim.<br>S.M.
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Ju, M. "Role of membrane cytoskeleton in fenestra biogenesis." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1383777/.
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Fenestrae are transcellular membrane pores that mediate blood-tissue exchange in highly specialized vascular endothelia such as in choroidal capillaries. Substances that traverse the pore never encounter the contents of the cytoplasm and are transported in a rapid and presumably energy-efficient manner. Fenestrae arise in attenuated regions of the endothelial cell periphery and are highly organized in clusters termed sieve plates. My PhD project was based on the identification of novel components of fenestrae and how these components contribute to mechanisms regulating fenestra formation. Using an in vitro biogenesis model coupled with proteomic analysis, we identified several proteins enriched in fenestrated plasma membranes. Localisation of candidate proteins was accomplished by immunolabelling, confocal microscopy, and transmission electron microscopy. Functional roles for the candidate proteins in fenestra biogenesis were probed through gain- and loss of function techniques. Coimmunoprecipitation was used to uncover protein-protein interactions, and biochemical reagents were applied to probe the signalling pathways involved in fenestra formation. Through extensive investigation, we identified the ERM (ezrin/radixin/moesin) protein moesin as a component of fenestral sieve plates. Inhibition of moesin function by expression of a dominant negative mutant or siRNA resulted in inhibition of fenestra formation, whereas knockdown of another regulator of the actin cytoskeleton, annexin II, led to a robust increase in fenestra formation. Biochemical and structural analyses showed that these modulators control the formation of an actin-fodrin submembrane cytoskeleton that is essential for sieve plate and fenestra formation, and that this cytoskeleton is directly linked to the fenestra pore protein PV-1. The transmembrane protein Na,K-ATPase is also a structural component of the submembrane complex, and functions as a regulator of fenestra formation in vitro and in vivo. These findings provide a conceptual framework linking the actin cytoskeleton to membrane remodeling during fenestra biogenesis and new molecular tools for probing fenestra structure and function.
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Dobbins, G. Clement. "The role of the cytoskeleton in AChR clustering." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2007p/dobbins.pdf.
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Esson, Heather Jean. "Morphological evolution and development of the euglenid cytoskeleton." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/6814.
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In an effort to better understand character evolution in the cytoskeleton (pellicle) of euglenid protists, I used comparative and descriptive methods to investigate the morphological diversity and development of pellicle surface patterns formed by differences in strip length at the anterior and posterior ends of the cell (strip reduction). By observing dividing Euglena gracilis cells with scanning electron microscopy (SEM) and integrating these data with previous evolutionary and developmental research, I showed that these patterns result from the semiconservative duplication and subsequent intermittent growth of pellicle strips during cytoskeletal replication and cytokinesis. Furthermore, simple changes in the developmental timing of this process (heterochrony) resulted in the diversity of posterior strip reduction patterns observed in phototrophic euglenids. This model was then used to interpret the results of two studies describing pellicle surface patterns in other photosynthetic taxa. The first was a morphological description of the complex linear pattern of posterior reduction in the benthic marine phototroph, Euglena obtusa. The second was an investigation of the evolution of bilaterally symmetrical, “clustered” strip reduction patterns in the rigid genus Phacus, examined in the context of maximum likelihood (ML) and Bayesian phylogenetic analyses of combined nuclear small subunit and partial large subunit ribosomal genes (SSU rDNA and LSU rDNA, respectively). These studies, taken together, show that strip length and other pellicle characters (such as pore placement) are strongly influenced by age and perhaps other developmental factors (such as parental strip identity and cell polarity), but the underlying genetics and molecular biology of these factors are completely unknown. Finally, SEM was used for the first time to describe prearticular strip projections, a pellicle character that has been extensively studied using transmission electron microscopy (TEM). The novel character state revealed by this study shows that the diversity of this pellicle character is still poorly understood. The structural complexity of the euglenid pellicle and the developmental and evolutionary processes that resulted in its astonishing diversity could make it an ideal model system for studying cytoskeletal evolution and development once a robust genetic research framework is constructed.
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Attaran, Amir. "CTL cytotoxicity and the cytoskeleton : a microscopical study." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308607.
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Carballido-Lopez, Rut. "Bacterial cytoskeleton : cell shape determination in Bacillus subtilis." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270007.
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Corben, Elizabeth Bower. "An immunological approach to the higher plant cytoskeleton." Thesis, University of East Anglia, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.540595.
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Cain, R. J. "Manipulation of the eukaryotic cytoskeleton by invasive Salmonella." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597214.
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Mechanical cell fractionation and immunofluorescence microscopy were applied to systematically investigate the subcellular localisation of epitope-tagged effectors in cultured cells after transfection or infection with wildtype <i>Salmonella </i>strains exogenously expressing individual effectors. Although five <i>Salmonella</i> effectors contain no apparent membrane-targeting domains, all six localised to the plasma membrane fraction and were visualised a the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated cytoplasmic effector pool was detectable. Parallel experiments with <i>Shigella </i>IpgD demonstrated analogous plasma membrane localisation. In agreement with their cellular location, <i>in vitro</i> reconstitution demonstrated that the prenylated cellular GTPase Cdc42 was necessary and sufficient for SopE and SptP membrane association. Three effector-augmented bacterial strains exhibited increased invasion compared to wildtype bacteria; SopE- and SopB- but not SipC-mediated events were inhibited by dominant negative Rho GTPase expression, demonstrating that effector directed actin rearrangements occur via both redundant and robust mechanisms. These data shoed that the host plasma membrane is a critical interface for effector-target interaction during both <i>Salmonella </i>and <i>Shigella</i> cell entry, and established versatile systems to further dissect effector interplay. <i>Salmonella</i> strains and effector transfected cells were combined in screens to assess potential pairwise interplay between effectors. These identified both synergistic and antagonistic effector protein pairs, namely three novel co-operativitive interactions (SipC:SopB, SipC:SopE, SipA:SopB) in addition to those previously demonstrated (SipA:SipC) or implied (SopE:SopB). A temporal requirement for effector activities was also revealed as SopB/IpgD or SptP transfection inhibited bacterial entry. Circuitry modelling of these data using logical gates was used analyse <i>Salmonella </i>effector activities during bacterial entry. The requirements for effector delivery were also investigated. Together these findings illustrate the complex cross-talk between host and bacterial factors and highlight the central role of the host plasma membrane as an interactive interface during bacterial invasion.
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Advani, Andrew. "The blood cell cytoskeleton in type 2 diabetes." Thesis, University of Newcastle Upon Tyne, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289211.
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Feret, Dorota. "Proteasomal control of the fission yeast microtubule cytoskeleton." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.704739.
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Speldewinde, Shaun. "Prions, autophagy, ageing and actin cytoskeleton in yeast." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/prions-autophagy-ageing-and-actin-cytoskeleton-in-yeast(03085d7f-283a-40e1-bcf7-d9533ff2e2fc).html.
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Prions are infectious protein entities capable of self-replication. Prions are the causal agents behind the transmissible spongiform encephalopathies causing neurodegeneration and death in affected organisms. Prions have been identified in yeast with the best-characterized prions being [PSI+] and [PIN+], whose respective native proteins are the Sup35 translation termination factor and Rnq1 (function unknown). Autophagy is a cellular housekeeping mechanism mediating the degradation of damaged proteins and superfluous organelles. It is a highly sequential process regulated by autophagy related genes (ATGs). Autophagy has also been implicated in the clearance of amyloidogenic proteins including prions. However, the mechanistic basis underlying this activity is poorly understood, and a key objective of this project was to characterize how autophagy prevents spontaneous prion formation. Our study found that the deletion of core ATGs correlated with an increase in de novo [PSI+] and [PIN+] formation as well as Sup35 aggregation. Enhancement of autophagic flux through spermidine treatment attenuated the increased levels of de novo [PSI+] formation in mutants that normally show elevated levels of [PSI+] formation. Defective autophagy correlated with increased oxidatively damaged Sup35 in an atg1 mutant whereas anaerobic growth abrogated the increased [PSI+] formation in the atg1 mutant to wild-type levels. Our data suggest that autophagy serves a protective role in the clearance of oxidatively damaged Sup35 proteins that otherwise has a higher propensity towards [PSI+] prion formation. We also investigated the role of prion formation and autophagy during yeast chronological ageing which is the time that non-dividing cells remain viable. Prion diseases are associated with advanced age which correlates with a decline in cellular protective mechanisms including autophagy. Our study found an age dependent increase in the frequency of de novo [PSI+] formation with chronological age of yeast cells, more so in an atg1 mutant relative to the wild-type. Autophagy competent cells carrying the [PSI+] and [PIN+] prions also had improved chronological lifespan relative to prion free cells and atg1 cells. Cells carrying the [PSI+] prion elicited elevated autophagic flux that may promote improved lifespan thus suggesting a beneficial role of the [PSI+] prion during chronological ageing. The actin cytoskeleton provides the structural framework essential for a multitude of cellular processes to occur. We investigated the role of the Arp2/3 complex responsible for branching of actin filaments towards prion formation. Knockout mutants of the nucleation promoting factors of the Arp2/3 complex, in particular the abp1 mutant, showed reduced de novo [PSI+] formation and Sup35 aggregation under basal and oxidative stress conditions. Similarly, treatment with latrunclin A, an actin monomer-sequestering drug also abrogated de novo [PSI+] formation. Colocalization studies revealed that Sup35 often does not colocalize with Rnq1, a marker for the insoluble protein deposit (IPOD) in an abp1 mutant. This suggests a role for the Abp1 protein in the efficient transport of Sup35 molecules to the IPOD that may facilitate de novo [PSI+] prion formation under vegetative states and oxidant challenges.
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Uppal, Sonal. "Studies of Microtubule Inhibitor Combinations on Cytoskeleton Architecture." Bowling Green State University / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1162842626.
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Turner, Jerrold Ross. "Secretory organelles and the cytoskeleton: Organization and interdependence." Case Western Reserve University School of Graduate Studies / OhioLINK, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=case1054668919.
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Kumar, Sanjay. "Molecular mechanisms of organization in the neurofilament cytoskeleton." Available to US Hopkins community, 2002. http://wwwlib.umi.com/dissertations/dlnow/3080703.
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Ricci, Mario. "The marsupial sperm tail cytoskeleton : a morphological and biochemical study /." Title page, table of contents and abstract only, 2004. http://web4.library.adelaide.edu.au/theses/09PH/09phr4911.pdf.
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Turina, Dean. "Propofol changes the cytoskeletal function in neurons : An experimental study in cortical cultures." Doctoral thesis, Linköpings universitet, Anestesiologi med intensivvård, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-77219.
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Every day, general anaesthetics are given to a large number of patients around the world but the cellular mechanisms of how anaesthetics act are still not clear. General anaesthetics cause the intended unconsciousness, amnesia and immobility in patients, but also side effects such as a decrease in mean arterial pressure and arrhythmia, both of which contribute to complications such as heart damage and stroke. With more knowledge of the mechanism of anaesthetic drugs, these complications could be reduced. It has been shown that anaesthetics cause a disruption of the thalamocortical connectivity and brain network connectivity. How the network communication is disrupted however is not known. Propofol and thiopental are both intravenous anaesthetic drugs used widely in clinical anaesthesia. They bind to the GABAA receptor and enhance its function. The cytoskeleton helps the cell to maintain its shape and participate in cellular movement and transport. Cellular transport to and from a neuron’s cell body and periphery is performed by motor proteins that move vesicles, organelles and proteins along cytoskeletal tracks. We have previously shown that propofol causes a reorganisation of the cytoskeleton protein actin in neurons, but we were further interested to study the effects of propofol and thiopental on the cytoskeletal function of cultured cortical rat neurons. Our results show that propofol and thiopental cause neurite (axon and dendrite) retraction. Propofol’s effects were time- and dose-dependent, and can be reversed when propofol is removed. We were able to inhibit propofolinduced neurite retraction if we stabilised actin by blocking either the motor protein myosin II or the GABAA receptor. We have previously shown that a small GTP-binding protein, RhoA, inhibits propofol-caused actin reorganisation. Propofol-induced neurite retraction was mediated via a downstream effector of RhoA, ROK, which induces phosphorylation of the myosin light chain and increases contractility. Furthermore, we have shown that propofol causes a switch from anterograde to retrograde transport and increases the average velocity of the moving vesicles in neurites. The propofol induced retrograde vesicle transport was GABAA receptor-mediated. Orexin A is a neuropeptide which regulates the sleep/awake cycle and has also been shown to reduce anaesthesia in animals when given intracerebroventricularly. We found that orexin A reverses propofol and thiopental-induced neurite retraction and actin reorganisation. Moreover, we have shown that the orexin A inhibition of propofol-induced neurite retraction is mediated via the PLD/PKC intracellular signalling pathway. Propofol and thiopental decreased the tyrosine phosphorilation of the intermediate cytoskeletal protein vimentin which is reversed by orexin A. Taken together, these results suggest that propofol causes a time- and dose-dependent, reversible and GABAAreceptor-mediated neurite retraction in cultured cortical rat neurons. Propofol also causes a switch from anterograde to retrograde vesicle transport in neurites. Orexin A reverses propofol and thiopental-induced neurite retraction and cytoskeletal reorganisation. Orexin A inhibits propofol-induced neurite retraction via the PLD/PKC intracellular signalling pathway.
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Jopson, Martin Frederick. "Plant microtubules, their associated proteins and the cell cycle." Thesis, University of East Anglia, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318090.
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Bellamy, Matthew Laurence. "A study of non-erythroid isoforms of protein 4.1." Thesis, University of Kent, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298166.
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Marks, John. "Cytological and biochemical investigation of actin in the fission yeast Schizosaccharomyces pombe." Thesis, University College London (University of London), 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337930.
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Wong, Chun-ming. "B-Catenin mutations and expression in hepatocellular carcinoma." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B24729942.
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Hoyos-Flight, Monica. "Wnt signalling and the regulation of the axonal cytoskeleton." Thesis, Imperial College London, 2005. http://hdl.handle.net/10044/1/11773.
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Flear, Andrea Karen. "The cytoskeleton during muscle cell fusion in early myogenesis." Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236303.
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Shuttleworth, Rebecca Jane. "Role of the chondrocyte cytoskeleton in health and disease." Thesis, Cardiff University, 2010. http://orca.cf.ac.uk/54356/.
Full textAbstract:
Introduction: Articular cartilage comprises a dense extracellular matrix (ECM) of primarily collagen, proteoglycans and water interspersed with the cartilage cell- the chondrocyte. Osteoarthritis (OA) is a disease characterised by articular cartilage degradation and a change in chondrocyte phenotype. Increased or abnormal joint loading is a risk factor for OA and can regulate chondrocyte phenotype. The chondrocyte cytoskeleton comprises actin microfilaments, tubulin microtubules and vimentin intermediate filaments and has been implicated in the propagation of physical signals to the chondrocyte nucleus, termed 'mechanotransduction'. In addition, the organisation of chondrocyte cytoskeletal networks has been observed to differ in both human OA and in a rat model of OA when compared with normal chondrocytes. We hypothesise that dysregulation of cytoskeletal networks prevents normal ECM-chondrocyte signalling and promotes a catabolic phenotype as in OA. Results: When compared with normal human chondrocytes, OA chondrocytes exhibited differences in the gene expression of components of the cytoskeleton and in the spatial organisation and architecture of the cytoskeleton, both in situ and in vitro. In normal and OA human chondrocytes cultured in agarose hydrogels, disruption of each of the three main cytoskeletal elements resulted in gene expression changes in both normal and OA cells. A number of gene responses to cytoskeletal disruption were similar in normal and OA cells, such as SOX9, MMP14, TGFB1, CASP3 and PTGS2 (COX-2). Other genes responded differently to the same treatment in normal versus OA cells, including ADAMTS5, COMP, FGFR3 and NOS2A (iNOS). Cyclic compression (15% strain, 0.5 Hz) for up to 40 minutes induced cytoskeletal reorganisation in normal and OA human chondrocytes and up-regulation of p-tubulin and destrin mRNA expression. Recovery in free swelling conditions for five hours post-load showed the chondrocyte phenotype was enhanced in OA chondrocytes. Cyclic compression in the presence of cytoskeletal disruption altered the transcriptional response of the actin depolymerising proteins cofilin and destrin in normal and OA chondrocytes, and the transcriptional response of SOX9 and the actin sequestering protein thymosin p4 in OA chondrocytes. Conclusions: Changes in the cytoskeleton of OA chondrocytes are not simply a result of the altered mechanical environment in OA articular cartilage. Changes in the cytoskeleton can affect chondrocyte phenotype and the response of chondrocytes to cyclic compression, therefore the observed differences in organisation and expression could result in the altered phenotype of OA chondrocytes. Differences between the effect of cyclic compression on normal and OA human chondrocytes support the existence of different or divergent mechanotransduction pathways that are mediated in part by elements of the chondrocyte cytoskeleton.
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Thodey, Catherine. "Actin cytoskeleton dynamics mediate sugar response in Arabidopsis thaliana." Thesis, University of East Anglia, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518364.
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Hawkins, Stephen Francis. "Studies relevant to the cytoskeleton in chronic lymphocytic leukaemia." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.539727.
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KleinJan, Fenneke [Verfasser]. "Viscoelastic and structural properties of the cytoskeleton / Fenneke KleinJan." Ulm : Universität Ulm, 2018. http://d-nb.info/1155827732/34.
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Tong, Peter. "Interactions between cell membrane dynamics, the cytoskeleton and insulin." Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242381.
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Price, Leo Sebastian. "Secretion and the actin cytoskeleton in rat mast cells." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307776.
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Filippi, Beatrice Maria. "Cellular effects of phosphoinositide derivatives on the actin cytoskeleton." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424620.
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Rocchetti, Alessandra. "Interactions between the plant Golgi apparatus and the cytoskeleton." Thesis, Oxford Brookes University, 2016. https://radar.brookes.ac.uk/radar/items/e035b419-1acc-4031-aadd-2cfc1f9ed3c8/1/.
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In animal cells, the relationship between the Golgi apparatus and cytoskeleton has been well characterised but not much is known in plants. The functions of the Golgi apparatus are conserved amongst eukaryotes. It is one of the main stations in the secretory pathway and is involved in protein processing and sorting to different destinations. In plants, it is also involved in trafficking and positioning of cell wall components. In tobacco epidermal cells, fluorescent labelling with Golgi marker proteins has shown that the Golgi apparatus is made of hundreds of individual units scattered in the cortical cytoplasm and moving on the actin cytoskeleton. The contribution of actin filaments to Golgi body motility in plant has been extensively described, but this actin-centric view has recently been challenged. Emerging evidence suggests that microtubules may contribute to short distance movement and 'fine tuning' of Golgi body displacement. Moreover, proteomic studies linking the actin- cytoskeleton to microtubules have demonstrated that these two components of the cytoskeleton are closely related and a role of the microtubules in Golgi movement cannot be excluded. In this thesis, automated tracking of Golgi bodies was used to understand and quantify the contribution of actin filaments and microtubules to the organelle dynamics. The tracking technique is also used to assess how the labelling of the cytoskeleton, with a novel fluorescent nanoprobe, affects the dynamics and stability of the actin filaments and the movement of Golgi bodies; FRAP analysis (fluorescent recovery after photo-bleaching) was also used to investigate the binding properties of the fluorescent nanoprobe to the actin filaments. The nanoprobe was compared with another cytoskeletal marker, Lifeact-GFP, to evaluate their suitability for studying the organelle's motility in relation to the actin-cytoskeleton. Micromanipulation of Golgi bodies with optical tweezers was used to test if there are physical links between the organelles and the cytoskeleton. The widely accepted model is that organelles move on actin filaments and movement is powered by myosins. The hypothesis that actin filaments slide one of top of the other, and drag the organelles along, was tested using the FRAP technique. Kinesin-13a is the only microtubule motor protein localized on Golgi bodies by immunochemical studies. Its localization was investigated in vivo to evaluate if it is involved in linking Golgi bodies to microtubules.
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Dickinson, Sarah. "Regulation of the actin cytoskeleton by ephrin-B signalling." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445415/.
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Ephrin ligands and their Eph receptors play an essential role in angiogenesis during development. Both ephrins and Eph receptors are membrane-tethered proteins and their interaction at sites of cell-cell contact triggers bi-directional signalling, with signals transduced from the receptor (forward signalling) and ligand (reverse signalling). I have used two model systems to study ephrin-B2 signalling: Swiss 3T3 fibroblasts expressing exogenous ephrin-B2 and Human Umbilical Arterial Endothelial Cells (HUAECs) endogenously expressing ephrin-Bs. Stimulation of ephrin-B2 with soluble EphB receptors, has enabled the characterisation of the cellular responses, and signalling pathways, triggered by ephrin-B2 activation. I have shown that clustering of expressed ephrin-B2 in cultured fibroblasts induces a loss of cell-cell contact, dependent on the presence of serum factors and independent of actin-myosin contractility. The intracellular domain of ephrin-B2 is essential: tyrosine phosphorylation of the ligand via Src, and binding of the adaptor protein Grb4 are required for loss of cell-cell contact. Stimulation of endogenous ephrin-B2 in cultured endothelial cells results in dramatic cell retraction, and in a proportion of cells membrane blebbing. I have shown that the small GTPase Rho and activation of its downstream effector ROCK are essential for membrane retraction to occur, which is driven by an actin-myosin contraction event. In addition, I find that the c-Jun amino terminal kinase (JNK) is required for retraction, acting upstream of Rho/ROCK, and retraction occurs independently of Grb4. The cell contraction response to ephrin-B2 activation is rapid and transient with cells recovering to re-spread lamellipodia within minutes. Re-spreading is coupled to a loss of actin stress fibres and concomitant with down regulation of Rho and ROCK activity.
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Fan, Yi. "POLARIZATION OF CYTOSKELETON-REGULATORY PROTEINS DURING ENDOTHELIAL CELL MIGRATION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1247148451.
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Caution, Kyle J. "Legionella pneumophila and caspases: modulation of the actin cytoskeleton." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449147516.
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Rodrigues, Joana Nogueira. "Dissecting the role of adducin in the axonal cytoskeleton." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/14771.
Full textAbstract:
Mestrado em Biologia Molecular e Celular<br>The neuronal cytoskeleton is an interconnected network of filamentous
polymers, having in its constitution three major components: actin, microtubules
and intermediate filaments. Up to the discovery of axon actin rings, the
neuronal actin cytoskeleton has gained relevance. Still, the molecular details of
the regulation of the actin cytoskeleton in neurons are largely unknown. Helping
in the actin cytoskeleton regulation and maintenance, there is adducin. Adducin
is organized in heterotetamers of heterodimers which comprises α/β and α/γ
subunits. In the nervous system, the depletion of α subunit results in an almost
complete absence of functional adducin. Given this, α-adducin KO mice arose
as relevant models to study the role of this protein in actin cytoskeleton.
Results from our group showed that α-adducin KO mice develop progressive
axon enlargement and degeneration. As defects in axonal transport have been
related to axon enlargement, we determined the importance of adducin in the
axonal cytoskeleton and, more specifically, in axonal transport. Although no
differences were found in the retrograde transport of CTB in the optic nerve, the
lack of adducin resulted in a decreased speed of axonal transport of
mitochondria and lysosomes. Several neurodegenerative disorders have been
associated with axonal transport deficits and, consequently, with alterations in
MT-based transport. Although no differences were found in the levels of
acetylated and de-tyrosinated tubulin, the levels of tyrosinated tubulin were
significantly decreased in α-adducin KO brains, suggesting a less dynamic
status of the MT cytoskeleton in the absence of adducin. Besides differential
PTMs of tubulin the decreased axonal transport speed may result from the
decreased levels of dynein and kinesin in α-adducin KO mice. Lastly, we
hypothesized that adducin might be involved in the organization and/or
plasticity of the AIS that requires actin dynamics. In α-adducin KO animals,
although the AIS forms normally, neurons do not have the ability to relocate it in
response to chronic depolarization. Still, the specific role of actin and its
associated proteins, like adducin, in this process remains unclear. In sum, with
this Thesis we contributed to understand the relevance of the actin in
cytoskeleton, more specifically, of the actin-binding protein adducin in neuron
biology.<br>O citoesqueleto neuronal é maioritariamente constituído por três componentes:
actina, microtúbulos e filamentos intermédios. Com a descoberta dos anéis de
actina presentes no axónio, o citoesqueleto neuronal de actina tem vindo a
ganhar bastante relevância. Contudo, os mecanismos moleculares envolvidos
na regulação da actina no citoesqueleto continuam por esclarecer. Nesta tese
focámo-nos no estudo da importância da aducina, uma proteína de ligação à
actina, na regulação do citoesqueleto neuronal. A aducina é uma proteína
constituída por heterotetrameros de heterodímeros das subunidades α/β e α/γ,
sendo que no sistema nervoso, a depleção da subunidade α resulta numa
completa ausência da proteína no seu estado funcional. Assim, murganhos KO
para α-aducina demonstraram ser um modelo animal relevante para o estudo
do papel desta proteína no citoesqueleto de actina. Resultados do nosso grupo
demonstraram que murganhos KO para α-aducina desenvolvem uma
degeneração progressiva e um aumento do calibre do axónio. Uma vez que
defeitos no transporte axonal têm vindo a ser relacionados com o alargamento
axonal, tornou-se importante determinar o papel da aducina no citoesqueleto
axonal, mais especificamente no transporte ao longo do axónio. Apesar de não
terem sido encontradas diferenças no transporte da toxina da cólera no nervo
óptico, a ausência da aducina resultou num decréscimo significativo na
velocidade de transporte axonal de mitocôndrias e lisossomas. Diversos
distúrbios neurodegenerativos têm sido associados com deficiências no
transporte axonal consequentes de alterações na maquinaria de transporte
incluindo microtúbulos e proteínas relacionadas. Apesar de não terem sido
encontradas diferenças nos níveis de acetilação e de-tirosinação da tubulina
em amostras de cérebro α-aducina KO, os níveis de tirosinação da tubulina
estão significativamente diminuídos quando comparados com aqueles
encontrados em amostras WT, sugerindo uma menor dinâmica dos
microtúbulos na ausência de aducina. Além das modificações da tubulina, a
diminuição da velocidade de transporte axonal poderá resultar também do
decréscimo dos níveis de ambos os motores moleculares dineina e cinesina
nos murganhos α-aducina KO. Por fim, sugere-se que a aducina poderá
também estar envolvida na organização e/ou plasticidade do segmento inicial
do axónio dada a sua ligação ao citoesqueleto de actina, importante para a sua
função e organização. Nos murganhos KO para α-aducina, foi verificado que
apesar da formação do segmento inicial ser normal, as células não têm a
capacidade para o relocalizar após uma depolarização crónica. Porém, o papel
específico da actina e das suas proteínas associadas, tal como a aducina,
neste processo deverá ser investigado com maior detalhe. Sumariamente, com
esta tese, foi possível contribuir para uma melhor compreensão da relevância
da actina, mais especificamente, da proteína de ligação à actina aducina, na
biologia de um neurónio.
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Pereira, José Carlos Ribeiro Ferreira. "Cytoskeleton regulation in bladder cancer cells after photodynamic treatment." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/21089.
Full textAbstract:
Mestrado em Biologia Molecular e Celular<br>A terapia fotodinâmica (PDT) é uma modalidade promissora para o tratamento do cancro. Esta terapia baseia-se na interação entre um composto químico (fotossensibilizador, PS), luz com um determinado comprimento de onda e oxigénio molecular para originar a produção de espécies reativas de oxigénio (ROS). Devido à sua elevada reatividade, estas espécies tóxicas podem causar danos severos conduzindo à morte celular. Atualmente, os PS disponíveis na clínica para o tratamento de tumores apresentam baixa seletividade para as células tumorais. Estudos anteriores do nosso grupo descreveram uma porfirina conjugada com unidades dendríticas de galactose (PorGal8) como um novo PS solúvel em solução aquosa, capaz de gerar ROS após fotoativação e com reconhecimento por parte de proteínas (galectina-1) que se encontram sobreexpressas nas células do cancro da bexiga. Vários estudos têm descrito alterações no citoesqueleto em resposta ao tratamento fotodinâmico. No entanto, a contribuição da desorganização do citoesqueleto na morte celular induzida por PDT encontra-se pouco esclarecida.
Neste trabalho, avaliámos de que forma alterações nos constituintes do citoesqueleto – filamentos de actina, filamentos intermédios e microtúbulos – estão relacionadas com morte celular induzida por PDT com PorGal8.
O uptake de PorGal8 em duas linhas celulares do cancro da bexiga derivadas de carcinoma de células transicionais (UM-UC-3 e HT-1376), foi dependente da concentração. O uptake celular de PorGal8 foi superior nas células UM-UC-3, que exibem níveis superiores da proteína galectina-1, comparativamente com as células HT-1376. PorGal8 mostrou não ser tóxico no escuro. A fotoativação da PorGal8 resultou numa fototoxicidade significativamente superior nas células UM-UC-3 relativamente às células HT-1376. A PorGal8 não induziu alterações significativas nos níveis de proteína α-tubulina nas células UM-UC-3. No entanto, observou-se uma redução significativa nos níveis de α-tubulina nas células HT-1376 vinte e quatro horas após tratamento com irradiação. Apesar de se ter observado uma recuperação na organização dos microtúbulos em algumas células, a intensidade da fluorescência diminuiu consideravelmente na maior parte das células HT-1376. Uma redução significativa nos níveis de proteína dos filamentos intermédios (vimentina) foi observada em ambas as linhas celulares vinte e quatro horas após irradiação. Trinta minutos após a irradiação, as células UM-UC-3 e HT-1376 apresentaram uma clara retração nos filamentos de actina com perda de fibras de stress. Ao contrário das células UM-UC-3 em que não se verificaram sinais de recuperação, em algumas células HT-1376 verificou-se uma certa reorganização dos filamentos de actina, com curtas fibras de stress, longas extensões, grandes filopodia, o que parece sugerir uma possível recuperação das células HT-1376. A RhoA, uma proteína da família de pequenas proteínas GTPases, descrita como estando relacionada com a expressão da galectina-1, foi adicionalmente avaliada. Resultados preliminares indicaram que a PorGal8 induziu uma tendência para aumentar os níveis de RhoA nas células HT-1376 vinte e quatro horas após tratamento com irradiação.
Concluindo, os nossos resultados contribuem para o esclarecimento dos mecanismos subjacentes dos efeitos fototóxicos da PorGal8. Uma melhor compreensão dos intervenientes e das alterações induzidas imediatamente após PDT nas estruturas do citoesqueleto em cancros resistentes à terapia, poderão contribuir para o desenvolvimento de novos agentes terapêuticos adjuvantes à PDT.<br>Photodynamic therapy (PDT) is a promising modality for the treatment of cancer that involves light of an appropriate wavelength and a photosensitizing drug (photosensitizer, PS), used in conjunction with molecular oxygen, leading to the production of reactive oxygen species (ROS). In a biological environment, these toxic species can interact with the cellular constituents eliciting cell death. Currently, the PS available show poor tumor specificity. Previous work from our research group reported a porphyrin conjugated with dendritic units of galactose (PorGal8) as a new water soluble PS, able to generate ROS after photoactivation and exhibiting increased selectivity to bladder cancer cells overexpressing galectin-1. Several studies reported cytoskeleton alterations derived from photodynamic treatments. However, the role of cytoskeleton disorganization in cell death induced by PDT remains unclear.
In this work we evaluated whether changes in the cytoskeletal constituents - actin filaments, intermediate filaments and microtubules - are correlated with cell death triggered by PDT with PorGal8.
The uptake of PorGal8 in two bladder cancer lines derived from transitional cell carcinoma (UM-UC-3 and HT-1376 cells), was concentration dependent. Cellular uptake of PorGal8 was higher in UM-UC-3 cells that express higher levels of galectin-1 protein than HT-1376 cells. PorGal8 was nontoxic in dark. Photoactivation of PorGal8 resulted in a significantly higher phototoxicity in UM-UC-3 cells than HT-1376 cells. PorGal8 did not change the α-tubulin protein levels in UM-UC-3 cells but reduced α-tubulin twenty-four hours after photodynamic activation in HT-1376 cells. Although a few cells showed a recovery in microtubules organization, the fluorescence intensity decreased noticeably in most of the HT-1376 cells. A significant decrease in intermediate filaments (vimentin) protein levels was exhibited in both cell lines twenty-hours after irradiation. Thirty minutes post-irradiation, UM-UC-3 and HT-1376 cells showed a clear retraction of actin filaments with loss of stress fibers. Although no recovery was observed in UM-UC-3 cells, some cells present some reorganization in actin filaments, presenting short stress fibers, long extensions, like large filopodia, suggesting a possible recovery in HT-1376 cells. A small GTPases family protein, RhoA, referred to be involved with galectin-1 expression, was also evaluated, with preliminary results indicating a tendency towards an increase in HT-1376 cells twenty-four hours after therapy.
Overall, our results give new insights into the mechanisms underlying the phototoxic effects of PorGal8. Better understanding the intrinsic web of events and alterations on cytoskeleton structures induced immediately after photodynamic treatment in resistant cancers may contribute to envisage new potential therapeutic adjuvants for PDT.
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Martin, Stuart S. "Phosphoinositide-dependent regulation of the actin cytoskeleton by insulin /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9904827.
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Zimmerman, Matthew J. "Regulation of the cytoskeleton in human microvascular endothelial cells /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9956453.
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