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Hebert, Alan. "Functional Relationship between Merlin and the ERM Proteins." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10567.
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The ability to spatially restrict specific activities across the cell cortex functionally defines individual cells and tissues. This is achieved, in part, via the assembly of protein complexes that link the plasma membrane to the underlying cortical actin cytoskeleton. The neurofibromatosis type 2 (NF2) tumor suppressor Merlin and closely related ERM proteins (Ezrin, Radixin and Moesin) are a special class of such membrane:cytoskeleton associated proteins that function to organize specialized cortical domains. In addition to their high degree of similarity, mounting evidence suggests that Merlin/ERMs share a functional relationship, which is largely unexplored. Unlike Merlin, the ERMs are not known to inhibit cell proliferation; in fact, Ezrin is thought to promote tumor metastasis. Defining the relationship between Merlin and the ERMs is essential to appreciating their respective roles in cancer development. Here I demonstrate a novel role for Merlin and the ERMs in generating cortical asymmetry in the absence of external cues. Our data reveal that Merlin functions to restrict the cortical distribution of Ezrin, which in turn positions the interphase centrosome in single epithelial cells and 3D organotypic cultures. In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles and aberrant epithelial architecture. Furthermore, in tumor cells with centrosome amplification, the failure to restrict cortical Ezrin abolishes centrosome clustering, yielding multipolar mitoses. Consistent with a functional relationship, I observe a strong genetic interaction between Nf2 and Ezrin in the mouse intestine in vivo. Finally, I begin to address the basis of their functional interaction by testing whether they are coordinately regulated by the Ste-20 like kinase SLK. Altogether, these data uncover fundamental roles for Merlin/ERM proteins in spatiotemporally organizing the cell cortex in vitro and in vivo and suggest that Merlin’s role in promoting cortical heterogeneity may contribute to tumorigenesis by disrupting cell polarity, spindle orientation and potentially genome stability.
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Machicoane, Michaël. "The role of ERM proteins in cell division." Paris 6, 2013. http://www.theses.fr/2013PA066285.
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Le contrôle de l’axe des divisions cellulaires assure le développement correct des organismes multicellulaires. Ce processus repose sur la localisation corticale de complexes protéiques capable de tirer sur les microtubules du fuseau mitotique. Notamment, le module conservé au court de l’évolution et formé des protéines Gαi / LGN / NuMA recrute le moteur Dynéine au cortex. De plus, des signaux intra- ou extracellulaires peuvent ensuite influencer ce complexe. Par exemple, l’organisation de l’actine corticale est essentielle à la localisation et l’activité des générateurs de force. L’identification des protéines capables d’organiser l’actine corticale en mitose est donc un enjeu majeur. Mon travail de thèse a consisté en l’exploration du rôle des ERM (Ezrine-Radixine-Moésine), des protéines liant l’actine à la membrane plasmique, dans l’orientation du fuseau mitotique. Nos travaux ont montré que, dans les cellules de mammifère, les ERM sont activés à l’entrée de mitose via une phosphorylation directe par la kinase SLK. Le rôle de l’activation des ERM dans l’orientation du fuseau mitotique a été démontré dans deux systèmes : des substrats adhésifs microfabriqués contrôlant l’adhésion des cellules en culture, et les progéniteurs apicaux du neuroépithélium murin. A l’échelle moléculaire, l’activation des ERM est nécessaire au recrutement cortical polarisé d’un membre des générateurs de force, NuMA. Ces travaux suggèrent donc que l’actine corticale et protéines organisatrices ERM jouent un rôle important au cortex mitotique, permettant le recrutement des générateurs de force à la membrane et en conséquence l’orientation correcte du fuseau mitotiqueThe control of cell division axis is crucial for embryogenesis, cell differentiation and adult tissue homeostasis, and relies on the cortical localization of protein complexes able to pull on the mitotic apparatus. One of these force generators is the evolutionary conserved Gαi / LGN / NuMA module, which recruits Dynein motors at the cortex. On top of this, intrinsic and extrinsic cues can then modulate the activity and localization of this complex. Particularly, the F-actin cortex has recently been involved in the dialogue between astral microtubules and force generators. Identifying the proteins involved in F-actin organization at the cortex is thus a major challenge. During my thesis work, I focused my attention on the proteins of the ERM (Ezrin-Radixin-Moesin) family. I investigated the role of these membrane-actin linkers in the orientation of the mitotic spindle during oriented cell division. Our work demonstrated that ERM are strongly and directly activated by the SLK kinase at mitotic entry in mammalian cells. Using micro-fabricated adhesive substrates to control the axis of cell division, we found that the activation of ERM plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of NuMA. We propose that F-actin and activated ERM at the mitotic cortex are critical for the correct localization of force generator complexes and hence for proper spindle orientation
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Matsui, Takeshi. "Activation of ERM (ezrin/radixin/moesin) proteins by the small GTP binding protein Rho." Kyoto University, 2000. http://hdl.handle.net/2433/180838.
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Bissen, Philippe [Verfasser], and Volker [Akademischer Betreuer] Gerke. "ERM proteins - regulators of mitosis? / Philippe Bissen ; Betreuer: Volker Gerke." Münster : Universitäts- und Landesbibliothek Münster, 2019. http://d-nb.info/1181188873/34.
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Zarrouk, Marouan. "The role of ezrin/radixin/moesin(ERM) proteins in L-selectin-dependent signalling." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510756.
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Olsson, Per-Anders. "MIR, a novel ERM-like protein in the nervous system." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5061-X/.
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Legg, James William. "Regulation of the hyaluronan receptor CD44 by the ezrin/radixin/moesin (ERM) family of proteins." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392688.
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Hayashi, Ken. "Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues : application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative." Kyoto University, 1999. http://hdl.handle.net/2433/181750.
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BRUNO, MARINA. "ROLE OF BIOACTIVE SPHINGOLIPIDS IN INNER EAR AND SKELETAL MUSCLE BIOLOGY." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1072579.
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Sphingolipids are a class of complex lipids known to be not only structural components of biological membranes, but also bioactive molecules involved in several processes, such as cell differentiation, proliferation, motility and cell survival. Between them, we focused on sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P). S1P is intracellularly produced by sphingosine kinase (SK) 1 and SK2 and exerts many of its action consequently to its ligation to S1P specific receptors (S1PR), S1P1–5, whereas C1P is generated by the action of ceramide kinase and it is able to via activation of different signalling pathways. Recent experimental findings demonstrate an emerging role for S1P signalling axis in the maintenance of auditory function. WHO reported that age-related sensorineural hearing loss (SNHL) affects more than 360 million people worldwide, and the current unique available treatment is cochlear implant, which has important use limitations. Our main aim was to investigate S1P signalling axis role in this biological context: in the first paper, we demonstrated that the fibroblast growth factor 2 (FGF2)-induced proliferative action in US/VOT-N33 auditory neuroblasts was dependent on SK1, SK2 as well as S1P1 and S1P2. Moreover, the pro-survival effect of FGF2 from apoptotic cell death induced by staurosporine treatment was dependent on SK but not on S1PR. In addition, ERK1/2 and Akt signaling pathways were found to mediate the mitogenic and survival action of FGF2, respectively. In the second paper, we focused on the emerging role of bioactive sphingolipids as regulators of ERM (ezrin-radixin-moesin) proteins. ERM are a family of cross-linker adaptors between plasma membrane and actin cytoskeleton, playing a crucial role in cell morphology and signal transduction. S1P was found to activate ERM in a S1P2-dependent manner in US/VOT-E36 auditory epithelial progenitors and S1P-induced ERM activation potently contributed to actin cytoskeletal remodeling and to the appearance of electrophysiological changes typical of more differentiated cells. Moreover, PKC and Akt activation was found to mediate S1P-induced ERM phosphorylation. Taken together, our findings demonstrate a crucial role for S1P signalling axis in inner ear biology and disclose potential innovative therapeutic approaches for hearing loss prevention and treatment. In order to develop new methodologies to solve the difficulties of getting drugs inside the inner ear, we studied solid lipid nanoparticles (SLN) as attractive biocompatible nanocarriers for the delivery of drugs with low solubility in aqueous media. In our third paper, we showed that SLN based on stearic acid are efficiently incorporated by HEI-OC1 cells and are not ototoxic at the doses studied. The SLN loaded with glucocorticoids were more effective in protecting cells by the cisplatin-induced damage than glucocorticoids alone. Preliminary in vivo studies also indicate that intratympanic SLN are able to reach the inner ear. These results indicate that SLN are highly efficient vehicles for inner ear drug-delivery and specifically for the administration of glucocorticoids. During my Ph.D. course I have also contributed to a parallel research focused on the role of sphingolipid signalling in skeletal muscle biology. Skeletal muscle is able to regenerate thanks to the presence of satellite cells that upon trauma enter into the cell cycle and start proliferating. Starting from the previously demonstrated positive role of C1P in myoblast proliferation, in the fourth paper we showed that C1P stimulates C2C12 myoblast proliferation via LPA signalling axis. Moreover, C1P via phospholipase A2 activation leads to LPA1 and LPA3 engagement, which in turn drive Akt and ERK1/2 activation, thus stimulating DNA synthesis. The present findings highlight a new key role for bioactive sphingolipids in skeletal muscle and provide further support to the possibility of using them as therapeutic targets for its regeneration.
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Tan, Yu Pei. "The development of Lactococcus lactis as an antimicrobial agent." Thesis, Queensland University of Technology, 2010. https://eprints.qut.edu.au/39143/1/Yu_Pei_Tan_Thesis.pdf.
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Non-pathogenic lactic acid bacteria are economically important Gram-positive bacteria used extensively in the food industry. Due to their “generally regarded as safe” status, certain species from the genera Lactobacillus and Lactococcus are also considered desirable as candidates for the production and secretion of recombinant proteins, particular those with therapeutic applications.
The hypothesis examined by this thesis is that Lactococcus lactis can be modified to be an effective antimicrobial agent. Therefore, the aims of this thesis were to investigate the optimisation of the expression, secretion and/or activities of potential heterologous antimicrobial proteins by the model lactic acid bacterium, Lactococcus lactis subsp. cremoris MG1363.
L. lactis strains were engineered to express and secrete the recombinant CyuC surface protein from Lactobacillus reuteri BR11, and a fusion protein consisting of CyuC and lysostaphin using the Sep promoter and secretion signal. CyuC has been characterised as a cystine-binding protein, but has also been demonstrated to have fibronectin binding activity. Lysostaphin is a bacteriolytic enzyme with specific activity against the Gram-positive pathogen, Staphylococcus aureus. These modified L. lactis strains were then investigated to see if they had the ability to inhibit the adhesion of S. aureus to host extracellular matrix (ECM) proteins. It was observed that the cell extracts of the L. lactis strain with the vector only (pGhost9:ISS1) was able to inhibit the adhesion of S. aureus to fibronectin, whilst the cell extracts of the L. lactis strain expressing lysostaphin was able to inhibit adhesion to keratin.
Finally, this thesis has identified specific lactococcal genes that affect the secretion of lysostaphin through the use of random transposon mutagenesis. Ten mutants with higher lysostaphin activity contained insertions in four different genes encoding: (i) an uncharacterised putative transmembrane protein (llmg_0609), (ii) an enzyme catalysing the first step in peptidoglycan biosynthesis (murA2), (iii) a homolog of the oxidative defence regulator (trmA), and (iv) an uncharacterised putative enzyme involved in ubiquinone biosynthesis (llmg_2148). The higher lysostaphin activity observed in these mutants was found to be due to higher amounts of lysostaphin being secreted.
The findings of this thesis contribute to the development of this organism as an antimicrobial agent and also to our understanding of L. lactis genetics.
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Gründel, Anne. "Funktion glykolytischer Enzyme von Mycoplasma pneumoniae in der Wirt-Erreger-Interaktion." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-213500.
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Mycoplasma pneumoniae ist ein parasitär lebendes Bakterium, das eine atypische Pneumonie beim Menschen verursacht. Aufgrund seiner geringen Genomgröße besitzt dieser Organismus einen eingeschränkten Metabolismus sowie eine limitierte Zahl an Pathogenitätsfaktoren. Dennoch ist dieser Mikroorganismus perfekt an seinen Wirt angepasst und es war zu vermuten, dass neben dem komplexen Adhäsionsapparat von M. pneumoniae auch glykolytische Enzyme eine Rolle bei der Interaktion mit humanen Zellen spielen. Diese Enzyme sind maßgeblich bei intrazellulär ablaufenden Stoffwechselprozessen beteiligt. Es wurde jedoch bereits bei anderen Bakterien gezeigt, dass glykolytische Enzyme ebenfalls auf der Bakterienoberfläche zu finden sind und dort mit Komponenten der extrazellulären Matrix des Wirtes interagieren können. Dieser Vorgang trägt offensichtlich zur erfolgreichen Kolonisation des Wirtes bei. Ziel dieser Arbeit war es, alle glykolytischen Enzyme von M. pneumoniae hinsichtlich ihrer Lokalisierung zu beschreiben und Teilaspekte ihrer Funktion in der Interaktion mit Wirtskomponenten zu analysieren.
Die glykolytischen Enzyme wurden rekombinant produziert und für die Herstellung von monospezifischen polyklonalen Antikörpern verwendet. Die Lokalisation der Enzyme wurde durch Nachweis in der Membran- und Zytosolfraktion des M. pneumoniae Gesamtantigens untersucht. Mittels Immunfluoreszenz, Colony Blot und Protease-Verdau intakter Bakterienzellen wurde bestätigt, dass acht (Glycerinaldehyd-3-phosphat-Dehydrogenase, Lactatdehydrogenase, Transketolase, Pyruvatdehydrogenase, Phosphoglyceratmutase und Pyruvatdehydrogenase Untereinheiten A-C) der 19 glykolytischen Enzyme mit der Bakterienoberfläche assoziiert vorkommen.
Die Untersuchung von Mutanten ergab, dass die Lokalisation der Enzyme nicht an das Vorkommen der für die Anheftung der Bakterien an Zielstrukturen wesentlichen Adhäsine wie die Proteine P1, P40 und P90 sowie das Oberflächenprotein P01, gekoppelt ist. Jedoch sind sowohl intakte Zellen von M. pneumoniae als auch die oberflächenlokalisierten glykolytischen Enzyme in der Lage, an verschiedene humane Zellen zu binden. Eine Analyse der nachweisbaren Proteine auf der Oberfläche der Zellen führte zur Auswahl von sechs humanen Proteinen für weiterführende Studien: Plasminogen, Vitronektin, Fibronektin, Fibrinogen, Laminin und Laktoferrin. Mittels ELISA wurde eine konzentrationsabhängige Bindung der oberflächenassoziierten Enzyme von M. pneumoniae mit Wirtsproteinen festgestellt, die hinsichtlich der Intensität jedoch Unterschiede aufwies. So konnten ausgeprägte Interaktionen aller Enzyme mit humanem Plasminogen und Vitronektin nachgewiesen werden. Die Bindung von Fibronektin und Laktoferrin ist dagegen nur für einen Teil der glykolytischen Enzyme zu bestätigen. Die Untersuchung verschiedener Einflussfaktoren ergab, dass alle Bindungen zwischen glykolytischen Enzymen und humanen Proteinen spezifisch durch die entsprechenden Antiseren gehemmt werden und dass der Großteil der Interaktionen ionischen Wechselwirkungen unterliegt. Die Bindung zu Plasminogen basiert überwiegend auf Lysin-Resten. Untersuchungen, ob sich die glykolytischen Enzyme gegenseitig in der Bindung zu Wirtsfaktoren beeinflussen, ergab ein komplexes Muster, das hinsichtlich Plasminogen, Fibronektin und Laminin für eine Überlagerung der für die Interaktion maßgeblichen Proteinbereiche spricht.
Die Untersuchung einer möglichen Aktivierung von inaktivem Plasminogen zu proteolytisch aktivem Plasmin ergab, dass in Gegenwart aller oberflächenlokalisierten glykolytischen Enzyme von M. pneumoniae Plasmin gebildet wird. Es wurden jedoch Unterschiede im Aktivierungspotenzial nachgewiesen. Die Pyruvatdehydrogenase Untereinheit B zeigte die höchste, die Pyruvatdehydrogenase Untereinheit C die geringste Plasminproduktion. Die Verwendung des gewebespezifischen Plasminogenaktivators führte zu einer höheren Aktivierung als der Urokinase-Typ Plasminogenaktivator. Die Variabilität der Plasminproduktion kann mit der unterschiedlichen Bindungsaffinität der glykolytischen Enzyme zu Plasminogen begründet werden. So besitzt die Pyruvatdehydrogenase Untereinheit B im Vergleich mit der Pyruvatdehydrogenase Untereinheit C ein höheres Bindepotenzial, das sich in der gemessenen Aktivierung widerspiegelt. Die Bildung von Plasmin kann zum Abbau verschiedener extrazellulärer Matrix-Proteine führen. Diese Prozesse sind physiologisch, z. B. in der Fibrinolyse, von Bedeutung. Während in Gegenwart der glykolytischen Enzyme die humanen Proteine Laktoferrin, Laminin und Fibronektin nicht abgebaut wurde, konnte Fibrinogen in Gegenwart der Pyruvatdehydrogenase Untereinheit B bzw. der Phosphoglyceratmutase und Vitronektin durch alle glykolytischen Enzyme (bis auf die Pyruvatdehydrogenase Untereinheit C) degradiert werden.
Mit der erstmals durchgeführten Analyse aller glykolytischen Enzyme eines Mikroorganismus hinsichtlich ihrer Lokalisation und der Bindung zu Komponenten der humanen extrazellulären Matrix wurde ein komplexes Netzwerk an Wirt-Erreger-Interaktionen nachgewiesen.
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Lo, Kin Ho. "Activation of signal transducer and activator of transcription 3 (STAT3) by G[alpha]16 and G[alpha]14 via a c-Src/JAK-and ERK-dependent mechanism /." View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20LO.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 92-111). Also available in electronic version. Access restricted to campus users.
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Owen, Jo. "Structural and functional studies of fibulin-1 EGF domains." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270656.
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Panaganti, Shilpa. "Parallel SVM with Application to Protein Structure Prediction." Digital Archive @ GSU, 2004. http://digitalarchive.gsu.edu/cs_theses/3.
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A learning task with thousands of training examples in Support Vector Machine (SVM) demands large amounts of memory and time requirements. SVMlight by Dr. Thorsten Joachims has been implemented in C using a fast optimizing algorithm for handling thousands of such support vectors. SVMlight solves the problem of classification, pattern recognition, regression and learning ranking function. The C code also provides methods for XiAlpha estimation of error rate and precision. Implementing these two methods leads to generalized performance of Support Vector Machine even for computation intensive text classification functions. SVMlight code allows users to define their own kernel functions. The SVMlight software employs an efficient algorithm and minimizes the cost, but it still takes considerable amount of time for computing thousands of support vectors and training examples. This time can be still reduced by parallelizing the code. In our work we refined the SVMlight code by removing unnecessary iterations and rewriting it as cost efficient. Then we parallelized the code individually using two different types, OpenMP and POSIX Threads shared memory parallelism. The code is parallelized for these two methods on Intel’s C compiler for Linux 7.1 using hyper threading technology. The parallelized code is tested for protein structure prediction. Different types of Protein Sequences are tested on these methods by varying the number of training examples and support vectors. The time consumption and speedup are calculated for both OpenMP and Pthreads. Implementation of OpenMP and Pthreads together showed good increase in speedup.
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El, Sabeh Rana. "Pleiotropism of MyD88, as Determined by its Multiple Protein-Protein Interactions." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10168.
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MyD88 est une protéine adaptatrice clé dans la signalisation des TLRs/IL-1R qui mène à l'activation de NF-KB et des MAPK, et à la production de cytokines inflammatoires. MyD88 participe à la tumorigénèse par le biais de son activité inflammatoire dans la signalisation des TLRs/IL-1R, et également via son interaction directe avec la kinase Erk dans la cellule cancéreuse. Dans cette thèse, nous identifions de nouveaux partenaires protéiques de MyD88 et nous examinons comment leurs interactions peuvent réguler sa fonction. Nous démontrons que MyD88 interagit avec Ubc9, ce qui conduit à sa sumoylation, et que cette modification posttraductionnelle régule négativement l'inflammation dépendante de MyD88. Nos résultats montrent également que MyD88 interagit avec le récepteur nucléaire, ER-α, et que cette interaction est nécessaire pour la réponse inflammatoire. Enfin, nous avons étudié l'importance de l'interaction MyD88/Erk dans le maintien de la transformation des tumeurs dépendant de l'oncogène Ras. Ces résultats pourraient éventuellement être exploités pour cibler MyD88 et ses interactions dans le traitement des maladies inflammatoires et le cancerMyD88 is a protein that is at the interface between inflammation and cancer. It is the key adaptor protein used by TLRs/IL-1R to mediate their downstream signaling, resulting in NF-κB and MAPK activation, and inflammatory cytokine production. MyD88 also plays a role in tumorigenesis via two mechanisms, an inflammatory one dependent on its function in TLRs/IL- 1R signaling, and an intrinsic, cell-autonomous mechanism mediated by its interaction with the kinase Erk. Based on the different roles played by MyD88, this thesis work consisted in studying how MyD88 protein-protein interactions can regulate its function. We show that MyD88 interacts with Ubc9, resulting in its sumoylation and subsequent negative regulation of MyD88- mediated inflammation. We also demonstrate that MyD88 interacts with the nuclear receptor ER-α, an interaction necessary for the inflammatory response. Finally, we have studied the importance of the MyD88/Erk interaction in the maintenance of the transformed phenotype of Ras-dependent tumors. These findings could eventually be exploited to target MyD88 and its interactions in the treatment of inflammatory disorders and cancer
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Pust, Sascha. "Einfluss der ERM-Proteine auf die Protrusion-Ausbildung und Zell-Zell-Ausbreitung von Listeria monocytogenes." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=967112273.
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Doarn, Michael C. "Altered differentiation in acute myeloid leukemias; role of ERG and FUS-ERG fusion protein." Connect to this title online, 2005. http://hdl.handle.net/1811/373.
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Thesis (Honors)--Ohio State University, 2005.Title from first page of PDF file. Document formattted into pages: contains, 45 p.; also includes graphics. Includes bibliographical references (p. 42-45). Available online via Ohio State University's Knowledge Bank.
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Samuels, Ivy S. "The roles of ERK₁ and ERK₂ MAP kinase in neural development and disease." Cleveland, Ohio : Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1214495630.
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Gandhi, Neha Sureshchandra. "Molecular modelling of the interactions of complex carbohydrates with proteins." Thesis, Curtin University, 2011. http://hdl.handle.net/20.500.11937/2027.
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Glycosaminoglycans (GAGs) are ubiquitous complex carbohydrate molecules present on the cell surfaces and in extracellular matrices (ECM) of vertebrate and invertebrate tissues. The interactions of sulphated GAGs such as heparin and heparan sulphate (HS) with numerous immunologically-relevant proteins is now attracting considerable interest as a source of new therapeutics for the treatment of infectious diseases, inflammation and allergies, and cancers. Various computational molecular modelling methods are being employed to determine the nature of the interactions of heparin oligosaccharides with various proteins in order to establish the structural requirements that determine their binding specificity and selectivity.The first part of this research focused on understanding the inflammatory cytokine CXCL-8 (Interleukin-8 or IL-8) and its interactions with GAGs. A variety of molecular modelling methods were used to investigate the binding of complex carbohydrates to this protein. A number of consensus heparin/HS binding motifs were predicted to be required for the binding of monomeric or oligomeric structures of CXCL-8. Bioinformatics analyses showed that the basic residues in the heparin binding site are highly variable within the CXC family and amongst various CXCL-8 species. Drug-like carbohydrate mimetic molecules (cyclitols) that bind optimally to CXCL-8 were identified and characterised. It was established that both an optimum number of sulphates and a certain length of alkyl spacers are required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Furthermore, explicit solvent molecular dynamics simulations of dimeric CXCL-8 showed how its two anti-parallel helices exhibit domain movements that can bring them in closer proximity. In addition, these simulations revealed shearing movements in the C-terminal helices in the CXCL-8 dimer. This inherent flexibility of the CXCL-8 dimer can be exploited in drug design as it plays an important role in the understanding of the interactions of molecules such as sulphated cyclitols with the two monomers.Structural bioinformatics and molecular modelling methods were used to generate and analyse a three-dimensional model of heparanase, an enzyme involved in metastasis and angiogenesis in cancer, in order to gain insight into its protein sequence, and its structural and functional relationships. The interactions of heparanase with disaccharide substrates and GAG mimetics were modelled to investigate the structural determinants of their protein binding specificity and selectivity. The choice of structural template for modelling the binding site of heparanase is very critical. Analyses of active-site similarity across groups of homologous template structures revealed that these bound oligosaccharides can block ligand binding to the catalytic and heparin binding sites of heparanase. Ligand-protein docking simulations further revealed the existence of a large binding site extending at least two saccharide units beyond the cleavage site (towards the non-reducing end) and at least three saccharides towards the reducing end (towards heparin-binding site 2). Extensive modelling of substrate and inhibitor interactions with the catalytically-active glutamic acids and the two binding sites for heparan sulphate of heparanase provides information useful for future drug discovery efforts focused on the identification of novel inhibitors of this enzyme.Free energy calculations of the binding of sGAGs to GAG-binding proteins were pioneered with the proteins PECAM-1 (platelet endothelial cell adhesion molecule) and Annexin, using the molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) method. Analysis of the free energy of binding components revealed that the major contributors to complex stability are electrostatic interactions, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large, unfavourable desolvation penalty due to the high charge density of sGAG. The calculated absolute free energies of binding of the molecules investigated were found to be inaccurate compared to experimental values, but the method performed well in discriminating weak and strong binding.A final focus of this research was to investigate the conformational properties of sulphated iduronic acid (IdoA2S), a hexopyranose present in heparin. IdoA2S adopts more than one conformation (skew boat and chair) when internally positioned within an oligosaccharide or when it interacts with proteins. The influence of the solvent on the flexibility and conformations of this saccharide is of significant interest given that its biomolecular interactions occur in an aqueous environment. Therefore, molecular dynamics simulations were used to investigate the ability of the GROMOS force field and the GLYCAM carbohydrate parameter set in the presence of explicit solvent to successfully predict rotamer populations for this ring system. Calculations of theoretical proton NMR coupling constants showed that the GROMOS96 force field can predict the skew-boat to chair conformational ratio in good agreement with experiment; however, the accuracy of the GLYCAM force field in representing ring conformer populations during unconstrained molecular dynamics simulations is still debatable.
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Lubart, Quentin. "Les protéines ERM , Interactions entre la membrane cellulaire et le cytosquelette : une approche biomimétique." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAI108/document.
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Les protéines ERMs (Ezrine, radixine et moésine) jouent un rôle central in cellulo, dans de nombreux processus cellulaires tels que les infections, la migration et la division cellulaire. Parmi celles-ci, la moésine est plus particulièrement impliquée dans la formation de la synapse immunologique, l’infection virale et bactérienne, et les métastases cancéreuses. D’un point de vue structural, les ERM peuvent être en conformation inactive (replies sur elles-mêmes) ou actives (ouvertes), ce qui permet leur interaction a la fois avec les constituants du cytosquelette (actine et tubuline) via leur domaine C-terminal et la membrane plasmique via leur domaine FERM. La liaison a la membrane plasmique se fait principalement et spécifiquement via un lipide de la famille des phosphoinositides, le phosphatidyl 4,5 bisphosphate (PIP2). De plus, les protéines peuvent être phosphorylées, ce qui contribue à leur ouverture structurale. Cependant, le rôle de la phosphorylation sur les interactions ERM/membrane et ERM/cytosquelette, bien que beaucoup étudié in cellulo, est peu compris au niveau moléculaire.Le but de cette thèse est précisément d’étudier, au niveau moléculaire et à l’aide de systèmes biomimétiques, les interactions entre des protéines recombinantes et des membranes biomimétiques contenant du PIP2. Pour cela, nous avons mis au point des membranes lipidiques sous forme de vésicules unilamellaires (petites ou larges) et de bicouches lipidiques supportées, qui permettent de caractériser les interactions entre protéines et membranes par des techniques biophysiques complémentaires, notamment la cosédimentation quantitative, la microscopie et spectroscopie de fluorescence, et la microbalance à cristal de quartz. Dans une première partie, nous avons étudié le rôle de la double phosphorylation de la moésine (réalisée par mutation sur site spécifique) sur les interactions moésine/membrane biomimétique, en comparaison de la protéine sauvage, les protéines recombinantes et les mutants ayant été produites et purifiées au laboratoire.Nos résultats mettent en évidence une interaction spécifique et coopérative pour le double mutant phosphomimétique alors que cette interaction est simple dans le cas de la protéine sauvage. Dans une seconde partie, nous avons employé les bicouches lipidiques supportées contenant le PIP2 pour étudier les mécanismes molécules d’adsorption de la protéine virale Gag et de ses mutants. Les méthodologies développées dans ce travail de thèse ouvrent des perspectives en biophysique moléculaires car elles sont facilement transposables à l’étude d’autres protéines sur des membranes lipidiques modèles contenant des phosphoinositides.Mots clés: Ezrine-Radixine-Moésine, phosphoinositides, PIP2, interactions protéine-lipide, membrane lipidique biomimétique, protéine virale Gag, cytosqueletteERM (ezrin, radixin, moesin) proteins play a central role in cellulo in a large number of physiological and pathological processes, including cell infection, migration and cell division. Among the ERMs, moesin is particularly involved in the formation of the immunological synapse, viral and bacterial infection, and cancer metastasis. From a structural point of view, ERMs can be in inactive (closed) conformation or active (open), which enable them to interact on one side with the cytoskeleton (actin and tubulin) via their C-terminal domain and on the other side with the plasma membrane via their FERM domain. Binding to the plasma membrane is mediated via a specific lipid of the phosphoinositide family, the phosphatidylinositol(4,5)bisphosphate (PIP2). In addition, ERM can be phosphorylated, which contribute to their structural opening. To date, the role of the phosphorylation in ERM/membrane and ERM/cytoskeleton interactions, although widely studied in cellulo, remains poorly understood at the molecular level.The aim of this PhD thesis is precisely to study, at the molecular level and using biomimetic systems, interactions between recombinant proteins and biomimetic membranes containing PIP2. To this end, we have engineered lipid membranes in the form of large and small unilamellar vesicles and supported lipid bilayers. These biomimetic membranes are used to characterize interactions between proteins and membranes by complementary biophysical techniques, notably quantitative cosedimentation, fluorescence microscopy and spectroscopy, and quartz crystal microbalance with dissipation monitoring. In a first part, we studied the role of double phosphorylation on moesin, achieved via a site-specific mutation on threonine residues, on moesin/biomimetic membrane interactions, in comparison to the wild type protein. The recombinant proteins and mutants were produced in our laboratory.Our results show that there is a specific and cooperative interaction for the double phosphomimetic mutant while interactions is 1:1 in the case of the wild type protein. In a second part, we used supported lipid bilayers containing PIP2 to study the molecular adsorption mechanism of the viral protein Gag and of its mutants. The methodologies that were developed in this work open perspectives in molecular biophysics since they are easily adaptable to other proteins on model lipid membranes containing phosphoinositidesKeywords: Ezrin-Radixin-Moesin, phosphoinositides, PIP2, protein/lipid interactions, biomimetic lipid membrane, Gag viral protein, cytoskeleton
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McCosker, Helen Clare. "Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients." Thesis, Queensland University of Technology, 2012. https://eprints.qut.edu.au/53580/1/Helen_McCosker_Thesis.pdf.
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Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM).
Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome.
To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins.
This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2).
Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models.
The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.
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Robinson, J. D. "G protein-coupled receptor kinase 2 is a Rho-dependent scaffold protein for the ERK MAPK cascade." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1336072/.
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The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs can also regulate signalling downstream of other families of receptors and are now known to have a number of non-receptor substrates and binding partners. Here I identify RhoAGTP, Raf1 and ERK2 as novel binding partners of GRK2 and report a previously unsuspected function for this kinase. GRK2 acts as a RhoA-activated scaffold protein for the ERK MAP kinase cascade downstream of the epidermal growth factor (EGF) receptor. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoAGTP binding to the catalytic domain of the kinase, however, while RhoAGTP binding is common to all of the ubiquitously expressed GRKs, the ability to act as a RhoA-regulated Raf/MEK/ERK scaffold is specific to GRK2. GRK2 over-expression in HEK-293 cells potentiates EGF-induced ERK activation in a Rho-dependent fashion. Conversely, depleting GRK2 expression by RNAi reveals that GRK2 is required for EGF-induced thymidine incorporation in vascular smooth muscle cells (VSMCs). Rho-dependent ERK MAP kinase scaffolding by GRK2 may therefore have an important role in the vasculature, where increased levels of GRK2 and RhoA have been associated with hypertension.
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Paunola, Eija. "Protein folding before and efter translocation into the yeast endoplasmic reticulum." Helsinki : University of Helsinki, 2000. http://ethesis.helsinki.fi/julkaisut/eri/biote/vk/paunola/.
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Dvořák, Pavel. "Biomedicínské aplikace polykaprolaktonových nanovlákenných membrán." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2021. http://www.nusl.cz/ntk/nusl-444549.
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The diploma thesis deals with the treatment of polycaprolactone (PCL) nanofibers. PCL fibers were subjected to the deposition of plasma amine polymers in a low pressure pulsed radiofrequency capacitively coupled discharge using cyclopropylamine monomer (CPA). Collagen as an extracellular matrix (ECM) protein was immobilized and cell proliferation on the modified nanofiber surface was monitored. Untreated PCL fibers were also subjected to the deposition of an antibacterial copper layer, and the fibers were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and energy dispersive spectroscopy (EDX).
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Mazzetti, Scott A. "Akt and ERK activation in human skeletal muscle : dose-dependency of responses to increasing muscle contractions." Virtual Press, 2003. http://liblink.bsu.edu/uhtbin/catkey/1259313.
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Akt activation mediates increases in glycogen synthesis in response to insulin in humans, while extracellular signal-regulated kinase (ERK) activation increases gene transcription and protein translation in response to endurance and resistance exercise. Akt activation increases only in response to intense muscle contractions and during hypertrophy in rats. No study has examined Akt and ERK activation with increasing numbers of intense muscle contractions in humans. Therefore, the primary objectives of this investigation were to determine if Akt activation increases in response to resistance exercise in humans, and to compare the changes in Akt and ERK activation in response to increasing numbers of muscle contractions.Akt and ERK activation were compared in muscle biopsy samples from 7 men before (Pre) and after (Post) knee extension and control protocols using enzyme linkedimmunosorbent assays. Baseline information was obtained including body composition and maximal strength (1-RM). Subjects were familiarized with knee extensions performed at 70% of 1-RM and a specified repetition cadence (2sec up, 2sec down). Once/wk, subjects performed one protocol in random order: 1 repetition (rep), 10reps, 3 sets of l0reps (3x10), or 6min of sitting. Akt activation decreased 42%, while ERK activation increased 108% in response to 3x10 (p<0.05). Akt and ERK activation did not change with 1 and 10reps, and thus their responses were not dose-dependent with resistance exercise in humans. The findings from this study represent the first indication that Akt activation is reduced in response to resistance exercise in human skeletal muscle, possibly to help mediate reductions in glycogen synthesis.Human Performance Laboratory
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Van, Kanegan Michael J. "Regulation of nerve growth factor signaling by protein phosphatase 2A." Diss., University of Iowa, 2008. https://ir.uiowa.edu/etd/279.
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The goal of this dissertation research is to determine novel regulatory mechanisms of neurotrophin signaling mediated by protein phosphatase 2A (PP2A). PP2A is a ubiquitous Ser/Thr phosphatase that removes phosphates from proteins to switch their activity on or off. The substrate specificity and subcellular localization of PP2A is determined by almost 20 regulatory subunits that associate with a core dimer built of catalytic and scaffold subunits. Since there are more than 48 possible heterotrimers, studying the function of PP2A poses many challenges. Therefore we have devised a strategy, using scaffold subunit knockdown and mutant replacement, to discern the function of specific families of regulatory subunits. With this approach, I have identified specific PP2A holoenzymes that modulate nerve growth factor (NGF) signaling pathways by positively regulating TrkA receptor tyrosine kinase activity. Many studies have shown that NGF is required for the survival and differentiation of sensory and sympathetic neurons. Additionally, NGF is implicated in many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease as well as neuropathic pain. NGF elicits its biological effect through sustained activity of the TrkA receptor and stimulated signaling cascades, including the MAP kinase pathway. Although PP2A has been shown to modulate the mitogen-activated protein (MAP) kinase pathway both positively and negatively at multiple levels, work described herein introduces yet another level of regulation. Specifically, I have shown that PP2A/B' holoenzymes complex with the TrkA neurotrophin receptor to potentiate receptor tyrosine kinase activity, downstream effector kinase activation, neurite outgrowth, and neuronal differentiation. On the other hand, extracellular signal regulated kinase (ERK), a terminal effector in the MAP kinase pathway was shown to phosphorylate a residue in the juxtamembrane region of TrkA and impose feedback inhibition of receptor activity. Collectively, these data suggest a model in which PP2A and ERK oppose each other in the regulation of TrkA receptor activity and downstream signaling cascades that govern neuronal differentiation and maintenance.
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Lam, King-yin Andy. "Differential regulation of FOXM1 isoforms by RaF/MEK/ERK signaling." Click to view the E-thesis via HKUTO, 2010. http://sunzi.lib.hku.hk/hkuto/record/B44251014.
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Lam, King-yin Andy, and 林敬賢. "Differential regulation of FOXM1 isoforms by RaF/MEK/ERK signaling." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44251014.
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SUWASTIKA, I. NENGAH. "Plant Specific Subcellular Targeting mechanism: studies on SYP 7s, Qc-SNARE Family Proteins, and on Plant Obg-Era Homologue of small GTPase Proteins." 京都大学 (Kyoto University), 2009. http://hdl.handle.net/2433/123813.
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Giollo, Manuel. "Computational Approaches to Address the Next-Generation Sequencing Era." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424280.
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In this thesis, I propose new algorithms and models to address biological problems. Computer science in fact plays a key role in proteomics and genetics research due to the advent of big datasets. In the context of protein study, I developed new methods for protein function prediction based on information retrieval principles. By using heterogeneous source of knowledge, like graph search and sequence similarity, I designed a tool called INGA that can be used to annotate entire genomes. It has been benchmarked during the Critical Assessment of Function Annotation challenge, and it proved to be one of the most effective approach for function inference.
To better characterize proteins from the structural point of view, I proposed a protein conformers detection strategy based on residue interaction network (RIN) data. RIN graphs were extended to deal with the time-dependent protein coordinate fluctuations, and were generated by clustering algorithms. An implementation called RING MD highlighted effectively the key amino acids known to be functionally relevant in Ubiquitin. These amino acids in fact are very important to explain the protein three-dimensional dynamics. With the same rationale, RIN graphs were used also to predict the impact of mutations within a protein structure. By combining information about a mutant node in the network and its features, an artificial neural network was trained to estimate the free Gibbs energy change of a protein. Extreme changes in the internal energy might lead to the protein unfolding, and possibly to disease. The reduction of a protein flexibility may hamper its function as well. As an example, the extreme fluctuations observed in intrinsically disordered proteins (IDPs) are fundamental for their activities. To better understand IDPs, I contributed in the collection of the largest dataset of disordered regions. In the following analysis, it was shown what are the typical functions of these sequences and the biological processes where they are involved. Due to the importance of their detection, a comprehensive assessment of disorder predictors was performed to show what are the state-of-the-art methods and their limitations.
In the context of genetics, I focused on phenotype prediction. During the Critical Assessment of Genome Interpretation (CAGI), I proposed new approaches for the analysis of exome data to prioritize the risk of Crohn's disease and abnormal cholesterol levels. These are often defined as complex disease, since the mechanism behind their insurgence is still unknown. In my study, human samples with an enrichment of mutations in critical genes were predicted to have an high genetic risk. In addition to disease associated genes, protein interaction networks were considered to better account for variants accumulation in biological pathways. Such strategy was shown to be among the best approaches by CAGI organizers. In the simpler case of Mendelian traits, with BOOGIE I designed a method for human blood groups prediction based on exome data. It uses a specialized version of nearest neighbor algorithm in order to match the gene variants in an unannotated exome with the ones available in a reference knowledge base. The most similar hit is used to transfer the blood group. With an accuracy above 90%, BOOGIE is a proof-of-concept that shows the potential applications of genetic prediction, and can be easily extended to any Mendelian trait.
To summarize, this thesis is a partial answer to the exponential growth of sequences available that need further experiments. By integrating heterogeneous information and designing new predictive models based on machine learning, I developed novel tools for biological data analysis and classification. All implementations are freely available for the community and might be helpful during future investigations like in drug design and disease studies.In questa tesi, vengono proposti nuovi algoritmi e modelli per affrontare problemi biologici. L'informatica svolge un ruolo chiave nella proteomica e nella ricerca genetica dovuto alla gestione delle grandi moli di dati biologici. Nel contesto dello studio di proteine, ho sviluppato nuovi metodi per la predizione delle loro funzioni basati su principi di reperimento dell'informazione. Utilizzando fonti eterogenee di conoscenza, come la ricerca su grafi e la similarità di sequenze, ho progettato uno strumento chiamato INGA che può essere utilizzato per annotare interi genomi. Questo è stato valutato imparzialmente dal Critical Assessment of Function Annotation, e ha dimostrato di essere uno degli approcci più efficaci per l'inferenza di funzione. Per meglio caratterizzare le proteine dal punto di vista strutturale, ho proposto una strategia di rilevamento delle conformazioni delle proteine basata su rete di interazione di residui (RIN). Le reti RIN sono state quindi estese per gestire le fluttuazioni temporali delle coordinate atomiche. Tali grafi sono stati infine generati automaticamente da algoritmi di clustering. Un'implementazione chiamata RING MD ha evidenziato efficacemente i principali amminoacidi noti per essere funzionalmente rilevanti nell'Ubiquitina. Questi aminoacidi sono infatti molto importanti per spiegare la dinamica strutturale della proteina. Con la stessa logica, sono stati usati i grafi RIN anche per prevedere l'impatto delle mutazioni all'interno di una struttura proteica. Combinando informazioni sul nodo mutante in una rete e le sue caratteristiche, una rete neurale artificiale è stata addestrata per stimare la variazione di energia libera di Gibbs all'interno di una proteina. Cambiamenti estremi nell'energia interna potrebbe portare all'unfolding della proteina, ed eventualmente ad una malattia. D'altro canto, anche la riduzione della flessibilità proteica può ostacolare la sua funzione. Ad esempio, le fluttuazioni estreme osservate nelle proteine intrinsecamente disordinate (IDP) sono fondamentali per le loro attività. Per studiare le IDP, ho contribuito alla raccolta del più grandi dataset di regioni disordinate mai esistito. Nella seguente analisi è stato dimostrato quali sono le funzioni tipiche di queste sequenze e i processi biologici in cui sono coinvolte. Data l'importanza della loro identificazione, una valutazione globale di predittori del disordine è stata eseguita per mostrare quali sono i metodi più efficaci e le loro limitazioni. Nel contesto della genetica, mi sono concentrato sulla previsione di fenotipi. Durante il Critical Assessment of Genome Interpretation (CAGI), ho proposto nuovi approcci per l'analisi dei dati dell'esoma progettati per valutare il rischio di morbo di Crohn e di ipercolesterolemia. Queste sono spesso definite come malattie complesse, dal momento che il meccanismo alla base della loro insorgenza è ancora sconosciuto. Nel mio studio, i campioni umani con un arricchimento di mutazioni in geni critici sono stati predetti come soggetti a rischio genetico elevato. Oltre ai geni associati alla malattia, le reti di interazione proteiche sono state considerate per valutare l'accumulo di varianti in pathway biologici. Tale strategia ha dimostrato di essere tra le migliori secondo gli organizzatori del CAGI. Nel caso più semplice dei tratti mendeliani, con BOOGIE ho progettato un metodo per la predizione dei gruppi sanguigni umani basata su dati di esoma. Esso utilizza una versione specializzata dell'algoritmo nearest neighbour al fine di far corrispondere le varianti genetiche in un esoma non annotato con quelle disponibili in una base di conoscenza di riferimento. L'esempio più simile è usato per trasferire il gruppo sanguigno. Con una precisione superiore al 90%, BOOGIE è un prototipo che mostra le potenziali applicazioni della predizione genetica, e può essere facilmente esteso a qualsiasi tratto mendeliano. Riassumendo, questa tesi è una risposta parziale alla crescita esponenziale di sequenze disponibili che necessitano ulteriori esperimenti. Integrando informazioni eterogenee e la progettazione di nuovi modelli predittivi basati su apprendimento automatico, ho sviluppato nuovi strumenti per l'analisi di dati biologici e per la loro classificazione. Tutte le implementazioni sono liberamente disponibili per la comunità e potrebbero essere utili durante indagini future come in studi di malattie e nella progettazione di farmaci.
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López, Ceballos Pablo. "Elucidating how protein turnover in cell-ECM adhesion stabilizes tissue structure during development." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57622.
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Morphogenesis is the process by which cells rearrange to form complex three dimensional structures. Cell to extracellular matrix (ECM) adhesion, primarily mediated by Integrins, is essential for the formation and maintenance of tissue architecture. A critical way to regulate cell-ECM adhesion is by modulating the turnover of Integrins and their adhesion complex, and thereby modulating the stability of Integrin-based adhesions. We previously showed that mechanical force stabilizes Integrin-based adhesions during development by modulating Integrin turnover. Here, we extend our studies to understand how mechanical stress impacts the dynamics of the cytoplasmic adaptor protein Talin, a critical regulator of Integrin function. Using Fluorescence Recovery After Photobleaching (FRAP) analysis in combination with a newly developed mathematical model that encompasses the complexities of Talin turnover, we determined that mechanical force stabilizes cell-ECM adhesion by increasing the rate of assembly of Talin-mediated adhesion complexes. To dissect the mechanisms that regulate Talin turnover downstream of mechanical force, we used point mutations of Talin which abrogate specific functions of the Integrin adhesion complex and measured turnover kinetics. We found that the activation of Integrins, resulting in increased affinity for ECM ligands, is a crucial process to regulate adhesion complex turnover. To further investigate the role of Integrin activation in regulating adhesion stability, we introduced small molecules known to induce “outside-in activation” of Integrins in vitro into live, intact embryos. This approach revealed that outside-in activation stabilizes cell-ECM adhesion by decreasing Integrin endocytosis; similarly to what we have previously seen when mechanical force is increased. Based on this finding, we propose that mechanical force may induce changes in Integrin activation in order to stabilize cell-ECM adhesions. Overall, we show that Integrin activation is a key mechanism that regulates cell-ECM adhesion stabilization during embryogenesis.Medicine, Faculty of
Graduate
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Leugers, Chad Jeremy. "Modulation of growth factor-induced ERK signaling by the microtubule associated protein tau." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/541.
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The microtubule-associated protein tau is known for its ability to bind to and stabilize microtubules and for its ability to nucleate microtubule assembly. In neurodegenerative tauopathies such as Alzheimer's disease, tau becomes hyperphosphorylated and loses the capacity for microtubule binding, possibly contributing to microtubule destabilization and axonal degeneration. However, evidence now indicates that soluble forms of hyperphosphorylated tau might have a toxic gain of function linked to abnormal signal transduction and cell cycle events in normally post-mitotic neurons. In support of this hypothesis, tau has been found to associate with numerous signaling proteins such as tyrosine kinases, adaptor proteins, and scaffold proteins. During early brain development, fetal tau is also more phosphorylated than tau in the adult brain and weakly binds microtubules, suggesting tau has functions in addition to microtubule stabilization.
The aim of this dissertation research is to investigate the possible role of tau in neuronal signaling, using tau-expressing and tau-depleted cell lines. Here, we provide evidence that during growth factor stimulation of neuronal cells, tau functions in advance of the neurite elongation stage. Tau is required for neurite initiation in a manner that does not require its microtubule binding function, and in addition, tau potentiates AP-1 transcription factor activation in response to nerve growth factor (NGF). The effect of tau on AP-1 activation is mediated through the enhanced activation of extracellular signal-regulated kinase (ERK), in response to both NGF and epidermal growth factor (EGF). We show that phosphorylation of tau at Thr231 also occurs in response to NGF and is required for tau to impact on ERK signaling, whereas the ability of tau to bind to microtubules is not required. Together, these findings indicate a new functional role for tau in neuronal signal transduction and have implications for tau function during early brain development and in neurodegenerative disease.
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Osta, Muhammad Samir Ahmed. "Characterisation of ECM protein processing mechanisms underlying simple peritoneal sclerosis and encapsulating peritoneal sclerosis." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/11406/.
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Introduction and hypothesis: Peritoneal dialysis (PD) is an important option for renal replacement therapy. Peritoneal sclerosis (PS) limits PD duration due to loss of ultrafiltration (UF) capacity, while about 3% of PD patients experience a condition termed encapsulating peritoneal sclerosis (EPS). In many fibrotic diseases reduced Extracellular matrix (ECM) breakdown due to lowered matrix metalloproteinase (MMP) activity occurs, often from over-expression of tissue inhibitors of MMP (TIMPs) that underlie fibrotic remodeling. Furthermore, recent application of 2D gel proteomics on peritoneal dialysis effluent (PDE) samples has identified several proteins that are elevated in patients with membrane damage. These observations have led to the hypothesis that: changes in proteins in PDE samples, in particular those associated with ECM breakdown have value as non-invasive biomarkers of PS and the switch to EPS. To test this hypothesis, PDE samples from 3 patient cohorts was analysed for ECM proteolytic activity. A range of ECM processing proteins and 3 proteins identified from previous proteomic studies of patients developing EPS (intellectin-1, dermatopontin and collagen α1 (I)) were analysed in PDE samples. Methods: Three patient cohorts were studied: two were from Sheffield Kidney Institute (SKI) that consisted of 32 spot PDE samples (SKI-1) that included 1 EPS patient & 51 PDE & plasma samples collected during a peritoneal equilibrium test (PET) with multiple dwell times in patients who did not have EPS (SKI-2). The third cohort consisted of 209 samples from the Global Fluid Study (GFS) including sequential samples from 12 EPS & 42 matched controls patients. MMP activity was assessed using the ENZchek assay system. Plasmin activity was assessed by using cleavage of the V0882 substrate. TIMPs, MMPs, intelectin-1, dermatopontin, and collagen (α1) I were quantified by commercial ELISA in PDE and plasma samples. PDE cytology (macrophages, leukocytes, fibroblasts and mesothelial cells) was performed to determine if changes in any protein could be associated with changes in cell types. Clinical data were recovered from either the peritoneal dialysis database (PDDB) at Sheffield or the GFS archives. The analysis was performed using Microsoft Excel 2010 software, SPSS, and Graphpad prism (prism 5.01 for windows). Results: Plasmin activity in PDE samples decreases with long duration of PD therapy. Minimal MMP activity was found in all PDE samples. In the SKI-1 cohort, MMP-1, -9, & -13 were almost undetectable with only MMP-2 & -3 being measurable with levels of ((mean±SD) 46±37 & 2.1±2.2 ng/mL respectively). In contrast TIMP-1 and TIMP-2 and to lesser extent TIMP-3 had significant levels in PDE samples from commencing PD (109±88, 17±12, and 0.28±0.33 ng/mL respectively). All TIMPs & MMP-2 were raised in the single patient who had a diagnosis of EPS. In samples from the GFS cohort, there was a rapid 6 fold increases in TIMP-1 within 100 days of the diagnosis of EPS, which when normalised to TIMP-2 levels was a good predictor of EPS. Calculation of the plasma to dialysate transfer rate by reference to that of circulating proteins with no peritoneal production and of known molecular weight (albumin, beta2microglobulin (B2M), transferrin, IgG, and creatinine) demonstrated that TIMPs & MMPs (especially TIMP-1 and MMP-2) have significant peritoneal production. Plasma levels for TIMP-1,-2, MMP-2,-3, and intelectin-1 (mean±SD) were 121±27, 85±16, 176±35, 11±5, and 374±136 ng/mL in healthy individuals respectively. Plasma levels in PD patients for TIMP-1,-2, MMP-2,-3, and intelectin-1 (mean±SD) were 297±78, 158±33, 309±112, 42±28, and 749 ±722 ng/mL respectively. None of the proteins identified by proteomics as predictors of EPS were able to be validated by ELISA. However TIMP-1,-2, MMP-2, intelectin-1, and collagen (α1) I in PDE samples had significant correlations with the loss of ultrafiltration and thus membrane damage. PDE cytology showed that peritoneal fibroblast and leukocyte numbers increase with time on PD, while peritoneal macrophage decreases with time on PD. There were no significant changes in mesothelial cells. Conclusions: Negligible MMP activity in PDE samples results from high TIMP levels which could underlie the development of PS. The rapid increase in TIMP-1 within 100 days of EPS development offers value as a diagnostic tool or a late biomarker. Plasma levels of TIMP-1,2, MMP-2,3, and intelectin-1 are higher in patients on PD compare to healthy individuals. The increase in peritoneal fibroblasts may be a source of TIMP-1.
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Parisis, Nikolaos. "Identification of PAR-2-regulated ERK substrates and (Beta)-arrestin-interacting proteins in invasive breast cancer cells." Thesis, University of Essex, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520107.
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Román, Pérez Erick. "The role of ERa, ERß and phytoestrogens from soy in P53-mediated response to DNA damage in mammary epithelium." Amherst, Mass. : University of Massachusetts Amherst, 2009. http://scholarworks.umass.edu/dissertations/AAI3372274/.
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Thesis (Ph. D.)--University of Massachusetts Amherst, 2009.On title page, the 'a' in ERa is symbolized by the Greek symbol for alpha. Includes bibliographical references (p. 108-124). Print copy also available.
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Conley, Travis B. "The influence of training status on ERK and AKT phosphorylation in human skeletal muscle." Virtual Press, 2005. http://liblink.bsu.edu/uhtbin/catkey/1319219.
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Exercise induces morphological and metabolic adaptations that are highly specific to the mode of exercise training. These specific phenotypical changes are due to an equally specific molecular response that may depend on the activation and coordination intramuscular signaling pathways. Just as metabolic and morphological changes are influenced by the mode of exercise training, the signaling pathways that mediate exercise adaptation may also be directly related to the training status of skeletal muscle. For example, pre-conditioned skeletal muscle may exhibit a specific intracellular signaling response to an acute bout of exercise that is dependent on past training history. Both Akt (protein kinase B) and extra-cellular signal-related kinase (ERK 1 /2) have been shown to be phosphorylated in response to an acute bout of resistance exercise in human skeletal muscle and have been suggested to mediate the adaptive response to exercise. The purpose of this investigation was to examine the response of Akt and ERKI/2 to an acute bout of resistance exercise in three groups with distinctly different exercise training backgrounds. Twenty one subjects performed 3 sets of 10 repetitions of knee extension exercise at 70% 1-RM. The subjects consisted of a resistance-trained group (RE) (n=7), endurance trained group (END) (n=7) and a sedentary group (SED) (n=7). Muscle biopsies were taken from the vastus lateralis muscle before, immediately after, and 10 min post-exercise and were analyzed for phosphorylation of Akt and ERK1/2. ERK1/2 phosphorylation increased 47%, and 54% from pre-exercise to immediately post-exercise in the SED and RE groups respectively (p < 0.05). ERK1/2 phosphorylation increased 95%, 196%, and 47% from pre-exercise to 10 min post-exercise in the SED, RE, and END groups, respectively. (p < 0.05). The magnitude of ERK1/2 phosphorylation 10 min post-exercise was different between each group and may be linked to the group's training status. (p < 0.05) Akt phosphorylation decreased 42% and 37% from pre-exercise to immediately post-exercise in the SED and END group, respectively (p < 0.05). There was a 40 % increase in Akt phosphorylation from immediate post-exercise to 10 min post-exercise in the END group. In conclusion, training status appears to influence the magnitude and time course of activation of both Akt and ERK1/2 in response to an acute bout of resistance exercise. The immediate response of both ERK1/2 and Akt may play a key role in the adaptive response of skeletal muscle ultimately resulting in metabolic and morphological changes that are dependent on the past training history of the individual.School of Physical Education, Sport, and Exercise Science
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Lundberg, Ida. "Fibroblasts and ECM in colorectal cancer : Analysis of subgroup specific protein expression and matrix arrangement." Thesis, Umeå universitet, Patologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-85606.
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The tumor microenvironment is important for tumor growth and progression, where the cancer associated fibroblast (CAF) is one central cell type. The CpG island methylator phenotype (CIMP) divides colorectal cancer (CRC) into three subgroups and in this study we investigate how the different CIMP-groups and adjacent fibroblasts affect each other. This was done with the aim of finding CIMP-specific markers and to see if different tumor-fibroblast interactions result in differently invasive tumors. Here we report that CIMP-negative tumors have increased expression of fibronectin, while CIMP-high tumors have reduced expression of E-cadherin, findings that were seen in both tumor tissue samples and tumor cell lines. We also show that CIMP-negative and CIMP-high cancer cells induce an alignment of the fibronectin fibers produced by the fibroblasts and that CIMP-high cancer cells migrate with directionality on these matrices. These findings indicate that the different tumor subgroups in fact induce different phenotypes in CAFs, resulting in CIMP-specific markers. They also indicate that CIMP-negative and CIMP-high tumors may induce an alignment of fibronectin in order to promote cancer cell migration and thereby also tumor invasion.
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Lee, Vivian Wing Yan. "Regulation of extracellular signal-regulated protein kinases (ERK) 1 and 2 in synaptic nerve terminals." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1446422/.
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Activation of extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2) is a key signalling event in the modulation of presynaptic function. This study looks at the upstream regulation of ERK 1 and 2 signalling in rat cerebrocortical synaptosomes. Kinase activation assays based on phospho-state specific antibodies revealed two major inputs for the activation of ERK 1 and 2: i) a neurotrophin-mediated signalling to ERK 1 and 2 which is dependent on the activation of small GTP-binding protein Ras and the presence of Ca ii) a Ca -dependent activation of ERK 1 and 2, stimulated by depolarisation or by the Ca ionophore ionomycin. Treatment of synaptosomes with Ca2+ chelators showed that basal ERK 1 and 2 activation was partly supported by Ca2+ from intracellular sources, whilst depolarisation-dependent activation of ERK 1 and 2 was entirely attributable to extracellular Ca influx. Like the BDNF-mediated activation, this Ca -dependent signalling to ERK was contingent on Ras, as evinced by the use of Ras inhibitor lovastatin. Using inhibitors of Ca /CaM-dependent protein kinase II (CaMKII) and phosphatidylinositol-3-kinase (PI3K), we next showed that both kinases are involved in mediating the Ca -dependent ERK 1&2 pathway. Furthermore, the Src family kinase (SFK) inhibitor, PP2, strongly reduced Ca -dependent ERK 1 and 2 activation, suggesting a role for non-receptor tyrosine kinases (nRTKs) in upstream signalling. Finally, using okadaic acid, roscovitine and baclofen respectively, we showed that the duration and efficacy of ERK 1 and 2 activation are determined by the function of protein phosphatase 2A (PP2A), cyclin- dependent kinase 5 (cdk5) and the presynaptic Gj/0-coupled GABAb receptors. Interestingly, our data demonstrate that baclofen-mediated inhibition of ERK 1&2 can be overridden by BDNF stimulation, revealing a potential feedback and cross-talk mechanism between the excitatory ERK and inhibitory GABA cascades. Together, these studies elucidate the role of ERK 1 and 2 as a hub for signalling in the nerve terminal.
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Kitajima, Sakihito. "Molecular analysis of gene expression of tobacco osmotin-like protein and its transcription factor ERF." Kyoto University, 1999. http://hdl.handle.net/2433/181925.
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Durrett, Timothy P. "Understanding Arabidopsis ion homeostasis in the post-genomic era assigning function to two proteins involved in iron metabolism /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4437.
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Thesis (Ph.D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 27, 2009) Vita. Includes bibliographical references.
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Shim, Hoon. "THE ROLE OF R7 REGULATORS OF G PROTEIN SIGNALING IN THE RETINA." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/580.
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The R7 regulators of G protein signaling (R7 RGS), namely RGS6, RGS7, RGS9 and RGS11, are expressed in the retina along with its binding partner Gβ5. The RGS9-1 isoform is expressed only in retinal photoreceptors and rate-limits the recovery of rod phototransduction by acting as a member of the transducin GAP complex (RGS9-1/Gβ5L/R9AP). The Gβ5L isoform is also only expressed in retinal photoreceptors and acts by stabilizing the GAP complex. The Gβ5S isoform differs from Gβ5L by the absence of exon 1 due to alternative splicing and is expressed in many other retinal cells. Gβ5L is barely detectable in RGS9-/- mice suggesting that Gβ5L has a protein degradation signal conferring instability in the absence of RGS9. To study the role of exon 1 of Gβ5L, we replaced Gβ5L with Gβ5S in rods by expressing transgenic Gβ5S under the control of the rhodopsin promoter within a Gβ5-/- mouse and determined that exon 1 of Gβ5L has two previously unidentified functions: (1) to increase the capacity of Gβ5L to bind to RGS9-1 and (2) to serve as a signal for rapid degradation of Gβ5L in RGS9-/- photoreceptors. Several groups have reported that RGS7 and RGS11 with Gβ5S reside in the dendritic tips of depolarizing bipolar cells (DBCs) and that they are involved in the mGluR6/Gαo/TRPM1 pathway, which mediates DBC light responses. The exact role of RGS7 in DBCs has not been unequivocally determined. We have contributed by making a true RGS7 null mouse line and found the RGS7-/- mice are viable and fertile, but have a small body size. Electroretinogram (ERG) b-wave implicit time in young RGS7-/- mice is prolonged at eye opening, but the phenotype disappears by 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7-/- retina, but the Gβ5S level is significantly reduced. We further generated a RGS7 and RGS11 double knockout (711dKO) mouse line and found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as well as the ERG b-wave. Ultrastructural defects similar to those of Gβ5-/- mice are present in 711dKO. Furthermore, in retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S becomes undetectable, while the photoreceptor-specific Gβ5L remains unaffected. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in the retina, the DBC-related defects found in Gβ5-/- mice appear to be caused solely by a combined loss of RGS7 and RGS11. The notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins is firmly established in this work. The availability of all four R7 RGS single knockout mouse lines enables future studies to further elucidate the roles of R7 RGS proteins in vision.
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Azzam, Diana Galil. "Extracellular signal regulated kinase/mitogen activated protein kinase (ERK/MAPK) regulation of the androgen receptor in breast cancer cells." University of Western Australia. School of Pathology and Laboratory Medicine, 2008. http://theses.library.uwa.edu.au/adt-WU2009.0024.
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[Truncated abstract] Androgens inhibit the growth of human breast tumours and have been successfully used to treat breast cancer in women. Expression of the androgen receptor (AR), which mediates androgen action, is upregulated in breast cancer cells and the AR is the most frequently expressed steroid hormone receptor in breast tumours. AR levels and activity are modulated by the activity of other signalling pathways, however interactions between the AR and signalling pathways and the consequent alterations to the androgen responsiveness of breast cancer cells are largely uncharacterised. The extracellular signal regulated kinase (ERK1/2) pathway is hyperactivated in ~30% of breast tumours and these tumours are often associated with low oestrogen receptor-a (ERa) levels, reduced responsiveness to antioestrogen therapies and an overall poorer prognosis. In this thesis, the MCF-7 human breast cancer cell line which expresses ERa, progesterone receptor (PR) and the AR, was used to investigate ERK1/2-mediated regulation of the AR and the androgen responsiveness of cells. Inhibition of ERK1/2 signalling was achieved by treatment of cells with U0126, an inhibitor of MEK1/2, the upstream activator of ERK1/2. Hyperactivation of ERK1/2 signalling was achieved by stably transfecting cells with a plasmid encoding a constitutively active form of the MEK1 protein (¿MEK1), resulting in the isolation of two clonal cell populations stably expressing ¿MEK1, ¿C3 and ¿6B, and a monoclonal cell line stably expressing the empty vector, MT3-1. Steady state AR mRNA levels, quantitated using real-time RT-PCR, were increased following U0126 treatment of MCF-7, MT3-1 and ¿6B cells. Conversely, treatment of cells with 10-8M 5a-dihydrotestosterone (DHT) for up to 72 hours decreased AR mRNA levels, indicating that ERK1/2 hyperactivation did not alter the androgenresponsiveness of AR mRNA. '...' Overall levels of AR phosphorylation were enhanced in ¿6B cells in the absence and presence of ligand, indicating that ERK1/2 hyperactivation either directly or indirectly induced receptor phosphorylation. The AR is localised in the cytoplasm in the absence of ligand and was more rapidly translocated to the nucleus in the presence of DHT in ¿C3 cells, an effect that was abrogated in the presence of U0126, thereby indicating an ERK1/2-specific mechanism. AR transcriptional activity, measured using androgen responsive reporter plasmids was not significantly altered in ¿6B cells in either the absence or presence of DHT, although the trend towards enhanced AR activity may be confirmed in future studies using optimised reporter assays. Consistent with the cell cycle regulatory functions of ERK1/2 signalling, proliferation of ¿C3 cells and ¿6B cells was increased in comparison to that of MT3-1 and MCF-7 cells. Treatment of ¿C3 cells and MCF-7 cells with 10-10 10-8M DHT produced similar inhibition of proliferation (~40%) during 8 days of culture, with no evidence of cytotoxicity. The results obtained in this thesis demonstrate that while ERK1/2 signalling regulates AR phosphorylation, processing and intracellular localisation, ERK1/2 hyperactivation in breast cancer cells does not inhibit the anti-proliferative effects of androgens. These findings support the development of tissue-specific androgenic treatments for breast tumours including poor prognosis tumours exhibiting ERK1/2 hyperactivation.
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Hodge, Jacob G. "Regulation of the MEK/ERK signaling cascade by ADAM12 in triple-negative breast cancer cells." Thesis, Kansas State University, 2016. http://hdl.handle.net/2097/35228.
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Master of ScienceBiochemistry and Molecular Biophysics Interdepartmental Program
Anna Zolkiewska
Mitogen-activated protein kinase (MAPK) signaling plays an important role in the proliferation, survival, and therapy resistance of breast cancer cells. Two important protein kinases involved in the MAPK pathway are MEK and ERK. The MEK/ERK signaling cascade can be stimulated by activation of the epidermal growth factor receptor (EGFR) upon binding of EGF-like ligands, which are released from cells by ADAM proteases. EGFR is frequently overexpressed in triple-negative breast cancer (TNBC), a particularly aggressive form of breast cancer. Our analysis of clinical data revealed that high expression of ADAM12, but not other ADAMs, in TNBC is associated with poor patient survival. Thus, we hypothesized that ADAM12 plays a critical role in the progression of TNBC, possibly by stimulating MEK/ERK activity in an EGFR-dependent manner. To test this hypothesis, ADAM12 was knocked-down (KD) in SUM159PT TNBC cells, which express high levels of the endogenous ADAM12 protein. An antibody array assay indicated a significant decrease in the activation of the MAPK pathway in SUM159PT cells after ADAM12 KD. The decrease in MAPK activity was further confirmed by Western blotting using phospho-MEK and phospho-ERK specific antibodies. Additionally, conditioned media from ADAM12-deficient SUM159PT cells failed to support the survival of MCF10A cells, suggesting that ADAM12 KD reduced the release of pro-survival growth factors from SUM159PT cells. Based upon this data, we propose that ADAM12 is a novel regulator of the MAPK pathway and a potential therapeutic target in breast cancer.
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Kisielnicka, Edyta. "SCF-mediated degradation of the two translational regulators, CPB-3 and GLD-1, during oogenesis in C. elegans." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-234186.
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The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-‐transcriptional level. This relies on RNA-‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-‐binding proteins abundance in the context of germ cell development.
This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-‐binding proteins, CPB-‐3 and GLD-‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-‐3/CPEB and GLD-‐1/STAR at the pachytene-‐to-‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-‐3 and GLD-‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-‐activated protein kinase, MPK-‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-‐1 signaling may couple RBP-‐mediated regulation of gene expression to progression through meiosis during oogenesis.
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Leandro, Luciana de Oliveira. "Controle de qualidade em imuno-histoquimica: o modelo de deteccao da oncoproteina C-erB-2." Sao Paulo : [s.n.], 2004. http://10.188.1.43/lildbi/docsonline/5/2/125-Tese%5FCIP%5FLeandro,%5FLuciana%5FO%5F2004.pdf.
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Salery, Marine. "Rôle de la protéine Arc (Activity-regulated cytoskeleton-associated protein) dans les adaptations moléculaires et comportementales induites par la cocaïne." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066400/document.
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Les adaptations cellulaires et moléculaires induites par les drogues jouent un rôle central dans les altérations comportementales à long terme observées dans l’addiction. Cette étude s’inscrit dans une démarche de compréhension des processus cellulaires rapidement mis en jeu par la cocaïne et susceptibles d’impacter durablement le fonctionnement neuronal et les comportements. La protéine Arc joue un rôle clé dans l’établissement de la plasticité synaptique à long-terme et la consolidation de la mémoire. Cette étude visait à caractériser l’induction de Arc dans le striatum en réponse à la cocaïne et d’analyser son rôle dans les réponses moléculaires et comportementales qu’elle induit. Notre étude a montré que l’expression de Arc est augmentée rapidement et transitoirement dans le striatum après une injection de cocaïne sous la dépendance de l’activation de la voie ERK. Nous montrons que la cocaïne induit une forte accumulation de la protéine Arc dans le noyau des neurones striataux où Arc se localise dans des zones actives de transcription, à proximité des histones H3 phosphorylées. In vitro, la surexpression de Arc diminue la phosphorylation des histones H3 induite par le glutamate indiquant qu’elle altère le remodelage de la chromatine. L’invalidation génétique de la protéine in vivo dans un modèle de souris transgénique conduit à une décompaction de la chromatine associée à une augmentation de l’activité de la RNA Polymerase II démontrant que Arc exerce un effet répresseur sur les mécanismes transcriptionnels. La perte totale d’expression de Arc favorise le développement d’altérations comportementales à long terme chez des animaux exposés à de faibles doses de cocaïneMolecular and cellular adaptations induced by drugs of abuse in the reward system play a key role in long-term behavioral alterations encountered in addiction. This work falls within an approach of understanding the cellular processes rapidly engaged by cocaine that could underlie the persistent alteration of neuronal physiology and behaviors. Arc protein is a major player in neuronal plasticity. Arc is induced in many behavioral paradigms and is essential for long-term synaptic plasticity and memory consolidation. The aim of this study was to characterize the profile and modality of Arc induction within the mouse striatum in response to cocaine administration. Our study shows that Arc expression is rapidly and transiently increased in the striatum after acute cocaine in an ERK-dependent fashion. This work revealed that cocaine-induced Arc protein rapidly and transiently accumulates in the nucleus of striatal neurons. In the nucleus, Arc is preferentially expressed in active transcription regions and localizes at the vicinity of phosphorylated histones H3. In vitro Arc overexpression decreased glutamate-induced Histones H3 phosphorylation showing that Arc interferes with activity-dependent chromatin remodeling. In vivo genetic invalidation of Arc expression in a transgenic mouse model was associated with a decreased chromatin compaction and increased RNA Polymerase II activity suggesting a repressive role of Arc on transcriptional mechanisms. Total Arc loss of expression leads to increased sensitivity to cocaine and promotes long-term behavioral alterations induced by low doses of cocaine
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Stevens, Payton D. "THE FUNCTION OF ERBIN, A SCAFFOLD PROTEIN, AS A TUMOR SUPPRESSOR IN COLON CANCER." UKnowledge, 2018. https://uknowledge.uky.edu/biochem_etds/36.
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Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that play important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colon cancer. Analysis of Erbin expression in patient specimens reveals that Erbin is downregulated at both mRNA and protein levels in tumor tissues. Functionally, knockdown of Erbin disrupts epithelial cell polarity and increases cell proliferation in 3D culture. In addition, silencing Erbin results in an increase in the amplitude and duration of signaling through Akt and RAS/RAF pathways. Moreover, Erbin-loss induces epithelial-mesenchymal transition (EMT), which coincides with a significant increase in cell migration and invasion. Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signaling propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promotes tumorigenesis and significantly reduces survival. Tumor organoids derived from Erbin/Apc double knockout mice have increased tumor initiation potential along with increased Wnt target gene expression as seen by qPCR. Collectively, the studies within this dissertation identify Erbin as a negative regulator of EMT and tumor progression by directly suppressing Akt and RAS/RAF signaling in vivo.
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Lok, Tsz-mei, and 駱芷薇. "Aberrant activation of ERK/FOXM1 signaling axis promotes cell migration/invasion in ovarian cancer." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45208104.
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Chen, Xi. "The role of PI3K and ERK/MAPK signal transduction cascades in long-term memory formation /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/6248.
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Novotny, Marian. "Applications of Structural Bioinformatics for the Structural Genomics Era." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7593.
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