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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

The Structure and Function of the TACC Protein Family in Neurodevelopment

Evans, Matt January 2015 (has links)
Thesis advisor: Laura Anne Lowery / Thesis advisor: Eric Folker / In order to form the exact synaptic connection required for proper neurological function, the growing tip of the neuron hosts an orchestra of hundreds of different proteins interacting with extracellular cues to steer neuron growth in the right direction. The goal of our current research is to study several of the components of this pathway, known as the TACC family. Here, we present a detailed structure/function analysis of the TACC family in regards to binding and activity with other proteins in the growth cone. We investigate the function of TACC3 in mediating neuron outgrowth and guidance in vivo. We have found structural elements of the TACC family that enable their activity. Studying these conserved structures and functions of the TACC family will enable greater understanding of the entire process of cytoskeletal regulation and neurodevelopment. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.
2

Functional analyses of microtubule and centrosome-associated proteins in Dictyostelium discoideum

Samereier, Matthias January 2011 (has links)
Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium. / Die Kenntnis der Funktion von Mikrotubuli-assoziierenden Proteinen (MAPs) ist von grundlegender Bedeutung für das Verständnis der Mikrotubuli-Dynamik und deren Regulation. Im Rahmen dieser Arbeit wurde die Rolle des Mikrotubuli-assoziierenden Proteins TACC (Transforming acidic coiled-coil), welches in vielen Organismen an der Stabilisierung und dem Wachstum von Mikrotubuli beteiligt ist, im Modellorganismus Dictyostelium discoideum untersucht. Das Dictyostelium TACC Protein konnte während des gesamten Zellzyklus am Centrosom nachgewiesen werden. Darüber hinaus wurde es an den Mikrotubuli-Plus-Enden vorgefunden, überraschenderweise jedoch ausschließlich während der Interphase. Die gleiche Zellzyklusabhängige Lokalisation wurde für CP224 beobachtet, einem Homologen der XMAP215 Proteine in Dictyostelium. Diese ubiquitären MAPs sind konservierte, direkte Interaktionspartner der TACC Proteine und spielen eine zentrale Rolle bei der Nukleation und der Polymerisation von Mikrotubuli. Durch diese Arbeit konnte erstmals in vivo gezeigt werden, dass TACC und XMAP215 Proteine während der Interphase und Mitose unterschiedlich stark mit Mikrotubuli-Plus-Enden assoziiert sein können. Durch Untersuchungen an Knockdown-Mutanten wurde ersichtlich, dass Dictyostelium TACC eine Rolle beim Mikrotubuli-Wachstum während der Interphase spielt und über weite Strecken der Mitose essentiell für die Ausbildung von astralen Mikrotubuli ist. In anderen Organismen konnte als Ursache instabiler Mikrotubuli in TACC Mutanten häufig unzureichendes Rekrutieren des jeweiligen XMAP215 Proteins an das Centrosom ausgemacht werden. Um entsprechende Auswirkungen auf die Lokalisation von CP224 durch den Knockdown von TACC in Dictyostelium zu untersuchen, wurden Fluorescence Recovery after Photobleaching (FRAP) Experimente durchgeführt. Diese ergaben, dass CP224 auch in Abwesenheit von TACC in vollem Umfang an die Centrosomen und Spindelpole rekrutiert wird. Anders als im Wildtyp, konnte in TACC Mutanten allerdings kein CP224 an den Mikrotubuli-Plus-Enden nachgewiesen werden. Somit konnte erstmals in vivo gezeigt werden, dass ein TACC Protein essentiell für die Assoziation eines XMAP215 Proteins mit den Mikrotubuli-Plus-Enden ist. Im Laufe der genannten Experimente stellte sich heraus, dass sich die GFP-TACC Stämme aufgrund ihrer markierten Plus-Enden sehr gut für Untersuchungen zur ungewöhnlichen Mikrotubuli-Dynamik in Dictyostelium eignen. Zwar weisen Mikrotubuli hier über die gesamte Länge ausgeprägte Krümmungs- und Seitwärtsbewegungen auf, es können jedoch im Vergleich zu anderen Organismen während der Interphase kaum Wachstums- oder Verkürzungsvorgänge beobachtet werden. Dennoch können Dictyostelium Mikrotubuli unter Verwendung von Agenzien wie Thiabendazol oder Nocodazol, welche ausschließlich auf dynamische Mikrotubuli wirken, signifikant verkürzt werden. Durch FRAP Experimente und Einsatz von 5D Fluoreszenz-Mikroskopie an lebenden Zellen konnte in dieser Arbeit erstmalig nachgewiesen werden, dass Dictyostelium Mikrotubuli nur in der Zellperipherie, nicht aber im pericentrosomalen Bereich dynamisch sind. Die Identifikation bislang unbekannter Bestandteile des Dictyostelium Centrosoms erfuhr in den vergangenen Jahren große Fortschritte. Ein von unserer Gruppe durchgeführter Proteomics-Ansatz brachte eine Vielzahl potentiell centrosomaler Proteine zu Tage, von welchen bereits viele am Centrosom nachgewiesen werden konnten. Der zweite Teil dieser Arbeit befasst sich mit der Charakterisierung dreier noch unbekannter Proteine aus dem Proteomics-Ansatz, Cenp68, CP103 und dem Dictyostelium Homologen des Spindle Assembly Checkpunkt Proteins Mad1. Hierbei zeigte sich, dass lediglich CP103 Bestandteil isolierter, Mikrotubuli-freier Centrosomen ist, während Cenp68 an die Centromere und Mad1 an die Kinetochoren lokalisieren. Die Charakterisierung von Cenp68 umfasste außerdem die Herstellung eines polyklonalen anti-Cenp68 Antikörpers, das Suchen nach Interaktionspartnern und die Erzeugung eines Cenp68 Knockout-Stammes. Letzterer wies jedoch keinen offensichtlichen Phänotyp auf. Das Verhalten des Dictyostelium Mad1 Proteins während der Mitose stimmte in großen Teilen mit dem anderer Mad1 Proteine überein, was auf die Existenz eines bislang unerforschten Spindle Assembly Chekpunkts in Dictyostelium hinweisen könnte.
3

Efficient state space exploration for parallel test generation

Ramasamy Kandasamy, Manimozhian 03 September 2009 (has links)
Automating the generation of test cases for software is an active area of research. Specification based test generation is an approach in which a formal representation of a method is analyzed to generate valid test cases. Constraint solving and state space exploration are important aspects of the specification based test generation. One problem with specification based testing is that the size of the state space explodes when we apply this approach to a code of practical size. Hence finding ways to reduce the number of candidates to explore within the state space is important to make this approach practical in industry. Korat is a tool which generates test cases for Java programs based on predicates that validate the inputs to the method. Various ongoing researches intend to increase the tools effectiveness in handling large state space. Parallelizing Korat and minimizing the exploration of invalid candidates are the active research directions. This report surveys the basic algorithms of Korat, PKorat, and Fast Korat. PKorat is a parallel version of Korat and aims to take advantage of multi-processor and multicore systems available. Fast Korat implements four optimizations which reduce the number of candidate explored to generate validate candidates and reduce the amount of time required to explore each candidate. This report also presents the execution time results for generating test candidates for binary tree, doubly linked list, and sorted singly linked list, from their respective predicates. / text
4

Molecular markers of gliomas : implications for diagnosis and new target therapies / Les marqueurs moléculaires de gliomes : implications pour diagnostics et nouvelles thérapies cibles

Di Stefano, Anna Luisa 21 February 2017 (has links)
Le travail de thèse est dédié à la caractérisation de fusions spécifiques oncogéniques entre les gènes FGFR et TACC dans les gliomes. Nous avons analysé 907 gliomes pour la présence du gène de fusion FGFR3-TACC3. Nous avons montré que les fusions FGFR3-TACC3 ne touchent que les gliomes IDH wild-type (3%), sont mutuellement exclusives avec l'amplification de EGFR et avec la forme tronquée EGFRvIII et inversement, sont associées à l'amplification de CDK4 et de MDM2 et à la délétion du 10q. Les fusions FGFR3-TACC3 sont associées à une expression intense et diffuse de FGFR3 en immunohistochimie (IHC) et l'IHC pour FGFR3 est un marqueur prédictif très sensible de la présence des fusions FGFR3-TACC3. Les patients porteurs d'une fusion FGFR3-TACC3 ont une survie globale significativement plus longue comparés aux patients avec gliome IDH wild-type. Nous avons traité deux patients porteurs d'un gène de fusion FGFR3-TACC3 avec un inhibiteur tyrosine-kinase (TK) spécifique pour FGFR et nous avons observé une stabilisation de maladie et une réponse mineur chez un patient. Dans la deuxième section nous avons optimisé une nouvelle séquence de spectroscopie différentielle-MEGA-PRESS-pour la détection de l'oncometabolite 2-hydroxyglutarate (2 HG) qui s'accumule de manière spécifique dans les gliomes IDH mutés. Nous avons analysé de façon prospective une cohorte de 25 patients avant chirurgie pour probable gliome de grade II et grade III. Nous avons trouvé que la MEGA-PRESS est hautement spécifique (100%) et sensible (80%) dans la prédiction de la présence de la mutation IDH. Son taux est corrélé aux concentrations de 2 HG mesurés sur tissu congelé par spectrométrie de masse (GC-MS/MS). / This work is devoted to the characterization of a specific oncogenic fusion between FGFR and TACC genes in gliomas. Overall, we screened 907 gliomas for FGFR3-TACC3 fusions. We found that FGFR3-TACC3 fusions exclusively affect IDH wild-type gliomas (3%), and are mutually exclusive with the EGFR amplification and the EGFR vIII variant, whereas it co-occurs with CDK4 amplification, MDM2 amplification and 10q loss. FGFR3–TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. We show that FGFR3 immunostaining is a sensitive predictor of the presence of FGFR3-TACC3 fusions. FGFR3-TACC3 glioma patients had a longer overall survival than those patients with IDH wild-type glioma. We treated two patients with FGFR3–TACC3 rearrangements with a specific FGFR-TK inhibitor and we observed a clinical improvement in both and a minor response in one patient. In the second section, we developed a non-invasive diagnostic tool by 1H-magnetic resonance spectroscopy in IDH mutant gliomas. We optimized a uniquely different spectroscopy sequence called MEGA-PRESS for the detection of the oncometabolite 2-hydroxyglutarate (2 HG) that specifically accumulates in IDH mutant gliomas. We analysed a prospective cohort of 25 patients before surgery for suspected grade II and grade III gliomas and we assessed specificity and sensitivity, correlation with 2 HG concentrations in the tumor and associations with grade and genomic background. We found that MEGA-PRESS is highly specific (100%) and sensitive (80%) for the prediction of IDH mutation and correlated with 2 HG levels measured by gas chromatography-tandem mass spectrometry (GC-MS/MS) in frozen tissue.

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