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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

Identification of cell cycle-regulated Drosophila microtubule-associated proteins using quantitative mass spectrometry

Syred, Heather January 2011 (has links)
The microtubule network is the central framework in multiple cellular processes. The microtubule array undergoes dramatic changes as cells progress through the cell cycle. In mitosis the interphase microtubule array is reorganised into the dynamic mitotic spindle which mediates chromosome segregation. This reorganisation is coordinated by microtubule associated proteins (MAPs). However, little is known about the cell cycle regulation of MAPs and how it plays a role in mitotic spindle formation. In this thesis, I describe the development of a method to determine the profiles and relative quantities of MAPs purified from mitotic and interphase Drosophila culture cells. This method utilises mass spectrometry combined with stable isotope labelling by amino acids in cell culture (SILAC) for protein quantification. This study identified MAPs whose association with microtubules increased during mitosis and revealed a new mitotic MAP, which I have named NuMAP. NuMAP localises to the nucleus in interphase and to microtubules only in mitosis, covering the entire spindle. Truncation analysis identified two protein domains sufficient but not essential for nuclear localisation and one C-terminal domain vital for microtubule localisation. Interestingly, creation of an interphase cytoplasmic pool indicated that the interphase form of NuMAP has low affinity for microtubules, suggesting a cell cycle-related post-translational modification. A deletion mutant of the NuMAP gene was generated by P element excision and will be valuable to define the role of NuMAP in fly development.
2

Studies on the resistance to antimicrotubular agents in cultured Chinese hamster ovary cells

Adams, K. January 1987 (has links)
No description available.
3

Studies on the molecular and cell biology of plant tubulin

Hussey, P. J. January 1986 (has links)
No description available.
4

Sequence and expression of an #alpha#-tubulin gene of Physarum polycephalum

Walden, P. D. January 1988 (has links)
No description available.
5

Regulation of Nucleoporins in Mitosis

Chakraborty, Papia 27 June 2007 (has links)
Nucleoporins mediate nucleocytoplasmic trafficking in interphase. In mitosis, upon nuclear envelope breakdown, the role and regulation of Nups remain to be elucidated. An important subcomplex of nucleoporins is the Nup107-160 complex, which, in mitosis, is involved in spindle assembly and nuclear pore re-assembly. Here we show that the level of a key constituent of the Nup107-160 complex- Nup96 is cell cycle regulated. We found that the mechanism involved in regulating Nup96 levels in mitosis is proteolysis by the anaphase-promoting complex (APC). Nup96 interacts with the APC, and its proteolysis can be regulated by Cdc20 and Cdh1. Like the Nup107-160 complex, the APC is localized at kinetochores, centrosomes, and spindles. Disruption of Nup96 levels led to an acceleration of prophase to prometaphase transition and, most importantly, resulted in a delay of G1 progression. Thus, regulation of Nup96 proteolysis in mitosis sets the stage for proper G1 progression. Additionally, we have observed differential regulation of members of the Nup107-160 complex during mitosis and have identified interacting partners of Nup96 at the centrosome which reveal a novel role of nucleoporins in regulating microtubule nucleation.
6

Exploration des propriétés dune protéine dont le gène est muté dans certaines familles atteintes dépilepsie myoclonique juvénile (EMJ) : EFHC1 ou myoclonine 1.

Léon, Christine 21 September 2010 (has links)
EFHC1 est une nouvelle protéine qui savère mutée dans certaines familles atteintes dEMJ. A ce jour, létude des mutations associées à différents types dépilepsie a souvent débouché sur lidentification de mutations au niveau de gènes codant des canaux ioniques. Cependant, dans ce cas, les résultats sont beaucoup plus inattendus ; la protéine impliquée nétant pas un canal ionique mais une protéine à domaine EF-hand. Lobjectif principal du présent travail est de mieux appréhender les propriétés biochimiques et fonctionnelles dEFHC1. Une telle démarche nécessite notamment didentifier les tissus et les cellules qui expriment la protéine, de déterminer la localisation cellulaire et enfin didentifier les partenaires protéiques. Dans la première partie de ce travail, après avoir observé la localisation dEFHC1 au niveau de lappareil mitotique et du centrosome dans différentes lignées cellulaires, nous nous sommes intéressés à létude de lexpression et de la localisation de lARN messager et de la protéine EFHC1 dans les cerveaux de souris adulte et embryonnaire (au 16ème jour embryonnaire). La seconde partie de ce travail consiste en lidentification des partenaires protéiques potentiels dEFHC1 grâce à des expériences dimmunoprécipitation. Les différentes protéines testées étant choisies sur bases de nos résultats antérieurs ainsi que sur les données disponibles dans la littérature. Dans la troisième partie de ce travail, nous nous sommes intéressés à létude de la localisation dans des cellules HEK-293 de quatre protéines à domaines DM10 homologues à EFHC1, à savoir EFHC2 humaine, Rib72 de Chlamydomonas reinhardtii, Defhc1 et Defhc2 de Drosophila melanogaster. Chaque chapitre fera lobjet dun rappel systématique du contexte scientifique qui a précédé notre démarche expérimentale, des objectifs poursuivis, des résultats obtenus et de leurs discussions. Nous rassemblerons ensuite ces résultats dans le cadre dune conclusion.
7

Molecular Interaction of Tau and Microtubule

Yen, Yi-Chen 21 August 2002 (has links)
Tau protein is one of the microtubule-associated proteins (MAPs) and mainly expressed in neuronal cells. It hasbeen demonstrated that Tau may play an important role in regulating microtubule dynamic in neurons. Structurally and functionally, Tau protein composed of regulatory projection domain in N-terninus and microtubule-binding domain in C-terminus. It has been shown that the biological function of Tau protein was regulated by phosphorylation and dephosphorylation. In Alzheimer¡¦s disease (AD) brain, hyperphosphorylated Tau caused by over active kinases may contribute to the disassociation of Tau from microtubule and form the pathologically hallmarker, paired helical filaments (PHFs). The reason to study cdc2 and GSK3£] is two folds. First, both cdc2 and GSK3£] activities are raised abbrently in AD brain. Second, the phosphorylation sites of cdc2 and GSK3£] have been identified as those in PHFs.These prompted us to regard cdc2 and GSK3£] as candidates that hyperphosphorylated Tau in AD. In the following study, we used immunofluorescence analysis, co-immunoprecipitation and GST-fusion protein pull down assay to clarify the subcellular localization of Tau. We also shown that the interaction between tubulin withfull length Tau (Tau WT) and some Tau mutants that we found that not only Tau WT, but also N-terminus of Tau (Tau-N) and C-terminus of Tau (Tau-C) can bind to tubulin. Surprisingly, we observed that a fragment of N-terminus, Tau 122-244, localized in nucleus. Furthermore, we used tubulin assembly assay to test if tau or its mutants can promote tubulin assembly in vitro. Results showed that only Tau WT can promote tubulin assembly in vitro but not Tau-N or Tau-C. Although Tau-N or Tau-C can bind to tubulin in vivo and in vitro, these mutants did not remain the ability to promote tubulin assembly that suggested both functional domains, N-terminus and C-terminus of Tau, are necessary and essential for the biological function of Tau. On the other hand, we used of phosphorylation assay and site directed mutagenesis to demonstrate that T231 of Tau is one of important phosphorylation sites of cdc2 and GSK3£]. Finally, we used tubulin assembly assay to show that phosphorylated Tau by GSK3£] can negatively regulate the ability of Tau to promote tubulin assembly that indicated that the phosphorylation at T231 may play a role in regulating Tau.
8

Les propriétés mécaniques des microtubules / Self-repair rejuvenates mechanically stressed microtubules

Schaedel, Laura 01 July 2016 (has links)
Les microtubules-qui définissent la forme des axones, des cils et des flagelles, et qui servent de rails pour le transport intracellulaire-subissent de fortes contraintes exercées par les forces intracellulaires. La structure des microtubules et leur rigiditépeuvent en théorie être affectées par des contraintes physiques. Cependant, il reste à établir comment les microtubules tolèrent de telles forces et quelles sont les conséquences de ces forces sur la structure des microtubules. En utilisant un dispositif demicrofluidique, j’ai pu montrer que la rigidité des microtubules diminue progressivementà chaque cycle de courbure induit par des contraintes hydrodynamiques.Comme dans d'autres exemples de fatigue des matériaux, l'application de contraintes mécaniques sur des défauts pré-existants le long des microtubules est responsable de la génération de dommages plus étendus. Ce processus rend les microtubules moins rigides.J’ai pu aussi montrer que les microtubules endommagés peuvent se réparer en intégrant de nouveaux dimères de tubuline à leur surface et de récupérer ainsi leur rigidité initiale. Nos résultats démontrent que les microtubules sont des matériaux biologiquesayant des propriétés d’auto-réparation, et que la dynamique des microtubules ne se produit pas exclusivement à leurs extrémités. La mise en évidence de ces nouvelles propriétés permet de montrer comment les microtubules peuvent s’adapter à des contraintesmécaniques. / Microtubules—which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport—can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules’ adaptation to mechanical stresses.
9

Novel concepts of microtubule regulation during axon growth and maintenance

Qu, Yue January 2015 (has links)
Axons are up-to-a-meter-long cable-like cellular processes of neurons. The proper function of nervous systems requires that axons grow and wire up correctly during development or regeneration. The uniquely challenging architecture of axons has to be sustained for an organism's lifetime, and renders them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Notably, axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. The structural backbones of axons are formed by parallel bundles of microtubules (MTs) which also provide the highways for life-sustaining long-distance transport between cell bodies and the growth cones or synaptic endings. To better understand axon development, regeneration, maintenance and degeneration during ageing, my PhD project has focused on mechanisms underpinning the regulation of MT bundles in axons. For this, I have capitalised on fast and genetically and experimentally amenable research possible in Drosophila neurons, both in primary culture and in vivo. I have used systematic combinatorial genetics and pharmacological approaches to unravel mechanisms and roles of actin as well as the cortical collapse factor Efa6 in MT regulation during axon formation and maintenance. I was able to gain a number of novel mechanisms contributing to the de novo alignment and maintenance of ordered MT bundles. First, it has been proposed that Spectraplakins (large actin-microtubule linkers) guide the extension of polymerising MTs along cortical F-actin, thus directly laying axonal MTs out into parallel bundles. Here, I have used manipulations of actin networks as well as hybrid constructs of Shot where the actin binding domain was replaced by actin associating domains of other molecules. My data strongly suggest that Shot's ABD domain has unique properties that can sense specific properties of F-actin networks, and this is important for its ability to appropriately regulate MT behaviours. Second, using combinations of actin and Shot manipulations, I found that Shot displays not only these actin-dependent guidance functions, but it displays novel actin-independent function in MT bundle maintenance for which I present a working hypothesis. Third, I found a novel and Shot-independent role of axonal actin in maintaining MTs and promoting axon growth, and my results suggest that these functions involve promotion of MT polymerisation. MT maintenance is therefore mediated through two complementary mechanisms involving Shot on the one hand and actin on the other, and simultaneous removal of Shot and actin leads to entire loss of axons. Finally, I have unravelled novel axonal functions of the cortical collapse factor Efa6 which serves as a check point in MT bundle maintenance by eliminating "off track" MTs that have escaped the axonal bundle organisation. In the absence of this factor, a gradual increase of disorganised, criss-crossed MTs occurs as a matter of days. These new mechanisms strongly suggest that different MT-regulatory mechanisms act in parallel in axons and complement each other in one common mechanism of MT bundle formation and maintenance. I propose here a local homeostasis model of axonal MT bundle maintenance which provides new ways to think about problems of ageing as well as a range of different neurodegenerative diseases.
10

Characterization of the thermostable nature of the alpha and beta tubulin proteins in Cyanidium caldarium and Cyanidioschyzon merolae

Arnold, Matthew Scott 26 March 2004 (has links)
Microtubules are critically important cytoskeletal elements. Together with microtubule associated proteins (MAPs), they form the latticework on which eukaryotic life exists. Simply put, microtubules are polymers of tubulin heterodimers, which are composed of the globular proteins alpha and beta tubulin. In vivo, these monomers associate with one another to form heterodimers, which then polymerize to form microtubules. In mammals, microtubule polymerization is a temperature-dependent process with an optimum of 37°C (Detrich et al., 2000). If temperatures exceed this optimal temperature by even a few degrees, the microtubule will begin to dissemble due to denaturation of the tubulin subunit and permanent loss of both shape and function will occur. This thermal barrier seems to be consistent in most eukaryotic organisms. Two exceptions are the thermophilic red algae, Cyanidium caldarium and Cyanidioschyzon merolae. These thermophilic acidophiles have been discovered in volcanic vents around the globe from Yellow Stone Park to Italy and grow at optimal temperatures of around 55°C. These organisms have been primarily studied in the context of evolutionary biology because of their primitive characteristics. Very little is known about the molecular biology of these organisms, and certainly nothing is known about how the biochemistry of these organisms brings about the ability to survive the harsh conditions of their environment. Currently, my hypothesis concerning the thermostable tubulin expressed within these organisms is that there may be key amino acid differences in the tubulin's primary structure that confer enhanced thermostability. I am testing this hypothesis by sequencing the alpha and beta tubulin genes of Cyanidium caldarium and Cyanidioschyzon merolae, generating homology models of the tubulin dimers, and comparing these models to a known mesophilic tubulin heterodimer structure in order to identify potential structural differences. / Master of Science

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