Dissertations / Theses on the topic 'Tubulins'
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1
Nacoulma, Aminata. "Reprogrammation métabolique induite dans les tissus hyperplasiques formés chez le tabac infecté par Rhodococcus fascians: aspects fondamentaux et applications." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209429.
Abstract:
Les pathosystèmes, plante-bactérie, aboutissent souvent au niveau de la plante à de profondes reprogrammations tant au niveau de la morphogenèse que du métabolome. Dans le cas de l’interaction plante-Rhodococcus fascians, une bactérie phytopathogène, il se développe au niveau du site d’infection, une structure morphologique particulière nommée « galle feuillée ». Au sein de cette hyperplasie, les altérations métaboliques induites concernent non seulement les produits du métabolisme primaire mais également le métabolisme secondaire et plus particulièrement des composés qui interviennent dans les mécanismes de défense ou qui affectent la prolifération cellulaire végétale.
Dans le cadre de notre travail de thèse, nous nous sommes fixé deux objectifs principaux qui sont de caractériser les altérations métaboliques au niveau des tissus hyperplasiques de tabac mais aussi de rechercher des applications potentielles du point de vue thérapeutique de cette interaction.
L’approche métabolomique globale basée sur une analyse comparative des spectres 1H-RMN d’extraits bruts de tissus infectés et de tissus non-infectés couplée à des analyses statistiques de données multivariées (ACP, OPLS-DA) a été utilisé pour l’étude de la reprogrammation métabolique. Le résultat indique une accumulation de composés phénoliques et des métabolites de la famille des diterpènes dans les tissus de la galle feuillée.
Les activités biologiques des extraits de la galle feuillée ont ensuite été évaluées, notamment des activités antioxydantes (DPPH, FRAP), anti-inflammatoire (15-LOX) et antiproliférative (MTT). Il ressort de cette analyse une augmentation du potentiel réducteur et anti-radicalaire des extraits de la galle feuillée, une activité inhibitrice de la lipoxygénase ainsi qu'une activité antiproliférative sur lignées tumorales humaines, comparée à la plante non infectée.
L’étude des composés affectant la prolifération des cellules cancéreuses humaines a aboutit à la mise en évidence d’un mélange de molécules (F3.1.1) appartenant au groupe des incensoles (cembrènoïdes). Ces composés ralentissent la durée de la division cellulaire, affectent la taille des cellules et induisent des anomalies de la karyokinèse et de la cytokinèse des cellules de glioblastome U373. La dynamique tubuline/microtubule est identifiée comme étant la cible des cembrènoïdes (F3.1.1). L’effet des ces composés est original comparé aux anti-tubulines usuels tel que la colchicine et le paclitaxel. Le mécanisme d’action des incensoles est unique et donc prometteur du fait que la dynamique des microtubules reste une cible de choix dans le traitement des cellules cancéreuses.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
2
Bladh, Håkan. "Structure-activity studies of novel colchicine analogs synthesis, conformation and tublin binding /." Lund : Lund University, 1998. http://books.google.com/books?id=1sBqAAAAMAAJ.
3
Lo, Wai Hong. "Biochemical, structural and functional characterization of the light chains of the microtubule-based motor dynein /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BICH%202003%20LO.
Abstract:
Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.Includes bibliographical references (leaves 133-154). Also available in electronic version. Access restricted to campus users.
4
Tang, Liang. "Characterization of tubulins from parasitic nematodes (Brugia malayi, B. pahangi and Nippostrongylus brasiliensis) and comparison with mammalian brain tubulin." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75933.
Abstract:
The properties of tubulins from Brugia malayi, B. pahangi, Nippostrongylus brasiliensis and rat brain were compared. Tubulins from all nematodes and rat brain were partially purified by polylysine agarose chromatography, those of brain also by cycles of assembly/disassembly, and all by taxol-induced assembly. The tubulins were compared with respect to concentration ($ mu$g tubulin/mg soluble protein), drugs binding and isoforms. The tubulins of B. malayi and B. pahangi were similar. However, the tubulin from these filariae were different from those of N. brasiliensis. Even larger differences were detected between the nematode tubulins and those of rat brain. However, all tubulins reacted to $ alpha$- and $ beta$-tubulin monoclonal antibodies, and had similar mobility on SDS-PAGE. Different peptide maps were obtained for N. brasiliensis tubulin compared with rat brain tubulin. Tubulins of N. brasiliensis bound more mebendazole than did those of Brugia nematodes (B$ sb{ rm max}$: pmoles/$ mu$g tubulin). The binding of benzimidazoles to nematode tubulins was much higher than to rat brain tubulin. Benzimidazole binding to brain tubulin was influenced by the degree of assembly of the tubulin. This did not appear to be the case for the nematode tubulins. In vitro translation of B. malayi mRNA resulted in two isoforms for both $ alpha$- and $ beta$-tubulins in contrast to the 4 $ alpha$- and 4-5 $ beta$-isoforms found naturally. This suggest post translational modification of tubulin may take place in B. malayi. This study has characterized some of the differences that exist between mammalian tubulins and those of nematodes on the one hand, and between the tubulins of a gastrointestinal nematode (N. brasiliensis) and those of filariae (B. malayi and B. pahangi) on the other hand.
5
Akkari, Yassmine M. Nazih. "Investigation of tubulins in Aspergillus nidulans and Cyanidium caldarium /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487942739806417.
6
Bittermann, Elizabeth A. "The Roles of Tubulins in the Developing Mouse Brain." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1523630790076922.
7
Cheung, Po Yan. "Interaction between MKK6 and p150 glued dynactin is required for microtubule-mediated p38 MAPK activation /." View Abstract or Full-Text, 2002. http://library.ust.hk/cgi/db/thesis.pl?BICH%202002%20CHEUNG.
Abstract:
Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002.On t.p. glued is superscript. Includes bibliographical references (leaves 84-94). Also available in electronic version. Access restricted to campus users.
8
Washington, Ashley L. "FUNCTIONAL TESTS OF β TUBULINS IN DROSOPHILA SPERM TAIL MORPHOLOGY." University of Dayton / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1229709260.
9
Washington, Ashley. "Functional tests of [beta] tubulins in Drosophila sperm tail morphology." Dayton, Ohio : University of Dayton, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1229709260.
10
Cushion, Thomas David. "Tubulin genes in human disorders of cerebral cortex development." Thesis, Swansea University, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.678290.
11
Bagonis, Maria M. "3D-QSAR of anti-mitotic, tubulin binding analogs using comparitive molecular field analysis (CoMFA)." Diss., Online access via UMI:, 2006.
12
Olson, Bradley Jesse Stanford Carnahan. "Biochemical analysis of the chloroplast division proteins FtsZ1 and FtsZ2." Diss., Connect to online resource - MSU authorized users, 2008.
Abstract:
Thesis (Ph.D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008.Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references (p. 222-243). Also issued in print.
13
Menon, Kathleen I. "Assessment of the antiprotozoal activity of some tubulin inhibitors following cyclodextrin complexation." Thesis, Menon, Kathleen I. (2002) Assessment of the antiprotozoal activity of some tubulin inhibitors following cyclodextrin complexation. PhD thesis, Murdoch University, 2002. https://researchrepository.murdoch.edu.au/id/eprint/201/.
Abstract:
The purpose of the present study was to evaluate the potential usefulness of tubulin inhibitors when complexed with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) against a range of protozoan parasites. This approach involved investigations into the complexation of these drugs with HP-beta-CD, and subsequent investigations of these drugs and their complexes in regard to cytotoxicity, pharmacokinetics, in vitro efficacy against Giardia, Cryptosporidium and rodent malaria (Plasmodium chabaudi), and their in vivo efficacy against Giardia and malaria.
Albendazole (ABZ) is a benzimidazole carbamate with a broad anti-parasite spectrum, while the dinitroanilines trifluralin (TF) and oryzalin (OZ) have recently been found to exhibit activity against certain parasites. All three compounds are microtubule antagonists in either nematodes or weeds and have poor aqueous solubility, with the solubility of ABZ and OZ dependent on pH. Cyclodextrins (CD) have a hydrophobic cavity that allows them to form inclusion complexes with hydrophobic drugs, resulting in increased drug aqueous solubility, and often, improved drug dissolution and bioavailability. Thus the complexation of these drugs with HP-beta-CD was investigated. All three compounds exhibited type AL phase solubility diagrams with HP-beta-CD complexation, with additional increases in ABZ and OZ solubility achieved through the manipulation of temperature and pH. OZ displayed a stronger interaction with HP-beta-CD when ionised over its neutral form. However, insufficient concentrations of the TF/HP-beta-CD complex were achieved for drug efficacy studies. The cytotoxicity of the drugs and their complexes was assessed using the assay kit Cytotox 96 with human carcinoma cells. This is a colourimetric assay that measures lactate dehydrogenase release as a consequence of compromised cellular and membrane integrity. Both ABZ and OZ are cytotoxic to rapidly proliferating and differentiating cells but are not cytotoxic to cells in the stationary phase. Complexation did not affect drug cytotoxicity.
In pharmacokinetic studies, complexation improved ABZ (and metabolites) bioavailability, but had no significant affect on OZ bioavailability. In vitro drug assessment studies found ABZ to be highly effective against Giardia, and effectiveagainst Cryptosporidium and malaria. OZ on the other hand exhibited no activity against Giardia, but was effective against Cryptosporidium and malaria. Complexation did not improve the antiprotozoal efficacy of either ABZ or OZ. In particular, excess HP-beta-CD decreased the antigiardial effects of ABZ, possibly due to competitive complex formation. In addition, complexation did not improve the antiprotozoal effects of ABZ in vivo.
However, the cytotoxic effect of the ABZ/HP-beta-CD complex was more evident in the treatment of malaria in vivo, resulting in increased anaemia and suppression in weight gain, due to the improved bioavailability of ABZ and metabolites. HP-beta-CD alone was found to be cytotoxic at greater than 2.5%, and inhibited Giardia both in vitro and in vivo at greater than 1% and 2% respectively. This was attributed to membrane disruption caused by the dissolution and removal of membrane components.
In comparison, malaria grew better in the presence of HP-beta-CD in vitro, with no detrimental effect observed at up to 8% HP-beta-CD. This was attributed to either the increased solubilization of a necessary media component, or the complexation and removal of an inhibitory compound from the cultivation medium. Therefore HP-beta-CD complexation did not improve the antiprotozoal activity of the tubulin antagonists ABZ and OZ. However, the results of the pharmacokinetic studies suggest that anthelmintic activity of ABZ, particularly against systemic infections, may be improved with oral administration of the ABZ/HP-beta-CD complex. In addition, the antiparasitic activity of HP-beta-CD alone may be promising, especially against intestinal infections. Finally, the improved in vitro cultivation of P. chabaudi in the presence of HP-beta-CD presents a promising approach to its potential long term cultivation.
14
Menon, Kathleen I. "Assessment of the antiprotozoal activity of some tubulin inhibitors following cyclodextrin complexation." Access via Murdoch University Digital Theses Project, 2002. http://wwwlib.murdoch.edu.au/adt/admin/view/adt-MU20040820.133836.
15
Ramirez, Daniel A. Kane Robert R. "Synthesis of protected amino thymidines and new thiol derivatives of the vascular targeting agent combretastatin A-4." Waco, Tex. : Baylor University, 2006. http://hdl.handle.net/2104/5008.
16
Dogra, Abhishek Pinney Kevin G. "Design and synthesis of dihydronaphthalene vascular disrupting agents and indolequinone-based bioreductives." Waco, Tex. : Baylor University, 2006. http://hdl.handle.net/2104/5018.
17
Liou, Anthony Kian-Fong. "Characterisation of the eukaryotic Chaperonin CCT." Thesis, Institute of Cancer Research (University Of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362743.
18
Rankin, Wendi Velando. "Evaluation of survivin, an inhibitor of apoptosis, in canine urinary bladder tissues." Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/5790.
Abstract:
Thesis (M.S.)--University of Missouri-Columbia, 2008.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. "August 2008" Includes bibliographical references.
19
Zheng, Yixian. "Identification and study of [gamma] tubulins in Drosophila melanogaster and Homo sapiens /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487841548269378.
20
Mou, Yi. "Molecular analysis of the roles of NRSF in TUBB3 transcription control." View the Table of Contents & Abstract, 2007. http://sunzi.lib.hku.hk/hkuto/record/B3742869X.
21
Mou, Yi, and 牟奕. "Molecular analysis of the roles of NRSF in TUBB3 transcriptioncontrol." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B38712799.
22
Giles, Natalie Lydia. "Exploitation of the protein tubulin for controlling African trypanosomiasis /." Access via Murdoch University Digital Theses Project, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20060315.191003.
23
Mugabe, Benon E. Trawick Mary Lynn. "Structure-activity relationships and thermodynamics of combretastatin A-4 and A-1 derivatives as potential inhibitors of tubulin polymerization." Waco, Tex. : Baylor University, 2005. http://hdl.handle.net/2104/3019.
24
Hall, John Jacobs Pinney Kevin G. Trawick Mary Lynn. "Inhibitors of tubulin, nitric oxide synthase, and HIF-1 alpha synthesis, biological, and biochemical evaluation /." Waco, Tex. : Baylor University, 2008. http://hdl.handle.net/2104/5193.
25
Pilhofer, Martin. "Elucidation of the cell division mechanism and characterization of tubulins in the bacterial phylum Verrucomicrobia." kostenfrei, 2008. http://mediatum2.ub.tum.de/doc/648344/648344.pdf.
26
Guénette, Suzanne. "Characterization of Brugia pahangi b-tubulin genes and gene products." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=70165.
Abstract:
The $ beta$-tubulin gene family of the parasitic nematode, Brugia pahangi consists of three to five $ beta$-tubulin sequences. Two genomic clones containing $ beta$-tubulin sequences were isolated and characterized. The $ beta$1-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mRNA of 1.7 kb which codes for a protein of 448 amino acids. A partial nucleotide sequence of the second clone confirmed the isolation of a distinct $ beta$-tubulin sequence, $ beta$2-tubulin. The $ beta$1-tubulin transcript is found in microfilariae and adult worms, whereas the $ beta$2-tubulin transcript is predominant in male adult worms but absent from microfilariae. Results of this study also indicate that the maturation of the $ beta$1-tubulin message involves the acquisition of the conserved nematode 22-nucleotide splice leader sequence. Antipeptide IgGs raised against the divergent carboxy-terminal region of $ beta$1-tubulin recognize the same $ beta$-tubulin isoform pattern as a phylum cross-reactive monoclonal antibody. This result suggests that the $ beta$1-tubulin is highly represented in B. pahangi adults and microfilariae.
27
Mackeh, Rafah. "Mécanisme de l’hyperacétylation de la tubuline en réponse aux stress." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA114852.
Abstract:
Au-delà de sa présence sur les microtubules stables, l'acétylation de l’-tubuline peut être augmentée après exposition des cellules aux UV ou après une carence en nutriments, phénomène que l’on appelle « hyperacétylation ». Cependant, le mécanisme d’induction de cette hyperacétylation est encore inconnu. Dans cette étude, nous montrons que l’hyperacétylation de la tubuline est une réponse générale aux stress cellulaire, et nous avons cherché à caractériser cette réponse, à identifier la voie de signalisation activée par le stress et conduisant à cette réponse, et à étudier la signification biologique de ce phénomène rapide et réversible. Nous avons trouvé que MEC-17/-TAT1, l’acétyltransférease majeure de l’ tubuline, est une enzyme nécessaire à l’induction de l’hyperacétylation en réponse aux stress, et qu'elle est régulée, à l’état basal par une autre acétyltransférase appelée p300. Au cours du stress, nous montrons que l'augmentation de la production des espèces réactives de l'oxygène (ROS), conduit à l'activation de la kinase « AMP-activated protein kinase (AMPK) », qui, à son tour provoque la phosphorylation de MEC-17, et probablement son activation. Enfin, nous montrons que l’hyperacétylation de la tubuline induite par le stress, participe à la survie des cellules dans des conditions de stress et à l'induction de l'autophagie de survieBeyond its presence in stable microtubules, -tubulin acetylation can be boosted after UV exposure or after nutrient deprivation but the mechanisms of this hyperacetylation are still unknown. In this study, we show that tubulin hyperacetylation is a general cell stress response, and aimed to characterize this response, to identify the stress-activated signaling pathway leading to its induction and the biological significance of this rapid and reversible phenomenon. We found that the major tubulin acetyltransferase MEC-17/-TAT1 is the main enzyme required for mediating tubulin hyperacetylation upon stress, and that it is regulated under normal conditions by the acetyltransferase p300. Upon stress, we show that the increased production of reactive oxygen species (ROS), leads to the activation of AMP-activated protein kinase (AMPK), which in turn mediates MEC-17 phosphorylation, and probably its subsequent activation. Finally, we show that tubulin hyperacetylation induced upon stress participate in cell survival under stress conditions and in the induction of protective autophagy
28
Moore, David Joseph. "Regional neuropathology and cognitive abilities in HIV infection /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2003. http://wwwlib.umi.com/cr/ucsd/fullcit?p3083453.
29
Xie, Yanqi. "SEMISYNTHETIC AURONES: A FAMILY OF NEWLY DISCOVERED TUBULIN INHIBITORS AS ANTINEOPLASTIC AGENTS." UKnowledge, 2019. https://uknowledge.uky.edu/biochem_etds/44.
Abstract:
Aurones belong to an uncommon class of plant flavonoids that provide the bright yellow coloration of some ornamental flowers and that possess a range of biological activities. Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the biologically active analogs developed in the course of this dissertation work were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5r). These two aurones 5a and 5r inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a for 18 days had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on a National Cancer Institute COMPARE analysis, studies using computer-based molecular docking and liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5r strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model. In summary, aurones possess a pharmacophore of considerable potential in the search for new antineoplastic agents for the clinical treatment of human cancers.
30
Low, Chee Kin Andrew. "Characterisation and evaluation of novel potential target (tubulin) for antimalarial chemotherapy." Thesis, Low, Chee Kin Andrew (2004) Characterisation and evaluation of novel potential target (tubulin) for antimalarial chemotherapy. PhD thesis, Murdoch University, 2004. https://researchrepository.murdoch.edu.au/id/eprint/165/.
Abstract:
Malaria has long affected the world both socially and economically. Annually, there are 1.5-2.7 million deaths and 300-500 million clinical infections (WHO, 1998). Several antimalarial agents (such as chloroquine, quinine, pyrimethamine, cycloguanil, sulphadoxine and others) have lost their effectiveness against this disease through drug resistance being developed by the malarial parasites (The- Wellcome-Trust, 1999). Although there is no hard-core evidence of drug resistance shown on the new antimalarial compounds (artemisinin and artesunate), induced resistant studies in animal models have demonstrated that the malarial parasites have capabilities to develop resistance to these compounds (Ittarat et al., 2003; Meshnick, 1998; Meshnick, 2002; Walker et al., 2000). Furthermore, a useful vaccine has yet to be developed due to the complicated life cycle of the malarial parasites (The- Wellcome-Trust, 1999). As such, the re-emergence of this deadly infectious disease has caused an urgent awareness to constantly look for novel targets and compounds.
In this present study, Plasmodium falciparum (clone 3D7) was cultured in vitro in human red blood cells for extraction of total RNA which was later reverse transcribed into cDNA. The alphaI-, alphaII- and beta-tubulin genes of the parasite were then successfully amplified and cloned into a bacterial protein expression vector, pGEX- 6P-1. The tubulin genes were then sequenced and analysed by comparison with previously published homologues. It was found that the sequenced gene of alpha-Itubulin was different at twelve bases, of which only six of these had resulted in changes in amino acid residues. alphaII- and beta-tubulin genes demonstrated 100% sequence similarity with the published sequences of clone 3D7, but differences were observed between this clone and other strains (strains NF54 and 7G8) of beta-tubulin. Nevertheless, the differences were minor in alphaI- and beta-tubulins and there was greater than 99% homology. Subsequently, all three Plasmodium recombinant tubulin proteins were separately expressed and purified. Insoluble aggregates (inclusion bodies) of these recombinant tubulins were also refolded and have been tested positive for their structural characteristics in Western blot analysis.
Both soluble and refolded recombinant tubulins of malaria were examined in a drugtubulin interaction study using sulfhydryl reactivity and fluorescence quenching techniques. Known tubulin inhibitors (colchicine, tubulozole-c and vinblastine) and novel synthetic compounds (CCWA-110, 239 and 443) were used as the drug compounds to determine the dynamics and kinetics of the interactions. In addition, mammalian tubulin was also used to determine the potential toxicity effects of these compounds. Similarities were observed with other published reports in the binding of colchicine with the recombinant tubulins, hence confirming proposed binding sites of this compound on the Plasmodium recombinant tubulins. Two synthetic compounds (CCWA-239 and 443) that have previously tested positive against P. falciparum in vitro were found to bind effectively with all three tubulin monomers, while displaying low binding interactions with the mammalian tubulin, thus indicating that these compounds have potential antimalarial activity. Therefore, this study has satisfied and fulfilled all the aims and hypotheses that have previously been stated.
31
Low, Chee Kin Andrew. "Characterisation and evaluation of novel potential target (tubulin) for antimalarial chemotherapy /." Access via Murdoch University Digital Theses Project, 2004. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050930.125714.
32
Cao, Luyan. "bases structurales de la motilité des kinésines." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS267/document.
Abstract:
Les kinésines sont des protéines moteur liées au cytosquelette de microtubules. Elles convertissent l’énergie provenant de l’hydrolyse de l’ATP en un travail mécanique. Leur fonction typique est de se déplacer le long du microtubule pour véhiculer des charges. La plupart des kinésines sont des dimères. Elles comprennent un domaine moteur, qui porte à la fois les sites de liaison du nucléotide et du microtubule, un domaine intermédiaire de dimérisation et une partie dite « queue » qui confère la spécificité des charges à transporter. Mon objectif est d’établir le mécanisme moléculaire à la base de la motilité, avec un intérêt particulier pour la détermination des variations structurales du domaine moteur de la kinésine le long de son cycle mécano-chimique. Au cours de ma thèse, mon objet d’étude principal a été la kinésine-1 humaine, encore appelée kinésine conventionnelle.J’ai étudié plus particulièrement deux aspects du cycle mécano-chimique de la kinésine-1, en combinant des approches de biologie structurale et l’étude de mutants. Les deux aspects concernent l’étude de la fixation de la kinésine-ADP au microtubule, conduisant à l’éjection du nucléotide et à une liaison forte de la kinésine au microtubule. Dans un premier temps, j’ai déterminé la structure du domaine moteur de la kinésine-1, dépourvue de nucléotide, et sous forme d’un complexe avec la tubuline. La tubuline est la protéine constitutive des microtubules. Cette structure était la donnée principale qui nous manquait dans le cycle structural de la kinésine. En comparant cette structure avec celle de la kinésine dans un état ATP, on peut rendre compte des changements de conformation de la kinésine selon le mouvement de trois sous-domaines du domaine moteur. Cette analyse explique notamment le lien entre la fixation de l’ATP et l’ouverture d’une poche hydrophobe distante de 28 Å du site du nucléotide. Cette cavité va accommoder le premier résidu du neck linker, conduisant à la stabilisation de ce peptide situé en partie C-terminale du domaine moteur. En s’ordonnant, le neck linker va faire avancer la charge ainsi que l’autre domaine moteur de la kinésine dimérique. Il lie ainsi la fixation de l’ATP au mouvement. L’étude de l’effet de mutations du neck linker montre aussi comment, réciproquement, le neck linker bloque la kinésine dans la conformation active pour l’hydrolyse de l’ATP. Ceci diminue la probabilité que l’ATP soit hydrolysé avant que l’étape mécanique se soit produite; cet aspect est essentiel pour rendre compte de la processivité de la kinésine-1.Ces données structurales suggèrent également comment la fixation de la kinésine-ADP au microtubule accélère l’éjection de l’ADP. Pour étudier cet aspect plus en détail, j’ai étudié l’effet de mutations sur la vitesse de largage de l’ADP. L’idée était de mimer à l’aide de mutations la fixation au microtubule. J’ai identifié ainsi deux séries de mutants qui présentent une vitesse accélérée de largage spontané de l’ADP, ce qui suggère deux voies pour interférer avec la fixation du nucléotide. J’ai ensuite déterminé la structure de deux de ces mutants dépourvus de nucléotide, ainsi que celle de la kinésine de départ également dans une forme apo, obtenue par digestion de l’ADP. En absence de microtubule, la kinésine dépourvue de nucléotide adopte une conformation soit à l’image de celle de la kinésine-ADP, ou proche de celle de la kinésine-apo liée à la tubuline. Dans un contexte naturel, seule la deuxième conformation est compatible avec la fixation au microtubule. L’ensemble de ces résultats suggère que le microtubule accélère l’éjection du nucléotide par un double mécanisme : en interférant avec la liaison du magnésium et en déstabilisant le motif P-loop de liaison du nucléotideKinesins are a family of microtubule-interacting motor proteins that convert the chemical energy from ATP hydrolysis into mechanical work. Many kinesins are motile, walking along microtubules to fulfill different functions. Most kinesins are dimers, the monomer comprising a motor domain, a dimerizing stalk domain, and a tail domain. The motor domain contains both the nucleotide-binding site and the microtubule-binding site. I am interested in the molecular mechanism of kinesin's motility. In particular I want to establish the structural variations of the kinesin motor domain along with the mechanochemical cycle of this motor protein. During my thesis, I have focused my work on the human kinesin-1, also named conventional kinesin, which is the best characterized kinesin.I have studied two aspects of the kinesin mechanochemical cycle, by combining structural and mutational approaches. Both aspects rely on the binding of ADP-kinesin to a microtubule, which leads to the release of the nucleotide and to a tight kinesin-microtubule association. First I determined the crystal structure of nucleotide-free kinesin-1 motor domain in complex with a tubulin heterodimer, which is the building block of microtubule. This structure represented the main missing piece of the structural cycle of kinesin. Three subdomains in the kinesin motor domain can be identified through the comparison of my structure with ATP-analog kinesin-1-tubulin structure. The relative movements of these subdomains explain how ATP binding to apo-kinesin bound to microtubule triggers the opening of a hydrophobic cavity, 28 Å distant from the nucleotide-binding site. This cavity accommodates the first residue of the “neck linker”, a short peptide that is C-terminal to the motor domain, allowing the neck linker to dock on the motor domain. The docking of the neck linker is proposed to trigger the mechanical step, i.e. the displacement of the cargo and the stepping of the dimeric kinesin. By studying mutants of the neck linker, I have shown that, reciprocally, this peptide locks kinesin in the ATP state, which is also the conformation efficient for ATP hydrolysis. Doing so, it prevents the motor domain from switching back to the apo-state. It prevents also an untimely hydrolysis of ATP, before the mechanical step has occurred. These features are required for movement and processivity.Second, these structural data also suggest how the binding of ADP-kinesin to tubulin enhances nucleotide release from kinesin. To further study this step of the kinesin cycle, I studied the effect of kinesin-1 mutations. These mutations were designed in isolated kinesin to mimic the state when kinesin is bound to a microtubule. I identified two groups of mutations leading to a high spontaneous ADP dissociation rate, suggesting that there are two ways to interfere with ADP binding. Then I determined the crystal structures of the apo form of two mutants as well as that of the nucleotide-depleted wild type kinesin. It showed that apo-kinesin adopts either and ADP-like conformation or a tubulin-bound apo-like one. In the natural context, the second one is stabilized upon microtubule binding. Overall, the mutational and structural data suggest that microtubules accelerate ADP dissociation in kinesin by two main paths, by interfering with magnesium binding and by destabilizing the nucleotide-binding P-loop motif
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Yakovich, Adam J. "Old targets and new beginnings a multifaceted approach to combating Leishmaniasis, a neglected tropical disease /." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1193247442.
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Nayak, Tania. "Investigations of the Functions of gamma-Tubulin in Cell Cycle Regulation in Aspergillus nidulans." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1218557154.
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Francisco, Samuel Nuno Furtado da Conceição. "Toxoplasma gondii Tubulin Cofactor B plays a key role in host cell invasion and replication." Doctoral thesis, Universidade de Lisboa, Faculdade de Medicina Vterinária, 2020. http://hdl.handle.net/10400.5/20149.
Abstract:
Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e BiomédicasTubulin cofactors participate in the folding, dimerization, and dissociation pathways of the tubulin dimer, being implicated in the control of tubulin proteostasis and consequently in the control of microtubule (MT) dynamics in vivo. We hypothesise that these proteins have a role in the regulation of MT cytoskeleton dynamics during Toxoplasma gondii host cell invasion. In this context, we characterized the Tubulin cofactor B (TBCB) in T. gondii. TBCB is a CAPGly domain-containing protein that together with TBCE, interact with and dissociate the tubulin dimer. The TBCB sub-cellular localization in T. gondii was studied using an in-house anti-TBCB serum. T. gondii lines overexpressing TBCB were obtained by random integration as well as TBCB conditional knockout lines by CRISPR/Cas9 system. TBCB transgenic clones were characterized by growing assays (plaque, invasion, replication and egress assays), western blot analysis and fluorescence microscopy (standard, confocal and super-resolution). TBCB showed a polarized localization, at the anterior region of the parasite, under the conoid and in close association with polar ring and subpellicular MTs. It did not present a clear co-localization with the apical complex secretory vesicles, although the interaction with rhoptries and micronemes cannot be excluded. TBCB overexpression lines showed a significant decrease in the capacity to form plaques, attributable to a proportional reduction in the capacity to invade. No differences were observed in replication and egress assays. The TgTBCB knockout line, showed a complete depletion of the protein and a viability no longer than a week. These lines showed a strong reduction in their capacity to invade the host cell and in their replication rate. In the absence of TBCB, cells have an altered axis of division resulting in abnormal division. Some parasites show the loss of the correct division axis and some parasites have four daughter cells forming inside instead of two. TBCB is a polarity marker in T. gondii and is involved in the invasion and replication processes. Its apical localization, together with TBCB mammalian partners already described (MT associated proteins) and the invasion phenotypes, suggest that TBCB can be involved in the intracellular traffic of secretory vesicles depending on MTs. Importantly, TBCB is an essential protein, constituting a good target for new control strategies.
RESUMO - O Cofactor B da Tubulina de Toxoplasma gondii tem um papel central na invasão da célula hospedeira e na replicação - Os parasitas protozoários pertencentes ao Filo Apicomplexa são agentes patogénicos responsáveis por um vasto leque de doenças. Apesar da grande biodiversidade deste filo, os mecanismos moleculares adjacentes ao processo de invasão das células hospedeiras parecem ser conservados entre as diferentes espécies. O processo de invasão das células hospedeiras tem gerado grande interesse em vários grupos, incluindo o nosso, visto ser um importante alvo para o delineamento de estratégias médicas profilácticas e terapêuticas. Assim, nos últimos anos o nosso grupo tem vindo a interessar-se pelo estudo e compreensão do envolvimento do citoesqueleto de microtúbulos, tanto do parasita como da célula hospedeira, no processo de invasão. Os nossos resultados anteriores em Besnoitia besnoiti mostraram que este parasita, aquando da interação com a célula hospedeira, sofre alterações dramáticas na sua forma e superfície, acompanhadas pela remodelação de estruturas específicas de microtúbulos (MTs), nomeadamente os MTs subpeliculares. Estas alterações foram evidenciadas através de uma marcação distinta da tubulina na zona posterior do parasita. Para além disso, o citoesqueleto de MTs da célula hospedeira também responde à entrada do parasita, resultados que, posteriormente, foram também obtidos em Toxoplasma gondii. Estudos anteriores em T. gondii demonstraram que os MTs subpeliculares são muito estáveis. Esta estabilidade está possivelmente relacionada com modificações pós-traducionais (MPT) da tubulina, uma vez que, ao contrário dos vertebrados, estes organismos possuem uma família multigénica de α- e β-tubulinas composta por um número reduzido de membros. As MPTs referidas parecem modelar a interação dos MTs com as proteínas que lhes estão associadas. Mais ainda, em T. gondii, foram descritas proteínas que cobrem os MTs, num padrão complexo e definido, e que são importantes para a estabilidade dos mesmos. Deste modo, as proteínas que interagem com os MTs podem desempenhar um papel crucial na regulação do citoesqueleto do parasita aquando da invasão da célula hospedeira. Outras proteínas importantes para a regulação da dinâmica do citoesqueleto de MTs são os cofactores da tubulina, os quais participam nas vias de “folding”, dimerização e dissociação do dímero de tubulina. Estes cofatores controlam a proteostase da tubulina, através do controlo da “pool” de tubulina solúvel, participando na regulação da dinâmica dos MTs in vivo. Consequentemente, estas proteínas são candidatas a desempenhar um papel crucial nas modificações observadas no citoesqueleto de MTs do parasita aquando da invasão da célula hospedeira. Neste contexto o nosso objetivo principal foi avaliar e caracterizar o papel do Cofator B da Tubulina (TBCB de “Tubulin-binding cofactor B”) em T. gondii. Esta é uma proteína relativamente pequena que possui um domínio CAP-Gly na sua extremidade C-terminal e um domínio semelhante à ubiquitina (UBL de “ubiquitin-like”) na extremidade N-terminal. Em conjugação com o Cofactor E da tubulina (TBCE de “Tubulin binding cofactor E”), o TBCB dissocia o dímero de tubulina, controlando desta forma a “pool” de tubulina solúvel disponível na célula e consequentemente a dinâmica do citoesqueleto de MTs. A escolha do parasita protozoário T. gondii como modelo biológico deve-se ao facto de o mesmo possuir um genoma totalmente sequenciado e bem anotado, juntamente com o vasto conjunto de ferramentas disponíveis para a sua manipulação genética. Neste trabalho identificámos o gene do Tbcb em T. gondii, analisámos os níveis de expressão por RT-PCR durante o processo de invasão da célula hospedeira e de replicação, estudámos a localização intracelular do TgTBCB usando um anticorpo produzido no nosso laboratório e recorrendo a microscopia confocal e de super resolução, examinámos o fenótipo de TBCB em excesso (sobre-expressão por integração ao acaso) e de ausência do TBCB (deleção do gene utilizando o sistema CRISPR/Cas9). Nestes dois últimos casos foram criadas e selecionadas linhas transgénicas de parasitas, as quais foram analisadas em ensaios de crescimento (formação de pacas, invasão, replicação e egresso) bem como por western blot e por microscopia de fluorescência. Da análise dos níveis da expressão do Tbcb de T. gondii durante o processo de invasão e de replicação do parasita na célula hospedeira, notámos uma diminuição significativa dos níveis de expressão às 4 horas após a invasão da célula hospedeira, à qual se seguiu uma fase de recuperação desses níveis. Quanto à localização sub-celular do TgTBCB, observámos que em T. gondii esta proteína tem uma localização polarizada, estando localizada essencialmente no polo anterior, junto do conoide, podendo, por vezes, ser também observada uma marcação menos abundante no polo posterior. Constatámos ainda que o TgTBCB co-localiza parcialmente com as proteínas 2 e 3 das micronemas e com a tubulina glutamilada. Foi ainda possível constatar que na região apical o TBCB em T. gondii parece co-alinhar com os MTs subpeliculares, MTs que afunilam para estarem ancorados ao anel polar. Desta forma, o TBCB também parece estar junto ou imediatamente abaixo ao anel polar apical. Observámos que o excesso de TgTBCB causa uma queda acentuada na capacidade de formar placas de lise em tapetes celulares, a qual foi acompanhada de forma proporcional por uma diminuição notória dos níveis de invasão de células pelos parasitas. Curiosamente, não verificámos qualquer alteração na replicação ou no egresso dos mesmos. Em relação à deleção do gene Tbcb do parasita, 72 horas após a indução da CRISPR/Cas9 comprovámos a completa ausência de TgTBCB por western blot. Observámos também que a viabilidade dos parasitas sem TgTBCB não supera uma semana e que após a indução da deleção do gene, os parasitas demonstraram uma enorme redução na capacidade de invasão e também de replicação. Isto é, os poucos parasitas que conseguiam invadir as células hospedeiras apresentavam enormes problemas na replicação. Por western blot, nos extratos proteicos insolúveis, notámos uma diminuição nos níveis de a-tubulina, tubulina acetilada e poliglutamilada. Estes resultados também foram confirmados por imunofluorescência. Constatámos ainda que os parasitas sem TgTBCB apresentavam vários problemas de divisão, entre eles a alteração do eixo de divisão, a perda do controlo da divisão e a formação de células com morfologia arredondada, compatível com a perda de polaridade. Por microscopia eletrónica observámos também a perda de polaridade dos parasitas bem como a presença de núcleos de dimensões muito superiores ao normal ou dois núcleos dentro da célula, sem que a divisão celular tivesse sido concluída. Concluindo, o TgTBCB é uma proteína com uma localização polar, sendo observada no polo anterior abaixo do conoide e junto ao anel apical polar, acompanhado os MTs subpeliculares na região apical. A sua co-localização parcial com as proteínas das micronemas e com os MTs subpeliculares, bem como os seus parceiros já descritos em células de mamífero (proteínas de ligação aos MTs), juntamente com o fenótipo de invasão, sugerem que esta proteína em T. gondii poderá estar envolvida no tráfego vesicular ao longo dos MTs subpeliculares. A sobre-expressão do TgTBCB demonstrou a importância desta proteína no processo de invasão e a sua deleção provou que é essencial quer para a invasão quer para a replicação do parasita, visto que na ausência de TgTBCB há um comprometimento irreversível do citoesqueleto de MTs do parasita, levando à morte em menos de uma semana. Este fenótipo, aparentemente, está associado à diminuição dos MTs subpeliculares bem como à impossibilidade de formar novos MTs nas células filhas. Em suma, o TgTBCB é uma proteína essencial em T. gondii, podendo constituir um novo potencial alvo para novas estratégias de controlo e tratamento do parasita.
N/A
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Gherbovet, Olga. "Synthèse d'hybrides vinblastine-phomopsine." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00925057.
Abstract:
La tubuline est une protéine essentielle de la cellule. En polymérisant sous forme de microtubules, elle crée notamment le fuseau mitotique le long duquel migrent les chromosomes pendant la mitose. Les médicaments qui inhibent la polymérisation et/ou la dépolymérisation de la tubuline sont des composés majeurs de la thérapie anticancéreuse. Les vinca-alcaloïdes en sont des représentants importants. Ils induisent la mort des cellules par apoptose, en inhibant la dynamique des microtubules. D'autres molécules d'origine naturelle, comme la phomopsine A, se fixent sur la tubuline à proximité ou dans le même site de fixation que celui des vinca-alcaloïdes. C'est la raison pour laquelle nous avons envisagé d'élaborer des composés antimitotiques hybrides entre la vinblastine et la phomopsine A. Dans ce contexte, deux séries de composés ont été conçues. La première série d'hybrides correspondant à des dérivés de l'anhydrovinblastine fonctionnalisés en position 7'. Cependant, aucune des trois stratégies étudiées n'a permis d'accéder à ces composés. La deuxième série d'hybrides, dérivés de la 7'-homo-anhydrovinblastine a pu être synthétisée grâce à une réaction originale d'insertion d'acétylènes activés au niveau du pont gramine de la vinorelbine, suivie d'une réduction avec un contrôle totale de la régio- et stéréoselectivité. Dans un premier temps, les réactions d'insertion et de réduction ont été mise au point. Ensuite, deux familles d'hybrides portant la chaîne latérale de l'octahydrophomopsine en position 8' ou 7' ont été synthétisés. La plupart des composés ainsi obtenus possédent une excellente activité sur la tubuline et sont très cytotoxique.
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Aillaud, Chrystelle. "Modifications post-traductionnelles de la tubuline : identification des tubulines carboxypeptidases et découverte de nouveaux variants." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV049.
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Perrin, Aurélie. "Rôle des alpha-tubulines fongiques dans la symbiose ectomycorhizienne et dans les interactions champignons plantes." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10019.
Abstract:
Les champignons ont développé diverses interactions avec les végétaux. Ces interactions peuvent être bénéfiques pour la plante dans le cas des champignons établissant des symbioses mutualistes ou néfastes si le champignon est pathogène. Elles reposent sur des mécanismes moléculaires mal élucidés. Des études réalisées sur le champignon mutualiste Hebeloma cylindrosporum associé au pin Pinus pinaster ont permis de créer une collection de mutants affectés dans leur capacité à interagir avec les plantes et à former l’organe mixte de la symbiose, l’ectomycorhize. L’objectif de ma thèse a été d’étudier un mutant affecté dans le gène codant une alpha-tubuline Hctubα2. Les tubulines sont des protéines présentes chez tous les Eucaryotes et permettent la formation des microtubules, des éléments clés du cytosquelette. Chez les champignons, on trouve une ou deux alpha tubuline(s). H. cylindrosporum en possède deux. J’ai étudié l’expression de ces deux tubulines lors l’établissement de l’interaction avec les racines de l’hôte. Les résultats indiquent que ces deux gènes sont différentiellement exprimés lors de l’interaction. J’ai étudié au niveau protéomique l’impact de la mutation en comparant les protéomes intracellulaires des deux souches. On retrouve deux alpha-tubulines chez certains champignons phytopathogènes comme Botrytis cinerea. L’hypothèse de l’implication de l’alpha-tubuline 2 dans l’établissement de la pathogénie a été émise. J’ai donc construit des mutants de Botrytis cinerea dans lesquels ce gène a été inactivé. J’ai également tenté de localiser à l’aide de fusions traductionnelles chacune des alpha-tubulines chez le champignon mycorhizien et chez le pathogèneIn all terrestrial ecosystems, plants live in close interaction with numerous fungi. The interaction has a negative or positive effect on host plant depending upon the pathogenic or symbiotic status of the fungus. The establishment of these interactions is based on a tightly regulated molecular dialog between symbiotic partners. Previous studies on the ectomycorrhizal fungi, Hebeloma cylindrosporum associated with maritime pine (Pinus pinaster), created a collection of mutants affected in their mycorrhizal abilitiy. The aim of my thesis was to characterize one of these mutants affected in a gene, Hctubα2, encoding an alpha tubulin. Tubulins are eukaryotic cytoskeletal proteins involved in microtubules formation. Fungi have one or two alpha-tubulin. For example, H.cylindrosporum has two alpha-tubulin. The site of mutagenic DNA insertion in fungal genome was characterized. I studied the expression of both alpha-tubulins during the establishement of mycorrhizal interaction. Results showed that the two genes are differentially expressed during the interaction with host plant. At proteomic level, I studied the impact of the mutation comparing the two strains using 2D gel electrophoresis and sequencing differentially accumulated spots. Pathogenic fungi also bear two alpha-tubulins, as Botrytis cinerea. The hypothesis of the involvement of the alpha-tubulin 2 in pathogenesis was investigated. I created Botrytis cinerea mutants deleted for this gene. I also created translational fusions in order to visualize both alpha-tubulins in Hebeloma cylindrosporum and in Botrytis cinerea
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Yagdi, Efe Esma. "Analyse du rôle des dérivés de polysulfanes de l’ail dans le réseau microtubulaire et l'autophagie : l’effet anticancéreux dans le cancer colorectal." Thesis, Université de Lorraine, 2017. http://www.theses.fr/2017LORR0281/document.
Abstract:
Le cancer colorectal est une cause majeure de morbidité et de mortalité dans le monde entier. Des études épidémiologiques révèlent une corrélation inverse entre le risque de développer un cancer du côlon et un régime alimentaire riche en ail. De nombreux travaux scientifiques rapportent l'activité anti-cancéreuse des polysulfures de diallyle (PSDA) dérivés de l'ail dans divers types de cancer in vitro et in vivo. Le mécanisme d'action le mieux connu repose sur l'induction de l'arrêt mitotique suivi de l'apoptose. La tubuline est identifiée comme nouvelle cible thérapeutique des PSDA. La tubuline est fondamental dans la progression de l'autophagie, source nutritionnelle essentielle pour le développement du cancer au stade avancé, et l'activation de l'autophagie joue un rôle de chimiorésistance dans le traitement du cancer du côlon. L'hypothèse de ce projet est que les PSDA dérivés de l'ail interagissent avec la tubuline pour altérer l'organisation du réseau microtubulaire responsable de l'inhibition de la prolifération cellulaire et de la modulation de l'autophagie dans le cancer du côlon. Dans un premier temps, nous avons analysé l'impact du TTSDA/TTSDB sur le réseau microtubulaire. Nous avons montré que le TTSDA/TTSDB interagissait avec la tubuline par spectrométrie en masse. Nous avons montré que l'organisation microtubulaire est altérée dans les trois lignées cellulaires : HT-29 (mutées BRAF), SW480 (mutées KRAS) et SW620 (mutées KRAS, métastatiques), plus sensibles au TTSDB que le TTSDA. Dans un deuxième temps, nous avons étudié le rôle anticancéreux du TTSDB dans le cancer du côlon. Nous avons montré que le TTSDB induisait un arrêt mitotique suivi de la mort cellulaire dans toutes lignées confondues. Son activité antiproliférative est validée dans un système de culture 3D et in vivo. Nous avons aussi montré que l'effet du TTSDB est comparable aux agents altérant les microtubules. Dans un troisième temps, nous avons évalué l'impact du TTSDB dans l'autophagie. L'inhibition de l'autophagie est accompagnée par l'accumulation de la protéine p62, qui joue un rôle de survie dans les cellules HT-29 uniquement. Ensemble, nous avons identifié l'autophagie comme mécanisme de survie lors de l'arrêt mitotique prolongé en fonction du type cellulaire. Cette étude permettra d'envisager un ciblage thérapeutique selon le profil génétique du cancer du côlonColorectal cancer is a major cause of morbidity and mortality worldwide. Epidemiological studies reveal an inverse correlation between the risk of developing colon cancer and a garlic-rich diet. Many scientific studies reported the anti-cancer activity of diallyl polysulfides (DAPS) derived from garlic in various types of cancer in vitro and in vivo. The best-known mechanism of action is the induction of mitotic arrest followed by apoptosis. Here tubulin is identified as a new therapeutic target for DAPS. Tubulin is fundamental in the progression of autophagy, an essential energy source for the development of advanced cancer, and autophagy activation plays a role of chemoresistance against the treatment of colon cancer.The hypothesis of this project is that garlic-derived DAPS interact with tubulin to alter the microtubule network organization responsible for the inhibition of cell proliferation and modulation of autophagy in colon cancer.First, we analyzed the impact of DATTS/DBTTS on the microtubular network. We have shown that DATTS/DBTTS interacts with tubulin by mass spectrometry. We have shown that the microtubule organization is altered in the three cell lines: HT-29 (BRAF mutated), SW480 (KRAS mutated) and SW620 (metastatic, KRAS mutated), which were more sensitive to DBTTS than DATTS. In a second step, we studied the anticancer activity of DBTTS in colon cancer. We showed that DBTTS induced mitotic arrest followed by cell death in all cell lines. Its anti-proliferative activity is validated in a 3D culture system and in vivo. We have also shown that the effect of DBTTS is comparable to microtubule altering agents. In a third step, we evaluated the impact of DBTTS in autophagy. Inhibition of autophagy is accompanied by accumulation of the p62 protein, which plays a survival role in HT-29 cells only.Altogether, we identified here autophagy as a survival mechanism during prolonged mitotic arrest depending the cell type. This study will allow us to consider targeted therapy according to the genetic profile of colon cancer
40
Bouissou, Anaïs. "Rôle de la tubuline gamma et des protéines associées dans la dynamique des microtubules." Toulouse 3, 2011. http://thesesups.ups-tlse.fr/1151/.
Abstract:
Les microtubules sont des polymères dynamiques, essentiels à la division cellulaire. Ils sont souvent organisés à partir du centrosome où se localise la tubuline Gamma. Cette protéine joue un rôle important dans la nucléation des microtubules. Elle est présente sous forme de deux complexes: un petit complexe (Gamma-TuSC) essentiel à la viabilité et à l'assemblage d'un fuseau bipolaire fonctionnel et un complexe d'organisation supérieure (Gamma-TuRC) nécessaire au déroulement optimal de la mitose. L'objectif de ma thèse a été de caractériser le rôle des protéines spécifiques du Gamma-TuRC. La stratégie a consisté à inhiber ces protéines « non essentielles » par RNAi dans les cellules de drosophile en culture et à analyser les conséquences sur l'organisation et la dynamique du cytosquelette. En interphase, et pour la première fois chez les métazoaires, j’ai mis en évidence un rôle du Gamma-TuRC dans la dynamique des microtubules. Localisé le long des microtubules, ce complexe agit comme un facteur stabilisateur. En mitose, le Gamma-TuRC est présent sur tous les types de microtubules y compris sur les microtubules astraux. La déplétion du Gamma-TuRC induit des défauts de positionnement du fuseau. Ces anomalies sont corrélées à une modification des propriétés dynamiques des microtubules astraux qui participent à la liaison fuseau/cortex. L'ensemble de mes résultats démontre que le Gamma-TuRC participe à la régulation de la dynamique des microtubules en interphase et en mitose. Ce rôle permettrait au complexe d'être impliqué dans des fonctions non-centrosomales, comme l'organisation de sous-réseaux de microtubules ou le positionnement du fuseauMicrotubules are highly dynamic polymers, essential in cell division. They are often organized from the centrosome where the protein Gamma-tubulin plays an important role in microtubule nucleation. Gamma-tubulin acts within two main complexes: a small Gamma-tubulin complex (Gamma-TuSC) is essential for viability and assembly of a functional spindle, and a larger complex (Gamma-TuRC) is required for efficient mitotic progression. The role of Gamma-TuRC-specific proteins is not well defined. Using RNAi-mediated depletion in Drosophila S2 cells, I studied the function of these non-essential Gamma-TuRC proteins in microtubule organisation and dynamics. In interphase, I show for the first time that Gamma-TuRCs, localized along microtubules, regulate microtubule dynamics, acting as pause factors. In mitosis, Gamma-TuRCs are associated with all microtubule subsets, including astral microtubules. The loss of Gamma-TuRCs alters astral microtubule dynamics, correlated with spindle positioning defects. Together, these results demonstrate that Gamma-TuRCs regulate microtubule dynamics in interphase and in mitosis. We propose that Gamma-TuRCs are essential to mediate non-centrosomal functions such as organization of cell type-specific microtubule networks or spindle positioning
41
Calligaris, David. "Caractérisation du système microtubulaire par analyse top-down et protéomique d'affinité." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22951.
Abstract:
Le cytosquelette microtubulaire est une des cibles majeures en thérapie anticancéreuse. La caractérisation des isoformes d'une protéine est une problématique complexe en analyse protéomique. Pourtant il est crucial de mettre en évidence les variations de séquence primaire et les modifications post-traductionnelles qui peuvent dans certains cas être corrélées avec un phénomène physiologique et dans d'autres avec un état pathologique. Ainsi, la surexpression de l'isotype βIII de la tubuline, protéine constitutive des microtubules, peut avoir des conséquences importantes sur la régulation des propriétés dynamiques des microtubules et donc sur la réponse des tumeurs aux agents anticancéreux. Chaque isotype de tubuline se distingue principalement par les derniers acides aminés composant leur extrémité C-terminale. L'analyse top-down par MALDI-ISD est une approche de choix pour la caractérisation des isotypes de tubuline dont βIII. De plus, les propriétés spécifiques de fragmentation de cette protéine rendent possible son étude in situ sur coupes de tissue. Le choix de la matrice ainsi qu'une connaissance de la séquence primaire des protéines sont des paramètres importants lors des analyses MALDI-ISD. Une protéine telle qu'EB1, présentant une forte homologie de séquence C-terminale avec l'isotype α1B de la tubuline, est un candidat intéressant pour ce type d'approche. Cette protéine associée aux microtubules est le centre d'un réseau protéique régulant la dynamique des microtubules tel que dans le cas de l'angiogenèse tumorale. L'interaction entre EB1 et les protéines à domaine CAP-Gly se réalise par l'intermédiaire de son motif C-terminal -EEY où la tyrosine semble en être l'élément régulateur. La mise au point d'une approche de protéomique d'affinité pour l'identification et la quantification par spectrométrie de masse des complexe interagissant avec EB1 semble donc être un prérequis pour l'élaboration de nouveaux composés inhibant ce type d'interaction dans des contextes biologiques précisMicrotubule is one of the major targets in cancer therapy. Protein isoforms characterization is a complex issue in proteomic analysis. Yet is is crucial to highlight primary sequence variations and posttranslational modifications that are associated with physiological processes or pathologies. Thus overexpression of βIII-tubulin isotype, protein that make up microtubules, can have consequences on the regulation of microtubules dynamic properties and therefore tumors response to anticancer agents. Each tubulin isotype is distinguished by the last amino acids of their C-terminus. MALDI-ISD top-down analysis is of interest for tubulin isotypes characterization as βIII. In addition, specific fragmentation properties of this protein make possible its in situ study on tissue sections. Matrix choice and knowledge of proteins primary sequence are important parameters for MALDI-ISD experiments. As protein such as EB1, that presents high sequence homology with α1B-tubulin isotype C-terminus, is an interesting candidate for this kind of approach. This microtubule associated protein is the core of a protein netword that regulates microtubule dynamics such as in the cas of tumor angiogenesis. The interaction between EB1 and proteins with CAP-Gly domain is realized through its C-terminal motif -EEY and tyrosine seems to be the regulatory element. The development of an approach by proteomics affinity for the identification and the quantification by mass spectrometry of complex interacting with EB1 appears to be a prerequisite for the development of new compounds that inhibit this peculiar mode of protein-protein interaction in specific biological contexts
42
Khelifi, Ilhem. "Conception, synthèse et évaluation de nouveaux composés hétérocycliques analogues de l'isoCombrétastatine A-4 : vers des composés antivasculaires à effets secondaires amoindris." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS280.
Abstract:
La Combrétastatine A-4 (CA-4), molécule naturelle isolée d’un saule d’Afrique du sud est le chef de file des agents antivasculaire qui détruit sélectivement le réseau vasculaire tumoral et qui conduit à une nécrose ischémique d’une tumeur solide. Sa prodrogue, la fosbrétabuline (CA-4P, First-in-class) a reçu en 2016 le statut de médicament orphelin aux USA et en Europe pour le traitement de tumeurs neuro-endocrines et des glioblastomes multiformes. Malgré un intérêt thérapeutique certain, la fosbrétabuline a montré une instabilité chimique (isomérisation de la double liaison Z conduisant à l’isomère inactif E) et à des effets indésirables de type cardiotoxique. Notre groupe a conçu l’isoCA-4, une forme plus stable et non isomérisable de la CA-4 qui a montré des activités biologiques similaires à la CA-4. Il a été supposé que la cardiotoxicité de la CA-4 et de ses analogues soit probablement due à la présence du groupement 3,4,5-triméthoxyphényle considéré comme crucial pour l’activité cytotoxique et antitubuline. Afin de surmonter ce problème et contrairement au dogme précédent, nos efforts se sont été concentrés sur le remplacement de ce cycle par divers hétérocycles. Nous avons identifié trois nouvelles classes d’agents antivasculaires hétérocycliques voire bis-hétérocycliques. Nous avons montré que ces molecules “drug-like” sont douées d’une excellente activité antiproliférative à des concentrations nanomolaires, d’une activité antivasculaire supérieure à celle de la CA-4 et possèdent un index de sécurité cardiaque très élevé. Ces résultats démontrent pour la première fois que le remplacement du cycle 3,4,5-triméthoxyphényle de l’isoCA-4 par un hétérocycle approprié est une approche prometteuse pour identifier de nouveaux agents antivasculaires ayant une faible cardiotoxicitéCombretastatin A-4 (CA-4), is a natural antivascular agent isolated from a South African Sallow tree that selectively destroys tumor vasculature leading to ischemic necrosis. In 2016, the prodrug fosbretabulin (CA-4P) obtained designation as orphan drug in USA and Europe for the treatment of neuroendocrine tumors and glioblastoma. Despite its importance as a therapeutic agent, fosbretabulin has shown chemical instability. In fact, the double bond form Z isomerizes to an inactive E form of the drug. Moreover, fosbretabulin is associated to several side-effects including cardiotoxicity. Our group succeeded in the design of a more stable and non-isomerizable form of CA-4 as isoCA-4 which exhibited similar biological activities as CA-4. It was thought that cardiotoxicity of CA-4 and analogs is probably due to the presence of the 3,4,5-trimethoxyphenyl A-ring however the latter seems to have an essential role for the cytotoxic and antitubulin activities of the drug. Despite the role of the trimethoxyphenyl ring, we have focused our challenges on the remplacement of this moiety by various heterocycles to reduce the cardiotoxicity and to put an end to this dogma. We have identified three new classes of heterocyclic and bis-heterocyclic antivascular agents. We have demonstrated that these "drug-like" molecules have excellent antiproliferative activities at nanomolar ranges, an antivascular activity superior to that of CA-4 and possesses a very high cardiac safety index. Regarding these results, we have been able to show for the first time that the replacement of the 3,4,5-trimethoxyphenyl ring of isoCA-4 by various heterocyclic systems is a promising approach to synthesize new antivascular agents having a low level of cardiotoxicity
43
Marzo, Más Ana. "Síntesis y evaluación biológica de análogos de colchicina." Doctoral thesis, Universitat Jaume I, 2017. http://hdl.handle.net/10803/400868.
Abstract:
Esta Tesis doctoral titulada "Síntesis y evaluación biológica de análogos de colchicina" se enmarca en el campo de la química médica. El objetivo principal es la síntesis de una serie de análogos de colchicina y su posterior evaluación biológica. En dichos análogos el residuo acetilo del átomo de nitrógeno de la colchicina es sustituido por grupos α-aminoacilo derivados de aminoácidos, por grupos acilo alifáticos de diversos tipos y por grupos aroílo. En cuanto a la evaluación biológica, se ha ensayado la citotoxicidad en diferentes líneas celulares, tanto tumorales como no tumorales, los efectos en la polimerización de tubulina, tanto a nivel de proteína como a nivel celular, y por último la capacidad antiangiogénica y la capacidad antitelomerasa en células tumorales. Como conclusión cabe destacar que se ha demostrado que la mayoría de los análogos sintéticos son más citotóxicos y más activos que la propia colchicinaEsta Tesis doctoral titulada "Síntesis y evaluación biológica de análogos de colchicina" se enmarca en el campo de la química médica. El objetivo principal es la síntesis de una serie de análogos de colchicina y su posterior evaluación biológica. En dichos análogos el residuo acetilo del átomo de nitrógeno de la colchicina es sustituido por grupos α-aminoacilo derivados de aminoácidos, por grupos acilo alifáticos de diversos tipos y por grupos aroílo. En cuanto a la evaluación biológica, se ha ensayado la citotoxicidad en diferentes líneas celulares, tanto tumorales como no tumorales, los efectos en la polimerización de tubulina, tanto a nivel de proteína como a nivel celular, y por último la capacidad antiangiogénica y la capacidad antitelomerasa en células tumorales. Como conclusión cabe destacar que se ha demostrado que la mayoría de los análogos sintéticos son más citotóxicos y más activos que la propia colchicina.This doctoral Thesis entitled "Synthesis and biological evaluation of colchicine analogues" is framed in the field of medical chemistry. The main objective of this Thesis is the synthesis of a series of colchicine analogues and their subsequent biological evaluation. So that, colchicine analogues in which the acetyl residue of the colchicine nitrogen atom is substituted by α-aminoacyl groups derived from amino acids, aliphatic acyl groups of various types and aroyl groups have been synthetized. As for the biological evaluation, the cytotoxicity has been tested in different cell lines, both tumor and non-tumoral, the effects on the polymerization of tubulin, both at protein and at the cellular level, and finally the antiangiogenic capacity and the antitelomerase capacity in tumor cells. To sum up it is worth to highlight that it has been shown that most synthetic analogues are more cytotoxic and more active than colchicine itself.
44
Quesnoit, Mélanie. "Régulation de la dynamique microtubulaire : implication de la tubuline GTP et de protéines associées aux microtubules." Paris 11, 2007. http://www.theses.fr/2007PA114811.
Abstract:
Les microtubules (MTs) sont un des composants clés du cytosquelette, dont la dynamique est indispensable à la fonction. Dans ce projet, nous cherchons à aborder sous plusieurs angles la régulation de la dynamique microtubulaire, liée au comportement de la tubuline elle-même ou par l'intermédiaire de protéines associées aux MTs. Nous avons construit un outil dans le but d'avancer dans la caractérisation de l'importance de l'hydrolyse du GTP par la tubuline pour la stabilisation du polymère. Cet outil est un anticorps recombinant qui permet de détecter la tubuline liée au GTP et nous a conduit à émettre des hypothèses quant aux mécanismes intrinsèques de régulation de la dynamique des MTs. Nous avons de plus étudié le rôle de protéines liées aux microtubules et avons montré que l'une (CLIP170) en association avec un moteur moléculaire, est indispensable à la mise en place du réseau microtubulaire alors que l'autre (CLIPR59) ralentit in vivo la croissance des MTsMicrotubules (MTs) are highly dynamic protein polymers essential for intracellular organization. In the work presented here we have studied different aspects of the mechanisms regulating dynamic instability of MTs. We have selected and characterised an antibody recognizing GTP-bound tubulin and the use of this tool has led us to propose a new model for the intrinsic mechanisms regulating dynamic instability of MTs. We also studied the influence of MT associated proteins and found that the molecular motor Kinesin-1 and the +end tracking protein CLIP-170 cooperate to build the interphase network whereas another protein of the CLIP family, CLIPR-59, preferentially binds unpolymerised tubulin and slows down MT growth in vivo
45
Karamtzioti, Paraskevi 1990. "Tubulin modifications in human gametes : from the oocytes spindle to the sperm flagellum : Characterization of tubulin post translational modifications in female meiosis and sperm pathologies." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/670643.
Abstract:
This thesis aimed to characterize the tubulin PTM profile of human oocytes and spermatozoa. Tubulin rich structures play critical roles in the cellular behavior of human gametes. Mutations in tubulin or related proteins can affect oocyte maturation and flagellum motility. We first focused on tubulin post-translational modifications (PTMs) in the oocyte spindle and sperm flagellum. We characterized the PTM spindle profile of MII oocytes cultured in vitro and matured in vivo, and compared PTM enzyme transcript levels with two additional groups: GV and failed to mature oocytes. Further determination of the transcripts’ translational fate was performed using the cytoplasmic polyadenylation element code with verification experiments on Xenopus oocytes. Additionally, we sought to deteremine the pattern and levels of tubulin PTMs along the sperm tail and correlate these profiles with pathologies like asthenozoospermia and teratozoospermia.Esta tesis tuvo como objetivo caracterizar el perfil de PTM de los microtúbulos de ovocitos y espermatozoides humanos. Las estructuras ricas en tubulina juegan un papel fundamental en el comportamiento celular de los gametos humanos. Las mutaciones en la tubulina o proteínas relacionadas pueden afectar la maduración de los ovocitos y la motilidad del flagelo. En primer lugar, nos centramos en las modificaciones posteriores a la traducción (PTM) de la tubulina en el huso del ovocito y el flagelo del esperma. Caracterizamos el perfil de PTM del huso en ovocitos de MII cultivados in vitro y madurados in vivo, y comparamos los niveles de transcripción de PTM enzimas con dos grupos adicionales: GV y ovocitos que no maduraron. Además se estudió la regulación de la transcripción de los RNA mensajeros por el código del elemento de poliadenilación citoplásmica con experimentos en oocitos de Xenopus. Además, investigamos el patrón y los niveles de PTM de tubulina a lo largo de la cola del esperma y su correlacioón potencial con patologías como la astenozoospermia y la teratozoospermia.
46
Chargari, Cyrus. "Evaluation préclinique de trois nouvelles stratégies de radiosensibilisation pharmacologique : modulation de p53/Mdm2, perturbation de la dynamique des microtubules et ciblage de MET/Aurora B." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T010.
Abstract:
Les résultats insuffisants de la radiochimiothérapie conventionnelle ont motivé l’évaluation de nouvelles cibles afin de moduler la radiosensibilité tumorale: voies intrinsèques impliquées dans la réponse aux rayonnements ionisants, vascularisation tumorale, stroma non vasculaire. A travers cette thèse, nous avons évalué trois nouvelles stratégies de radiosensibilisation pharmacologique. Nous avons d’abord étudié en association à la radiothérapie l’intérêt de la modulation de l’axe p53/Mdm2 par le JNJ26854165, un inhibiteur de la dégradation de p53 par le protéasome. Les résultats in vitro et in vivo dans des xénogreffes sous-cutanées de cancers bronchiques non à petites cellules (CBNPC) montrent que cette stratégie permet d’améliorer significativement l’efficacité de la radiothérapie. Nous avons également rapporté des résultats encourageants in vitro dans plusieurs lignées cellulaires tumorales avec un nouvel agent antivasculaire ciblant la tubuline, l’EHT 6706. Cette stratégie augmentait l’efficacité de l’irradiation et potentialisait l’effet antiprolifératif de certains agents de chimiothérapie conventionnelle. Enfin, le développement le plus abouti a consisté en l’évaluation de l’association d’un triple inhibiteur de MET/AXL/FGFR en association à l’irradiation in vitro et dans des modèles de CBNPC implantés en xénogreffes sous-cutanées, mais également sous forme de tumeurs pulmonaires orthotopiques. Cet agent pharmacologique potentialisait l’efficacité de la radiothérapie dans des lignées ne surexprimant pas MET. Il est apparu que l’activité de la drogue faisait intervenir, au moins partiellement, l’inhibition de l’activité d’acteurs de la cytocinèse. Ces trois évaluations, qui s’inscrivent dans la recherche translationnelle, montrent l’importance de la recherche préclinique pour les études d’association aux rayonnements ionisants. Seul un développement préclinique rationnel permettra de faire émerger de nouveaux standards dans le domaine de la biomodulation pharmacologique de la radiosensibilité tumoraleInsufficient results of conventional chemoradiation have encouraged assessment of new targets for radiosensitization: intrinsic cellular pathways involved in radiation response, tumor angiogenesis, and nonvascular stroma. We have investigated these three strategies for pharmacological radiosensitization. First, we examined the usefulness of targeting p53/Mdm2 pathway in combination with irradiation. In vitro and in vivo results obtained in non-small cell lung carcinoma (NCSLC) showed that this strategy was promising for enhancing radiation efficacy. We also found encouraging results within several cell lines with a novel vascular disrupting agent targeting tubulin. This strategy enhanced radiation effects and also increased the antiproliferative effects of various chemotherapeutics. Finally, the most advanced preclinical development was obtained with a novel MET/AXL/FGFR inhibitor, which improved effectiveness of radiation therapy in vitro and in subcutaneous and orthotopic models of non MET-dependent cell cancer lines. This effect was not only related to an inhibition of stroma/cancer cell interactions, as it probably involved activity toward actors of cytocinesis. These studies, which are part of translational research, highlight the importance of preclinical investigations in the area of radiation research. Only rationale preclinical development will allow new standards to emerge for pharmacological modulation of tumor radiosensitivity
47
Hage-Sleiman, Rouba. "Impact of tululin binding cofactor C (TBCC) on microtubule mass and dynamics, cell cycle, tumor growth and response to chemotherapy in breast cancer." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10085/document.
Abstract:
La mise en conformation de l’α et β tubulines en hétérodimeres polymérisables nécessite l’intervention de cinq protéines « Tubulin Binding Cofactors » (TBCA a TBCE) dont TBCC qui joue un rôle indispensable. Dans des cellules humaines d’adénocarcinome mammaire, nous avons modifié le niveau d’expression de TBCC et nous avons montre que ceci avait un impact sur le contenu des fractions de tubuline, la dynamique des microtubules ainsi que sur le phénotype et chimiosensibilité des cellules. La distribution en cycle cellulaire et les durées de la mitose et de la phase S ont été altérées. La modification de TBCC avait un faible effet sur la vitesse de prolifération in vitro par contre les cellules présentaient des différences significatives de croissance tumorale in vivo. Les réponses aux agents antimicrotubulaires et à la gemcitabine ont montrées une chimiosensibilité dépendante de la distribution en cycle cellulaire. Tous ces résultats montrent l’importance de la régulation du contenu en tubulines et l’impact de ceci sur le comportement de la cellule en général et vis-à-vis des traitementsThe proper folding pathway of α and β-tubulin into the α/β-tubulin heterodimers involve five Tubulin Binding Cofactors (TBCA to TBCE). TBCC plays a crucial role in the formation of polymerization-competent the α/β-tubulin heterodimers. To evaluate the impact of microtubule mass and dynamics on the phenotype and chemosensitivity of breast cancer cells, we targeted TBCC in human breast adenocarcinoma and developed variants of breast cancer cells with modified content of TBCC. We have shown that the modifications in TBCC expression level influenced tubulin fraction distribution and microtubule dynamics. Cell cycle distribution and the durations of mitosis and S-phase were altered. The proliferation rate in vitro was slightly modified whereas in vivo the TBCC variants presented major differences in tumor growth capacity. Chemosensitivity to antimicrotubule agents (paclitaxel and vinorelbine) as well as to gemcitabine was observed to be dependent on the cell cycle distribution of the TBCC variants. These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype, cell cycle progression and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterization of resistant phenotypes
48
Jürgens, Lukas Julian Christoph [Verfasser], Kristen [Gutachter] Rak, and Michael [Gutachter] Sendtner. "Spatio-temporale Distribution der Tubuline und Tubulin spezifischen Chaperone im sensorischen Epithel der murinen Cochlea / Lukas Julian Christoph Jürgens ; Gutachter: Kristen Rak, Michael Sendtner." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1213247543/34.
49
Bosson, Anouk. "A la recherche de l'enzyme de détyrosination du C-terminus de l'α-tubuline." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV025.
Abstract:
Dans les cellules, les microtubules interviennent dans de nombreux événements comme le maintien de l'architecture, la division du matériel génétique, la migration cellulaire ou encore le transport de vésicules et d'organites. Les modifications post-traductionnelles du le C-terminus de la tubuline, bloc de base des microtubules, apparaissent comme très impliquées dans la régulation de ces fonctions car elles régulent le recrutement de leurs nombreux partenaires protéiques. Durant ma thèse, je me suis particulièrement intéressée à une de ces modifications post traductionnelles : le cycle de détyrosination/tyrosination du C-terminus de l'α-tubuline. Ce cycle implique deux enzymes la Tubuline CarboxyPeptidase (TCP), qui clive la tyrosine à l'extrémité de l'α-tubuline, et la Tubuline Tyrosine Ligase (TTL) qui ré-additionne une tyrosine à la tubuline détyrosinée. Des études menées sur ce cycle et notamment la découverte de la TTL ont permis de montrer que la présence d'une tyrosine à l'extrémité C-terminale de l'α-tubuline est indispensable au développement neuronal et que son absence favorise la progression tumorale. La TCP quant à elle est encore inconnue et sa découverte apparaît essentielle afin de pouvoir appréhender le cycle de détyrosination/tyrosination dans sa globalité. Avec pour fil rouge l'identification de la TCP, mon travail s'est déroulé en trois temps. Je me suis tout d'abord intéressée à la protéine EB1. Cette protéine se lie à l'extrémité positive des microtubules où elle recrute de nombreux partenaires microtubulaires. EB1, présente le même C-terminus que l'α-tubuline et notamment une tyrosine terminale indispensable à la liaison des protéines à CAP-Gly. Dans des cellules et des tissus sains j'ai montré qu'EB1 n'existe pas sous forme détyrosinée ce qui souligne la spécificité de la TCP pour l'α-tubuline. Dans un second temps, j'ai participé à l'étude d'une famille de carboxypeptidases cytosoliques impliquées dans la neurodégénérescence, les CCPs, parmi lesquelles nous pensions trouver la TCP. Nous avons montré que quatre de ces enzymes (CCP1, 4, 5, et 6) retirent les glutamates présents de manière latérale au C-terminus de la tubuline. Les CCP1, 4 et 6 peuvent également cliver le dernier glutamate de la tubuline détyrosiné générant de l'α-tubuline où les deux derniers acides aminés ont été clivés (tubuline-Δ2). Aucune de ces carboxypeptidases ne se révélant être la TCP, j'ai mis en place une méthode biochimique dans le but de purifier cette enzyme. Après plusieurs étapes de purification à partir de cerveaux de souris, des préparations enrichies en activité carboxypeptidase ont été obtenues. Les analyses spectrométriques et bioinformatiques de ces préparations ont permis d'isoler des candidats TCP actuellement testés pour leur potentielle activité de détyrosination du C-terminus de l'α-tubuline. Si la TCP n'est pas présente parmi eux, les outils développés lors de cette étude devraient permettre une très prochaine identification de cette enzyme essentielleMicrotubules control many aspects of cellular function. Those polymers of tubulin are involved in numerous events ranging from the maintenance of cell architecture to cell division and migration through the transport of vesicles and organelles. The post-translational modifications of the C-terminus of tubulin appear to be involved in the regulation of microtubule functions by recruiting different effectors at the growing end of microtubules. During my PhD, I focused on one of these post-translational modifications: the detyrosination/tyrosination cycle of the C-terminal α-tubulin. This cycle involves the enzymatic removal of the C-terminal tyrosine of α-tubulin by an uncharacterized tubulin carboxypeptidase (TCP) and the re-addition of a tyrosine residue by the Tubulin-Tyrosine-Ligase (TTL) isolated in 1975. On one hand, tubulin tyrosination is important in neuronal organization whereas TTL suppression in human cancers is associated with tumor aggressiveness. Those defects are in part due to the failure of microtubule partners to bind detyrosinated microtubules. My project was divided into three main parts. I have first studied EB1, a microtubule plus end tracking protein which recruits many proteins at the microtubule plus end. This protein ends with the same amino acids as does α-tubulin. As in tubulin, the tyrosine terminal is important for the binding of EB1 partners. I showed that EB1 does not exist under detyrosinated form underlying the TCP specificity. Then, I collaborated to identify the function of a carboxypeptidase family within which we thought we could find the TCP. Four members of this family are deglutamylating enzymes (CCP1, CCP4, CCP5 and CCP5). Three of them (CCP1, CCP4 and CCP6) can cleave the last glutamate of detyrosinated tubulin to generate tubulin without the last two C-terminus amino acids (Δ2-tubulin). However, none of them was identified as the TCP. I consequently developed a biochemical approach to find this enzyme. Extracts enriched in carboxypeptidase activity after purification steps from mice brain were analyzed by mass spec and bioinformatics. Some candidates are currently tested for their potential C-terminus α-tubulin detyrosinating activity. The tools developed here should allow for pending identification of the TCP
50
Mouton, Carole. "La podophyllotoxine et ses dérivés inhibant la polymérisation de la tubuline et/ ou l'ADN-topoisomérase II." Paris 5, 1994. http://www.theses.fr/1994PA05P153.