Dissertations / Theses on the topic 'Tubulin'

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

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 survie
Beyond 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

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 nucléotide
Kinesins 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

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas
Tubulin 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

Les microtubules sont un élément important du cytosquelette et de réaliser une variété de fonctions essentielles. La diversité fonctionnelle des microtubules provient de différentes diverses - et-tubuline isotypes qui sont exprimés dans la cellule, et une vaste gamme de réversibles modifications post-traductionnelles. tyrosination Tubulin est l'un de ces modifications exécutées par la tubuline tyrosine ligase (TTL). TTL est le membre fondateur du 14 tyrosine ligase membres tubuline comme (TTLL) de la famille. Wasylyk laboratoire a trouvé TTLL12 être exprimés de manière différentielle dans la tête et du cou et le cancer de la prostate. L'analyse fonctionnelle sur TTLL12 révélé qu'il peut réguler nitrotyrosination tubuline. Nitrotyrosine - un analogue structural de la tyrosine est présent dans les cellules dans des conditions pathologiques et est incorporé sur le α-tubuline ce qui entrave le fonctionnement normal des microtubules. nitrotyrosination Tubulin est préjudiciable à la croissance des cellules. Nous montrons que la surexpression de TTLL12 conduit à diminuer en α-tubuline nitrotyrosination et vice versa. Nous montrons que nitrotyrosination α-tubuline affecte la croissance cellulaire dans les A549 et cellules HEp-2. On voit aussi que TTLL12 peut modifier α-tubuline nitrotyrosination et affecter la croissance des cellules Hep-2 et des cellules DU145. Ainsi TTLL12 pourrait jouer un rôle important dans la régulation de la croissance cellulaire ou la survie des cellules dans les tumeurs avec des niveaux accrus de nitrotyrosine. Nous avons développé une analyse à haut débit de trouver des composés qui peuvent augmenter nitrotyrosination tubuline par TTLL12, TTL ou d'autres mécanismes. Le criblage d'une bibliothèque de 10000 composés donné lieu à deux tubes potentiels qui ont augmenté nitrotyrosination tubuline. Des études plus approfondies de ces visites sur la croissance cellulaire en présence de nitrotyrosine et mécanisme d'action est en cours
Microtubules are an important component of the cytoskeleton and carry out a variety of essential functions. Functional diversity of microtubules comes from various different - and -tubulin isotypes that are expressed within the cell, and an extensive array of reversible post-translational modifications. Tubulin tyrosination is one of such modifications executed by tubulin tyrosine ligase (TTL). TTL is the founding member of 14 member tubulin tyrosine ligase like (TTLL) family. Wasylyk’s laboratory found TTLL12 to be differentially expressed in head and neck cancer and prostate cancer. Functional analysis on TTLL12 revealed that it can regulate tubulin nitrotyrosination. Nitrotyrosine - a structural analogue of tyrosine is present in cells in pathological conditions and is incorporated on the α-tubulin thus hampering the normal functioning of microtubules. Tubulin nitrotyrosination is detrimental to cell growth. We show that over expression of TTLL12 leads to decrease in α-tubulin nitrotyrosination and vice versa. We show that α-tubulin nitrotyrosination affects cell growth in A549 and HEp-2 cells. We further show that TTLL12 can alter α-tubulin nitrotyrosination and affect the cell growth in HEp-2 and DU145 cells. Thus TTLL12 could play an important role in the regulation of cell growth or cell survival in tumors with increased levels of nitrotyrosine. We developed a high throughput assay to find compounds which can increase tubulin nitrotyrosination via TTLL12, TTL or other mechanisms. Screening a library of 10000 compounds resulted in two potential hits which increased tubulin nitrotyrosination. Further investigations of these hits on cell growth in the presence of nitrotyrosine and mechanism of action is in progress

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.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.
Includes bibliographical references.
The heterodimerization of α- and β-tubulin represents a critical early step in microtubule morphogenesis. In vitro studies have defined a pathway that mediates the incorporation of monomeric tubulin polypeptides into heterodimer. The components of this pathway, tubulin cofactors, are dispensable for growth in Saccharomyces cerevisiae under laboratory conditions. Yet, these proteins are required for survival under conditions of stress or in the presence of a weakened tubulin heterodimer. This finding suggests cofactors may function in vivo to promote reformation of dissociated tubulin heterodimer. In order to carry out this activity, cofactors are thought to facilitate the association of tubulin monomers without likewise promoting the dissociation of tubulin heterodimer. However, the mechanism of cofactor activity in vivo and the method by which these proteins achieve vectorial catalysis of heterodimerization has remained obscure. In this study, we present evidence that several endogenous tubulin cofactors associate with one another in vivo and bind tubulin monomer under conditions of stress. We also provide physical and genetic data suggesting that Cin4p, an ARF family GTPase, associates with the tubulin cofactor Cin1 p (cofactor D) and promotes tubulin heterodimerization by modulating Cin1 p's association with β-tubulin. Through site-directed mutagenesis, we conclude that Cin4p GTPase activity is important for these functions. These data support a model in which the production of tubulin heterodimer via a putative cofactor complex is coupled to nucleotide hydrolysis by a small GTPase. The linkage of these reactions could serve to impart directionality to the activity of tubulin cofactors, allowing them to selectively promote tubulin heterodimerization without also catalyzing heterodimer dissociation.
by John M. Doll.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1988.
On t.p. [alpha] appears as the original Greek letter.
Includes bibliographies.
by Peter Joseph Schatz.
Ph.D.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.
Includes bibliographical references.
A variety of cellular factors regulate the formation of complex microtubule structures in the cell. Most of these factors modulate microtubule structures by affecting the polymerization of [alpha]/[beta]-tubulin heterodimer subunits to form microtubules. However, there is another class of factors that affect microtubule formation by regulating the steps that precede the polymerization of [alpha]/[beta]-tubulin heterodimer. One of these factors is Rbl2p. Rbl2p was originally isolated in a screen for genes that when overexpressed can rescue the lethality of [beta]-tubulin overexpression. The mammalian homolog of Rbl2p, Cofactor A, was isolated as a non-essential cofactor in an in vitro tubulin folding reaction (Tian et al., 1996). Both Rbl2p and Cofactor A form a heterodimer with monomeric [beta]-tubulin that excludes [beta]-tubulin (Archer et al., 1995; Melki et al., 1996). The assays that identified Rbl2p and its homolog Cofactor A suggest two possible models to explain the ability of Rbl2p/CofactorA to protect cells from free [beta]-tubulin in vivo. First if unfolded [beta]-tubulin is toxic, Rbl2p could protect cells from free [beta]-tubulin by catalyzing the folding of [beta]-tubulin1 as proposed in the in vitro tubulin folding assay. Second, Rbl2p could bind to and sequester free [beta]-tubulin into a Rbl2p/[beta]-tubulin heterodimer. Our data suggest that Rbl2p's in vivo function is more complicated than predicted by either of these two models. Rbl2p binds transiently to a subpopulation of the free [beta]-tubulin and prevents it from interacting with the target of [beta]-tubulin toxicity until it associates with an aggregate of [beta]-tubulin. Cells expressing an allele of [alpha]-tubulin, tubl-729, arrest in the cold as largebudded cells with either short spindles or no microtubules. The spindle defect checkpoint proteins Bublp and Bub3p suppress the cold-sensitivity of tubl-729 cells (Guenette et al., 1995). We tested whether other proteins involved in the mitotic checkpoints can suppress tubl-729 cells. Our results show that an extra genomic copy of MPSl also suppresses the cold-sensitivity of tubl-729 cells, but that genomic copies of BUB2 and MADsl-3 do not. Bub3p rescues the coldsensitivity of tubl-729 cells in which deletion of MAD2 has eliminated the spindle defect checkpoint. This suggests that Bub3p does not act through the spindle defect checkpoint to suppress tubl-729 cells. We present preliminary evidence in support of a model proposing that tubl-729 cells are defective in kinetochoremicrotubule attachment and that Bublp, Bub3p, and Mpslp may suppress the phenotypes of tubl-729 cells by strengthening these attachments.
by Katharine Boyer Compton Abruzzi.
Ph.D.

タウオパチー変性神経細胞では、本来軸索に局在する微小管結合タンパク質タウが細胞体や樹状突起に蓄積するとともにチューブリン・微小管が減少する。この広範な神経細胞骨格系の変性は、タウの異常リン酸化に起因すると考えられてきた。しかし、タウの異常性獲得と微小管の消失との関係は未だ不明な状態にある。本研究では、初代培養神経細胞でαチューブリンの分子シャペロンであるTubulin-specific chaperon Eの発現抑制を行うことで、チューブリンの恒常性破綻を誘導するともに、それとタウの異常性獲得の関連性について検討した。
Tauopathy is a type of neurodegenerative disorder including Alzheimer's disease defined by formation of tau filamentous inclusion in neurons. Tau is a microtubule associated protein localized in axon and assumed to promote microtubule stabilization in healthy neuron. In contrast, accumulation of hyperphosphorylated tau in somatodendrite and loss of microtubules (tubulin) are observed in tauopathy neuron. Although it is believed that abnormal phosphorylation of tau results in neurodegeneration, the relation with tau abnormalities and microtubule loss remains unclear. To investigate whether disruption of tubulin homeostasis induce tau abnormalities, we performed a miRNA-mediated knockdown of tubulin-specific chaperon E, an essential factor for the formation of alpha and beta tubulin heterodimeric complex, in mouse primary hippocampal neuron.
博士(理学)
Doctor of Philosophy in Science
同志社大学
Doshisha University

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 cardiotoxicité
Combretastatin 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

Tubulin, microtubules and associated proteins were studied theoretically, computationally and experimentally in vitro and in vivo in order to elucidate the possible role these play in cellular information processing and storage. Use of the electric dipole moment of tubulin as the basis for binary switches (biobits) in nanofabricated circuits was explored with surface plasmon resonance, refractometry and dielectric spectroscopy. The effects of burdening the microtubular cytoskeleton of olfactory associative memory neurons with excess microtubule associated protein TAU in Drosophila fruitflies were determined. To investigate whether tubulin may be used as the substrate for quantum computation as a bioqubit, suggestions for experimental detection of quantum coherence and entanglement among tubulin electric dipole moment states were developed.

Microtubules are a highly dynamic component of the cytoskeleton, which are crucial for many cellular processes. Microtubules are comprised of α/β-tubulin heterodimers, which are subject to multiple post-translational modifications including the detyrosination/tyrosination cycle. This cycle involves the removal of an RNA-encoded C-terminal α-tubulin tyrosine residue by tubulin carboxypeptidase, followed by the reattachment of the tyrosine residue by tubulin tyrosine ligase. This research project is focused on the development of an inhibitor against tubulin tyrosine ligase and tubulin carboxypeptidase. The precise function of this cycle has yet to be determined; an inhibitor could function as a chemical biology tool that could be used to study the physiological effects of the detyrosination/tyrosination cycle. This thesis details the design and synthesis of phosphinic acid and phosphonic acid peptide analogue inhibitors. Progress towards the synthesis of a dipeptide phosphinic acid is reported; due to complications in phosphorus-carbon bond forming reactions the total synthesis was not completed. The focus of the research project changed to the synthesis of phosphonic acid peptide analogue inhibitors. A dipeptide phosphonic acid inhibitor was successfully synthesized, but was inactive against tubulin tyrosine ligase. Progress towards the synthesis of a tripeptide phosphonic acid inhibitor is reported; due to complications in the final deprotection steps the total synthesis was not completed. A pentapeptide phosphonic acid inhibitor was successfully synthesized, and it showed moderate inhibitor activity against tubulin tyrosine ligase.
Science, Faculty of
Chemistry, Department of
Graduate

Chromosome segregation is the crucial step for cell division in most living organisms. In order to segregate the chromosomes equally into mother and daughter cells, the cell needs to build a spindle, which comprises three major components: chromosomes, microtubules and microtubule organizing center (centrosome in animal cells or spindle pole body in fungal cells). The building and normal execution of spindle function are tightly linked to cell cycle control system. gamma-Tubulin is an evolutionally conserved protein with its canonical function as a microtubule nucleator. Here we report that gamma-tubulin is phosphorylated at residue S360, a Cdk1/Cdc28 site. Phosphorylation of S360 in vivo was identified in a global analysis of the phosphoproteome of the spindle pole body (J. Keck and M. Jones, et al.). We confirmed Cdc28-Clb2 can phosphorylate S360 by in vitro kinase assay and peptide identification by mass spectrometry, and in vivo assay using two dimensional-polyacrylamide gel electrophoresis (2D-PAGE). A phospho-mimetic mutation (tub4-S360D) causes mitotic delay but does not inhibit recruitment of the gamma-tubulin complex (reported by GRIP Spc97-EGFP) to spindle poles. Analysis of synthetic genetic interactions and live cell imaging revealed that cytoplasmic microtubule function is normal in tub4-S360D cells but spindle microtubule function is perturbed. High-resolution analysis of spindle dynamics revealed fluctuations in length in metaphase and anaphase spindles. The velocities of spindle elongation in anaphase are similar in S360D and WT spindles, but the initial phase of rapid elongation in anaphase is prolonged in the S360D mutant, and spindle breakdown is delayed. Interpolar microtubules play a central role in spindle assembly and are actively responsible for spindle elongation with sliding forces generated from microtubule cross-linking proteins such as kinesin-5 (Cin8 in budding yeast). Genetic analysis indicates that mutations in proteins that act redundantly to Cin8 enhance the growth defect in S360D, and S360A can partially suppress deletion of Cin8. High-resolution tomography analysis of S360D mutant and WT cells reveals that the interpolar microtubules organization in S360D is completely perturbed, as almost none overlapping of interpolar microtubules were observed.Therefore, we conclude that S360 phosphorylation of gamma-tubulin/Tub4 by Cdk1/Cdc28 plays an important role in the control of spindle microtubule organization during spindle assembly, through docking and/or monitoring critical microtubule associated proteins, for example Cin8, at microtubule minus end hence regulating their functions on the plus end.
La ségrégation des chromosomes est une étape cruciale pour la division des cellules de la plupart des être vivants. Pour que les chromosomes ségrégent également entre les cellules mère et fille, la cellule doit produire un fuseau comprenant trois composants : les chromosomes, les microtubules et un centre d'organisation des microtubules (le centrosomes chez les cellules animales ou le spindle pole body chez les cellules fongiques). La production et l'exécution normale de la fonction du fuseau sont étroitement couplées au système de contrôle du cycle cellulaire.La gamma-tubuline, une protéine très conservée dans l'évolution, a une fonction de nucléation des microtubules. Nous reportons ici que la gamma-tubuline est phosphorylée au résidu S360, un site pour Cdk1/Cdc28. Initialement identifiée in vivo lors d'une analyse globale du phosphoprotéome du spindle pole body (J. Keck and M. Jones, et al.), la phosphorylation de S360 par Cdc28-Clb2 est confirmée par nos travaux in vitro grâce à des essais kinase suivis d'identification de peptides par spectroscopie de masse et in vivo grâce à l'électrophorèse bidimensionnelle en gel de polyacrylamide (2D-PAGE). Une mutation phospho-mimétique (tub4-S360D) provoque un délai mitotique mais n'inhibe pas le recrutement du complexe de gamma-tubuline (détecté grâce à GRIP Spc97-EGFP) aux pôles du fuseau. Les études des interactions génétiques synthétiques et d'imagerie sur cellules vivantes révèlent que la fonction cytoplasmique des microtubules est normale chez le mutant tub4-S360D alors que la fonction des microtubules au niveau du fuseau est perturbée. Une analyse dynamique à haute résolution révèle des fluctuations de la longueur des fuseaux en métaphase et en anaphase. Bien que les vitesses d'élongation du fuseau durant l'anaphase soient similaires chez le mutant S360D et chez le sauvage, la phase initiale d'élongation rapide en anaphase est prolongée et la déconstruction du fuseau est retardée chez la mutant S360D.Les microtubules interpolaires jouent un rôle central dans l'assemblage du fuseau est sont activement responsables de l'élongation du fuseau grâce aux forces de glissement générées par les protéines liant les microtubules telle la kinesine-5 (Cin8 dans la levure de boulangerie). Une analyse génétique indique que des mutations de protéines redondantes avec Cin8 augmentent le défaut de croissance du mutant S360D alors que la mutation S360A supprime partiellement la délétion de Cin8. L'analyse tomographique à haute résolution de cellules du mutant S360D et de cellules sauvages révèle une perturbation extrême de l'organisation des microtubules interpolaires du mutant S360D avec très peu de microtubules interpolaires chevauchants observés.En conséquence, nous concluons que la phosphorylation du résidu S360 de la gamma-tubuline/Tub4 par Cdk1/Cdc28 joue un rôle important dans le contrôle de l'organisation des microtubules au cours de l'assemblage du fuseau, via l'arrimage et/ou le contrôle de protéines associées aux microtubules à l'extrémité (-) des microtubules, comme Cin8, et la régulation de leur fonction à l'extrémité (+) des microtubules.

YB-1 est un régulateur important de l’expression des gènes dans les cellules eucaryotes. En plus de son rôle dans la transcription, YB-1 joue un rôle clé dans la traduction et la stabilisation des ARN messagers. Nous avons identifié plusieurs nouveaux partenaires de la protéine YB-1 par chromatographie d’affinité à partir de différents extraits tissulaires. Parmi ces partenaires, nous avons démontré que YB-1 interagit avec la tubuline et les microtubules et stimule fortement l'assemblage des microtubules in vitro. Les microtubules assemblés en présence de YB-1 ont une ultrastructure normale, et les données montrent que YB-1 recouvre probablement la surface extérieure des microtubules. De la même façon YB-1 stimule aussi l'assemblage de la tubuline-MAP qui est plus proche des complexes protéiques qui existent dans la cellule, et de la tubuline clivée par subtilisine ce qui suggère que son interaction avec la tubuline ne relève pas seulement d’effets électrostatiques. Nous avons enfin découvert que la tubuline interfère avec la formation des complexes ARNm:YB-1. Ces résultats suggèrent que YB-1 peut réguler l'assemblage des microtubules in vivo et que son interaction avec la tubuline peut contribuer à la régulation de la traduction des ARN messagers. En effet, in vivo, la traduction des mRNPs dépend de l’état de saturation de l’ARN messager par YB-1. Nous avons montré ici que lorsque le rapport YB-1:ARNm est faible, les complexes mRNPs possèdent des structures non-compactes, alors que les mRNPs saturés sont compacts. Ce changement structural est observé de façon parallèle à l'inhibition de la traduction des ARN messagers lorsqu’ils passent des polysomes (traduits) aux mRNPs libres (non traduits). De façon intéressante, nous avons découvert que les mRNPs saturés se lient aux microtubules via des interactions protéine:protéine et ont tendance à former des agrégats sur la surface des microtubules. Cette dernière propriété pourrait contribuer à la formation de granules de stress et à la localisation des mRNPs dans le cytoplasme. Finalement, un modèle de diffusion facilité a été développé pour expliquer l'assemblage des microtubules orchestré par les polyamines naturelles (telles que YB-1 qui sont positivement chargées dans la cellules). L’ensemble de ces données contribuent à une meilleure compréhension de processus biologiques fondamentaux concernant l’assemblage de la tubuline en microtubules et le trafic des ARN dans la cellule. Ils pourraient avoir un intérêt pour développer de nouveaux médicaments qui ciblent les microtubules
YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs. We identify several novels YB-1 protein partners by affinity chromatography of different tissue extracts. We observed that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly in vitro. Microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure where YB-1 probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Additionally, we demonstrated that tubulin interferes with mRNA:YB-1 complexes. These results suggest that YB-1 may regulate microtubule assembly in vivo and that its interaction with tubulin may contribute to the control of mRNA translation. The translational status of mRNPs in vivo depends on amount of YB-1 associated with mRNA. We show here that at low YB-1:mRNA ratios mRNP complexes possess an incompact structures, whereas saturated mRNPs are compact. This structural change corresponds to translation inhibition when mRNA moves from polysomal (translatable) to free (untranslatable) mRNPs. Saturated mRNPs bind to microtubules via protein:protein interactions and tend to self-aggregate on microtubule surface. This property could contribute to stress granule formation, mRNPs traffic and localization of translation apparatus within cytoplasm. Finally, the facilitated diffusion model was developed to explain enhancement of microtubule assembly by positively charged natural polyamines in living cells. Altogether our data contribute to the understanding of fundamental biological processes

Thèse de doctorat : Biologie cellulaire et moléculaire : Evry-Val d'Essonne : 2008. Thèse de doctorat : Biologie cellulaire et moléculaire : Institute of protein Research Russian Academy of Sciences RUSSIE : 2008.
Titre provenant de l'écran-titre.

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.

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 pathogène
In 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

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 côlon
Colorectal 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

Mutations in LRRK2 are a common cause of Parkinson’s disease (PD). LRRK2 encodes leucine-rich repeat kinase 2 (LRRK2), a ROCO protein. It has an enzymatic core consisting of a Ras of complex proteins (Roc) GTPase domain and kinase domain, surrounded by protein-protein interaction regions. Pathogenic LRRK2 mutations modify activity in these enzymatic domains, but how this leads to neurodegeneration is still to be elucidated. One of the few confirmed LRRK2 interactors is tubulin, the main constituent of microtubules (MTs) and part of the cytoskeletal network. Disease-causing mutations in LRRK2 alter this network, reducing neurite outgrowth and leading to accumulation of hyperphosphorylated MT-associated protein (MAP) tau. Meanwhile changes in post-translational modifications of tubulin and MAPs alter the dynamic instability of MTs, leading to aberrant axonal transport, synaptic dysfunction and axonal degeneration. I investigated the LRRK2-tubulin interaction. Using yeast two-hybrid I demonstrated that the interaction is conferred by the LRRK2 Roc domain and the C-terminus of the β-tubulin isoforms TUBB, TUBB4 and TUBB6. The interaction requires Lys362 and Ala364 and is blocked in isoforms expressing a serine at these positions. This site is on the luminal face of MT protofibrils, close to the paclitaxel binding site and α-tubulin Lys40 acetylation site, both of which are involved in MT stability. This location is poorly accessible within mature, stabilised MTs but exposed in dynamic MT populations. Consistent with this finding, endogenous LRRK2 located to dynamic growth cone MTs in SH-SY5Y cells. Overexpression and knock-out studies in HEK cells and mouse embryonic fibroblasts showed that LRRK2 is associated with reduced α-tubulin acetylation. These results demonstrate the specificity of the LRRK2-tubulin interaction, suggesting LRRK2 distribution at the cytoskeleton is determined by the tubulin composition and may vary between cell types. Changes in MT acetylation in the presence of disease-causing LRRK2 mutations could contribute to pathogenic mechanisms, with altered MT stability implicated in PD neurodegeneration. As mutations affecting the β-tubulin C-terminal residues could disrupt the LRRK2 interaction without compromising MT integration, a cohort of late-onset familial PD cases was also screened for mutations within the cytoskeleton.

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.

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.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

The mature nervous system is composed largely of two cell types, glial cells and postmitotic neurons. All postmitotic neurons of the mature nervous system derive from proliferating neural precursor cells. To generate a neuron, a precursor must cease dividing and express a number of genes that are characteristic of the neuronal phenotype. How these changes in cell behaviour and phenotype are brought about in mammals is still poorly understood.
This thesis describes experiments that were designed to explore cell intrinsic mechanisms regulating the generation of neurons from neural precursor cells. Specifically, the regulatory region of the rat Talpha1 alpha-tubulin gene, which encodes an isoform of alpha-tubulin expressed in neurons throughout the nervous system immediately following cell cycle exit, was analyzed to identify DNA sequences directing early neuronal gene expression.
A novel 10-nucleotide regulatory sequence, named the neuronal restriction element (NRE), has been identified. In the context of the Talpha1 gene, the NRE inhibits precocious expression in neural precursor cells. Interestingly, the NRE is conserved in the alpha-1 alpha-tubulin gene and is found in a number of neural genes expressed widely and early in development. As such, the NRE may affect the onset time of a battery of neuronal genes and modulate the timing of neuronal differentiation. In vitro , the NRE binds Su(H), a highly conserved transcription factor involved in the repression of neuronal differentiation.
A second novel regulatory element has been identified, the forebrain response element (FRE), which acts to enhance gene expression specifically in the neocortex. The FRE overlaps the NRE and also contains a conserved 30-nucleotide sequence constituting a putative homeodomain recognition sequence. We speculate that the FRE consists of two subelements that act synergistically to promote gene expression in newborn and mature neocortical neurons.

Regulation of tubulin gene expression in embryos of the sea urchin Lytechinus pictus has been experimentally investigated by use of cloned recombinant tubulin DNA and anti-tubulin antiserum. Tubulin synthesis appears to be autogenously regulated at the level of tubulin mRNA stability by the level of unpolymerized tubulin; i.e., the more unpolymerized tubulin, the less stable the tubulin mRNA. Destabilization of tubulin mRNA requires continued protein synthesis. Most of tubulin stored in eggs is unpolymerized; during embryogenesis the mass of tubulin per embryo changes little, but unpolymerized tubulin is increasingly polymerized into microtubules. There is a transcriptional stimulation of tubulin genes at the time of ciliogenesis but thereafter autoregulation by the ontogenetic decrease of the level of unpolymerized tubulin plays a predominant role for an increasing accumulation of tubulin mRNA. Deciliation results in a transient enhancement of transcription of tubulin genes, which is independent of the level of unpolymerized tubulin and does not require protein synthesis.

The main aim of the study described in this thesis is the development of new anticancer agents. The first chapter is a general introduction to cancer, and the development of chemotherapy anticancer agents during the course of the years. The following four chapters briefly introduce the biological targets in the authors study. Chapter Two describes a general introduction to tubulin and microtubules as anticancer targets. A discussion of those compounds most relevant to this thesis is provided. Chapter Three describes Signal Transducers and Activator of Transcription 3 (STAT3) proteins, their role in cancer and the advances in the search of anticancer agent inhibitors of the STAT3 signalling pathway. Chapter Four focuses on the src homology 2 (SH2) domain containing tyrosine phosphatases SHP-2, a protein-tyrosine phosphatase implicated in pathogenesis of cancer and other human diseases. A brief discussion of the SHP-2 inhibitors is provided. Chapter Five describes the role of proteins Aurora kinases in cancer, promising targets for anticancer drug development, and the advances in the search of their inhibitors targeting the kinase activity at the ATP binding site. The following chapters (6-11) describe the authors own findings. Chapter six focuses on the design and synthesis and biological evaluation of novel styrylchromones, styrylquinazolones, and quinazolones as inhibitors of tubulin polymerization. Styrylchromones Styrylquinazolones Quinazolones Two series of isomeric styrylchromones were initially synthesized in order to establish the methoxy substitution pattern on the A ring favorable for optimal activity. The structure activity relationship on the B ring is also reported. Next, our strategy focused on identifying a chromone core replacement with improved potency. We directed our chemical efforts toward the synthesis of novel styrylquinazoline analogs. The quinazoline core would also provide easy access to the preparation of diverse sets of N-substituted derivatives (methyl and ethyl derivatives). Finally, a novel series of quinazolines were synthesized as conformationally-restricted analogs of chalcones. SAR was conducted around the quinazoline spacer between the aryl rings and systematically investigating the substituent effect in the B ring. Among the synthesized compounds we selected those analogues showing significant cytotoxicity (generally defined as IC50 value < 1.5 uM), and evaluated for activity in vitro tubulin polymerization inhibition assay. Chapter Seven focused on the identification of novel inhibitors of STAT3 dimerization. Computational analyses led us to the development of a T-shape model of molecules that can >2 occupy the pTyr-binding pocket of STAT3 SH2 domain. The rN"s' conjugate addition of nitromethane to a series of amides and the l/ J reduction of the nitro group were combined to give an easy route to onh j. the target T-shape molecules in a combinatorial fashion. The ksJJ methodology was also extended to amides activated by a nitro group. co2r observed a dramatic change in the course of the reaction, which Scaffold of T-shape molecules afforded a mixture of unexpected and unknown products, that each possessed an additional methylene group. A brief study into the mechanism was also conducted. Chapter Eight, Nine and Ten focuss on the development of Aurora kinases and SHP-2 inhibitors. Oxindole derivatives HL10581 and NSC117199 emerged as lead compounds from a high throughput screen for Aurora-A and SHP-2, respectively. f f Chapter Eight describes the synthesis of n-nfh Cl nnhN 2 several derivatives of HL10581 and H03SYYLo H03SYr>o NSC117199, directed to exploration of h h SAR around the oxindole moiety to HLiow.AAKfci-SMM nscii7,99,shp-2,,c5047 mM determine the structural features that are responsible for the activity. Chapters nine and ten report the biological evaluation of oxindole derivatives as inhibitors SHP-2 and Aurora kinases, respectively.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

This thesis presents the results of an investigation into the structural protein, tubulin, as a potential target for anti-trypanosomatid drug discovery and vaccine development. Recombinant alpha- and beta- tubulin proteins from Trypanosoma brucei rhodesiense were expressed as soluble fusion proteins in an E. coli expression system. The recombinant alpha- and beta- tubulins were used to determine the nature of binding of novel trifluralin analogues EPL-AJ 1003, 1007, 1008, 1016 and 1017. Native tubulin from rats was used to determine the extent of binding to mammalian tubulin. The results of this study clearly demonstrate two important aspects of the binding of trifluralins to tubulin. Firstly, they have specific affinity for trypanosomal tubulin compared with mammalian regardless of the chemical composition of the trifluralin analogue tested. Secondly, they have a demonstrably stronger affinity for alpha-tubulin compared with beta-tubulin. In addition, compounds 1007, 1008, 1016 and 1017 have strong binding affinities for alpha-tubulin, with limited binding affinity for mammalian tubulin, which indicates that these compounds selectively bind to trypanosomal tubulin. The morphology of bloodstream forms of T. b. rhodesiense exposed to trifluralin analogues was studied using electron microscopy and immunofluorescence to determine the ultrastructural changes these compounds induce as a result of binding to tubulin. All compounds tested induced severe irreparable damage in T. b. rhodesiense, including perturbation of subpellicular microtubules, extensive cytoplasmic swellings, axoneme and paraflagellar rod malformation, disconfiguration around the flagellar pocket and membrane disintegration. These results suggest that the mechanism of action of these trifluralin analogues is through the disruption of polymerization of tubulin into microtubules as a result of binding to alpha-tubulin. The potential for recombinant trypanosomal tubulins to be used as vaccine candidates was assessed by monitoring parasitaemia and length of survival of mice immunised with the proteins and challenged with a lethal infection of T. b. rhodesiense. Although all the mice vaccinated with recombinant tubulin developed a patent parasitaemia and did not survive, they were partially protected because their patency period and length of survival were significantly greater than the control groups. Furthermore, plasma collected from mice immunised with recombinant trypanosomal tubulin contained antibodies that recognized tubulin in a soluble extraction from T. b. rhodesiense. The results of this thesis confirm the potential for the structural protein, tubulin, to be used as a target for anti-trypanosomatid drug discovery and vaccine development.

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 traitements
The 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