The conserved human protein KANK1 has been identified as a tumour suppressor and its expression is down-regulated in several tumour types. Roles for this protein in actin regulation, cell migration and cell polarity have been documented in cultured mammalian cells. In C. elegans the KANK1 orthologue, VAB-19, is required for normal development as it helps stabilise attachment structures between muscle and epidermal cells. Despite these studies, the precise cellular role of KANK remains elusive. It was found that the Drosophila KANK orthologue binds directly to EB1, a crucial regulator of microtubule plus-end dynamics. I aimed to determine the role of KANK with respect to this indirect microtubule interaction using Drosophila. I identified residues which mediate the interaction between KANK and EB1, and showed they are essential for localisation of KANK to microtubule plus-ends in Drosophila culture cells. I found that KANK expression increases during embryogenesis and peaks in the late embryonic development when KANK is shown to localise to sites of attachment between muscle and epidermal cells. This suggests a role for the protein in stabilisation of muscle attachment during embryonic development, a process previously shown to require EB1. I generated a KANK deletion mutant and found they are viable and fertile but show a mild neuronal phenotype, specifically early branching of the neurons and less organised neuron bundles. My results suggest previously unknown roles for KANK in myogenesis and neurogenesis in Drosophila embryogenesis.

A number of viruses exhibit microtubule-based bidirectional transport following cell entry. This behavior raises three questions: First, what mediates their transport along microtubules? Second, how do viruses recruit the motor proteins? Finally, how do they go to the right place by bidirectional transport in a variety of cell types with different microtubule organizations? We studied these questions with Adenovirus 5 (Ad5), a virus with well characterized, dynein-mediated minus transport mechanism. One form of plus end directed motor, Kif5C, has been reported to disrupt Ad5 capsids at the Nuclear Pore Complexes(NPC), but the mechanisms and roles of microtubule plus end-directed Ad5 transport prior to this stage are largely unknown. Here we performed a RNAi screen of 38 microtuble plus end-directed kinesins, which implicated Kif5B (kinesin-1 family) in plus-end directed Ad5 transport, along with several other forms of kinesin. Kif5B knockdown caused an accumulation of Ad5 particles near the centrosomes in human pulmonary epithelial A549 cells. This effect was strongly enhanced by blocking Ad5 nuclear pore targeting with Leptomycin B and supports a role for Kif5B in Ad5 transport prior to NPC docking. Kif5B RNAi was rescued by expression of any of the three Kif5 orthologues. We also found that Ad5 directly interacts with kinesin-1 via the capsid subunit Penton Base in a PH-independent manner. Together with our earlier studies, these findings reveal that Ad5 has evolved distinct recruitment mechanisms for cytoplasmic dynein and at least one form of kinesin-1 during early infection. Despite clear evidence for short-range linear microtubule-associated Ad5 transport, we found the overall behavior of most Ad5 particles to be stochastic at a larger time scale, by mean-square-displacement (MSD) analysis. We named this behavior "assisted diffusion''. In consistent with this mechanism, Ad5 was able to maintain a normal nuclear targeting after we displaced centrosomes away from the nucleus by inhibiting CDK1 in late G2 cells. We also directly observed Ad5 switching from microtubule based transport to nuclear targeting from a microtubule near the nucleus. Kif5B RNAi dramatically inhibited this novel microtubule-based random-walk/“assisted-diffusion” mechanism. By super resolution microscopy, we found a more local distribution of NPC attached Ad5 over the entire nuclear surface under conditions of Kif5B knock down. We propose that adenovirus uses independently-recruited kinesin and dynein to fully explore the cytoplasm to search for and dock at the nucleus, a mechanism of potential importance for physiological cargoes as well.

Microtubules are a major constituent of the cytoskeleton in all eukaryotic cells. They are essential for cell morphogenesis and motility. Specifically in the dividing cells, microtubules form the spindle which segregates chromosomes. Microtubule plus ends constantly switch between phases of growth and shrinkage which is necessary for microtubule reorganization and thus their function. Importantly, microtubule dynamics are highly regulated by microtubule-associated proteins (MAPs). EB1 and Mini spindles (Msps) are unique amongst MAPs because they bind and track growing microtubule plus ends autonomously. Although essential for cell division and thus highly expressed in dividing cells, EB1 and Msps are also abundant in differentiated cells. However, to identify post-mitotic roles of proteins essential for cell division, particularly in context of a multicellular organism, is a challenge requiring new tools which I aimed to develop in my project. Since EB1 acts by recruiting MAPs to the microtubule plus ends, I generated short peptides which bind to Drosophila EB1 to block interactions with these MAPs. I showed that an EB1-MAP interaction was disturbed in Drosophila S2 cultured cells and expressing these peptides in developing Drosophila reduced fly viability. Further screening and analysis of peptides interacting with fly EB1 and its human homologues uncovered sequence determinants promoting strong binding and specificity. To uncover Msps function, I generated a msps temperature sensitive mutant and found that Msps is essential for neuromuscular function in developing Drosophila. This study showed that the regulation of microtubule dynamics has crucial functions at the whole organism level. These new tools allow the roles of microtubule regulation to be dissected in developing organisms.

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.

Thesis submitted to the Division of Veterinary and Biomedical Sciences. Bibliography: leaves 237-283.

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

Growth anisotropy, in which cells grow predominantly in one direction, is common in plant cells, and an essential event for plant form and function. The direction and degree of growth anisotropy are governed by the mechanical properties of the primary cell wall. When aligned in a parallel manner, cellulose microfibrils accommodate great resistance in the direction of their alignment to expansion driven by isotropic turgor pressure. Using the Arabidopsis thaliana inflorescence stem as a model system, field emission scanning electron microscopy (FESEM) analysis demonstrated that the establishment of parallel arrangement of microfibrils is closely correlated with anisotropic cell expansion. In the novel anisotropy 1 (any1) mutant allele of the primary cellulose synthase CesA1, growth defects were correlated with random cellulose microfibril patterns in some inflorescence stem tissues. Microtubules have been considered to be the most likely candidates for controlling the orientation of cellulose microfibrils. Recent studies have indeed demonstrated a close association of the plasma membrane-localized cellulose-synthase-complexes (CSCs) that produce cellulose and cortical microtubules. Despite this close association, microtubule disruption did not cause cellulose microfibrils to lose parallel alignment in the radial and inner periclinal walls of cells in the inflorescence stem, suggesting that microtubules influence mechanical properties of cellulose microfibrils other than orientation. X-ray diffraction analysis demonstrated that cellulose crystallinity in wild-type plants declines at the growth-promoting temperature of 29°C, whereas crystallinity fails to adapt and remains high in mor1-1, the temperature-sensitive mutant whose microtubule arrays become disorganized at its restrictive temperature (29°C). This finding suggests that organized microtubules are involved in reducing cellulose crystallinity that normally accompanies increased cell expansion. Live-cell imaging of CSCs by tracking a yellow fluorescent protein (YFP)-tagged CesA6 subunit in hypocotyl cells demonstrated that dynamic and well-organized microtubules affect the velocity, the direction of movement, and the density of CSCs, suggesting that there is a close relationship between microtubules and CSCs. Together with the finding that microtubules also control the distribution of COBRA, a GPI-anchored wall protein that is essential for growth anisotropy, I discuss the variety of roles microtubules play in anisotropic growth.

L'angiogènesis, el desenvolupament de nous vasos sanguinis a partir de vasculatura preexistent, és un procés complex que involucra múltiples productes gènics expressats per diferents tipus cel.lulars, i tots ells contribueixen a una seqüència integrada de fenòmens. L'angiogènesis és necessària per tal de permetre el creixement tumoral més enllà d'una certa mida. La hipòxia, un fenomen inherent en els tumors, és un dels factors principals que desencadena el procés angiogènic. En concordança amb el fet que la hipòxia juga un paper clau en tot el procés, un nombre elevat de gens involucrats en diferents processos de l'angiogènesis, incloent el factor de creixement de l'endoteli vascular (VEGF), responen de manera independent a la hipòxia. El principal mecanisme de resposta dels tumors a nivells reduïts d'oxigen és a través de l'activació del factor induïble per hipòxia-1 (HIF-1). HIF-1 és un heterodímer format per dues subunitats; el factor sensible a la hipòxia HIF-1, i el factor no regulable HIF-1. En presència d'oxigen, HIF-1 és hidroxilat i això permet la seva interacció amb la proteïna supressora de tumors Von Hippel Lindau (VHL) que produirà la seva subseqüent i ràpida degradació a través del proteasoma. En absència d'oxigen, HIF-1 és estabilitzat i és transportat cap al nucli cel.lular on heterodimeritza amb HIF-1, unint-se llavors als llocs de resposta a la hipòxia (HRE) i activant la transcripció de nombrosos gens que juguen un paper fonamental en la supervivència tumoral, l'adaptació metabòlica i l'angiogènesis.Per tant, el desenvolupament d'agents capaços d'inhibir l'angiogènesis ha esdevingut una important modalitat terapèutica. Fruit dels bons resultats en estudis preclínics, diversos agents antiangiogènics sols o en combinació amb teràpies convencionals estan en fase de desenvolupament clínic. Entre aquests agents trobem el 2-Metoxiestradiol (2ME2), un metabolit natural de l'estradiol, actualment en avaluació clínica com a inhibidor de l'angiogènesis tumoral. Diversos estudis han demostrat que el 2ME2 és una molècula petita, mot ben tolerada i que posseeix activitats antitumorals i antiangiogèniques en diferents models in vivo. In vitro, el 2ME2 és capaç d'unir-se a la tubulina al lloc d'unió a la colchicina i despolimeritzar els microtúbuls en interfase, produint arrest en mitosi i la posterior mort cel.lular en línies cel.lulars tumorals. A més a més, s'ha mostrat l'abilitat de diferents agents amb capacitat per inhibir la funció dels microtúbuls, sobretot d'aquells que s'uneixen al lloc d'unió a la colchicina, per fer desaparèixer ràpidament la vasculatura tumoral. Tot i això, encara que s'han proposat diferents mecanismes d'acció per al 2ME2, el seu mecanisme d'acció encara no està ben definit. La nostra hipòtesi és que el 2ME2 inhibeix l'angiogènesis tumoral mitjançant la inhibició del factor de transcripció HIF-1 un cop ja ha exercit els seus efectes sobre el citoesquelet de microtúbuls. Així doncs, l'objectiu del nostre estudi va ser I) caracteritzar el mecanisme a nivell molecular pel qual el 2ME2 inhibeix el HIF-1 i l'angiogènesis, i II) investigar si hi ha cap relació causa-efecte entre les propietats antiangiogèniques i anti-tubulina del 2ME2. El nostre treball demostra que el 2ME2 inhibeix l'angiogènesis tumoral mitjançant la inhibició dels nivells proteics del factor HIF-1 així com la seva activitat transcripcional, incloent la transcripció del VEGF, en diverses línies cel.lulars. El 2ME2 no indueix la degradació proteica a través del proteosoma ni tampoc redueix els nivells d'ARNm del HIF-1, sinó que més aviat inhibeix la síntesi proteica de novo. Notablement, hem mostrat que el 2ME2 inhibeix el factor HIF-1 amb posterioritat a la disrupció del citoesquelet de microtúbuls aportant sòlides proves que afavoreixen una relació funcional entre els efectes anti-angiogènics i la disrupció dels microtúbuls. Aquests efectes no són una propietat única del 2ME2 sinó que és compartida per altres agents que actuen sobre els microtúbuls, com ara el taxol i la vincristina, suggerint un mecanisme d'acció semblant per a tots aquests agents. Finalment, la nostra investigació demostra per primera vegada que el 2ME2 inhibeix l'angiogènesis en dosis que són eficaces despolimeritzant els microtúbuls in vivo. Així doncs, el nostre treball aporta la primera demostració d'una associació mecanística entre la disrupció del citoesquelet de microtúbuls i la inhibició de l'angiogènesis a través del factor de transcripció HIF-1 i dóna suport a la hipòtesis que els agents que actuen sobre la tubulina inhibeixen tant el creixement tumoral com la vascularització tumoral amb posterioritat a la disrupció del citoesquelet de microtúbuls. Una caracterització més exhaustiva dels senyals que tenen lloc a nivell molecular entre la disrupció del citoesquelet de microtúbuls i l'angiogènesis mediada pel factor de transcripció HIF-1, molt possiblement doni lloc a la identificació de noves dianes per al desenvolupament de noves estratègies terapèutiques. / Angiogenesis, the development of new vessels from preexisting vasculature, is a complex process involving multiple gene products expressed by different cell types, all contributing to an integrated sequence of events. Angiogenesis is required to support tumor growth beyond a certain threshold size. Inherent tumor hypoxia is one of the major factors triggering angiogenesis and consistent with a major role for hypoxia in the overall process, a large number of genes involved in different steps of angiogenesis, including vascular endothelial growth factor (VEGF), are independently responsive to hypoxia. The primary mechanism of tumor response to reduced oxygen levels is via activation of hypoxia inducible-factors-1 (HIF-1). HIF-1 is a heterodimer consisting of two subunits; the labile hypoxic responsive factor, HIF-1, and the non-regulated factor HIF-1β. In the presence of oxygen, HIF-1a is hydroxylated, allowing its interaction with the tumor suppressor protein Von Hippel Lindau (VHL) and its subsequent and rapid degradation by the proteasome. Following hypoxic stabilization, HIF-1 is translocated to the nucleus. There it heterodimerizes with HIF-1 and binds to hypoxia-response elements (HREs), activating the transcription of numerous genes important for cancer survival, metabolic adaptation to hypoxia and angiogenesis. Therefore, the development of agents that inhibit angiogenesis are attractive therapeutic options. Based on successful preclinical data, several anti-angiogenic agents alone or in combination with conventional therapies are now in clinical trials. Among these agents is 2-methoxyestradiol (2ME2), a natural occurring derivative of estradiol, currently undergoing clinical evaluation as an inhibitor of tumor angiogenesis. 2ME2 has been shown to be a well-tolerated small molecule posses antitumor and antiangiogenic activity in different in vivo models. In vitro, 2ME2 has been shown to compete with colchicine for tubulin binding and to disrupt interphase microtubules leading to mitotic arrest and cell death in cultured cancer cells. Furthermore, the ability of microtubule-targeting agents, especially those binding to the colchicine site, has been shown to rapidly shut down existing tumor vasculature. Although several mechanism have been proposed for 2ME2 activity, its mechanism of action still remains unclear. Our hypothesis is 2ME2 inhibits tumor angiogenesis by targeting the HIF pathway downstream of its effects on the microtubule cytoskeleton. Therefore, in this study we seek to I) characterize the molecular mechanism by which 2ME2 inhibits HIF-1 and angiogenesis, and II) Investigate whether there is cause-effect relationship between 2ME2's antiangiogenic and anti-tubulin properties. Our work demonstrated that 2ME2 inhibits tumor angiogenesis by effectively inhibiting HIF-1α levels and transcriptional activity, including transcription of VEGF, in a variety of human cancer cell lines. 2ME2 does not induce proteasomal degradation of HIF-1 nor does it reduces HIF-1 mRNA levels, but rather inhibits the de novo HIF-1 protein synthesis. Notably, we showed that 2ME2 inhibits HIF-1α downstream of disruption of the microtubule cytoskeleton, providing solid proof of a functional relationship between the antitubulin and antiangiogenic effects of 2ME2. These effects of 2ME2 are not unique but rather shared by other microtubule-targeting drugs, such as taxol or vincristine, suggesting a common mechanism of action for all microtubule-targeting drugs. Finally, our findings demonstrate for the first time that 2ME2 inhibits angiogenesis at doses are efficacious disrupting microtubules in vivo. Herein, our work provides the first demonstration of a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the HIF-pathway and supports the hypothesis that tubulin-targeting drugs inhibit both tumor cell growth and tumor vascularization following disruption of the microtubule-cytoskeleton. The further characterization of the molecular signal linking the disruption of the microtubule-cytoskeleton with the downregulation of the HIF-mediated angiogenesis, are likely to identify novel targets for the development of new anticancer therapies.

The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.

Reproduction de : Thèse de doctorat : Cancérologie : Lyon 1 : 2007. / Titre provenant de l'écran titre. Bibliogr. f. [214]-[245].