β-Glucosidase zymograms of certain maize genotypes (nulls) do not show any activity bands after electrophoresis. We have shown that a chimeric lectin called β-glucosidase aggregating factor (BGAF) is responsible for the absence of β-glucosidase activity bands on zymograms. BGAF specifically binds to maize β-glucosidase isozymes Glu1 and Glu2 and forms large, insoluble complexes. Furthermore, we have previously shown that the N-terminal (Glu⁵⁰-Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions contain residues that make up the BGAF binding site on maize Glu1. However, sequence comparison between sorghum β-glucosidases (dhurrinases, Dhr1 and Dhr2), to which BGAF does not bind, and maize β-glucosidases, and an examination of the 3-D structure of Glu1 suggested that the BGAF binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²-Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹-Thr²⁹, together with C-terminal region Phe⁴⁶⁶-Ala⁵¹², affects the size of Glu1-BGAF complexes. To determine the importance of each region for binding, we determined the dissociation constants (K_d) of chimeric β-glucosidase-BGAF interactions. The results demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²-Thr⁸² on Glu1 for BGAF binding, we constructed chimeric Dhr2 (C-11, Dhr2 whose Val⁷²-Glu⁸² region was replaced with the Ile⁷²-Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²-Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding. We also constructed mutant β-glucosidases to identify and define the contribution of individual amino acids in the above three regions to BGAF binding. In the N-terminal region (Ile⁷²-Thr⁸²), critical region for BGAF binding, Glu1 mutants K81E and T82Y failed to bind BGAF in the gel-shift assay and their frontal affinity chromatography (FAC) profiles were essentially similar to that of sorghum β-glucosidase (dhurrinase 2, Dhr2), a non-binder, indicating that these two amino acids within Ile⁷²-Thr⁸² region are essential for BGAF binding. In the extreme N-terminal (Ser¹-Thr²⁹) and C-terminal (Phe⁴⁶⁶-Ala⁵¹²) regions, N481E [substitution of asparagine-481 with glutamic acid (as in Dhr)] showed lower affinity for BGAF, whereas none of the single amino acid substitutions in the Ser¹-Thr²⁹ region showed any effect on BGAF binding indicating that these regions play a minor role. To further confirm the importance of lysine-81 and threonine-82 for BGAF binding, we produced a number of Dhr2 mutants, and the results showed that all four unique amino acids (isoleucine-72, asparagine-75, lysine-81, and threonine-82) of Glu1 in the peptide span Ile⁷²-Thr⁸² are required to impart BGAF binding ability to Dhr2. The sequence comparison among plant β-glucosidases supports the hypothesis that BGAF binding is specific to maize β-glucosidases because only maize β-glucosidases have threonine at position 82. / Ph. D.

Les β-arrestines (Arrbs) régulent diverses voies de signalisation dont la voie Wnt/β-caténine (Wnt), un acteur clé dans le cancer colorectal. Le but de mon projet était d'étudier l'implication et les mécanismes régulés par les Arrbs dans la tumorigenèse intestinale dépendante de la voie Wnt. L'inhibition de l'expression des Arrbs partielle ou totale dans des souris ApcΔ14/+ montre que seules les souris invalidées pour l'Arrb2 développent 33% des tumeurs détectées chez les souris ApcΔ14/+ ; Arrb2+/+. Ces tumeurs ont une croissance normale. Cependant, l'analyse de leur transcriptome montre qu'elles expriment notamment certains gènes liés au système immunitaire, alors que les tumeurs dépendantes de l'Arrb2 expriment des gènes différents impliqués entre autres dans la voie Wnt. L'invalidation de l'Arrb2 réduit l'expression de gènes cibles de la voie Wnt dans les cellules isolées de 12 sur 18 tumeurs de souris ApcΔ14/+, et inhibe l'augmentation d'activité Wnt et la formation de colonies en agar mou induite par l'invalidation d'Apc dans des cellules murines ApcMin/+. L'Arrb2 est donc essentielle pour l'initiation et la croissance des tumeurs intestinales présentant une activité Wnt élevée. Pour comprendre les mécanismes régulés par l'Arrb2 dans ce contexte, les complexes protéiques associés à l'Arrb2 ont été analysés par protéomique dans des cellules humaines de cancer colorectal SW480 exprimant ou non un dominant négatif de Tcf4. 132 partenaires de l'Arrb2 potentiellement imbriqués dans un réseau de 917 protéines, ont été identifiés dans les cellules où la voie Wnt est active. Une baisse de 80% de l'activité Wnt entraine la disparition de 41 protéines avec 256 interactions potentielles alors que 42 protéines apparaissent avec 244 interactions potentielles. Le rôle clé d'Arrb2 dans le cancer colorectal s'expliquerait par la connexion d'au moins une quarantaine de protéines dépendantes de l'activité Wnt à un réseau de signalisation complexe dont l'analyse est en cours. / Β-arrestins (Arrbs) participate in the regulation of multiple signaling pathways, including Wnt/β-catenin (Wnt), the major actor in human colorectal cancer. The aim of my project was to study the involvement of Arrbs and the mechanisms they regulate in Wnt-dependent intestinal tumorigenesis. The partial or total inhibition of Arrbs in ApcΔ14/+ mice showed that only mice with Arrb2 depletion developed only 33% of the tumors detected in their Arrb2-WT littermates. These remaining tumors grow normally and are Arrb2–independent. Transcriptomic analysis showed that they overexpressed genes that reflect a high interaction with the immune system, whereas those overexpressed in Arrb2–dependent tumors are predominantly involved in Wnt signaling. Moreover, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from ApcΔ14/+ mice, completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment in ApcMin/+ cells. Therefore, Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity. To better understand the mechanisms involved in this context, Arrb2 protein complexes were analyzed by a differential systematic proteomic approach in SW480 human colorectal carcinoma cells expressing or not a Tcf4 dominant negative. 132 Arrb2 partners potentially involved in a signaling network of 917 proteins were identified in cells with a high Wnt activity. Upon a 80% decrease of this activity 41 partners disappeared with their 256 potential interactions whereas 42 partners appeared with 244 new possible interactions. Arrb2 key role in colorectal cancer could be explained by the cross-talk of about 40 proteins dependent of Wnt activity with a highly complex signaling network that is currently analyzed.

Mycobacterium abscessus est une mycobactérie responsable principalement d’infections pulmonaires, en particulier chez les patients atteints de mucoviscidose ou de dilatation des bronches. M. abscessus est naturellement résistante aux antituberculeux, laissant peu d’options thérapeutiques. Le traitement de référence associait classiquement un aminoside, un macrolide (clarithromycine) et une β-lactamine (céfoxitine ou imipénème), avec un taux de succès d’environ 50 %. Cependant, des souches résistantes à la clarithromycine sont fréquemment isolées, remettant en cause l’utilisation de cet antibiotique. M. abscessus produit naturellement une β-lactamase à large spectre (BlaMab) mais les mécanismes d’action des β-lactamines n’ont pas été étudiés chez cette espèce, ce qui constitue une entrave à l’optimisation des traitements par cette classe d’antibiotiques. Le premier objectif était d’identifier et de caractériser les cibles des β-lactamines chez cette espèce. Inhibant la dernière étape de polymérisation du peptidoglycane, les cibles potentielles des β-lactamines sont trois familles d’enzymes : les D,D-transpeptidases et les D,D­carboxypeptidases appartenant à la famille des protéines de liaison à la pénicilline (PLP), ainsi que les L,D-transpeptidases qui sont majoritairement responsables de cette dernière étape chez cette espèce. Pour identifier les cibles, des mutants résistants aux β-lactamines ont été sélectionnés à partir de la souche de référence M. abscessus CIP104536 et d’un dérivé portant une délétion du gène blaMab (∆blaMab). Pour les deux souches, l’émergence de la résistance aux β-lactamines a requis de multiples étapes, ce qui constitue un atout pour leur utilisation thérapeutique. Pour les mutants obtenus à partir de la souche CIP104536, les analyses phénotypiques ont montré que la résistance aux β-lactamines n'est pas due à une augmentation de l’efficacité catalytique de BlaMab, à une surproduction de cette enzyme, ou à une diminution de la perméabilité. Le séquençage des génomes de mutants résistants n’a pas révélé de mutations dans les gènes codant pour les L,D-transpeptidases, mais des mutations ont été trouvées dans des gènes codant pour deux PLP. D’autres mutations se situent dans des gènes codant en particulier pour des protéines non caractérisées. L’acquisition de la résistance pourrait donc dépendre de mutations affectant des facteurs essentiels à l’activité des cibles des β­lactamines. Le deuxième objectif était d’étudier et de comparer l’activité in vitro des β-lactamines sur M. abscessus. Des expériences de bactéricidie et d’activité intracellulaire chez le macrophage infecté ont été effectuées pour les souches CIP104536 et ∆blaMab. Parmi les antibiotiques étudiés (amikacine, céfoxitine, imipénème, ceftaroline, et amoxicilline), l’imipénème est le plus efficace sur les deux souches. Sur la souche ∆blaMab, l’association d’imipénème et d’amikacine est bactéricide. En l’absence de BlaMab, l’amoxicilline est aussi efficace que l’imipénème. L’avibactam augmente l’activité de la ceftaroline mais l’inhibition de BlaMab est seulement partielle en intracellulaire. Les résultats obtenus in vitro montrent que l’imipénème est supérieur à la céfoxitine pour des concentrations atteignables dans le sérum. L’inhibition de BlaMab pourrait augmenter l’efficacité de l’imipénème et d’autres composés utilisés pour traiter les infections pulmonaires à M. abscessus. / Mycobacterium abscessus is an important pathogen responsible for pulmonary infections in cystic fibrosis patients or in patients suffering from bronchiectasis. The treatment of infections due to M. abscessus is complicated since this bacterium is naturally resistant to the anti­tuberculous agents. The recommended treatment includes an aminoglycoside, a macrolide (clarithromycin) and a β-lactam (cefoxitin or imipenem), with a success rate of about 50 %. However, strains resistant to clarithromycin are frequently isolated, questioning the use of this antibiotic. M. abscessus naturally produces a broad spectrum β-lactamase (BlaMab) but the mechanisms of action of the β-lactams have not been studied in this species, impairing the optimization of the treatment by these antibiotics. The first objective was to identify and characterize the targets of β-lactams antibiotics in this species. Inhibiting the final stage of the peptidoglycan polymerization, the potential targets of β-lactams are three families of enzymes: the D,D-transpeptidases and D,D­carboxypeptidases belonging to the family of penicillin-binding proteins (PBP), and the L,D-transpeptidases which are mainly responsible for this final stage in this species. To identify the targets, mutants resistant to β-lactams have been selected from the reference strain M. abscessus CIP104536 and from its β-lactamase-deficient derivative ΔblaMab. For both strains, the emergence of resistance to β­lactams has required multiple steps, which is an advantage for the therapeutic use of these antibiotics. For the mutants derived from the strain CIP104536, phenotypic analyzes showed that the resistance to β-lactams is not due to an increase in the catalytic efficiency of BlaMab, to an overproduction of this enzyme, or to a decrease in permeability. Genomes sequencing of the resistant mutants did not reveal mutations in the genes encoding the L,D-transpeptidases, but mutations have been found in genes encoding two PBPs. Other mutations have been detected in genes encoding uncharacterized proteins. Acquisition of resistance could therefore depend on mutations affecting key factors essential for the activity of β-lactams targets. The second objective was to study and compare the in vitro activities of β-lactams against M. abscessus. Bactericidal experiments and intracellular activity in the infected macrophage were performed for the strains CIP104536 and ΔblaMab. Among the antibiotics tested (amikacin, cefoxitin, imipenem, ceftaroline, and amoxicillin), imipenem is the most effective agent against the two strains. Combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. In the absence of BlaMab, amoxicillin was as active as imipenem. Avibactam increased the intracellular activity of ceftaroline but inhibition of BlaMab was only partial intracellularly. Evaluation of the killing and intracellular activities of β-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drug potentially useful to treat pulmonary infections.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

Summary : Enterococcus faecium possesses a low affinity PBP5 that, like in other enterococci, is an important factor contributing to the intrinsic resistance to penicillin. The structure of PBP5 soluble form in complex with benzylpenicillin has been solved at 2.1 Å resolution. On the basis of this 3D structure, it was proposed that the I623KEKQDEKG631 turn connecting β3 and β4 strands and constituting one wall of the active site, as well as, R464 involved in a salin-bridge near the active site could be responsible for the low β-lactam affinity of PBP5fm The k2/K of sPBP5fm acylation by various b-lactams have been determined and, as expected for a low affinity PBP, they are very low. Mutants were constructed to determinate the role of the residues pointed in the 3D structure as related to the observed low affinity. For the sPBP5fm∆I7G mutant, the acylation rate constants are similar to those measured for sPBP5fm. However, when compared to the wild type, the mutants without salin-bridge show a higher affinity that could be explained as the consequence of an easier accessibility to the active site for the inactivator. This study allows mapping active site cavity and determines the importance of structural elements to understand the low affinity of PBP5. Furthermore, three news β-lactams (ceftaroline, ceftobiprole and ME1036) were tested against PBP5fm and they all exhibit inhibitory activity against the protein. Ceftaroline appears as the best inhibitor of sPBP5fm as well as the best inhibitor while tested on Enterococcus faecium cultures. Résumé : Enterococcus faecium possède une protéine PBP5 de faible affinité, qui est un facteur important de la résistance intrinsèque à la pénicilline. La structure de la forme soluble dans PBP5 complexés avec la benzylpénicilline a été résolue à 2,1 Å. Sur la base de cette structure 3D, il a été proposé que la boucle I623KEKQDEKG631 reliant brins β3 et β4 et pouvant constituer une barrière pour atteindre le site actif, ainsi que, R464 impliqué dans un pont-salin près du site actif qui pourrait être responsable de la faible affinité β-lactamines de PBP5fm. Dans un 1er temps, les k2/K de sPBP5fm sauvage par diverses β-lactamines ont été déterminées, et comme prévu, elles sont très faibles. Dans un 2nd temps, les mutants ont été produits, purifiés et caractérisés de manière à déterminer le rôle des résidus choisis dans la faible affinité. Pour le mutant sPBP5fmΔI7G, les constantes de vitesse acylation sont similaires à ceux mesurée pour sPBP5fm sauvage. Tandis que les mutants sans le pont salin R464-D481 montrent une relative plus grande affinité par rapport au type sauvage, qui pourrait être expliqué par un accès plus facile de linhibiteur au site actif. Cette étude permet une première cartographie de la cavité du site actif de PBP5fm et qui détermine limportance ou non déléments de structure pour comprendre la faible affinité. Trois nouvelles β-lactamines (ceftaroline, ceftobiprole et ME1036) ont été testés contre PBP5fm et elles présentent une activité inhibitrice de PBP5fm. La ceftaroline apparaît comme le meilleur inhibiteur de PBP5fm ainsi que le meilleur inhibiteur lors des essais sur les cultures Enterococcus faecium.

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs.

Acidified dairy products are one of the oldest types of food products. Unfortunately all of them are low in dietary fibre. Thus, to improve health benefit of these products the idea of fortifying them with dietary fibre seems attractive. However dairy products enriched with Glucagel (a commercial product that is high in barley β-glucan) were found to suffer from textural defects. When the Glucagel concentration exceeded a certain value (5 g/L), dramatic phase separation was observed in set yogurt and yogurt drink with volume fraction of casein micelles greater then 0.108. To investigate interactions of β-glucan polymers and casein micelles in the milk prior to setting of yogurt, mixtures of yogurt milk and Glucagel were systematically studied. Depending on the volume fraction of casein micelles and the Glucagel concentration, a stable phase or a gel or a sedimented material could exist. The driving force for phase separation was depletion flocculation of casein micelles in the presence of β-glucan. The phase separation responsible for textural defects in yogurt systems supplemented with high amounts of Glucagel can be avoided by the reduction of β-glucan molecular weight, a process that limits the range of attraction between micelles. Incubation of Glucagel with lichenase for 90 min resulted in homogeneous (stable) yogurt systems with Glucagel concentrations as high as 10 g/L.

Acidified dairy products are one of the oldest types of food products. Unfortunately all of them are low in dietary fibre. Thus, to improve health benefit of these products the idea of fortifying them with dietary fibre seems attractive. However dairy products enriched with Glucagel (a commercial product that is high in barley β-glucan) were found to suffer from textural defects. When the Glucagel concentration exceeded a certain value (5 g/L), dramatic phase separation was observed in set yogurt and yogurt drink with volume fraction of casein micelles greater then 0.108. To investigate interactions of β-glucan polymers and casein micelles in the milk prior to setting of yogurt, mixtures of yogurt milk and Glucagel were systematically studied. Depending on the volume fraction of casein micelles and the Glucagel concentration, a stable phase or a gel or a sedimented material could exist. The driving force for phase separation was depletion flocculation of casein micelles in the presence of β-glucan. The phase separation responsible for textural defects in yogurt systems supplemented with high amounts of Glucagel can be avoided by the reduction of β-glucan molecular weight, a process that limits the range of attraction between micelles. Incubation of Glucagel with lichenase for 90 min resulted in homogeneous (stable) yogurt systems with Glucagel concentrations as high as 10 g/L.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

Tese (doutorado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2015. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2016-03-28T20:21:25Z No. of bitstreams: 1 2015_PauloHenriqueSilvaRibeiro.pdf: 10102032 bytes, checksum: 686e512f84476c253b39b708c60a2c9a (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2016-04-11T18:29:06Z (GMT) No. of bitstreams: 1 2015_PauloHenriqueSilvaRibeiro.pdf: 10102032 bytes, checksum: 686e512f84476c253b39b708c60a2c9a (MD5) / Made available in DSpace on 2016-04-11T18:29:06Z (GMT). No. of bitstreams: 1 2015_PauloHenriqueSilvaRibeiro.pdf: 10102032 bytes, checksum: 686e512f84476c253b39b708c60a2c9a (MD5) / O presente trabalho investigou a composição química dos óleos essenciais (OE) das folhas das espécies Eugenia langsdorffii (ELA), Eugenia dysenterica (EDY) e Eugenia lutescens (ELU) coletados nos períodos chuvoso (PC) e seco (PS) dos anos de 2012 e 2014. Os OE foram extraídos por hidrodestilação e os rendimentos percentuais observados para EDY e ELU, foram muito semelhantes nos dois anos/períodos de coleta e os melhores rendimentos foram observados em PC. Ao contrário, foi observada uma grande variação percentual para ELA, que exibiu maiores teores em PS. A composição química foi determinada por cromatografia em fase gasosa acoplada à espectrometria de massa (CG-EM), onde se observou uma grande variabilidade sazonal intraespécie no quantitativo de monoterpenos e sesquiterpenos. De forma geral, os OE apresentaram notáveis teores de β-cariofileno, δ-cadineno, α e β-pineno, limoneno, biciclogermacreno e espatulenol. O sesquiterpeno β-cariofileno foi encontrado com elevado percentual em todos OE, independente da fase de coleta, podendo ser considerado como um marcador químico das espécies. Os OE obtidos no ano de 2012 foram testados contra Tetranychus urticae (ácaro rajado) e, interessantemente, todos apresentaram atividade acaricida, sendo que E. langsdorffii (PS/2012) exibiu o melhor resultado (CL50 = 1,58 μL.L-1 de ar). β-Cariofileno, um dos componentes majoritário e comum a todas as espécies, foi isolado do OE de E. dysenterica por cromatografia em coluna dopada com nitrato de prata e comparado com padrão adquirido comercialmente por IV, CG-EM, RMN 1H e 13C bem como testado sua atividade acaricida, exibindo CL50 = 4,7 x 10-2 μL.L-1 de ar. Adicionalmente, o β-cariofileno comercial foi submetido a diversas modificações químicas para fornecer cinco derivados, os quais foram caracterizados por métodos espectrométricos como sendo óxido de cariofileno; 4,5-dihidróxi-cariofil-8(15)-eno; acetato de 4,8-dihidróxi-cariof-5-ila; aldeído β-nocariofilon e ácido β-nocariofilônico. A atividade acaricida destes derivados somente foi observada na CL95 do β-cariofileno (97,78 ± 1,11 % de letalidade), sendo o melhor resultado de letalidade observada para o aldeído β-nocariofilon (5,71 1,20 %). Além da determinação da composição química, este estudo mostrou os períodos de maior produção e atividade acaricida dos OE das três espécies de Eugenia. / The present work investigated the chemical composition of the leaf essential oils (EO) of the species Eugenia langsdorffii (ELA), Eugenia dysenterica (EDY) and Eugenia lutescens (ELU) collected during the rainy (RS) and dry (DS) seasons of the years 2012 and 2014. The EO were extracted by hydrodistillation and the percentage yields observed for EDY and ELU were very similar in both years/collection periods and the best yields were observed in RS. Unlike, it was observed a large variation for ELA which exhibited higher amount in DS. The chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS), where it was observed a large seasonal variability intra species. The global data show that the chemical profile of the EO varied in quantity of monoterpenes and sesquiterpenes. Overall, the EO featured notable amount of β-caryophyllene, δ-cadinene, α and β-pinene, limonene, bicyclogermacrene and spathulenol. The sesquiterpene β-caryophyllene can be considered as a chemical marker for the species, as it was found with high percentage in all EO, independent of the collection phases. The EO obtained in 2012 were tested against Tetranychus urticae (spotted spider mite) and, interestingly, all showed acaricide activity, being E. langsdorffii (DS/2012) that exhibited best result (CL50 = 1,58 μL.L-1 of air). The β-caryophyllene, one of the major component and common to all species, isolated from E. dysenterica by chromatography column doped with silver nitrate, compared to stand purchased commercially by IR-FT, GC-MS, and NMR (1H and 13C) and also tested for acaricide activity, displaying CL50 = 4,7 x 10-2 μL.L-1 of air. Additionally, the commercial β-caryophyllene was subjected to various chemical modifications to afford five derivatives, which were characterized by spectrometric methods as being caryophyllene oxide; 4,5-dihydroxy-caryophyl-8(15)-ene; caryoph-4,8-dihydroxy-5-yl acetate; β-nocaryophyllone aldehyde and β-nocaryophyllonic acid. The acaricide activity against T. urticae of these derivatives was only observed at β-caryophyllene CL95 (97,78 ± 1,11% of lethality), and the best result of lethality was for the β-nocaryophyllone aldehyde (5,71±1,20%). Beyond the chemical composition, this study showed periods of increased oil production and acaricidal activity.