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.

Insulin resistance is the major metabolic defect in obesity and Type 2 diabetes. Increased lipid accumulation is strongly associated with insulin resistance. A significant component of insulin resistance is thought to be a reduced ability of insulin to activate the cascade of phosphorylation events that lead to the metabolic effects of this hormone. The broad aims of this thesis were to examine the effect of high-fat diets containing different fat subtypes on in vivo insulin signalling, under conditions normally used to detect whole body insulin resistance, and to compare the effects of acute and chronic lipid oversupply on insulin signalling in vivo. Time-course and dose-response effects of insulin stimulation on site-specific phosphorylation of key signalling proteins were studied in rat tissues in vivo, to establish an appropriate experimental system to examine the onset of activation of the insulin signalling pathway. It was determined that short insulin infusions with concurrent glucose infusion, similar to the beginning of a euglycaemic-hyperinsulinaemic clamp, significantly increased the phosphorylation of major intermediates of the insulin signalling pathway in important tissues of insulin action (skeletal muscle [RQ], liver [LIV] and white adipose tissue [EPI]). These experiments provided a platform to study insulin signalling under the same conditions used to study lipid-induced insulin resistance. The provision of diets enriched in polyunsaturated or saturated fatty acids (FA) resulted in the corresponding enrichment of these fat subtypes in rat plasma and tissues. However, the effects on insulin signalling were essentially the same. Both fat diets induced defects in sitespecific phosphorylation of insulin receptor substrate (IRS)-1 and protein kinase B (PKB) in RQ and LIV, but not EPI. This suggests that the amount of fat in the diet, rather than enrichment in a particular fat subtype, had a greater impact on the development of signalling defects and that the response to high-fat feeding was tissue-specific. A 3hr elevation of circulating FA (using a lipid/heparin infusion), to a level that is relevant in clinical Type 2 diabetes, impaired insulin-stimulated PKB phosphorylation with no significant effect on IRS-1 phosphorylation. This suggests that there may be differences in the way acute and chronic exposure to increased FA impair insulin signalling. The phosphorylation defects observed in both chronic and acute studies did not seem to be associated with activation of major stress signalling pathways (JNK and NFkB), which have been suggested to have a role in lipidinduced insulin resistance. In conclusion, these studies demonstrate that impaired IRS-1 and PKB phosphorylation do have a role in the reduced insulin action observed with lipid oversupply in vivo, because the changes were detected under similar conditions as those used to determine whole body insulin resistance.

The objective of this Ph.D. research was to identify new and novel mechanisms of wild radish (Raphanus raphanistrum L.) resistance to photosystem II (PSII) inhibitors, auxinics, and acetohydroxyacid synthase (AHAS) inhibitors. PSIIinhibitor resistance was demonstrated to be target-site based, and conferred by a Ser264 to Gly substitution of the D1 protein. Auxinic resistance was associated with reduced herbicide translocation to the meristematic regions of resistant wild radish plants. Two new resistance mutations of wild radish AHAS were discovered, including one encoding the globally rare Asp376 to Glu substitution, and another encoding an Ala122 to Tyr substitution, which has never been identified or assessed for resistance in plants previously. Characterization of the frequency and distribution of AHAS resistance mutations in wild radish from the WA wheatbelt revealed that Glu376 was widespread, and that some mutations of AHAS are more common than others. Computer simulation was used to examine the molecular basis of resistance-endowing AHAS target-site mutations. Furthermore, through the computer-aided analysis, residues were identified with the potential to confer resistance upon substitution, but which have not previously been assessed for this possibility. Results from this Ph.D. research demonstrate that diverse, unrelated mechanisms of resistance to PSII inhibitors, auxinics, and AHAS inhibitors have evolved in wild radish of the WA wheatbelt, and that these mechanisms have accumulated in some populations.

The molecular events that promote or inhibit specific training adaptations (i.e. skeletal muscle hypertrophy or mitochondrial biogenesis) are not completely understood. Accordingly, there is a need to better define both the acute and chronic responses to divergent exercise stimuli in order to elucidate the specific molecular mechanisms that ultimately determine skeletal muscle phenotype. Therefore, the primary aims of the studies undertaken for this thesis were to examine the acute molecular adaptation responses in skeletal muscle following resistance and endurance training. In order to determine the acute molecular events following repeated bouts of exercise, the study described in Chapter Two compared a high-frequency stacked training regimen designed to generate a summation of transient exercise-induced signalling responses with a conventional-frequency resistance training protocol. Groups (n= 6) of Sprague-Dawley rats performed either high-frequency training (four exercise bouts consisting of 3 - 10 repetitions separated by 3 h) or conventional-frequency training (three exercise bouts consisting of 4 - 10 repetitions with 48 h between sessions). Protocols were matched for total work, and repetitions were performed at 75% one-repetition maximum with 3 min recovery between sets. White quadriceps muscle was extracted 3 h after every training bout, and 24 and 48 h following the final exercise session of each protocol. AKT phosphorylation was significantly decreased 3 h following the 2nd bout of high-frequency training, an effect that persisted until 48 h after the final exercise bout (P less than 0.05), while the phosphorylation state of this kinase was unchanged with conventional training. These results suggest that high-frequency training suppressed IGF-1 mediated signalling. Furthermore, high-frequency training generated sustained and coordinated increases in TNFá and IKK phosphorylation (P less than 0.05), indicating an extended response of inflammatory signalling pathways. Conversely, and irrespective of an initial increase after the first bout of exercise, TNFá signalling ultimately returned to control Abstract values by DAY 5 of conventional-frequency training, indicative of a rapid adaptation to the exercise stimulus. Notably, despite differential AKT activation there were similar increases in p70 S6K phosphorylation with both training protocols. These results indicate high-frequency resistance training extends the transient activation of inflammatory cytokine-mediated signalling and results in a persistent suppression of AKT phosphorylation, but these events do not appear to inhibit kinase activity proximal to translation initiation. The aim of the study described in Chapter Three was to determine the effect of prior training history on selected signalling responses after an acute bout of resistance and endurance exercise. Following 24 h diet / exercise control 13 male subjects (7 strength-trained and 6 endurance-trained) performed a random order of either resistance (8 x 5 maximal leg extensions) or endurance exercise (1 h cycling at 70% peak O2 uptake). Muscle biopsies were taken from the vastus lateralis at rest, immediately and 3 h post-exercise. AMPK phosphorylation increased after cycling in strength-trained, but not endurance-trained subjects (P less than 0.05). Conversely, AMPK was elevated following resistance exercise in endurance-, but not strength-trained subjects (P less than 0.05). Thus, AMPK was elevated only when subjects undertook a bout of exercise in a mode of training to which they were unaccustomed. Surprisingly, there was no change in AKT phosphorylation following resistance exercise regardless of the training background of the subjects. In the absence of increased AKT phosphorylation, resistance exercise induced an increase in p70 S6K and ribosomal S6 protein phosphorylation in endurance-trained but not strength-trained subjects (Pless than 0.05). AKT phosphorylation was increased in endurance-trained, but not strength-trained subjects after cycling (P less than 0.05). These results show that a degree of signalling

Antimicrotubule drugs are used in the treatment of childhood neuroblastoma and acute lymphoblastic leukaemia (ALL). Resistance to these agents can be a major clinical problem and mechanisms mediating resistance are not fully understood. Previous studies have reported an association between the actin cytoskeleton and resistance to antimicrotubule drugs. Thus, the aim of this study was to investigate the role of the actin regulating proteins, LIM kinases (LIMK1 and LIMK2) in drug resistance. In addition, the role of ?? actin, a major actin isoform, in drug resistance was also examined. Chapter 1 reviewed the known mechanisms of antimicrotubule drug resistance and the interaction between the microtubules and actin cytoskeleton. The methodologies used in this study are described in chapter 2. LIMKs are known to regulate the actin cytoskeleton via phosphorylation of cofilin. Real Time RT PCR and western blotting was used in chapter 3 and showed that expression of LIMKs and their downstream target cofilin was altered in antimicrotubule resistant neuroblastoma and leukaemia cells. Moreover, altered LIMK expression was detected in in vivo derived vincristine resistant ALL xenografts and ALL clinical samples, further demonstrating that alterations in LIMKs and cofilin are associated with antimicrotubule drug resistance. Importantly, in chapter 4, gene silencing and drug treated clonogenic assays were performed to elucidate the functional role of LIMK1 and LIMK2 in drug response. Silencing of LIMK1 and/or LIMK2 increased sensitivity of neuroblastoma cells to microtubule targeting drugs and DNA damaging agents, suggesting that LIMKs may be useful targets to improve the efficacy of anticancer drugs. ??-Actin has been associated with drug resistance and chapter 5 used gene silencing and drug treated clonogenic assays to show that decreased ?? actin expression conferred resistance to anitmicrotubule drugs but not to DNA damaging agents. Microscopy and tubulin polymerisation assays showed that reduced ??-actin protects microtubules from paclitaxel induced polymerisation. This data supports a functional role for ?? actin in antimicrotubule drug action. In conclusion, this study showed that LIMKs and ?? actin mediate the action of antimicrotubule drugs and other anticancer agents, demonstrating that the actin cytoskeleton may serve as a useful drug target to improve the efficacy of anticancer drugs.

As anti-cancer agents, retinoid induce cell growth arrest and differentiation, while HDACIs cause cell differentiation, growth inhibition, death and inhibit angiogenesis in many cancer types. However, a proportion of patients respond poorly to these therapies. My studies, presented here, aimed to improve the anti-cancer effects of these agents by identifying key factors which mediate cancer cell sensitivity or resistance to their action. In this study I have found that the clinically used retinoid, 13-cis RA, exerts its anti-cancer signal in a manner similar to atRA, by modulating the transcriptional response of retinoid-regulated genes. HDACI-induced cytotoxicity is significantly enhanced when combined with IFNα in 8 out of 9 cancer cell lines from various organ origins. Sensitivity to the combination treatment correlated with an absence of basal p21 protein expression, and cell cycle arrest. Knocking-down p21 gene expression further sensitized cancer cells to the combination therapy. Moreover, IFNα and HDACI co-operatively inhibited pro-angiogenic gene expression in cancer cells, and the combination therapy decreased endothelial cell migration, invasion, and capillary tubule formation. Further experiments on p21 as a resistance factor to anti-cancer treatment demonstrated that conditioned media from breast cancer MCF-7 cells transfected with p21 siRNA, induced significantly less endothelial cell migration, invasion and vascular sprouting, compared with media from cells transfected with scrambled siRNA. LC/MS analysis of the conditioned media revealed that Trx secretion was significantly reduced after p21 knockdown. The reduction in Trx secretion following p21 knockdown was due to a direct effect of p21 siRNA on the expression of intracellular TBP2 in neuroblastoma, prostate and lung cancer cells. Consistent with this result, media from MCF7 cells transfected with TBP2-specific siRNA alone, promoted endothelial cell invasion and vascular sprouting, Trx knockdown resulted in opposite effects, and the anti-angiogenic effect of p21 siRNA was offset by simultaneous TBP2 siRNA transfection. ChIP assay revealed that p21 directly bound to an E2F1-bindng site in the TBP2 gene promoter. These data indicate that p21 promoted tumour-driven angiogenesis through transcriptional repression of TBP2. Collectively, my experiments indicate several potential treatment targets directed toward enhancing the effectiveness of HDACIs and retinoids.

Widespread tetracycline resistance (TcR) has limited the clinical use of Tc for the treatment of bacterial infections. Tet(O) protein is present in many bacteria and is the major transmissible TcR determinant in Campylobacter jejuni, a common cause of acute bacterial diarrhea worldwide. Tet(O) protects ribosomes against the inhibition of protein synthesis by Tc. Tet(O) binds to the ribosome at a similar site as EF-G, a structural homologue of Tet(O) with GTPase activity that is required for protein elongation. EF-G interfered with the kinetics of Tet(O)-mediated Tc release suggesting that EF-G competes with Tet(O) for ribosome binding. Indirect assessment of EF-G and Tet(O) binding to 70S ribosomes by GTP hydrolysis was unable to clearly demonstrate competition for binding. This thesis contributed to the further understanding of the kinetics of Tc release by Tet(O), and may facilitate the development of novel strategies to overcome Tet(O)-mediated TcR in bacteria which cause human infections.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 118-125).

Thesis (M.Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

When I first arrived in Liège, the scientific activities of our laboratory focused on the identification of candidate-genes whose different alleles interfere with the resistance/susceptibility of animals against infectious diseases. Two genes were being intensively studied (Mx and OAS) due to their theoretical potential in interfering with the replicative cycle of several viruses responsible for bovines viral pneumonias (Baise et al., 2004 ; Gerardin et al., 2004 ; Leroy et al., 2005 ; 2006). My work consisted then, in the identification of other genes potentially implicated in the resistance against the Paramyxoviruses. The Paramyxoviridae family includes some of the great and ubiquitous disease-causing viruses of animals, including the bovine parainfluenza type 3, the bovine respiratory syncytial virus, the Newcastle disease virus, the distemper virus, etc. Evidence was accumulating that genetic factors were involved both in the control of infectious diseases (Abel et al., 1991 ; 1995 ; Alcais et al., 1997 ; Jin et al., 1999 ; Martinson et al. 1997 ; Shaw et al., 1995) and in the regulation of infection levels and clinical presentation (Garcia et al., 1999 ; Plancoulaine et al., 2000 ; 2003). Thus, identifying genes that control the organism response to paramyxoviruses was a crucial step in elucidating how they might affect the pathophysiological processes underlying the severity of the disease induced. Other experiences done in this laboratory showed us how risky and difficult was any extrapolation of the mouse results to another species if these results were brought through infection with a heterologous virus, so we decided to implement this strategy with Sendai virus (SeV), the archetype organism of the Paramyxoviridae family, from which most of the basic biochemical, molecular and biologic properties of the whole family were derived from (Chanock et al., 2001). With this goal in mind my thesis was divided in five successive steps: In order to establish a standard model of SeV infection, the first step consisted in determining the best volume of inoculum that was needed to achieve a safe, reproducible pulmonary deposition of Sendai virus in the mice lungs. Secondly we developed a murine model of SeV infection using a series of different and sophisticated procedures that allowed a quantitative assessment of disease severity and progression. Then we compared SeV infections among 6 strains of mice that were deliberately chosen because they originated from different lineages, as deduced from known genealogical and phylogenetic data. Applying these procedures to distinct inbred strains of mice, revealed highly significant differences in susceptibility between them. More specifically 129/Sv were highly susceptible while BALB/c were particularly resistant, BALB/c exhibiting a benign and asymptomatic affection of the epithelium of the airways, with no functional impact, generating slight mononucleated cell infiltration, in which viral replication is repressed and the virus swiftly eliminated. As a result, in the fourth part of my study we discussed a series of hypotheses that should be tested in the future to improve our understanding of why BALB/c is so resistant to SeV infection. Practically speaking, our studies led to the gathering of a genomic DNA collection from the parental extreme lines in terms of susceptibility (129/Sv) and resistance (BALB/c) and their F1 and F2 offspring. Within this bank, each of the 263 DNA sample is associated with a portfolio of phenotypic values that are estimators of the resistance each mouse opposed to the SeV. We hope that, between expert hands, this bank will allow the detection of genes of which the alleles contribute, at least in part, to the spectacular resistance of BALB/c. The last part of my thesis consisted in applying our model to establish if the receptor TLR4 influenced the pathophysiology of the Paramyxoviridae in general. Because the role of this receptor had already been excluded for SeV, we tested another virus of the same family and homologous for the mice, the PVM (standing for pneumonia virus of mice). This work showed, in contradiction of what had been found in heterologous models in the past, that TLR4 is not involved in host defense against respiratory tract infection with the Paramyxoviridae.