Дисертації з теми "Nox inhibition"

Dans le monde, 350 000 leucémies sont diagnostiquées chaque année. La rechute reste un problème majeur des leucémies aiguës myéloïdes (LAM) et le métabolisme oxydatif pourrait jouer un rôle essentiel dans la réponse au traitement. Un faible niveau des espèces réactives de l’oxygène (ROS) est associé à des propriétés des cellules souches leucémiques et la quiescence alors qu’un niveau plus élevé caractérise les leucoblastes proliférants. L’homéostasie des ROS repose sur un équilibre entre les systèmes oxydants et antioxydants. Les antioxydants sont bien documentés dans les LAM alors que les connaissances sur l’activité oxydante sont encore limitées. Dans ce travail nous avons choisi d’étudier les sept complexes NADPH oxydases (NOX) dans 25 lignées issues de LAM humaines et des LAM primaires. L’analyse des ARNm et des protéines montre des profils d’expression variables entre les lignées avec une expression plus forte des sous-unités du complexe NOX2 dans les lignées correspondant à des stades de différenciation myéloïde plus avancés. L’activité enzymatique des NOX est cependant équivalente entre les lignées. Deux inhibiteurs, DPI et VAS3947, ont été utilisés pour connaître la contribution des NOX à la production des ROS cellulaires. Alors qu’ils ont inhibé l’activité, ils ont aussi généré un stress oxydatif majeur conduisant à une diminution de la prolifération cellulaire et une forte apoptose, le DPI en augmentant les ROS mitochondriaux et VAS3047 les ROS cytoplasmiques. Afin de connaitre les sous-unités impliquées et de mieux comprendre les mécanismes, les sous-unités NOX2 et p22phox ont été inhibée par ARN interférence. Celle-ci n’ont pas affecté la prolifération mais ont montré des effets compensatoires. Nos data montrent qu’inhiber les NOX pourrait s’avérer une stratégie thérapeutique en augmentant le stress oxydatif dans les cellules leucémiques
350,000 leukaemia are diagnosed each year worldwide. In acute myeloid leukaemia (AML), relapse remains a major problem and the oxidative metabolism might play a crucial role in the therapeutic response. Low level of reactive oxygen species (ROS) is associated with properties of leukemic stem cells and quiescence whereas higher level promotes leukoblasts proliferation. ROS homeostasis relies on a tightly regulated balance between the oxidant and antioxidant systems. Although the antioxidant system is extensively studied in AML, the oxidant system remains poorly documented. In this work we aimed to study the seven NADPH oxidases (NOX) complexes in 25 AML human cell lines and primary samples. NOX transcriptional and protein profiles are variable with a higher expression of NOX2 in cell lines belonging to mature differentiation stages. An equivalent level of enzymatic activity was observed across all the cell lines. To reveal the contribution of NOX to global ROS production in the cells, two NOX inhibitors, DPI and VAS3947, were then used. Although both inhibitors efficiently blocked NOX activity they unexpectedly triggered strong oxidative stress leading to reduced cell proliferation and strong apoptosis, DPI by increasing mitochondrial ROS while VAS3947 by increasing cytoplasmic ROS production. To highlight which of the subunits were involved and to understand the mechanisms, NOX2 and p22phox subunits were inhibited using shRNA strategy. These did not affect cell proliferation but revealed a compensation effect. Our data suggest that NOX inhibition might be potential therapeutic strategy by increasing oxidative stress in leukemic cells

The aim of the research in this thesis was to study how environmentally friendly corrosion inhibitors for cooling water systems might be developed and used. Firstly, reduced toxicity inorganic corrosion inhibitors (i.e. nitrite/molybdate) were considered. Secondly, non-toxic inhibitors based on mono and di-basic salts of carboxylic acids were studied systematically as a function of carbon chain length. For nitrite inhibitor alone, a concentration of 7 mM NaNO2 was effective to inhibit carbon steel in chloride media of 10 mM NaCl, while 10 mM nitrite was needed in sulphate media of 3.66 mM Na2SO4. However, it was found possible to significantly reduce the concentration of nitrite by adding molybdate in synergy. This was attributed to the nitrite passivation combined with ferrous molybdate salt film pore plugging thus promoting a continuous and protective film on the material within these media. Thus, in pH 6-10 an inhibition efficiency of 97% was recorded with a mixture of 3 mM nitrite/2 mM molybdate in both chloride and sulphate media and at 25°C and 60°C. However as the solution pH decreased below pH 4 the inhibition efficiency decreased to about 47%.In the second part of the study, the use of sodium salts of carboxylic acids with different chain lengths has been investigated. In this part a summary of the performances and limitations of both mono- and di-sodium carboxylate inhibitors are presented. For mono-carboxylates, the inhibition efficiency reached a maximum value of 95% in stagnant aerated solutions at a chain length of C=4 with a critical inhibition concentration of 6 mM in 10 mM NaCl solution. However the inhibition efficiency gradually decreased as the number of carbon atoms in the chain length increased to more than 8, or less than 4, and this was in agreement with surface hydrophobicity and contact angle results. For lower chain lengths, the carboxylate anion becomes more acidic and complexing of the metal ion while for longer chain lengths, the carboxylate anion becomes less soluble and tends to micellise wherby the active groups are no longer available for surface adsorption. For di-carboxylates the inhibition efficiency improved in 10 mM NaCl at a given chain length compared with mono-carboxylates, and continued to increase to C=8 (sebacate), which achieved excellent inhibition efficiency. However, sebacate is costly so a blend with ethyl hexanoate was found to be economically favoured.

Modern, large scale Cheddar cheese manufacture is dependent on reliable acid production by Lactococcus lactis subspecies cremoris and subspecies lactis starter cultures. Any inhibition of acid production may affect cheese quality, disrupt production schedules and reduce profitability. The presence of antibiotic residues in manufacturing milk resulting from the treatment of mastitis in lactating cattle is a potential source of starter culture inhibition. Therefore, a range of antibiotic concentrations was assessed for measurable inhibitory effects on acid production and compared to the minimum detectable concentrations by approved screening test procedures. Antibiotics were selected from formulations approved for use on lactating cattle for the treatment of mastitis. Novobiocin, lincomycin, oleandomycin and oxytetracyline HCl, all non-b-lactam antibiotics, inhibited acid production of one or more L. lactis strains at antibiotic concentrations below the detectable limit of standard screening procedures.
Depending on the antibiotic, either or both the Bacillus stearothermophilus (var. calidolactis) disk assay and/or the Delvo SP assay were ineffective at detecting the antibiotics at concentrations required to inhibit the starter strains. Consequently, antibiotic residues below the detectable limits of these testing procedures could cause significant starter culture inhibition, disrupting cheese making schedules. Another potential source of starter culture inhibition is related to raw milk quality and the practice of refrigerated storage prior to processing. Previous studies differed as to whether the growth of psychrotrophic organisms would have a detrimental impact on subsequent acid production by starter bacteria employed in cheese manufacture. In this study, no inhibition of acid production by a commercial L. lactis subsp. cremoris strain was evident when grown in milk that had undergone short term temperature abuse. Antimicrobial systems native to bovine milk may also have an adverse impact on starter culture performance. The present study assessed the inhibitory effect of an activated lactoperoxidase system (LPS) on a range of L. lactis cultures. All of the strains were significantly inhibited when grown on reconstituted skim milk in the presence of an active LPS. Inhibition of acid production by strains grown on glucose was also observed, leading to further investigations to describe the inhibitory process. A non-phosphoenolpyruvate phosphotransferase (PEP/PTS) dependent glucose transport system, first observed in 1980 in one L. lactis subsp. lactis strain, was hypothesised as a link in strain variations in LPS sensitivity. However, the LPS sensitive L. lactis subsp. cremoris strains tested did not take up glucose in a PEP depleted state, most likely due to their inability to utilise arginine as an ATP generating energy source. The questions remain unanswered whether cremoris strains possess this glucose transport mechanism and whether it could contribute to strain variations in LPS sensitivity.
In a subsequent investigation, galactose phosphotransferase system (PTS) deficient L. lactis strain ATCC 7962 demonstrated log phase growth inhibition when grown on galactose in the presence of the model LPS. Previously reported LPS mediated effects on the glycolytic enzyme hexokinase do not appear to explain this result. The present study confirmed strain variability in sensitivity to the model LPS among both Lactococcus lactis subspecies lactis and subspecies cremoris strains. Further, the observation that dithiothreitol significantly alleviated the inhibition of a highly sensitive cremoris strain, implicated the involvement of sulphydryl groups as the target of the transient inhibitory factors. Data collected excluded the possibility that portions of the metabolic pathways involved in fructose and galactose metabolism are sensitive to the LPS in cells possessing PEP/PTS capability. This study also identified potential directions of further work to elucidate the mechanism(s) of LPS inhibition.

Cerebral stroke has become one of the leading causes of death and disability worldwide. During an ischemic stroke, oxygen and nutrient deprivation occurs, which combined lead to cell starvation, anoxia, and eventually cell death. However, when blood flow is restored, reperfusion damage occurs resulting in increased cell death through several mechanisms. One of the main reasons behind ischemia/reperfusion damage is oxidative stress due to elevated production of reactive oxygen species (ROS) during reperfusion. There are several proteins and processes that are thought to be involved in elevated oxidative stress and the formation of ROS during reperfusion, among which the NADPH oxidase (Nox) family is suggested to be the main contributor of ROS.To examine this hypothesis, in the present work, we inhibited activity of the Nox2 and Nox4 enzymes during ischemia/reperfusion with the Glucox Biotech AB (Sweden) inhibitors M4, M107, and M114 to evaluate whether reducing Nox activity could reduce the ischemia/reperfusion-induced cell death, hence be used as a potential stroke treatment, the cell viability was measured with MTS after ischemia/reperfusion induction and treatment with the Nox substances. We also examined the gene expression levels of the Nox enzymes Nox2 and Nox4 with qPCR after induced ischemia/reperfusion in the neuroblastoma cell line NB69.Our results showed a decrease in Nox4 gene expression after 1h ischemia/8h reperfusion and an increased expression after 1h ischemia/24h reperfusion in NB69 cells. Treatment with M114 resulted in increased cell viability after 2h ischemia/72h reperfusion. However, the toxic effect of ischemia/reperfusion-induced response was found to be inadequate, as indicated by extensive proliferation and lack of cell death. This unfavorable outcome is suggested to be excess of oxygen in medium, metabolization of L-glutamine, and effects of growth factors in the serum used in cell culture medium during the ischemic phase. Therefore, the cell culture protocol was modified to the use of PBS instead of glucose-free medium under serum-free condition during the ischemia. The altered ischemic conditions resulted in continuous reduction in cell viability at increasing ischemic time points with total cell death at 2h ischemia, suggesting applicable conditions for ischemia/reperfusion studies. Even though a conclusion could not be made about the inhibitors M4, M107, and M114 as the cell viability assay was performed under insufficient conditions; the Nox inhibitors shows high potential as future ischemic stroke treatments, which may help save lives and improve life quality for affected patients.

Central to human adaptive behaviour is the ability to update one’s motor actions in the face of environmental changes, for which a key component is the ability to inhibit ongoing actions that are no longer appropriate. A substantial body of previous research has implicated the right inferior frontal gyrus (rIFG) and the pre-supplementary motor area (pre-SMA) as plausible sources of inhibitory control, but it remains unclear whether these regions host a specialised inhibitory control mechanism or instead support a more general system of action updating. This uncertainty stems from the limited number of studies that have controlled for non-inhibitory processes in response inhibition research. The overarching aim of this thesis was to resolve this ambiguity by studying behaviour, neurophysiology and neurochemistry during action updating in the presence and absence of inhibition. For the key experiments, detailed methods and hypotheses were pre-registered prior to data collection to minimise research bias and ensure transparent discrimination of confirmatory and exploratory inferences.

Mechanisms underlying relaxation in response to inhibitory NANC nerve stimulation and putative neurotransmitters of these nerves have been examined in the guinea-pig internal anal sphincter (IAS) and compared with those in the bovine retractor penis muscle (ERP) and guinea-pig taenia caeci. Two types of techniques were employed. One which measured the effects of nerve stimulation and drugs on electrical membrane properties where intracellular microelectrode and simultaneous mechanical recording techniques were used. Drugs, for example ATP or cromakalim were applied by perfusion in the Krebs' solution, microinjection into the bath, or by hydrostatic pressure ejection. A second method assessed the underlying biochemical changes accompanying relaxation by measuring alterations in second messenger systems, for example cyclic AMP and cyclic GMP using radiomimmunoassay techniques. Electrical events were clearly an important accompaniment to mechanical inhibition in the IAS. Field stimulation (single pulse and 5 pulses at 5, 10 and 20 Hz; 0.5ms; supramaximal voltage) produced large inhibitory junction potentials of up to 15mV in amplitude which accompanied relaxation of 80% of muscle tone. Indeed, hyperpolarising electrotonic current passed into the IAS produced relaxation. The neurotransmitter which is released by field stimulation of the inhibitory nerves is probably ATP since exogenous application of purine by hydrostatic pressure ejection (5.8x10-4M; 10-55ms) produced a dose-dependent hyperpolarisation. The membrane potential change was similar in size, rate of decline and duration to the ijp. Neither hyperpolarisation nor relaxation could be achieved with the P2x-purinergic agonist, betaMeATP (10-5-10-3M) or the P2-purinergic agonist adenosine (10-3M) thus ATP was acting on the P2y-purinergic receptor. Inhibitory NANC neurotransmission was not peptidergic since VIP (10-7-10-5M), bradykinin (10-3M), neuropeptide Y (10-5M), bombesin (10-5M), leu-enkephalin (1.8x10-4M), met-enkephalin (1.8x10-5M), somatostatin (10-6-10-3M) and substance P (7.6x10-6 - 7.6x10-4M) each had no effect on the membrane potential of the IAS. There is also evidence that stimulation of -adrenoceptors by isoprenaline (10^-9 - 10^-5M) produced relaxation which was accompanied hyperpolarisation of the IAS. In all cases where hyperpolarisation and relaxation are associated in the IAS, the mechanism underlying the electrical change appeared to be an increase in K^+ conductance. Apamin (4.5 x 10^-6M) which blocks certain Ca^2+-mediated K^+ channels, antagonised the electrical and mechanical responses produced by field stimulation and ATP. Similarly, TEA (8x10^-2M), which blocks most K^+ channels, antagonised the hyperpolarisations and relaxations produced by field stimulation, ATP and isoprenaline. Indeed, the K^+ channel activator cromakalim (10^-9-10^-5M) produced hyperpolarisation and relaxation of the IAS suggesting that an increase in K^+ conductance is important in the mediation of mechanical inhibition of the IAS. Relaxation of the IAS was also produced without a significant change in membrane potential by altering the levels of cyclic nucleotides within the smooth muscle cells of the IAS. Forskolin (10^-9-10^-5M), which activates adenylate cyclase with a subsequent increase in cyclic AMP, relaxed the IAS. Similarly, sodium nitroprusside (10^-9-10^-4M) - a cyclic GMP phosphodiesterase inhibitor, and 8-bromo-cyclic GMP (10^-4M) each increased cyclic GMP and produced relaxation of the IAS. Direct measurement of cyclic nucleotide levels of the IAS showed that field stimulation (80 pulses at 8H_Z; 0.5ms; supramaximal voltage) and ATP (10^-4) elevated the cyclic AMP and cyclic GMP contents of the IAS. All other stimuli which produced slow, prolonged electrical and mechanical changes increased the level of only one cyclic nucleotide. Isoprenaline (10^-4M), cromakalim (10^-5M) and forskolin (10^-5) increased cyclic AMP content while sodium nitroprusside (10^-5M) increased the cyclic GMP content. Further investigation of other second messenger systems involved in relaxation of the IAS showed that increase in inositol phosphate turnover was not associated with stimulation of inhibitory P_2y-purinoceptors by ATP (10^-2M) in the IAS. However, an increase in inositol phosphate accumulation was produced by noradrenaline (10^-4M) and associated with contraction. A method was devised to measure the intraluminal pressure changes of the internal anal spincter in the anaesthetised guinea-pig using a Millar pressure transducer. Using this method the in vitro results were largely confirmed by this in vivo study. Basal intraluminal sphincter pressure was increased by noradrenaline acting on -adrenoceptors and decreased by isoprenaline acting on -adrenoceptors, ATP on P_2y-purinoceptors and 2-chloroadenosine on P_1-purinoceptors.

Alors que le traitement de nombreuses tâches ordinaires (e.g., parler à son passager, répondre à un e-mail) est en cours, l'apparition d'un stimulus important (e.g., le klaxon d'un voiture) peut nécessiter un traitement prioritaire. Paradoxalement, l'investigation de ce constat dans le champs de la psychologie expérimentale ne rend compte que très rarement compte de la possibilité d'interrompre une action en cours en vue de donner la priorité à une autre réponse motrice. Il apparait donc heuristique d'approfondir les limites et les effets de cette possibilité relativement répandue d'un point de vue écologique. Pour ce faire, nous avons articulé les problématiques inhérentes à l'interférence en double tâche et à l'inhibition motrice afin de mettre au point une série de quatre volets expérimentaux lors desquels la possibilité d'interrompre une réponse non prioritaire en vue de donner la priorité à une autre réponse motrice fut systématiquement donnée aux participants. L'ensemble des résultats convergent vers une diminution de la probabilité d'interruption de la réponse motrice non prioritaire lorsque le stimulus prioritaire apparaît tardivement et peu fréquemment. De plus, le caractère prioritaire d'une tâche motrice ne semble pas constituer un paramètre suffisant permettant d'isoler les processus correspondants de ralentissement. Corollairement, l'interruption de la réponse motrice non prioritaire ne semble pas non plus totalement prémunir le traitement de la tâche prioritaire de ralentissement notamment lorsqu'il n'est pas possible de prédire avec certitude l'apparition du stimulus correspondant
While many ordinary task processes are ongoing (e.g., to talk with a passenger, answer an e-mail), the apparition of an important stimulus (e.g., a car honking at us) can urge a prioritized process. Paradoxically, investigations about this observation in experimental psychology's field give very few account of the possibility to interrupt a processing action in order to give all priority to another one. Thus, it seems heuristic to deepen the limits and the effects of this widespread possibility from an ecological perspective. To do so, we articulated dual task and motor inibition problematics in order to set up a serie four experimental parts where the possibility to interrupt a low priority response in order to prioritize another response was systematically given to the participants. The whole results converge toward a diminution of the capability to interrupt a low prioritized motor response when the prioritized stimulus appears belatedly and rarely. Moreover, the prioritized nature of a motor task seems to not represent a sufficient parameter alowing isolation of its processes from slowing. As a corollary, interruption of the low prioritized response seems to not prevent the prioritized process from slowing especially when corresponding stimulus apparition is not predictable

Les pensées répétitives négatives (PRN) sont considérées comme un des processus transdiagnostiques impliqués dans le développement, le maintien et la récurrence de plusieurs troubles psychologiques tels que les troubles de l'humeur, les troubles anxieux ou les addictions. Une des priorités dans les recherches actuelles sur les PRN est de déterminer quel processus contribue au développement et au maintien des PRN inadaptés. La littérature suggère que les déficits d’inhibition et de désengagement attentionnel sont deux facteurs potentiellement impliqués dans la récurrence des PRN. L’objectif de la présente thèse de doctorat était, premièrement de systématiser les recherches antérieures sur le lien entre les PRN et l’inhibition. Deuxièmement nous avons testé dans des études expérimentales comment l’induction des PRN affectait l’inhibition et le désengagement attentionnel.Le premier article expérimental présente une série d’études testant l’impact des PRN sur l’efficacité de l’inhibition. Le second article expérimental montre comment l’induction des PRN affecte le désengagement attentionnel. Une plus-value des études présentées dans cette thèse est la différenciation faite entre les PRN constructives (concrètes expérientielles) et non constructives (abstraites analytiques) dans la procédure d’induction.Les résultats suggèrent que les pensées analytiques abstraites interfèrent avec la régulation émotionnelle dans une situation de problème non résolu. Contrairement aux prédictions, il semble que les pensées abstraites favorisent les processus inhibiteurs et attentionnels dans le traitement des stimuli verbaux.Les résultats sont discutés selon la perspective de la théorie du mode de traitement et de la théorie de dérégulation du niveau objectif/action. Une nouvelle approche des fonctions exécutives dans les PRN est également proposée : l’hypothèse de l’allocation des ressources cognitives dirigée par le mode de traitement
Repetitive negative thinking (RNT) is one of the transdiagnostic processes involved in development, recurrence and relapse of various psychological disorders, i.e. mood disorders, or anxiety disorders. One of the priorities in the current RNT research is to identify the mechanisms responsible for RNT development and maintain. RNT theory and previous research identified inhibition and attentional disengagement impairment as two potential factors of RNT recurrence.The first aim of the dissertation was to systematize previous research exploring the relation between RNT and inhibition. The second aim was to experimentally test how RNT induction affects both, inhibition and attentional disengagement. The first two chapters present RNT concept itself and a systematic literature review on the links between RNT and inhibition. The following chapters are composed of two empirical articles. The first article presents three experimental studies exploring the impact of RNT induction on inhibition efficiency. The second article tests experimentally how RNT affects attentional disengagement. An important contribution of these experimental studies lays also in testing separately constructive (concrete experiential) and unconstructive (abstract analytic) RNT processing mode.The results suggest that abstract analytic thinking impairs emotional regulation in a non-resolved problem situation – a situation predicted to activate RNT. Contrarily to the predictions, it seems that abstract analytic processing enhances inhibition and attentional performance for verbal stimuli comparing to concrete processing.These results are discussed at the theoretical level in the processing mode theory and deregulation of goal/action level perspective. We provide also methodological recommendation for the further research on the link between RNT and executive functions. Finally, we propose a new framework for the hypothesis of processing mode driven resource allocation in RNT

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is one of the leading causes of death due to a single infectious agent and results in more than 1 million human deaths every year. M.tb infection of the host initiates a local inflammatory response, resulting in the migration of a number of host plasma protein and non-protein factors to the site of infection. In addition, some of these factors are also produced locally at the site of infection. It is envisaged that these host factors are likely to come in direct contact with M.tb and immune cells and may modulate the outcome of the infection. In this study, a number of host factors including transferrin, lactoferrin, fibrinogen, C-reactive protein, alpha-2-macroglobulin (α2M), vitronectin, plasminogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), serotonin, L-alpha dipalmitoyl phosphatidylcholine (DPPC) and platelet activating factor C-16 (PAF C-16) were screened in vitro for their direct effect on the growth of mycobacteria using M.smegmatis as a model. As a result of this screening, PAF C-16, a phospholipid compound was identified that directly inhibited the growth of M.smegmatis and M.bovis BCG in a dose and time-dependent manner. Use of a range of PAF C-16 structural analogues, including Lyso-PAF, PAF C-18, Hexanolamino PAF, 2-O-methyl PAF & Pyrrolidino PAF, revealed that small modifications in structure did not alter the direct growth inhibition property of PAF C-16 and similar levels of M.smegmatis and M.bovis BCG growth inhibition were observed as compared to PAF C-16. Structural dissection of PAF C-16 suggested that the attachment of carbon tail to the glycerol backbone via ether bond at sn-1 position was important for its direct growth inhibition activity against mycobacteria. Microscopy and flow cytometry with PAF C-16 treated M.smegmatis and M.bovis BCG showed damage to the bacterial cell membrane. The addition of membrane-stabilizing agents, α-tocopherol, tween-80 and tween-20, partially mitigated the growth inhibitory effect of PAF C-16. These results suggested that the growth inhibition activity of PAF C-16 against mycobacteria is most likely due to its detergent-like effect, resulting in damage to the bacterial cell membrane. PAF C-16 and its structural analogues were also investigated for their effect on the growth of intracellular M.smegmatis inside THP1 cells. In vitro, PAF C-16, PAF C-18 and Hexanolamino PAF inhibited the growth of intracellular M.smegmatis, whereas, analogues such as Lyso-PAF and 2-O-methyl PAF failed to show any growth inhibitory effect, suggesting that the presence of acetyl group at sn-2 position was important for growth inhibition of intracellular M.smegmatis. Use of PAF receptor antagonists partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M.smegmatis, suggesting this inhibition was through receptor-mediated signalling pathways. Blocking of PAF C-16 signalling pathway components such as phospholipase C and phospholipase A2, resulted in the increased survival of intracellular M.smegmatis. Arachidonic acid, a product of PAF C-16 signalling pathway directly inhibited the growth of M.smegmatis. Furthermore, inhibition of iNOS enzyme and antibody-mediated neutralization of TNF-α partially mitigated the inhibitory effect of PAF C-16 on intracellular M.smegmatis growth, suggesting that the production of NO and TNF-α were also involved in PAF C-16 induced intracellular growth inhibition. Overall, this study has identified PAF C-16, its structural analogues such as Lyso-PAF, PAF C-18, Hexanolamino PAF and other compounds including 1-O-hexadecyl-sn-glycerol, miltefosine and hexadecyl lactate with novel anti-mycobacterial activity. Further investigations are needed to demonstrate their effectiveness against M.tb both in vitro and in animal models to assess their therapeutic potential as anti-TB drugs.

Cardiovascular disease is the leading cause of death worldwide and ischemic heart disease is the most common manifestation. Despite improved outcomes during the last decades, patients with acute coronary syndromes (ACS) are still at substantial risk of recurrent ischemic events and mortality. The aims of this thesis were to investigate the effect of the novel antiplatelet agent ticagrelor versus clopidogrel in patients with non-ST-elevation ACS (NSTE-ACS), overall and in relation to initial revascularization, and to explore this effect in relation to cardiac biomarkers. The impact of timing of revascularization in non-ST-elevation myocardial infarction (NSTEMI) was also studied, by assessing risk of mortality and recurrent myocardial infarction in relation to delay of percutaneous coronary intervention (PCI) in a nation-wide cohort. Finally, a novel clinical prediction model based on angiographic findings, biomarkers, and clinical characteristics was developed to estimate risk of ischemic events after performed revascularization. Ticagrelor treatment compared with clopidogrel was associated with a reduction in the composite endpoint of cardiovascular death/myocardial infarction/stroke and mortality alone, without any increase in overall major bleeding, but increased non-CABG-related major bleeding. The effect of ticagrelor over clopidogrel was consistent independent of initial revascularization. Elevated high-sensitivity cardiac troponin-T predicted benefit of ticagrelor over clopidogrel, while no difference between treatments was detected at normal levels. In patients with NSTEMI, PCI treatment within two days after hospital admission was associated with lower risk of all-cause death and recurrent myocardial infarction compared with delayed PCI. The new clinical prediction model included the following variables: prior vascular disease, extent of coronary artery disease, level of N-terminal pro-B-type natriuretic peptide and estimated glomerular filtration rate; and showed good discriminatory ability for the risk prediction of cardiovascular death/myocardial infarction/stroke and cardiovascular death alone. In conclusion, these results show that ticagrelor reduces the risk of recurrent ischemic events and mortality in patients with NSTE-ACS when compared with clopidogrel, and this effect seems independent of performed revascularization. The results also indicate that biomarkers could be used to select patients who would benefit most from more intense platelet inhibition. Furthermore, early PCI in NSTEMI seems to be associated with improved outcome. Finally, the novel clinical prediction model based only on four variables showed good discriminatory ability, which makes it a potentially effective and simple tool for tailored treatment based on individual risk of recurrent events.

The cell cycle is tightly regulated to safeguard the complete and equal division of the nuclear contents between the daughter cells. To ensure accurate segregation of the chromatin, DNA catenation generated during replication must be resolved by topoisomerase IIα (TopoIIα). In some tumour-derived cells, Protein kinase C epsilon (PKCε) has been shown to play a crucial role in mediating a delay signal at the metaphase-to-anaphase transition when catenation is still present, allowing time for catenation resolution prior to anaphase entry and sister chromatid separation. PKCε has also been shown to affect the decatenation process itself, with an increase in metaphase catenation being observed upon PKCε knock down or inhibition. Active PKCε is predominantly membrane associated as lipid binding is required for canonical activation. However, a lipid-independent activation pathway has been observed which involves complex formation with the scaffold protein 14-3-3. In this thesis, we have identified a second lipid-independent pathway. In mitosis, we have identified a caspase-mediated, proteolytic cleavage of PKCε within a chromatin-associated sub-compartment. The cleavage occurs at two distinct sites, one of which leads to the generation of a free kinase domain. Blocking PKCε cleavage at this site, via mutation or caspase-7 inhibition, results in a reduction in the PKCε dependent delay to the metaphase-to-anaphase transition. This can be rescued by artificially recapitulating the cleavage event. Cells expressing a non-cleavable PKCε also retain larger amounts of metaphase catenation, indicating that cleavage is required to support resolution as well as the delay. As both of these phenotypic responses require active PKCε this proteolytic cleavage represents a novel, non-canonical activation pathway. The mechanism by which the free kinase domain generated influences the response to TopoIIα inhibition is still to be fully resolved but phosphorylation of the key mitotic regulator Aurora B and subsequent phosphorylation of TopoIIα have been shown to play a crucial role. Due to an increased dependence on this response in a number of tumour-derived cells, identifying the pathways involved in its regulation may offer a potentially novel target for cancer therapeutics.

Nicht-kleinzelliger Lungenkrebs (NSCLC) hat bis heute einen hohen medizinischen Bedarf an effektiveren Therapien. Inhibitoren der Bromodomain and extra-terminal domain (BET) Familie wie JQ1 wirken in verschiedenen Krebsarten, einschließlich Lungenkrebs. Während ihre Aktivität auf die Expression von Onkogenen wie c-Myc in vielen Studien untersucht wurde, bleibt der Effekt von BET-Inhibition auf den Apoptose Signalweg weitgehend unbekannt. In dieser Arbeit wurde die Aktivität von BET-Inhibitoren auf den Zellzyklus und auf Komponenten der Apoptose-Antwort der Zelle untersucht. Genomweite Transkriptionsanalysen haben zusammen mit Chromatin Immunpräzipitation und anschließender Sequenzierung geholfen das MYC Gen und dessen assoziierte Super-enhancer als primäres Ziel des BET-Inhibitors JQ1 zu identifizieren. Mittels einer Gruppe von NSCLC Modellen belegt diese Arbeit, dass Zelllinien die auf die BET-Inhibitoren reagieren in Apoptose gehen und eine Reduktion der S-Phasen Population zusammen mit gleichzeitiger de-regulation der c-Myc Expression aufwiesen. Andererseits konnte die ektopische Überexpression von c-Myc der anti-proliferativen Wirkung entgegenwirken. Die Auswirkung von BET-Inhibition auf die Expression von 370 Genen, die in der Apoptose Regulation involviert sind, wurde in sensitiven und resistenten Zellen verglichen und dabei wurde die starke BET-Abhängigkeit der Expression von zwei Schlüsselgenen der Apoptose FLIP und XIAP festgestellt. Die Kombination von JQ1 mit dem tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) oder dem Chemotherapeutikums Cisplatin die verstärke die Induktion von Apoptose in sowohl BET-Inhibitor sensitiven als auch in resistenten Zellen. Des Weiteren zeigte die Kombination einen verbesserten Antitumor-Effekte in A549 tumortragenden Mäusen. Insgesamt zeigen diese Ergebnisse, dass die Identifizierung von BET-abhängigen Genen unterstützend für die Wahl von therapeutischen Kombinationspartnern in der Krebsbehandlung sein kann.
Non-small cell lung cancer (NSCLC) has a high medical need for more effective therapies. Small molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1 are active in different cancer types, including lung cancer. While their activity on oncogene expression such as c-Myc has been addressed by many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. This work evaluates the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptotic response. Genome-wide transcriptional analyses together with chromatin immunoprecipitation followed by sequencing helped to identify the MYC gene and associated super-enhancers as a primary target of JQ1. Using a panel KRAS-mutated NSCLC models, it was found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with down-regulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and the expression of the two key apoptosis regulators FLIP and XIAP was found to be highly BET-dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Furthermore the combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment.

Protocole d'entente entre l'Université Laval et l'Université du Québec à Chicoutimi
Protocole d'entente entre l'Université Laval et l'Université du Québec à Chicoutimi
La littérature scientifique abordant les relations entre motricité, attention et inhibition est plutôt rare. L’étude des relations permet une meilleure compréhension des interactions et de leurs répercussions comportementales. Le projet de recherche vise à documenter les relations chez un groupe d’adolescents, en plus d’un sous-groupe rapportant un TDA/H. Les fonctions cognitives et motrices de 219 adolescents furent évaluées. Inhibition et attention étaient liés à la motricité par plusieurs déterminants moteurs. Ces relations étaient fortement modulées par le sexe des sujets. L’exploration chez le sous-groupe rapportant un TDA/H montrait un profil neuropsychologique typique et des fonctions motrices normales. De plus, les interactions entre fonctions motrices et cognitives de cette population étaient moins nombreuses et confinées au domaine de l’agilité. Des études ultérieures devront aborder la différence entre les sexes et les relations en présence d’un TDA/H diagnostiqué.
La littérature scientifique abordant les relations entre motricité, attention et inhibition est plutôt rare. L’étude des relations permet une meilleure compréhension des interactions et de leurs répercussions comportementales. Le projet de recherche vise à documenter les relations chez un groupe d’adolescents, en plus d’un sous-groupe rapportant un TDA/H. Les fonctions cognitives et motrices de 219 adolescents furent évaluées. Inhibition et attention étaient liés à la motricité par plusieurs déterminants moteurs. Ces relations étaient fortement modulées par le sexe des sujets. L’exploration chez le sous-groupe rapportant un TDA/H montrait un profil neuropsychologique typique et des fonctions motrices normales. De plus, les interactions entre fonctions motrices et cognitives de cette population étaient moins nombreuses et confinées au domaine de l’agilité. Des études ultérieures devront aborder la différence entre les sexes et les relations en présence d’un TDA/H diagnostiqué.
Scientific literature addressing the association between motor functions, attention and inhibition is scarce. Studying these relations helps better understand the interactions and their behavioural repercussions. The research project aims to document these relations in a group of teenagers and in a subgroup reporting ADHD. The cognitive and gross motor functions of 219 teenagers were assessed. Inhibition and attention were linked to several determinants of motor skills. These relations were strongly modulated by gender. The exploration using the subgroup with ADHD indicated typical neuropsychological profile and normal gross motor functions. In this sample, there were fewer interactions between gross motor skills and cognitive functions, specifically related to the agility domain. Future studies should address the effect of gender on gross motor skills and explore the association with cognitive functions in teenagers with ADHD.
Scientific literature addressing the association between motor functions, attention and inhibition is scarce. Studying these relations helps better understand the interactions and their behavioural repercussions. The research project aims to document these relations in a group of teenagers and in a subgroup reporting ADHD. The cognitive and gross motor functions of 219 teenagers were assessed. Inhibition and attention were linked to several determinants of motor skills. These relations were strongly modulated by gender. The exploration using the subgroup with ADHD indicated typical neuropsychological profile and normal gross motor functions. In this sample, there were fewer interactions between gross motor skills and cognitive functions, specifically related to the agility domain. Future studies should address the effect of gender on gross motor skills and explore the association with cognitive functions in teenagers with ADHD.

Des petits ARN non codants dérivant d'ARN de transfert (tRF) ont été identifiés dans tous les domaines de la vie, et de plus en plus de fonctions importantes leur sont attribuées chez de nombreux organismes. Dans ce travail mené sur la plante modèle Arabidopsis, nous avons d’abord montré que la population en tRF varie en fonction des tissus et des conditions de stress. Concernant leur biogenèse, les endoribonucléases responsables du clivage des ARNt ont été identifiées, il s'agit des RNases T2, RNS1, 2 et 3. Afin de réaliser une étude structure/fonction, une approche d’expression en système de levure a été initiée pour permettre l’obtention de quantité suffisante de RNS1 purifiée. L’étude des fonctions des tRF montre que certains d’entre eux sont associés à AGO1, qu'ils semblent cibler entre-autres des éléments transposables et qu’ils pourraient avoir une localisation nucléaire. Enfin, deux tRF, le tRF-5D (Ala) et le tRF-5D (Asn) inhibent efficacement la traduction in vitro. Une association de tRF-5D (Ala) aux polyribosomes de plantules d'Arabidopsis a pu être visualisée, suggérant que certains tRF puissent agir en tant que régulateur global de la traduction
Small non-coding RNAs derived from transfer RNAs (tRFs) have been identified in all domains of life, and more and more important functions are attributed to them in many organisms. In this work on the model plant Arabidopsis, we first showed that the tRF population varies according to tissues and stress conditions. With regard to their biogenesis, the endoribonucleases responsible for tRNA cleavage were identified, it is the RNases T2, RNS1, 2 and 3. In order to carry out a structure / function analysis, heterologous expression in yeast has been developed with the hope to get sufficient amount of purified RNS1. The question of tRF functions has also been studied. It has been shown that some tRFs are associated with AGO1, that they often seem to target transposable elements and could have a nuclear localization. Finally, the study of the involvement of the tRFs in the regulation of translation was tackled. Two tRFs, tRF-5D (Ala) and tRF-5D (Asn) efficiently inhibit translation in vitro. An association of tRF-5D (Ala) with polyribosomes of Arabidopsis seedlings could be visualized suggesting that some plant tRFs could as global regulator of the translation process

Le biais attentionnel observé dans l'anxiété pourrait être le résultat d'un déficit au niveau de l'inhibition des distracteurs négatifs. Une façon d'analyser ce déficit a consisté, dans les trois premières expériences à dissocier dans le temps les composantes sémantique et chromatique de la tâche de Stroop. Les résultats observés dans ces expériences ont principalement montré que les sujets induits dans un état anxieux se différenciaient des sujets contrôles au niveau du décours temporel de l'interférence mot/couleur et que le biais attentionnel associé à l'anxiété était le reflet d'un dysfonctionnement attentionnel général à l'égard de l'information distractrice et pas seulement d'un déficit au niveau de l'inhibition sélective. Une quatrième expérience, dans laquelle nous avons utilisé un paradigme d'amorçage négatif ne confirme pas l'existence d'un déficit des mécanismes inhibiteurs chez les personnes anxieuses. En référence aux travaux de Fox (1994) qui laissent supposer que les personnes anxieuses auraient des difficultés à inhiber l'information négative, lorsqu'un processus de recherche visuelle est engagé, nous avons conçu trois autres expériences, impliquant un traitement spatial. Les expériences 5 et 6 n'ont montré aucun biais attentionnel associé à la valence des mots chez les personnes induites dans un état émotionnel. Cet échec pourrait s'interpréter par le fait que le traitement émotionnel ne fait pas l'objet d'une interférence mesurable dans ces expériences car d'autres types de traitement interviendraient et neutraliseraient l'activation de cette information. Nous avons donc mis en place une autre expérience, qui prendrait plus en compte le traitement émotionnel de l'information. Les résultats vont dans le sens d'une difficulté à inhiber l'information distractrice de valence négative chez les personnes induites dans un état anxieux, lorsqu'une recherche visuo-spatiale est impliquée.

Over the past twenty years, the Approximate Number System (ANS), a cognitive system for representing non-symbolic quantity information, has been the focus of much research attention. Psychologists seeking to understand how individuals learn and perform mathematics have investigated how this system might underlie symbolic mathematical skills. Dot comparison tasks are commonly used as measures of ANS acuity, however very little is known about the cognitive skills that are involved in completing these tasks. The aim of this thesis was to explore the factors that influence performance on dot comparison tasks and discuss the implications of these findings for future research and educational interventions. The first study investigated how the accuracy and reliability of magnitude judgements is influenced by the visual cue controls used to create dot array stimuli. This study found that participants performances on dot comparison tasks created with different visual cue controls were unrelated, and that stimuli generation methods have a substantial influence on test-retest reliability. The studies reported in the second part of this thesis (Studies 2, 3, 4 and 5) explored the role of inhibition in dot comparison task performance. The results of these studies provide evidence that individual differences in inhibition may, at least partially, explain individual differences in dot comparison task performance. Finally, a large multi-study re-analysis of dot comparison data investigated whether individuals take account of numerosity information over and above the visual cues of the stimuli when comparing dot arrays. This analysis revealed that dot comparison task performance may not reflect numerosity processing independently from visual cue processing for all participants, particularly children. This novel evidence may provide some clarification for conflicting results in the literature regarding the relationship between ANS acuity and mathematics achievement. The present findings call into question whether dot comparison tasks should continue to be used as valid measures of ANS acuity.

The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.

A partial nitritation-anammox continuous flow reactor (CFR) was operated for eight months demonstrating that a mixture of large anammox-supported aerobic granules (ASAGs) and small conventional aerobic granules (CAGs) can be maintained stably for extended periods of time. The influent NH4+ was kept at 50 - 60 mg N L-1 to verify that the upper range of total ammonia nitrogen (TAN) for domestic wastewater can supply an inhibitory level of free ammonia (FA) for nitrite oxidizing bacteria (NOB) suppression in CFRs at pH around 7.8. The ammonia oxidizing bacteria (AOB):NOB activity ratio was determined for a series of granule sizes to understand the impact of mass diffusion limitation on the FA inhibition of NOB. When dissolved oxygen (DO) limitation is the only mechanism for NOB suppression, the AOB:NOB ratio was usually found in previous studies to increase with the granule size. However, the trend is reversed when FA has an inhibitory effect on NOB, as was observed in this study. The decrease in AOB:NOB ratio indicates that the resistance to the diffusion of FA along the granule radius limited its ability to inhibit NOB. This means smaller granules, e.g. diameter < 150 microns, are preferred for nitrite accumulation when high FA is present, e.g. in the partial nitritation-anammox process. The trend was further verified by observing the increase in the apparent inhibition coefficient, KI,FAapp, as granule size increased. This study for the first time quantified the effect of diffusion limitation on the KI,FAapp of NOB in granules and biofilms. A mathematical model was then utilized to interpret the observed suppression of NOB. The model predicted that NOB suppression was only complete at the granule surface. The NOB that did survive in larger granules was forced to dwell within the granule interior, where the FA concentration was lower than that in the bulk solution. This means FA inhibition can be taken advantage of as an effective means for NOB suppression in small granules and thin biofilms. Further, FA and DO were found to be both required for the stratification of AOB and NOB in partial nitritation-anammox CFRs. The structural stratification commonly observed in granules is then concluded to be a consequence but not a cause of the NOB suppression.
MS
A partial nitritation-anammox continuous flow reactor (CFR) was operated for eight months demonstrating that granular sludge can be maintained stably for extended periods of time. In this approach, NH3 is only partially converted to NO2 - (partial nitritation), and the conversion to NO3 - is prevented by the suppression of nitrite oxidizing bacteria (NOB). NH3 and NO2 - are then utilized by anammox bacteria to create N2 gas. The influent NH4 + fed to the reactor was kept at 50 to 60 mg N L-1 to verify that the upper range of total ammonia nitrogen (TAN) for domestic wastewater can supply a sufficiently high level of free ammonia (FA) to inhibit NOB growth in CFRs at a pH around 7.8. It is expected that the penetration of a substrate into granule sludge will experience diffusional resistance as it moves from water to denser solid material and is consumed by bacteria. The ammonia oxidizing bacteria (AOB):NOB activity ratio was determined for a series of granule sizes to understand the impact of mass diffusion limitation on the FA inhibition of NOB. When dissolved oxygen (DO) limitation is the only mechanism for NOB suppression, the AOB:NOB ratio was usually found in previous studies to increase with the granule size. However, the trend is reversed when FA has an inhibitory effect on NOB, as was observed in this study. The decrease in AOB:NOB ratio indicates that the resistance to the diffusion of FA, which increases with increasing granule size, along the granule radius limited its ability to inhibit NOB. This means smaller granules, e.g. diameter < 150 µm, are preferred for NO2 - accumulation when high FA is present. The trend was further verified by observing the increase in the apparent inhibition coefficient, KI,FAapp, as granule size increased. This coefficient quantifies the effectiveness of an inhibitor, with larger values indicating weaker inhibition. This study for the first time quantified the effect of diffusion limitation on the KI,FAapp of NOB in granules and biofilms. A mathematical model was then utilized to interpret the observed suppression of NOB. The model predicted that NOB suppression was only complete at the granule surface. The NOB that did survive in larger granules was forced to dwell within the granule interior, where the FA concentration was lower than that in the bulk solution. This means FA inhibition can be taken advantage of as an effective means for NOB suppression in small granules and thin biofilms. Further, FA and DO were found to be both required for the stratification of a layer of AOB at the surface over a layer of NOB in partial nitritation-anammox CFRs. The structural stratification commonly observed in granules is then concluded to be a consequence but not a cause of the NOB suppression.

BACKGROUND: HSP90 is a molecular chaperone which supports the maturation of numerous client proteins, many of which are involved in oncogenic signaling pathways such as PI3K/AKT, MAPK and JAK/STAT. HSP90 is an appealing target for small molecule inhibitors due to its ability to simultaneously downregulate multiple oncogenic conduits. Results in the clinic have been mixed and biomarkers of response to HSP90 inhibitors for patient stratification remain obscure. RESULTS: 1. EML4-ALK is a HSP90 client which degrades in response to HSP90 inhibition. Variants of EML4-ALK exist with disparate structures and stabilities. This chapter demonstrates differential stability of EML4-ALK variants and cell line sensitivity in response to HSP90 inhibition. A RT-PCR test to identify variants from FFPE tumour tissue is developed for tissues from the CHIARA trial to link variant status and response to ganetespib. 2. Characterisation of a cell line resistant to ganetespib, and verification that high UGT1A levels prevent ganetespib from inhibiting HSP90 in a NSCLC context. 3. Array-based analysis of genome-wide copy number alterations in the GALAXY-1 trial, which examined ganetespib plus docetaxel (G-D) in the second line setting against docetaxel alone (D), following platinum-based chemotherapy in NSCLC patients. Three CNAs were associated with sensitivity to ganetespib plus docetaxel: loss in cytoband 18q23, and gains in 11q13.3 and 16q22.3. Patients with multiple CNAs may represent a group of patients who perform badly on docetaxel but respond much better with the addition of ganetespib. SUMMARY: Several putative biomarkers which are predictive of response to ganetespib are identified in this study. The relationship between EML4-ALK variant stability and ganetespib response may shed light on HSP90 client selection. Genome wide CNA analysis associated with randomized clinical trial survival data has identified putative biomarkers of efficacy to ganetespib plus docetaxel.

Non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes are increasingly being associated with outbreaks of foodborne infections and illness. This study evaluated the in vitro probiotic characteristics of Lactobacillus plantarum strain B411 and dominant lactic acid bacteria (LAB) isolated from traditional African fermented maize gruel (ogi). Subsequently, the effects of acid adaption and those potential probiotic bacteria on the growth and survival of non-O157 STEC strains in fermented goat s milk and traditional African fermented cereal-based foods were investigated. Two of the 14 lactic acid bacteria (LAB) strains that were isolated from ogi together with L. plantarum strain B411 possess hydrophobic cell surface with ability to coaggregate with pathogens as well as antimicrobial activities against non-O157 STEC strains and E. coli ATCC 25922. The three strains with in vitro probiotic attributes also exhibited high level of adhesion to Caco-2 cells and the two LAB strains from ogi spontaneous fermentation were genetically similar to other reported potential probiotic bacteria. Though prior adaptation to acid enhanced acid tolerance of non-O157 STEC strains in Brain Heart Infusion (BHI) broth at low pH, it was detrimental to the survival in fermented goat s milk. The growth of both acid adapted (AA) and non-acid adapted (NAA) non-O157 STEC strains was not inhibited by the single strain potential probiotic L. plantarum B411 in fermented goat s milk. However, combination of yoghurt starter culture and potential probiotic L. plantarum B411 for the fermentation of the goat s milk had a bacteriostatic effect on the NAA non-O157 STEC strains while the growth of AA non-O157 STEC strains were substantially inhibited. Non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes are increasingly being associated with outbreaks of foodborne infections and illness. This study evaluated the in vitro probiotic characteristics of Lactobacillus plantarum strain B411 and dominant lactic acid bacteria (LAB) isolated from traditional African fermented maize gruel (ogi). Subsequently, the effects of acid adaption and those potential probiotic bacteria on the growth and survival of non-O157 STEC strains in fermented goat s milk and traditional African fermented cereal-based foods were investigated. Two of the 14 lactic acid bacteria (LAB) strains that were isolated from ogi together with L. plantarum strain B411 possess hydrophobic cell surface with ability to coaggregate with pathogens as well as antimicrobial activities against non-O157 STEC strains and E. coli ATCC 25922. The three strains with in vitro probiotic attributes also exhibited high level of adhesion to Caco-2 cells and the two LAB strains from ogi spontaneous fermentation were genetically similar to other reported potential probiotic bacteria. Though prior adaptation to acid enhanced acid tolerance of non-O157 STEC strains in Brain Heart Infusion (BHI) broth at low pH, it was detrimental to the survival in fermented goat s milk. The growth of both acid adapted (AA) and non-acid adapted (NAA) non-O157 STEC strains was not inhibited by the single strain potential probiotic L. plantarum B411 in fermented goat s milk. However, combination of yoghurt starter culture and potential probiotic L. plantarum B411 for the fermentation of the goat s milk had a bacteriostatic effect on the NAA non-O157 STEC strains while the growth of AA non-O157 STEC strains were substantially inhibited. Non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes are increasingly being associated with outbreaks of foodborne infections and illness. This study evaluated the in vitro probiotic characteristics of Lactobacillus plantarum strain B411 and dominant lactic acid bacteria (LAB) isolated from traditional African fermented maize gruel (ogi). Subsequently, the effects of acid adaption and those potential probiotic bacteria on the growth and survival of non-O157 STEC strains in fermented goat s milk and traditional African fermented cereal-based foods were investigated. Two of the 14 lactic acid bacteria (LAB) strains that were isolated from ogi together with L. plantarum strain B411 possess hydrophobic cell surface with ability to coaggregate with pathogens as well as antimicrobial activities against non-O157 STEC strains and E. coli ATCC 25922. The three strains with in vitro probiotic attributes also exhibited high level of adhesion to Caco-2 cells and the two LAB strains from ogi spontaneous fermentation were genetically similar to other reported potential probiotic bacteria. Though prior adaptation to acid enhanced acid tolerance of non-O157 STEC strains in Brain Heart Infusion (BHI) broth at low pH, it was detrimental to the survival in fermented goat s milk. The growth of both acid adapted (AA) and non-acid adapted (NAA) non-O157 STEC strains was not inhibited by the single strain potential probiotic L. plantarum B411 in fermented goat s milk. However, combination of yoghurt starter culture and potential probiotic L. plantarum B411 for the fermentation of the goat s milk had a bacteriostatic effect on the NAA non-O157 STEC strains while the growth of AA non-O157 STEC strains were substantially inhibited. Non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes are increasingly being associated with outbreaks of foodborne infections and illness. This study evaluated the in vitro probiotic characteristics of Lactobacillus plantarum strain B411 and dominant lactic acid bacteria (LAB) isolated from traditional African fermented maize gruel (ogi). Subsequently, the effects of acid adaption and those potential probiotic bacteria on the growth and survival of non-O157 STEC strains in fermented goat s milk and traditional African fermented cereal-based foods were investigated. Two of the 14 lactic acid bacteria (LAB) strains that were isolated from ogi together with L. plantarum strain B411 possess hydrophobic cell surface with ability to coaggregate with pathogens as well as antimicrobial activities against non-O157 STEC strains and E. coli ATCC 25922. The three strains with in vitro probiotic attributes also exhibited high level of adhesion to Caco-2 cells and the two LAB strains from ogi spontaneous fermentation were genetically similar to other reported potential probiotic bacteria. Though prior adaptation to acid enhanced acid tolerance of non-O157 STEC strains in Brain Heart Infusion (BHI) broth at low pH, it was detrimental to the survival in fermented goat s milk. The growth of both acid adapted (AA) and non-acid adapted (NAA) non-O157 STEC strains was not inhibited by the single strain potential probiotic L. plantarum B411 in fermented goat s milk. However, combination of yoghurt starter culture and potential probiotic L. plantarum B411 for the fermentation of the goat s milk had a bacteriostatic effect on the NAA non-O157 STEC strains while the growth of AA non-O157 STEC strains were substantially inhibited. Non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes are increasingly being associated with outbreaks of foodborne infections and illness. This study evaluated the in vitro probiotic characteristics of Lactobacillus plantarum strain B411 and dominant lactic acid bacteria (LAB) isolated from traditional African fermented maize gruel (ogi). Subsequently, the effects of acid adaption and those potential probiotic bacteria on the growth and survival of non-O157 STEC strains in fermented goat s milk and traditional African fermented cereal-based foods were investigated. Two of the 14 lactic acid bacteria (LAB) strains that were isolated from ogi together with L. plantarum strain B411 possess hydrophobic cell surface with ability to coaggregate with pathogens as well as antimicrobial activities against non-O157 STEC strains and E. coli ATCC 25922. The three strains with in vitro probiotic attributes also exhibited high level of adhesion to Caco-2 cells and the two LAB strains from ogi spontaneous fermentation were genetically similar to other reported potential probiotic bacteria. Though prior adaptation to acid enhanced acid tolerance of non-O157 STEC strains in Brain Heart Infusion (BHI) broth at low pH, it was detrimental to the survival in fermented goat s milk. The growth of both acid adapted (AA) and non-acid adapted (NAA) non-O157 STEC strains was not inhibited by the single strain potential probiotic L. plantarum B411 in fermented goat s milk. However, combination of yoghurt starter culture and potential probiotic L. plantarum B411 for the fermentation of the goat s milk had a bacteriostatic effect on the NAA non-O157 STEC strains while the growth of AA non-O157 STEC strains were substantially inhibited.
Thesis (PhD)--University of Pretoria, 2015.
Food Science
PhD
Unrestricted

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

Diabetes mellitus (DM) is an established cause of increased mortality. Most deaths linked to DM are attributable to cardiovascular disease, and hyperglycaemia alone is not sufficient to explain the increased cardiovascular risk associated with DM. It is accepted that insulin resistance, the principal underlying cause of the most common form of DM, is independently linked to CVD. Prior research demonstrates insulin resistance is associated with excessive production of reactive oxygen species (ROS), in part via the enzyme NADPH oxidase 2 (NOX2). We hypothesised that reduction of NOX2-derived ROS represents a translationally promising strategy to improve the diminished vascular repair and regeneration observed in insulin resistance. This project explores the effect of NOX2 inhibition on angiogenesis and vascular repair in murine models of whole-body and endothelial-specific insulin resistance. Mice with haploinsufficiency of the insulin receptor (IRKO), and those expressing a kinase dead human insulin receptor transgene in the endothelium (ESMIRO), were bred with NOX2 deficient mice. NOX2 knockdown was associated with significant improvement in vascular repair following arterial injury in ESMIRO mice. Subsequent work provided two potential explanations: improved endothelial cell migration, and an increased abundance of circulating progenitor cells. Pharmacological NOX2 inhibition partly recapitulated these findings in vitro, as treatment of insulin resistant human umbilical vein endothelial cells with the NOX2 inhibitor GP91-ds tat was associated with improved cell migration. Whilst this project yielded a number of promising findings, it also highlighted pitfalls associated with manipulation of ROS. We corroborated two existing concerns relating to NOX2 inhibition: the potential to compromise immune function, and the risk of inciting a pro-inflammatory state. Although NOX2 remains a potentially suitable target for therapeutic intervention, greater understanding of NOX2 biology is required. We hope the work carried out in this project can be built upon to facilitate that understanding, ultimately generating novel therapeutic agents.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第17830号
農博第2015号
新制||農||1016(附属図書館)
学位論文||H25||N4787(農学部図書室)
30645
京都大学大学院農学研究科農学専攻
(主査)教授 稲村 達也, 教授 冨永 達, 教授 奥本 裕
学位規則第4条第1項該当

Les ARNm codent pour les protéines, tandis qu'un grand nombre d'ARNs nommés longues ARNs non codants (ARNlnc) ne sont pas traduites en protéines. Les deux types d’ARNs existent en isoforms qui se distinguent à cause de l’épissage alternatif. Certains des ARNlnc jouent des rôles importants dans la croissance et différentiation cellulaire. Cependant, leurs fonctions dans la cytotoxicité de la chimiothérapie anti-cancéreuse médicamenteuse utilisant le 5-fluorouracile (5-FU) sont encore inconnues. Pendant mes travaux j'ai trouvé que le traitement par le 5-FU cause l’accumulation des ARNlnc. Ce phénomène est parfois, sous forme d’ARN double brin (ARNds) formé par une paire de transcrits chevauchant, corrélé négativement avec le niveau de la protéine codée par l'ARNm. Cette inhibition potentielle de la traduction des régulateurs du cycle cellulaire clés et les gènes essentiels en formant des l'ARNds peut éventuellement empêcher la progression du cycle cellulaire. Nos analyses prometteuses devraient inspirer des études approfondies des ARNlnc dans la cytotoxicité du 5-FU chez la levure et l’homme afin d’'améliorer la chimiothérapie. J'ai trouvé que la surexpression de RRP6, peut conduire à une résistance accrue au traitement par le 5-FU. Je démontre ensuite que l’ARNlnc MUT1312 forme des ARNds avec RRP6 qui sont négativement corrélés avec le niveau de la protéine Rrp6. Par ailleurs, la surexpression de MUT1312 pendant la mitose et associé avec une diminution d’Rrp6. Ainsi, mon étude suggère que MUT1312 soit impliqué dans la régulation de Rrp6 pendant la differentiation cellulaire. Mes recherches de MUT477/SWI4 indiquent la function importante de la méiose induite à long ARN non codantes en tant que forme d'ARN double brin potentiellement réguler la traduction. J'ai trouvé que SUT200 pourrait inhiber la transcription de CDC6 durant la méiose par read-through. Un cas comparable est MUT1465 et CLN2. J’ai fait un criblage in silico pour trouver des facteurs de transcription qui activent des MUTs durant la méiose. J’ai trouvé que la plupart des MUTs sont induites par Ndt80. MUT1465 est parmi eux : il pourrait être induite par Ndt80 ce qui inhiberait l’expression de CLN2 après l’initiation de la méiose. J’ai trouvé que la répression de certains MUTs par le complexe Ume6/Rpd3 en mitose est différemment régulée entre JHY222 et SK1. MUT100 qui ne possède pas l'élément USR1 fixé par Ume6, et qui est donc une cible indirecte, est déréprimé dans JHY22 ume6 mais pas dans SK1 ume6. Pour la régulation de l'étude de isoforme méiose, Nous avons trouvé que le complexe histone déacétylase Rpd3/Sin3/Ume6, empêche également l'induction de l'isoforme longue de BOI1 dans la mitose par liaison directe de liaison Ume6 à sa cible de URS1. Orc1 est importante pour la réplication de l'ADN. J’ai démontré que mORC1 est une cible directe de l'activateur Ndt80 et que son motif de fixation (MSE) est nécessaire pour l'induction de l’isoforme mORC1 et du gene méiotique SMA2 transcrit de façon divergente. J’ai trouvé qu'une souche incapable d’induire mORC1, contient des niveaux anormalement élevés d’Orc1 pendant la gamétogenèse, ce qui corréle mORC1 avec la baisse de la protéine Orc1. En conclusion, mes études au cours du doctorat révèlent des nouvelles cibles et ainsi offrent des nouvelles perspectives de l’amélioration de la chimiothérapie par le 5-FU. Les mécanismes incluent la formation d'un ARN double brin avec son ARNm anti-sens pour potentiellement inhiber la traduction de l'ARNm, et inhibition en aval de l'ARNm par transcription read-through d’une ARNlnc. Mon travail a également révélé un mécanisme de régulation des ARNlnc et les isoforms d’ARN pendent la croissance et la différentiation cellulaire
RNAs are molecules with important functions in diverse cellular processes. mRNAs encode proteins, while a large number of RNAs called long noncoding RNAs (lncRNAs) are not translated into proteins. Both types of RNAs exist in various isoforms due to alternative splicing.Some of lncRNA play important roles in cell growth and differentiation. However, their functions in the cytotoxicity of the drug anticancer chemotherapy using 5-fluorouracil (5-FU) are still unknown. During my research I found that treatment with 5-FU causes accumulation of lncRNA. Acuumulated antisense lncRNA form double stranded RNA with the mRNAs , negatively correlated with the level of the protein encoded by the mRNA. This potential inhibition of translation of key cell cycle regulators and essential genes by forming dsRNA may possibly prevent the progression of the cell cycle. My results suggest that lncRNA are likely to play an important role in the cytotoxicity of 5-FU. Our promising testing should inspire in-depth studies of lncRNA in the cytotoxicity of 5-FU in yeast and humans to improve chemotherapy.Rrp6 is a 3'-5 'exoribonuclease, which plays an important role in the regulation and modification of rRNA, mRNA and lncRNA. I found that overexpression of RRP6, the homologue of the yeast EXOSC10 gene in mammals, can lead to increased resistance to treatment with 5-FU. I found that the lncRNA MUT1312 form dsRNA with RRP6 that are negatively correlated with the level of Rrp6 protein. Furthermore, overexpression of MUT1312 during mitosis and associated with a decrease of Rrp6. Thus, my study suggests that MUT1312 may involved in the regulation of Rrp6 during cell differentiation. I further explored the function of the double-stranded RNA in meiosis. My research about SWI4/MUT477 indicates the important function of meiosis induced long noncoding RNA as a form of double-stranded RNA potentially regulate translation. Another aspect of the function of lncRNA is to regulate the transcription of downstream mRNA. I found SUT200 could inhibit transcription of CDC6 during meiosis by read-through. A similar case is CLN2/MUT1465. I did an in silico screening to find transcription factors that activate MUTs during meiosis. I found that most MUTs are induced by Ndt80. MUT1465 is among them: it could be induced by Ndt80 which inhibit the expression of CLN2 after initiation of meiosis. I found that repression of certain MUTs by the Ume6 / Rpd3 complex in mitosis is regulated differently between JHY222 and SK1. MUT100 which does not have the Ume6 binding site URS1 element, and is therefore an indirect target is derepressed in JHY22 ume6 but not in SK1 ume6. For the study about regulation of meiosis isoform, we have found that the histone deacetylase complex Rpd3 / Sin3 / Ume6 prevents the induction of long isoform BOI1 in mitosis by direct binding Ume6 binding to its target URS1.Orc1 is important for DNA replication. I have demonstrated that mORC1 is a direct target of the Ndt80 activator and its binding motif (MSE) is required for induction of isoform mORC1 and meiotic gene SMA2 divergently transcribed. I found that a strain incapable of inducing mORC1 contains abnormally high levels of Orc1 during gametogenesis, which correlates with mORC1 declining Orc1 protein. Since eukaryotic genes often encode multiple transcripts with 5'-UTR of variable length, the findings are likely relevant to gene expression during development and disease in higher eukaryotes. In conclusion, my studies during PhD reveal new targets and thus offer new prospects for improving chemotherapy with 5-FU. Mechanisms include (1) the formation of a double strand with its antisense mRNAs to potentially inhibit translation of mRNA, and (2) downstream inhibition of mRNA transcription read-through of a lncRNA. My work also revealed a lncRNA regulatory mechanism and RNA isoforms dangling growth and cell differentiation

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第13887号
農博第1702号
新制||農||955(附属図書館)
学位論文||H20||N4354 [所蔵なし](農学部図書室)
UT51-2008-C803
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 伏木 亨, 教授 河田 照雄, 教授 安達 修二
学位規則第4条第1項該当

Masters of Science
The aim of this study was therefore to evaluate the antioxidant and anti-diabesity potential of a hot water extract of C. maculata in non-cell based assays and correlate the activities with phenolic composition. Total antioxidant capacity (TAC) was assessed in terms of free radical scavenging and iron reducing ability. The DPPH, ABTS, ORAC and FRAP assays were employed. Anti-diabesity potential was assessed in terms of the inhibition of the digestive enzymes, α-glucosidase and pancreatic lipase

BACKGROUND: Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models. METHODS: A diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ. RESULTS: CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC₅₀ values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC₅₀ values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP). CONCLUSIONS: CFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

The research investigated consumers' decision-making process during pre-adoption and consumption stages of consumer-based technologies via the context of mobile apps. In an attempt to integrate consumer resistance in predicting the end-decisions to adopt/not adopt or continue/discontinue the use of a technology, the study presented some interesting findings. Employing the theoretical framework of cognitive appraisal theory, the study integrated the TAM, paradoxes of technology, and coping strategies to propose and empirically validate a process-based model of decision-making.Data were collected via a self-administered web-based survey. Two versions of the questionnaire were used to elicit consumers' responses from adopters and non-adopters of mobile apps. A total of 646 smartphone owners responded to the survey, of which, 375 respondents had downloaded apps in the past and 271 respondents had not downloaded any apps. The proposed hypotheses were tested using structural equation modeling.Results demonstrated that most part of the TAM3 framework is replicable in a consumer-based setting. Additional findings provided evidence for the strong role of goal relevance in the TAM framework. The study also supported the effect of perceived usefulness and perceived ease-of-use on different technology paradoxes. The factor structure of the technology paradoxes suggested three distinct dimensions. Consumers' evaluation of control, freedom, newness, assimilation, and fulfillment of need as derived from the use of mobile apps was captured by Perceived Benefits. The construct, Perceived Apprehension, comprised of consumers' assessment of the chaos, enslavement, obsolesce, isolation, and creation of needs as a result of using mobile apps. Finally, the factor, Perceived Obscurity, investigated the confusion and/or ambiguity within individuals by measuring their perceived inefficiency and incompetence in using mobile apps.Most importantly, separate investigations of the pre-adoption and consumption stages highlighted consumers' use of varying degrees of resistance as influenced by their appraisal of the technology. The non-adopters resisted the use of mobile apps by either being indifferent towards it or postponing the decision to adopt. The adopters of mobile apps were also found to reject its use by distancing, abandoning, or neglecting the apps. The role of positive coping investigated the positive behavioral tendencies employed by consumers to overcome the challenges of using mobile apps. Managerial implications are discussed.

A growing body of literature suggests Breast Cancer-Associated Protein 1 (BRCA1) is important not only as a cause, but also as a target in the quest for cancer treatment. BRCA1 deficient cells treated with radiation as well as PARP inhibitors and other chemotherapeutics demonstrate a greater sensitivity than cells with wild type BRCA1. Inhibitors of BRCA1 would take advantage of this synthetic lethality and represent a significant advance in cancer treatment as well as an understanding of the biology of DNA repair. Despite significant study of BRCA1 protein and function, it is a large protein (220 KDa) that is still largely uncharacterized, but its N- and C-terminal domains have been described by significant structural data. The BRCT (BRCA1 C-Terminal) Domain is a phosphoprotein binding domain that is commonly mutated or lost in cancers and has a binding cleft seemingly very suitable for drug design. Small molecule screens have been conducted against this domain, but the resulting hits with moderate affinity have not been shown to induce BRCA1 deficient phenotypes. Phosphopeptides have also been studied as potential BRCA1 inhibitors, yet despite some having affinities in the mid-nanomolar range the presence of a phosphate is not without its pharmacologic challenges. We generated an mRNA display library with 1.3 x 10^13 cyclized peptides covalently attached to the mRNA that encoded them. Eight rounds of selection exposing the library to a GST-BRCT fusion resulted in selection of non-phosphorylated peptides that bind to a BRCT domain of BRCA1. The sequences resulting from the selection have common homologies and initial characterization has shown that these peptides may be the first viable non-phosphoserine containing inhibitors of BRCA1.