A discrete formalism for reasoning about action and change

This paper presents a discrete formalism for temporal reasoning about actions and change, which enjoys an explicit representation of time and action/event occurrences. The formalism allows the expression of truth values for given fluents over various times including nondecomposable points/moments and decomposable intervals. Two major problems which beset most existing interval-based theories of action and change, i.e., the so-called dividing instant problem and the intermingling problem, are absent from this new formalism. The dividing instant problem is overcome by excluding the concepts of ending points of intervals, and the intermingling problem is bypassed by means of characterising the fundamental time structure as a well-ordered discrete set of non-decomposable times (points and moments), from which decomposable intervals are constructed. A comprehensive characterisation about the relationship between the negation of fluents and the negation of involved sentences is formally provided. The formalism provides a flexible expression of temporal relationships between effects and their causal events, including delayed effects of events which remains a problematic question in most existing theories about action and change.

A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2. HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target.

Comparison of the modes of action of Apremilast and Roflumilast

The phosphodiesterase 4 (PDE4) family are cAMP specific phosphodiesterases that play an important role in the inflammatory response and is the major PDE type found in inflammatory cells. A significant number of PDE4 specific inhibitors have been developed and are currently being investigated for use as therapeutic agents. Apremilast, a small molecule inhibitor of PDE 4 is in development for chronic inflammatory disorders and has shown promise for the treatment of psoriasis, psoriatic arthritis as well as other inflammatory diseases. It has been found to be safe and well tolerated in humans and in March 2014 it was approved by the US food and drug administration for the treatment of adult patients with active psoriatic arthritis. The only other PDE4 inhibitor on the market is Roflumilast and it is used for treatment of respiratory disease. Roflumilast is approved in the EU for the treatment of COPD and was recently approved in the US for treatment to reduce the risk of COPD exacerbations. Roflumilast is also a selective PDE4 inhibitor, administered as an oral tablet once daily, and is thought to act by increasing cAMP within lung cells. As both (Apremilast and Roflumilast) compounds selectively inhibit PDE4 but are targeted at different diseases, there is a need for a clear understanding of their mechanism of action (MOA). Differences and similarity of MOA should be defined for the purposes of labelling, for communication to the scientific community, physicians, and patients, and for an extension of utility to other diseases and therapeutic areas. In order to obtain a complete comparative picture of the MOA of both inhibitors, additional molecular and cellular biology studies are required to more fully elucidate the signalling mediators downstream of PDE4 inhibition which result in alterations in pro- and anti-inflammatory gene expression. My studies were conducted to directly compare Apremilast with Roflumilast, in order to substantiate the differences observed in the molecular and cellular effects of these compounds, and to search for other possible differentiating effects. Therefore the main aim of this thesis was to utilise cutting-edge biochemical techniques to discover whether Apremilast and Roflumilast work with different modes of action. In the first part of my thesis I used novel genetically encoded FRET based cAMP sensors targeted to different intracellular compartments, in order to monitor cAMP levels within specific microdomains of cells as a consequence of challenge with Apremilast and Roflumilast, which revealed that Apremilast and Roflumilast do regulate different pools of cAMP in cells. In the second part of my thesis I focussed on assessing whether Apremilast and Roflumilast cause differential effects on the PKA phosphorylation state of proteins in cells. I used various biochemical techniques (Western blotting, Substrate kinase arrays and Reverse Phase Protein array and found that Apremilast and Roflumilast do lead to differential PKA substrate phosphorylation. For example I found that Apremilast increases the phosphorylation of Ribosomal Protein S6 at Ser240/244 and Fyn Y530 in the S6 Ribosomal pathway of Rheumatoid Arthritis Synovial fibroblast and HEK293 cells, whereas Roflumilast does not. This data suggests that Apremilast has distinct biological effects from that of Roflumilast and could represent a new therapeutic role for Apremilast in other diseases. In the final part of my thesis, Phage display technology was employed in order to identify any novel binding motifs that associate with PDE4 and to identify sequences that were differentially regulated by the inhibitors in an attempt to find binding motifs that may exist in previously characterised signalling proteins. Petide array technology was then used to confirm binding of specific peptide sequences or motifs. Results showed that Apremilast and Roflumilast can either enhance or decrease the binding of PDE4A4 to specific peptide sequences or motifs that are found in a variety of proteins in the human proteome, most interestingly Ubiquitin-related proteins. The data from this chapter is preliminary but may be used in the discovery of novel binding partners for PDE4 or to provide a new role for PDE inhibition in disease. Therefore the work in this thesis provides a unique snapshot of the complexity of the cAMP signalling system and is the first to directly compare action of the two approved PDE4 inhibitors in a detailed way.

Investigating the mode of action of AN5568, a novel therapeutic against African trypanosomiasis

No abstract available.

The military in the Chamber of Deputies 1851-1870 – corporative lines of action in defense of the army

Based on our reading of the minutes of the parliamentary sessions, we shall attempt in this study to analyze the performance of army officers in the Chamber of Deputies, which, from 1851 to 1870, represented one of the most important settings for the military’s participation in politics

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Candidacidal potential of licorice phenolic extract: emphasis on its mode of action

Medicinal plants bave gained a special attention in the last years, due to its renowned health benefits, such as antimicrobial effects [I]. In fact, several natural matrices bave been increasingly studied, namely for its antifungal activity against opportunistic fungi [2,3]. Candida species, although commensa! microorganisms, have caused severe organic dysfunctions to the host, once current antifungal agents have lost their recognized efficiency [2]. So, numerous studies have been carried out focusing the mechanisms of acquired drug-resistance by Candida species [of an efficient multi-drug therapy (i.e. chemical drugs and also natural extracts combination), the discovery of the involved mechanisms of actions, morphological changes and related kinetic parameters are of major importance. Glycyrrhiza glabra L. (licorice) hydromethanolic extract have evidenced promissory candidacidal effects, and therefore, the involved mechanisms of action need to be clarified. Thus, in the present study these modes of action were assessed, by using flow cytometry.Overall, the licorice extract induced significant and irreversible primary damages on Candida cells, being membrane disruption and consequent unviability one of the main targets. In fact, after membrane destabilization, cells lost their proper homeostasis, their metabolic functions were blocked and, consequently cells lost functionality. The relevance and interest of the achieved results open new insights towards the upcorning use of the present phenolic matrix, being important to evaluate its in viva efficacy. Therefore, further studies are necessary to deepen knowledge on this field, aiming not only to establish therapeutic and prophylactic doses, but also to improve the clinical intervention in Candida infections.

Fish Reproduction Is Disrupted upon Lifelong Exposure to Environmental PAHs Fractions Revealing Different Modes of Action

Polycyclic aromatic hydrocarbons (PAHs) constitute a large family of organic pollutants emitted in the environment as complex mixtures, the compositions of which depend on origin. Among a wide range of physiological defects, PAHs are suspected to be involved in disruption of reproduction. In an aquatic environment, the trophic route is an important source of chronic exposure to PAHs. Here, we performed trophic exposure of zebrafish to three fractions of different origin, one pyrolytic and two petrogenic. Produced diets contained PAHs at environmental concentrations. Reproductive traits were analyzed at individual, tissue and molecular levels. Reproductive success and cumulative eggs number were disrupted after exposure to all three fractions, albeit to various extents depending on the fraction and concentrations. Histological analyses revealed ovary maturation defects after exposure to all three fractions as well as degeneration after exposure to a pyrolytic fraction. In testis, hypoplasia was observed after exposure to petrogenic fractions. Genes expression analysis in gonads has allowed us to establish common pathways such as endocrine disruption or differentiation/maturation defects. Taken altogether, these results indicate that PAHs can indeed disrupt fish reproduction and that different fractions trigger different pathways resulting in different effects.

Antitumor effect and mechanism of action of polysaccharides extracted from Polygonum perfoliatum L whole plant in human lung carcinoma A549 cell line

Purpose: To optimize the extraction conditions of polysaccharides from Polygonum perfoliatum L. (PSDP) and to evaluate their anti-tumor activities on A549 cell line. Methods: Extraction of PSDP was optimized using Box-Behnken design (BBD). Three factors of response surface methodology (RSM) including extraction time, ratio of water to raw material and number of extractions were employed to optimize the yield of PSDP. The cytotoxic effect of PSDP on human lung carcinoma A549 cell line was evaluated in vivo, while its effects on expressions of caspase3, caspase-9, Bcl-2 and Bax were determined by western blot assay. Result: BBD was significant and applicable to PSDP extraction. Based on the contour plots, response surface plots and variance analysis, it predicted that the optimum conditions for PSDP extraction were: 1.58 h (extraction time); 30.18 mL/g (ratio of water to raw material); and 2.02 (number of extractions). PSDP had significant inhibitory effect on the growth of A549 cells in a concentration- and timedependent manner (p < 0.05). After treatment with PSDP, caspase-3, caspase-9 and Bax were significantly up-regulated (p < 0.05), whereas Bcl-2 was down-regulated, all concentration-dependently. Conclusion: RSM analysis is an appropriate method to optimize PSDP extraction. The results also indicate that PSDP has significant anti-tumor effect against A549 cells, most likely via inducing mitochondria-mediated apoptosis.

Anticonvulsant and sedative effect of Fufang Changniu pills and probable mechanism of action in mice

Purpose: To investigate the anticonvulsant and sedative effects of Fufang Changniu Pills (FCP) and its probable mechanism of action in mice. Methods: The water decoction of FCP was prepared and the main constituents were determined by high performance liquid chromatography (HPLC). The anticonvulsant activities of FCP were evaluated by maximal electroshock (MES) and pentylenetetrazole (PTZ)-induced seizures in mice. Pentobarbital sodium-induced sleeping time and locomotor activity measurements were performed to evaluate the sedative effects of FCP in mice. Finally, PTZ-induced chronic seizures were established, and expressions of gamma-aminobutyric acid A receptor (GABA-A) and glutamic acid decarboxylase 65 (GAD65) in the brains of the mice were assayed by western blot in order to explore the probable mechanisms of action of the drug. Results: Gallic acid, liquiritin, cinnamyl alcohol, cinnamic acid and glycyrrhizic acid were detected in FCP decoction. FCP (50, 100 and 200 mg/kg) showed significant anticonvulsant and sedative effects on epileptic mice induced by MES (p < 0.05) and PTZ (p < 0.05). Moreover, pentobarbital sodium-induced sleeping time and locomotor activity tests showed that FCP possesses sedative effect (p < 0.05). Western blot data indicate that FCP significantly up-regulated GABA-A and GAD 65 in the brains of chronic epileptic rats (p < 0.05). Conclusion: FCP has significant anticonvulsant and sedative effects, and the mechanism of its action may be related to the up-regulation of GABA-A and GAD 65 in mice brain.

Antiviral activity and mechanism of action of arbidol against Hantaan virus infection

Purpose: To investigate the activity and mechanism of action of arbidol against Hantaan virus (HTNV) activity by modulating inflammation via TLR-4 pathway. Methods: HUVEC cells infected with HTNV 76-118 were treated with serially diluted arbidol solutions at -2h (2 h before viral infection, pre-treatment mode), 0 h (at the same time as viral infection, simultaneous treatment mode) or 2 h (2 h after viral infection, post-treatment mode). The transcript levels of TLR4 were detected by semi-quantitative reverse transcription-PCR (RT-PCR) at 6, 12, 18, and 24 h later. The levels of iNOS and TNF-α were examined using enzyme-linked immunosorbent assay (ELISA). Results: Pre-treatment with arbidol, rather than simultaneous treatment or post-treatment, effectively inhibited up-regulation of cellular TLR4 expression (up to 40 ± 6.1 % inhibition) and activity of supernatant iNOS induced by HTNV infection(up to 44.1 ± 9.4 % inhibition), as well as in a LPSstimulated inflammatory endothelial cell. Arbidol decreased the elevated TNF-α levels induced by LPS stimulation. Conclusion: These results are the first evidence that arbidol modulates viral PRRs signaling and its consequential inflammatory cytokine/chemokine response during hantavirus infection.

Ethical-moral courses of action and active citizenship in health students

Abstract Introduction: The practice of active citizenship, ethical-moral courses of action and civic, moral and ethics education are essentials for ethical decision making in health. Objetive: To determine if gender influences students’ ethical- moral course of action. Methods: Descriptive study with a non-probabilistic sample of 85 students enrolled in the 1st cycle of the health degree. Results: Of the participants surveyed 61.2% were found to say that action should take into account their moral principles, with ethical/ moral subjectivism prevailing; 44.7% consider that one should “Do what will have the best consequences for the greatest number of people”, with the principle of utilitarianism being significant; 55.3% think that “An action is ethically good” if “It is in accordance with morality”, thereby highlighting subjectivism/relativism; 45.9% believe that “ethical-moral values” “are relative and vary from society to society” agreeing with relativism as an explanatory principle for action. Males showed a greater tendency to support their decision-making with the principle of objectivism, (Fischer=.010). Conclusion: The results suggest that students’ ethical-moral education is required to promote an ethical-moral course of action in their professional practice. Thus, universities with their health courses should be at the forefront of this education, making their graduates ambassadors/interveners of a way of knowing and of being as well as promoters of the dignity of the citizen of the modern world.

An investigation of the possible health-promoting modes of action of regular- and super- doses of phytase in the broiler chicken

The overall objective of this thesis was to study the effects of regular and high (super-) doses of phytase in the gut of broilers, with the aim of documenting the mechanism of their action leading to improvements in animal health. Phytase is often supplemented to commercial broiler diets to facilitate the hydrolysis of plant phytate and release of phosphorus for utilisation. Although not the original intention of its addition, phytase supplementation leads to improvements in growth performance parameters and enhanced nutrient utilisation. Further benefits have also been observed following the addition of super-doses of phytase which are not explained by an increase in phosphorus release, and thus have been termed ‘extra-phosphoric effects’. Using diets formulated to be adequate or marginally deficient in available phosphorus (aP; forming the negative control, NC), phytase was supplemented at 1,500 and 3,000 FTU/kg phytase in the first study (both super-doses) and the partitioning of nutrients within the body was investigated. It appeared that there were some metabolic changes between 1,500 and 3,000 FTU/kg, switching between protein and fat accretion, potentially as a consequence of nutrient availability, although these changes were not reflected by changes in growth performance parameters. However, the loss of the NC treatment without phytase on day 12 limits the comparison of the phytase within the NC treatment, but does allow for comparison of each dose at adequate or low dietary aP levels. As expected, a greater degree of phytate hydrolysis was achieved with 3,000 than with 1,500 FTU/kg phytase, but changes in carcass accretion characteristics were greater with 1,500 than 3,000 FTU/kg. Using these findings and the observation that there were no further changes in the parameters measured by increasing phytase from 1,500 to 3,000 FTU/kg (aside from phytate hydrolysis), 1,500 FTU/kg phytase was selected as the super-dose to be used in subsequent studies. The next study considered the influence of regular (500 FTU/kg) and super doses (1,500 FTU/kg) of phytase from within the gut. Overall, it was observed that changes were occurring to the gut environment, which ultimately would influence the absorptive capacity and conditions for further phytate hydrolysis. Dietary treatment influenced gut conditions such as pH, intestinal morphology and bacterial populations which can subsequently influence nutrient utilisation and potential for growth. The subsequent study was designed to investigate the effects within the gut in more detail. The release of nutrients from phytate hydrolysis and their bioavailability within the digesta can influence conditions within intestine, facilitating enhanced absorption. One of the parameters investigated was the expression of genes involved in the transport of nutrients in the intestine. Overall, there were few significant dietary treatment influences on gene expression in the intestine, however there was a dose-dependent response of phytase on the expression of the jejunual divalent mineral transporter. This indicates a change in divalent mineral bioavailability in the intestine, with correlations with inositol phosphate esters (IPs) being identified. This is likely explained by the IPs produced by phytase hydrolysis and accumulating in the digesta, differing between regular and high doses of phytase. It became apparent that interactions between the products of phytate hydrolysis (IP3, IP4) and minerals in the digesta had the potential to influence the gut environment and subsequent nutrient bioavailability and overall phytase action. The final study was designed to increase the content of the IPs, and investigate the influence of phytase under these conditions. As the complete hydrolysis of phytate to myo-inositol has been reported to be beneficial due to its proposed insulin mimetic effects, myo-inositol was also supplemented to one of the diets to see if any further benefits would be observed when supplemented alongside super-doses of phytase. Neither increased concentrations of the higher IP esters (IP6, IP5 and IP4) nor myo-inositol (myo-) had any effect on broiler growth performance, however there were still apparent beneficial influences of phytase supplementation. The results suggest considerable and important interactions between minerals and IP esters within the digesta, which ultimately have the potential to influence gut conditions and thus nutrient utilisation and growth performance. Reduced concentrations of blood glucose in the high IP ester diet with additional phytase supplementation suggest some insulin-like effects of myo- production. Additionally, the lack of effect of myo- supplementation on blood glucose and insulin concentrations suggests a difference between the structure of phytase-produced myo- and supplemented myo-. Although there were no improvements in growth performance by increasing phytase from 500 to 1,500 FTU/kg, there were changes occurring at the level of the gut and expression of genes in the intestine, influencing nutrient utilisation and the partitioning of nutrients within the body. There are many factors to be considered when supplementing phytase, with dietary nutrient content and nutrient release and IP production during phytate hydrolysis having an influence on phytase action, nutrient absorption and conditions within the gut. Super-doses of phytase may be beneficial for maintaining optimal gut conditions, clearing IP esters from the digesta, reducing their potential to form complexes with minerals and other nutrients, ultimately influencing the efficiency of production.