Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana

The plant-signaling molecules salicylic acid (SA) and jasmonic acid (JA) play an important role in induced disease resistance pathways. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. We investigated possible antagonistic interactions between the SA-dependent systemic acquired resistance (SAR) pathway, which is induced upon pathogen infection, and the JA-dependent induced systemic resistance (ISR) pathway, which is triggered by nonpathogenic Pseudomonas rhizobacteria. In Arabidopsis thaliana, SAR and ISR are effective against a broad spectrum of pathogens, including the foliar pathogen Pseudomonas syringae pv. tomato (Pst). Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection against Pst. In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene PR-1 in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. SAR and ISR both require the key regulatory protein NPR1. Plants expressing both types of induced resistance did not show elevated Npr1 transcript levels, indicating that the constitutive level of NPR1 is sufficient to facilitate simultaneous expression of SAR and ISR. These results suggest that the enhanced level of protection is established through parallel activation of complementary, NPR1-dependent defense responses that are both active against Pst. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control.

Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative β isoform: A mechanism for the generation of glucocorticoid resistance

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-κB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRα), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRβ), which does not bind known ligands and attenuates GRα action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-κB DNA binding site 5′ to the hGR promoter that leads to a 1.5-fold increase in GRα mRNA and a 2.0-fold increase in GRβ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-α treatment disproportionately increased the steady-state levels of the GRβ protein isoform over GRα, making GRβ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-α or IL-1. The increase in GRβ protein expression correlated with the development of glucocorticoid resistance.

Evolution of resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum

The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum malaria. Although the molecular basis for parasite resistance is known, the factors promoting the development and transmission of these resistant parasites are less clear. This paper reports the results of a quantitative comparison of factors previously hypothesized as important for the development of drug resistance, drug dosage, time of treatment, and drug elimination half-life, with an in-host dynamics model of P. falciparum malaria in a malaria-naive host. The results indicate that the development of drug resistance can be categorized into three stages. The first is the selection of existing parasites with genetic mutations in the dihydrofolate reductase or dihydropteroate synthetase gene. This selection is driven by the long half-life of the sulfadoxine-pyrimethamine combination. The second stage involves the selection of parasites with allelic types of higher resistance within the host during an infection. The timing of treatment relative to initiation of a specific anti-P. falciparum EMP1 immune response is an important factor during this stage, as is the treatment dosage. During the third stage, clinical treatment failure becomes prevalent as the parasites develop sufficient resistance mutations to survive therapeutic doses of the drug combination. Therefore, the model output reaffirms the importance of correct treatment of confirmed malaria cases in slowing the development of parasite resistance to sulfadoxine-pyrimethamine.

Abrasion resistance of fibre reinforced concrete floors

This thesis focuses on the investigation of the abrasion resistance of fibre reinforced concrete floors at both the macro and micro levels. A literature review of the available literature concerning subjects allied to the current project is included. This highlights themes relevant to wear mechanisms and the factors influencing it: factors that affect the abrasion resistance of concrete and several test methods for assessing it; and the historical development of fibres and the properties of different fibre types and their influence on concrete. Three accelerated abrasion testers were compared and critically discussed for their suitability for assessing the abrasion resistance of concrete floors. Based on the experimental findings one accelerated abrasion apparatus was selected as more appropriate to be used for carrying out the main investigations. The laboratory programme that followed was undertaken to investigate the influence of various material and construction factors on abrasion resistance. These included mix variations (w/c ratio), fibre reinforcement, geometry, type and volume, curing method and superplasticizing agents. The results clearly show that these factors significantly affected abrasion resistance and several mechanisms were presumed to explain and better understand these observations. To verify and understand these mechanisms that are accountable for the breakdown of concrete slabs, the same concrete specimens that were used for the macro-study, were also subjected to microstructutural investigations using techniques such as Microhardness examination, Mercury intrusion porosimetry and Petrographic examination. It has been found that the abrasion resistance of concrete is primarily dependent on the microstructure and porosity of the concrete nearest to the surface. The feasibility of predicting the abrasion resistance of fibre reinforced concrete floors by indirect and non-destructive methods was investigated using five methods that have frequently been used for assessing the quality of concrete. They included the initial surface absorption test, the impact test, ball cratering, the scratch test and the base hardness test. The impact resistance (BRE screed tester) and scratch resistance (Base hardness tester) were found to be the most sensitive to factors affecting abrasion resistance and hence are considered to be the most appropriate testing techniques. In an attempt to develop an appropriate method for assessing the abrasion resistance of heavy-duty industrial concrete floors, it was found that the presence of curing/sealing compound on the concrete surface at the time of accelerated abrasion testing produces inappropriate results. A preliminary investigation in the direction of modifying the Aston accelerated abrasion tester has been carried out and a more aggressive head has been developed and is pending future research towards standardisation.

The Effect of low-grade aggregates on the abrasion resistance of concrete

Three types of crushed rock aggregate were appraised, these being Carboniferous Sandstone, Magnesian Limestone and Jurassic Limestone. A comprehensive aggregate testing programme assessed the properties of these materials. Two series of specimen slabs were cast and power finished using recognised site procedures to assess firstly the influence of these aggregates as the coarse fraction, and secondly as the fine fraction. Each specimen slab was tested at 28 days under three regimes to simulate 2-body abrasion, 3-body abrasion and the effect of water on the abrasion of concrete. The abrasion resistance was measured using a recognised accelerated abrasion testing apparatus employing rotating steel wheels. Relationships between the aggregate and concrete properties and the abrasion resistance have been developed with the following properties being particularly important - Los Angeles Abrasion and grading of the coarse aggregate, hardness of the fine aggregate and water-cement ratio of the concrete. The sole use of cube strength as a measure of abrasion resistance has been shown to be unreliable by this work. A graphical method for predicting the potential abrasion resistance of concrete using various aggregate and concrete properties has been proposed. The effect of varying the proportion of low-grade aggregate in the mix has also been investigated. Possible mechanisms involved during abrasion have been discussed, including localised crushing and failure of the aggregate/paste bond. Aggregates from each of the groups were found to satisfy current specifications for direct finished concrete floors. This work strengthens the case for the increased use of low-grade aggregates in the future.

Resistance Performances: (Re)constructing Spaces of Resistance and Contention in the 2010-2011 University of Puerto Rico Student Movement

On the night of April 20, 2010, a group of students from the University of Puerto Rico (UPR), Río Piedras campus, met to organize an indefinite strike that quickly broadened into a defense of accessible public higher education of excellence as a fundamental right and not a privilege. Although the history of student activism in the UPR can be traced back to the early 1900s, the 2010-2011 strike will be remembered for the student activists’ use of new media technologies as resources that rapidly prompted and aided the numerous protests. This activist research entailed a critical ethnography and a critical discourse analysis (CDA) of traditional and alternative media coverage and treatment during the 2010 -2011 UPR student strike. I examined the use of the 2010-2011 UPR student activists’ resistance performances in constructing local, corporeal, and virtual spaces of resistance and contention during their movement. In particular, I analyzed the different tactics and strategies of resistance or repertoire of collective actions that student activists used (e.g. new media technologies) to frame their collective identities via alternative news media’s (re)presentation of the strike, while juxtaposing the university administration’s counter-resistance performances in counter-framing the student activists’ collective identity via traditional news media representations of the strike. I illustrated how both traditional and alternative media (re)presentations of student activism developed, maintained, and/or modified students activists’ collective identities. As such, the UPR student activism’s success should not be measured by the sum of demands granted, but by the sense of community achieved and the establishment of networks that continue to create resistance and change. These networks add to the debate surrounding Internet activism and its impact on student activism. Ultimately, the results of this study highlight the important role student movements have had in challenging different types of government policies and raising awareness of the importance of an accessible public higher education of excellence.

Kinome-wide CRISPR-Cas9 screen to identify kinases involved in Taxol resistance in breast cancer

Breast cancer is the most frequently diagnosed cancer in women, accounting for over 25% of cancer diagnoses and 13% of cancer-related deaths in Canadian women. There are many types of therapies for treatment or management of breast cancer, with chemotherapy being one of the most widely used. Taxol (paclitaxel) is one of the most extensively used chemotherapeutic agents for treating cancers of the breast and numerous other sites. Taxol stabilizes microtubules during mitosis, causing the cell cycle to arrest until eventually the cell undergoes apoptosis. Although Taxol has had significant benefits in many patients, response rates range from only 25-69%, and over half of Taxol-treated patients eventually acquire resistance to the drug. Drug resistance remains one of the greatest barriers to effective cancer treatment, yet little has been discerned regarding resistance to Taxol, despite its widespread clinical use. Kinases are known to be heavily involved in cancer development and progression, and several kinases have been linked to resistance of Taxol and other chemotherapeutic agents. However, a systematic screen for kinases regulating Taxol resistance is lacking. Thus, in this study, a set of kinome-wide screens was conducted to interrogate the involvement of kinases in the Taxol response. Positive-selection and negative-selection CRISPR-Cas9 screens were conducted, whereby a pooled library of 5070 sgRNAs targeted 507 kinase-encoding genes in MCF-7 breast cancer cells that were Taxol-sensitive (WT) or Taxol-resistant (TxR) which were then treated with Taxol. Next generation sequencing (NGS) was performed on cells that survived Taxol treatment, allowing identification and quantitation of sgRNAs. STK38, Blk, FASTK and Nek3 stand out as potentially critical kinases for Taxol-induced apoptosis to occur. Furthermore, kinases CDKL1 and FRK may have a role in Taxol resistance. Further validation of these candidate kinases will provide novel pre-clinical data about potential predictive biomarkers or therapeutic targets for breast cancer patients in the future.

DATA DRIVEN APPROACHES TO IDENTIFY DETERMINANTS OF HEART DISEASES AND CANCER RESISTANCE

Cancer and cardio-vascular diseases are the leading causes of death world-wide. Caused by systemic genetic and molecular disruptions in cells, these disorders are the manifestation of profound disturbance of normal cellular homeostasis. People suffering or at high risk for these disorders need early diagnosis and personalized therapeutic intervention. Successful implementation of such clinical measures can significantly improve global health. However, development of effective therapies is hindered by the challenges in identifying genetic and molecular determinants of the onset of diseases; and in cases where therapies already exist, the main challenge is to identify molecular determinants that drive resistance to the therapies. Due to the progress in sequencing technologies, the access to a large genome-wide biological data is now extended far beyond few experimental labs to the global research community. The unprecedented availability of the data has revolutionized the capabilities of computational researchers, enabling them to collaboratively address the long standing problems from many different perspectives. Likewise, this thesis tackles the two main public health related challenges using data driven approaches. Numerous association studies have been proposed to identify genomic variants that determine disease. However, their clinical utility remains limited due to their inability to distinguish causal variants from associated variants. In the presented thesis, we first propose a simple scheme that improves association studies in supervised fashion and has shown its applicability in identifying genomic regulatory variants associated with hypertension. Next, we propose a coupled Bayesian regression approach -- eQTeL, which leverages epigenetic data to estimate regulatory and gene interaction potential, and identifies combinations of regulatory genomic variants that explain the gene expression variance. On human heart data, eQTeL not only explains a significantly greater proportion of expression variance in samples, but also predicts gene expression more accurately than other methods. We demonstrate that eQTeL accurately detects causal regulatory SNPs by simulation, particularly those with small effect sizes. Using various functional data, we show that SNPs detected by eQTeL are enriched for allele-specific protein binding and histone modifications, which potentially disrupt binding of core cardiac transcription factors and are spatially proximal to their target. eQTeL SNPs capture a substantial proportion of genetic determinants of expression variance and we estimate that 58% of these SNPs are putatively causal. The challenge of identifying molecular determinants of cancer resistance so far could only be dealt with labor intensive and costly experimental studies, and in case of experimental drugs such studies are infeasible. Here we take a fundamentally different data driven approach to understand the evolving landscape of emerging resistance. We introduce a novel class of genetic interactions termed synthetic rescues (SR) in cancer, which denotes a functional interaction between two genes where a change in the activity of one vulnerable gene (which may be a target of a cancer drug) is lethal, but subsequently altered activity of its partner rescuer gene restores cell viability. Next we describe a comprehensive computational framework --termed INCISOR-- for identifying SR underlying cancer resistance. Applying INCISOR to mine The Cancer Genome Atlas (TCGA), a large collection of cancer patient data, we identified the first pan-cancer SR networks, composed of interactions common to many cancer types. We experimentally test and validate a subset of these interactions involving the master regulator gene mTOR. We find that rescuer genes become increasingly activated as breast cancer progresses, testifying to pervasive ongoing rescue processes. We show that SRs can be utilized to successfully predict patients' survival and response to the majority of current cancer drugs, and importantly, for predicting the emergence of drug resistance from the initial tumor biopsy. Our analysis suggests a potential new strategy for enhancing the effectiveness of existing cancer therapies by targeting their rescuer genes to counteract resistance. The thesis provides statistical frameworks that can harness ever increasing high throughput genomic data to address challenges in determining the molecular underpinnings of hypertension, cardiovascular disease and cancer resistance. We discover novel molecular mechanistic insights that will advance the progress in early disease prevention and personalized therapeutics. Our analyses sheds light on the fundamental biological understanding of gene regulation and interaction, and opens up exciting avenues of translational applications in risk prediction and therapeutics.

Single gene resistance to Fusarium oxysporum f. sp. cubense Race 4 in the wild banana Musa acuminata subsp. malaccensis

Fusarium wilt of banana, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. A particularly virulent strain of the pathogen, tropical race 4 (TR4), presents an emerging threat to banana producing regions throughout the world. No commercially acceptable banana cultivar is resistant to TR4 and, as with all strains of the Fusarium wilt pathogen, there is no effective chemical control. Genetic resistance to TR4 has been observed in the diploid wild banana Musa acuminata subsp. malaccensis, which has consequently received attention as a potential source of Fusarium resistance genes. The aim of this research was to determine the pattern of inheritance of the resistance trait by screening plants for resistance to Foc subtropical race 4 (SR4) and TR4. Our results showed that the F1 progeny of self-fertilized malaccensis plants challenged in pot trials against SR4 (VCGs 0120, 0129, 01211) and TR4 (VCG 01213/16) segregated for resistance according to a Mendelian ratio of 3:1 which is consistent with a single dominant gene hypothesis.

Improving DPA and DFA resistance of circuits using asynchronous logic

International audience

Chemical resistance of lime-based grouts for masonry strengthening

The stone masonry walls are present in many buildings and historical monuments, with undeniable asset value, but also in old buildings housing both in Portugal and in Europe. Most of these buildings in masonry are in certain cases in a high state of degradation needing urgent intervention. This requires the identification of deficiencies and the application of appropriate intervention techniques. One of the possible techniques for structural consolidation works of stone masonry walls is the injection of fluid mortars currently called grouts. The choice of grouts is very important with regard in particular to their chemical and physical properties. In this study, carried out under the Master of Chemical Engineering, two types of lime-based grouts were used, in order to evaluate and compare their chemical resistance due to the crystallization of soluble salts. One of the grouts is a pre-dosed blend commercially available, Mape-Antique I from company Mapei (CA), and the second grout is a mixture prepared in the laboratory (LB), comprising metakaolin, cement, hydrated lime, water and superplasticizer. With the purpose of evaluating the action of sulphates on these grouts, a series of samples underwent several wetting-drying cycles using two different temperatures, 20 °C and 50 °C. During the experiment it was determined the change of weight and compressive strength in the analyzed grouts, as well as the sulphate ion concentration and pH of the solution in which the samples were dipped. The commercial grout (CA) apparently has a greater chemical resistance to sulphates. However grout LB showed to have positive results in some parameters.

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis , can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus . The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance: ermA, ermB, ermC, msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistant S. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination was ermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

Prediction of mode I delamination resistance of z-pinned laminates using the embedded finite element technique

A new finite modelling approach is presented to analyse the mode I delamination fracture toughness of z-pinned laminates using the computationally efficient embedded element technique. In the FE model,each z-pin is represented by a single one-dimensional truss element that is embedded within the laminate. Each truss is given the material, geometric and spatial properties associated with the global crackbridging traction response of a z-pin in the laminate; this simplification provides a computationally efficient and flexible model where pin elements are independent of the underlying structural mesh for thelaminate. The accuracy of the FE modelling approach is assessed using mode I interlaminar fracture toughness data for a carbon-epoxy laminate reinforced with z-pins made of copper, titanium or stainless steel. The model is able to predict with good accuracy the crack growth resistance curves and fracture toughness properties for the different types of z-pinned laminate.

Resistance performances: (Re)constructing spaces of resistance and contention in the 2010-2011 University of Puerto Rico student movement

On the night of April 20, 2010, a group of students from the University of Puerto Rico (UPR), Río Piedras campus, met to organize an indefinite strike that quickly broadened into a defense of accessible public higher education of excellence as a fundamental right and not a privilege. Although the history of student activism in the UPR can be traced back to the early 1900s, the 2010-2011 strike will be remembered for the student activists’ use of new media technologies as resources that rapidly prompted and aided the numerous protests. ^ This activist research entailed a critical ethnography and a critical discourse analysis (CDA) of traditional and alternative media coverage and treatment during the 2010 -2011 UPR student strike. I examined the use of the 2010-2011 UPR student activists’ resistance performances in constructing local, corporeal, and virtual spaces of resistance and contention during their movement. In particular, I analyzed the different tactics and strategies of resistance or repertoire of collective actions that student activists used (e.g. new media technologies) to frame their collective identities via alternative news media’s (re)presentation of the strike, while juxtaposing the university administration’s counter-resistance performances in counter-framing the student activists’ collective identity via traditional news media representations of the strike. I illustrated how both traditional and alternative media (re)presentations of student activism developed, maintained, and/or modified students activists’ collective identities. ^ As such, the UPR student activism’s success should not be measured by the sum of demands granted, but by the sense of community achieved and the establishment of networks that continue to create resistance and change. These networks add to the debate surrounding Internet activism and its impact on student activism. Ultimately, the results of this study highlight the important role student movements have had in challenging different types of government policies and raising awareness of the importance of an accessible public higher education of excellence.^

Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock

UNLABELLED Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.