High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.

We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance. A. thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3. Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB). Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested. Interestingly, a hypersensitive-like response was still induced by several of the strains. The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica. Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III. Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.

Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway.

Plants can recognize and resist invading pathogens by signaling the induction of rapid defense responses. Often these responses are mediated by single dominant resistance genes (R genes). The products of R genes have been postulated to recognize the pathogen and trigger rapid host defense responses. Here we describe isolation of the classical resistance gene N of tobacco that mediates resistance to the well-characterized pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator (Ac) transposon. We confirmed isolation of the N gene by complementation of the TMV-sensitive phenotype with a genomic DNA fragment. Sequence analysis of the N gene shows that it encodes a protein with an amino-terminal domain similar to that of the cytoplasmic domains of the Drosophila Toll protein and the interleukin 1 receptor in mammals, a putative nucleotide-binding site and 14 imperfect leucine-rich repeats. The presence of these functional domains in the predicted N gene product is consistent with the hypothesis that the N resistance gene functions in a signal transduction pathway. Similarities of N to Toll and the interleukin 1 receptor suggest a similar signaling mechanism leading to rapid gene induction and TMV resistance.

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

Background: Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions: Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.

Fire-resistance, physical, and mechanical characterization of particleboard containing Oceanic Posidonia waste

In this work, particleboards manufactured with Oceanic Posidonia waste and bonded with cement are investigated. The particleboards are made with 3/1.5/0.5 parts of cement per part of Posidonia waste. The physical properties of bulk density, swelling, surface absorption, and dimensional changes due to relative humidity as well as the mechanical properties of modulus of elasticity, bending strength, surface soundness, perpendicular tensile strength and impact resistance are studied. In terms of the above properties, the best results were obtained for particleboards with high cement content and when the waste “leaves” are treated (crushed) before board fabrication, due to internal changes to the board structure under these conditions. Based on the results of fire tests, the particleboard is non-flammable without any fire-resistant treatment.

The incorporation of construction and demolition wastes as recycled mixed aggregates in non-structural concrete precast pieces

Concern for the environment has lately heightened awareness about the need for recycling in the construction industry. However, some standards, such as the Spanish standard, only accept the recycling of aggregates derived from concrete, which limits the extensive use of construction and demolition waste, which are produced in much bigger volumes. The aim of this work was to explore the possibility of using recycled mixed aggregates (RMA) in the preparation of precast non-structural concretes. To that end different percentages of natural aggregate were replaced by RMA in non-structural elements (25, 50, 75 and 100%). Contents of cement, water, and the dosages commonly used by companies were unchanged by the introduction of RMA. The characterization of the prepared elements has been done using the specific tests for each type of non-structural element (terrazzo for indoor use, hollow tiles, kerbstones and paving blocks): compression and flexural strength, water absorption, dimensional tolerances, abrasion and slipping resistance. The paving blocks, kerbstones, and hollow tiles prepared were tested for 360 days. The stability of the tested properties confirmed the possibility of using these wastes on an industrial scale satisfying the standard requirements. However, the surface of terrazzo with RMA is not as good as that prepared with natural aggregate.

Biologic therapies and non-melanoma skin cancer rates in three chronic immune-mediated diseases

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.

Serotype/serogroup-specific antibiotic non-susceptibility of invasive and non-invasive Streptococcus pneumoniae, Switzerland, 2004 to 2014.

Concurrent analysis of antibiotic resistance of colonising and invasive Streptococcus pneumoniae gives a more accurate picture than looking at either of them separately. Therefore, we analysed 2,129 non-invasive and 10,996 invasive pneumococcal isolates from Switzerland from 2004 to 2014, which spans the time before and after the introduction of the heptavalent (PCV7) and 13-valent (PCV13) conjugated pneumococcal polysaccharide vaccines. Serotype/serogroup information was linked with all antibiotic resistance profiles. During the study period, the proportion of non-susceptible non-invasive and invasive isolates significantly decreased for penicillin, ceftriaxone, erythromycin and trimethoprim/sulfamethoxazole (TMP-SMX). This was most apparent in non-invasive isolates from study subjects younger than five years (penicillin (p = 0.006), erythromycin (p = 0.01) and TMP-SMX (p = 0.002)). Resistant serotypes/serogroups included in PCV7 and/or PCV13 decreased and were replaced by non-PCV13 serotypes (6C and 15B/C). Serotype/serogroup-specific antibiotic resistance rates were comparable between invasive and non-invasive isolates. Adjusted odds ratios of serotype/serogroup-specific penicillin resistance were significantly higher in the west of Switzerland for serotype 6B (1.8; 95% confidence interval (CI): 1.4-4.8), 9V (3.4; 95% CI: 2.0-5.7), 14 (5.3; 95% CI: 3.8-7.5), 19A (2.2; 95% CI: 1.6-3.1) and 19F (3.1; 95% CI: 2.1-4.6), probably due to variations in the antibiotic consumption.

Genotypic Resistance Tests Sequences Reveal the Role of Marginalized Populations in HIV-1 Transmission in Switzerland.

Targeting hard-to-reach/marginalized populations is essential for preventing HIV-transmission. A unique opportunity to identify such populations in Switzerland is provided by a database of all genotypic-resistance-tests from Switzerland, including both sequences from the Swiss HIV Cohort Study (SHCS) and non-cohort sequences. A phylogenetic tree was built using 11,127 SHCS and 2,875 Swiss non-SHCS sequences. Demographics were imputed for non-SHCS patients using a phylogenetic proximity approach. Factors associated with non-cohort outbreaks were determined using logistic regression. Non-B subtype (univariable odds-ratio (OR): 1.9; 95% confidence interval (CI): 1.8-2.1), female gender (OR: 1.6; 95% CI: 1.4-1.7), black ethnicity (OR: 1.9; 95% CI: 1.7-2.1) and heterosexual transmission group (OR:1.8; 95% CI: 1.6-2.0), were all associated with underrepresentation in the SHCS. We found 344 purely non-SHCS transmission clusters, however, these outbreaks were small (median 2, maximum 7 patients) with a strong overlap with the SHCS'. 65% of non-SHCS sequences were part of clusters composed of >= 50% SHCS sequences. Our data suggests that marginalized-populations are underrepresented in the SHCS. However, the limited size of outbreaks among non-SHCS patients in-care implies that no major HIV outbreak in Switzerland was missed by the SHCS surveillance. This study demonstrates the potential of sequence data to assess and extend the scope of infectious-disease surveillance.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

‘All of this Belongs to Us’: Land, Horses, and Indigenous Resistance on the Yakama Indian Reservation, 1900-1950

In the early 1900s, the Yakima Indian Agency welcomed non-Native ranching operations onto Yakama tribal lands, taxing rangelands, and resulting in widespread overgrazing. By the 1920s, agency concern for the welfare of ranchers facilitated a need to gain access to tribal grazing lands sustaining Yakama horses. As a result, agency officials launched systematic assaults on Yakama horse herds, citing horses as culprits of overgrazing and land degradation. However, Yakamas showed little interest in removing their horses, and instead actively opposed settler encroachment on tribal grazing lands. Through analyzing archival sources, conducting interviews, and reviewing scholarly sources, I argue that Yakamas and settlers used horses as a terrain of struggle, whereby they asserted competing claims to Indigenous lands and resources. Examining horses as a tool of resistance provides a useful lens for understanding forms of Native opposition to colonial hegemony, while interrogating problematic tropes settlers utilized to justify divesting Native communities.

Mollusca Non-Grata: The influence of top-down control and residence time on the abundance, distribution, and behavior of non-native marine snails in Washington State

Thesis (Ph.D.)--University of Washington, 2016-06

Antimicrobial resistance in Staphylococcus aureus in Australian teaching hospitals, 1989-1999

An annual survey of antimicrobial resistance in clinical isolates of Staphylococcus aureus was conducted in 21 Australian teaching hospital microbiology laboratories in eight major cities from 1989 to 1999. A total of 19,000 isolates were tested for susceptibility to 18 antimicrobials, with 3795 being methicillin-resistant (MRSA). Resistance to ciprofloxacin in MRSA increased from 4.9% to 75.9%. The proportion of MRSA resistant to erythromycin decreased significantly (99.0%-88.9%), as did that to trimethoprim (98.4%-82.4%) and to tetracycline (96.5%-80.1%). The proportion of MRSA isolated increased in Sydney, Melbourne, Canberra, Adelaide, Perth, and Darwin, but not in Brisbane. The proportion in Hobart peaked in 1994. MRSA in Perth were predominantly non-multiresistant (nmMRSA) throughout the survey (i.e., resistant to less than three of eight indicator antibiotics) due mainly to local strains that originated in the community. The proportion of nmMRSA increased to modest levels in the other cities. In eastern cities, this was due to the appearance of strains closely related to nmMRSA seen in other countries of the southwestern Pacific.