Molecular address tags for vaccines

Antibodies are neutralising proteins produced by plasma cells of the mammalian immune system to combat invading pathogens. The specificity of antibodies towards antigenic epitopes is paramount to successful vaccination therapies. This thesis set out to enhance antibody specificity by introduction of a 'molecular address tag' (MAT) to vaccines for targeting antigens to cells of the immune system. The aim was to produce antibodies more specific and more potent towards antigenic epitopes, eliminating the need for adjuvants. β-1,3-Glucan tags of 15 degrees of polymerisation were synthesised for use as address tags, along with laminarin, a commercially available storage polysaccharide of brown alga consisting of β-1,3-glucan backbone with β-1,6-glucan side chains. Following conjugation to BSA, MATs were studied for their ability to interact with Dectin-1, a C-type lectin receptor with an affinity for β-(1-3)-glucans, and to stimulate a Dectin-1-mediated signalling response, by utilising a Dectin-1-expressing reporter cell line. This study revealed a signalling response triggered by conjugated β-(1-3)-glucans with β-(1-6)-side chains, and particulate β-(1-3)-glucans of DP15 either free or unconjugated. The latter half of this thesis focused on antigen generation and immunisation trials. Fungal β-(1-2)-mannan antigens of DP3 and DP4 were synthesised enzymatically and isolated via gel permeation chromatography. However, conjugation efforts were unsuccessful and time did not allow for further optimisation. Two vaccines to protect against the causative agent of Q fever, Coxiella burnetii, were generated. Both vaccines comprised of a virus-like-particle core to which lamarin address tags were conjugated as well as a virenose antigen. The antigen comprise of either α-Vir(1-4)-α-Vir or β-Vir(1-4)-β-Vir, depending on the vaccine. Immunisation trials were performed in rabbits, and serum analysis performed by ELISA, revealing a specificity towards both α and β-virenose antigens when immunised with β-Vir(1-4)-β-Vir, but a specificity towards α-vir when immunised with α-Vir(1-4)-α-Vir. These finding

Alien Registration- Winsbury, William F. (Madison, Somerset County)

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Novel Antimicrobial Activities of Trichoderma hamatum GD12 Following Deletion of Heterochromatin Protein 1

Heterochromatin Protein 1 (HEP1) is a highly conserved, chromatin re-modelling protein involved in activation and repression of secondary metabolite producing gene clusters. In-house genome sequencing of the plant growth promoting and biocontrol fungus Trichoderma hamatum GD12 has shown that ~40 % of the genome is unique to GD12 compared to its closest relatives, suggesting enormous genetic potential to encode novel bioactive compounds with antimicrobial and PGP activities. It is apparent that under axenic conditions, a substantial proportion of the bioactive potential of the fungus is not expressed. We therefore hypothesised that loss of HEP1 would lead to activation of cryptic gene clusters responsible for the production of novel bioactive secondary metabolites. Identification of compounds with antimicrobial activities might benefit a growing population faced with numerous multidrug resistant microorganisms. HEP1 was inactivated in T. hamatum GD12 using the split-marker method of homologous recombination and ΔThhepA::hph strains were confirmed via DIG-labelled Southern blot analysis. Phenotypic analysis revealed significantly reduced hyphal growth of hepA mutants compared to GD12. Confrontation assays of GD12 and three independent ΔThhepA::hph strains against fungal pathogens revealed a change in the biocontrol activities, with a zone of inhibition surrounding mutant strains suggesting the secretion of inhibitory bioactive compound(s). Liquid chromatography-mass spectrometry was used to determine the secretome profiles of hepA mutants. Analysis of the data revealed a number of key features which are differentially expressed in hepA mutants. One such feature of particular interest is Brefeldin A, which functions as an antimicrobial agent. This project would benefit from characterisation of key features to determine their antimicrobial potentials

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A situational comedy horror in which new hire Alison investigates mysterious happenings at the local Spendsaver convenience store, and finds that everything is even less normal than it seems

Investigating the beneficial traits of Trichoderma hamatum GD12 for sustainable agriculture-insights from genomics.

This is the final version of the article. Available from the publisher via the DOI in this record.Trichoderma hamatum strain GD12 is unique in that it can promote plant growth, activate biocontrol against pre- and post-emergence soil pathogens and can induce systemic resistance to foliar pathogens. This study extends previous work in lettuce to demonstrate that GD12 can confer beneficial agronomic traits to other plants, providing examples of plant growth promotion in the model dicot, Arabidopsis thaliana and induced foliar resistance to Magnaporthe oryzae in the model monocot rice. We further characterize the lettuce-T. hamatum interaction to show that bran extracts from GD12 and an N-acetyl-β-D-glucosamindase-deficient mutant differentially promote growth in a concentration dependent manner, and these differences correlate with differences in the small molecule secretome. We show that GD12 mycoparasitises a range of isolates of the pre-emergence soil pathogen Sclerotinia sclerotiorum and that this interaction induces a further increase in plant growth promotion above that conferred by GD12. To understand the genetic potential encoded by T. hamatum GD12 and to facilitate its use as a model beneficial organism to study plant growth promotion, induced systemic resistance and mycoparasitism we present de novo genome sequence data. We compare GD12 with other published Trichoderma genomes and show that T. hamatum GD12 contains unique genomic regions with the potential to encode novel bioactive metabolites that may contribute to GD12's agrochemically important traits.This work was supported by a Biotechnology and Biological Sciences Research Council grant BB/I014691/1 to Murray Grant and Chris R. Thornto

Investigating the beneficial traits of Trichoderma hamatum GD12 for sustainable agriculture : insights from genomics

Trichoderma hamatum strain GD12 is unique in that it can promote plant growth, activate biocontrol against pre- and post-emergence soil pathogens and can induce systemic resistance to foliar pathogens. This study extends previous work in lettuce to demonstrate that GD12 can confer beneficial agronomic traits to other plants, providing examples of plant growth promotion in the model dicot, Arabidopsis thaliana and induced foliar resistance to Magnaporthe oryzae in the model monocot rice. We further characterize the lettuce-T. hamatum interaction to show that bran extracts from GD12 and an N-acetyl-β-D-glucosamindase-deficient mutant differentially promote growth in a concentration dependent manner, and these differences correlate with differences in the small molecule secretome. We show that GD12 mycoparasitises a range of isolates of the pre-emergence soil pathogen Sclerotinia sclerotiorum and that this interaction induces a further increase in plant growth promotion above that conferred by GD12. To understand the genetic potential encoded by T. hamatum GD12 and to facilitate its use as a model beneficial organism to study plant growth promotion, induced systemic resistance and mycoparasitism we present de novo genome sequence data. We compare GD12 with other published Trichoderma genomes and show that T. hamatum GD12 contains unique genomic regions with the potential to encode novel bioactive metabolites that may contribute to GD12's agrochemically important traits. Read Full Tex

Transcriptional reprogramming underpins enhanced plant growth promotion by the biocontrol fungus Trichoderma hamatum GD12 during antagonistic interactions with Sclerotinia sclerotiorumin soil

The free-living soil fungus Trichoderma hamatum strain GD12 is notable amongst Trichoderma strains in both controlling plant diseases and in stimulating plant growth, a property enhanced during its antagonistic interactions with pathogens in soil. These attributes, alongside its markedly expanded genome and proteome compared to other biocontrol and plant growth promoting Trichoderma strains, imply a rich potential for sustainable alternatives to synthetic pesticides and fertilisers for controlling plant disease and increasing yields. The purpose of this study was to investigate the transcriptional responses of GD12 underpinning its biocontrol and plant growth promotion capabilities during antagonistic interactions with the pathogen Sclerotinia sclerotiorum in soil. Using an extensive mRNA-seq study capturing different time points during the pathogen-antagonist interaction in soil, we show that dynamic and biphasic signatures in the GD12 transcriptome underpin its biocontrol and plant (lettuce) growth promotional activities. Functional predictions of differentially expressed genes demonstrate the enrichment of transcripts encoding proteins involved in transportation and oxidation-reduction reactions during both processes and an over-representation of siderophores. We identify a biphasic response during biocontrol characterised by a significant induction of transcripts encoding small-secreted cysteine rich proteins, secondary metabolite producing gene clusters and genes unique to GD12. These data support the hypothesis that Sclerotinia biocontrol is mediated by the synthesis and secretion of antifungal compounds and that GD12's unique reservoir of uncharacterised genes is actively recruited during effective biological control of a plurivorous plant pathogen. This article is protected by copyright. All rights reserved

Characterisation and development of histopathological lesions in a guinea pig model of Mycobacterium tuberculosis infection

Tuberculosis (TB) remains a very significant infectious disease worldwide. New vaccines and therapies are needed, even more crucially with the increase of multi-drug resistant Mycobacterium tuberculosis strains. Preclinical animal models are very valuable for the development of these new disease control strategies. Guinea pigs are one of the best models of TB, sharing many features with the pathology observed in human TB. Here we describe the development of TB lesions in a guinea pig model of infection. We characterise the granulomatous lesions in four developmental stages (I–IV), using histopathological analysis and immunohistochemical (IHC) techniques to study macrophages, T cells, B cells and granulocytes. The granulomas in the guinea pigs start as aggregations of macrophages and few heterophils, evolving to larger lesions showing central caseous necrosis with mineralisation and abundant acid-fast bacilli, surrounded by a rim of macrophages and lymphocytes in the outer layers of the granuloma. Multinucleated giant cells are very rare and fibrotic capsules are not formed in this animal model

Algorithms, governance, and governmentality:on governing academic writing

Algorithms, or rather algorithmic actions, are seen as problematic because they are inscrutable, automatic, and subsumed in the flow of daily practices. Yet, they are also seen to be playing an important role in organizing opportunities, enacting certain categories, and doing what David Lyon calls ‘‘social sorting.’’ Thus, there is a general concern that this increasingly prevalent mode of ordering and organizing should be governed more explicitly. Some have argued for more transparency and openness, others have argued for more democratic or value-centered design of such actors. In this article, we argue that governing practices—of, and through algorithmic actors—are best understood in terms of what Foucault calls governmentality. Governmentality allows us to consider the performative nature of these governing practices. They allow us to show how practice becomes problematized, how calculative practices are enacted as technologies of governance, how such calculative practices produce domains of knowledge and expertise, and finally, how such domains of knowledge become internalized in order to enact self-governing subjects. In other words, it allows us to show the mutually constitutive nature of problems, domains of knowledge, and subjectivities enacted through governing practices. In order to demonstrate this, we present attempts to govern academic writing with a specific focus on the algorithmic action of Turnitin

End-results of plastic operations on the kidney pelvis for hydronephrosis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32613/1/0000755.pd