Aspergillus flavus infections in Galleria mellonella : a pathogen-host model system for the study of emerging diseases

To study emerging diseases, I employed a model pathogen-host system involving infections of insect larvae with the opportunistic fungus Aspergillus flavus, providing insight into three mechanisms ofpathogen evolution namely de novo mutation, genome decay, and virulence factoracquisition In Chapter 2 as a foundational experiment, A. flavus was serially propagated through insects to study the evolution of an opportunistic pathogen during repeated exposure to a single host. While A. flavus displayed de novo phenotypic alterations, namely decreased saprobic capacity, analysis of genotypic variation in Chapter 3 signified a host-imposed bottleneck on the pathogen population, emphasizing the host's role in shaping pathogen population structure. Described in Chapter 4, the serial passage scheme enabled the isolation of an A. flavus cysteine/methionine auxotroph with characteristics reminiscent of an obligate insect pathogen, suggesting that lost biosynthetic capacity may restrict host range based on nutrient availability and provide selection pressure for further evolution. As outlined in Chapter 6, cysteine/methionine auxotrophy had the pleiotrophic effect of increasing virulence factor production, affording the slow-growing auxotroph with a modified pathogenic strategy such that virulence was not reduced. Moreover in Chapter 7, transformation with a virulence factor from a facultative insect pathogen failed to increase virulence, demonstrating the necessity of an appropriate genetic background for virulence factor acquisition to instigate pathogen evolution.

An analysis of the systemic aspects of rights training for people with intellectual disabilities

Individuals with intellectual disabilities (ID) as a group have been subject to abuse. Individuals with ID need to be made aware of their rights. The 3Rs: Rights, Respect and Responsibility Human Rights Project is promoting rights awareness in individuals with ID, their caregivers and family members. To be effeCtive, abuse prevention must include support from the whole organization and its processes. This research evaluated the impact of the 3Rs initiative on the organization. It focused particularly on descriptions of organizational change perceived by full-time staff and managers in response to the initiation of the 3Rs Project. Behavioural interviews were conducted and a thematic analysis was used to describe changes in the organizational culture and behavioural mechanisms maintaining these changes. Systemic barriers to change were also explored. The results indicate that the Association is effectively implementing and supporting the rights-based philosophy.

The acute effects of systemic cytokines on peripheral nerve function in humans

Cytokines have been shown to cause a reduction in nerve conduction when examined using animal models. Such effects, if shown in humans, could result in detrimental effects to physical function during periods heightened systemic cytokine concentrations. The study investigated the acute effects of cytokines on nerve conduction velocity (NCV) and functional measures. Measures were taken under both basal and elevated cytokine concentrations to determine any corresponding changes to NCV. A significant positive correlation was found between the cytokine IL-6 and NCV at 2 hours post-exercise (r=0.606, p=0.048). A significant negative correlation was found between IL-1ra and NCV at 24 hours post-exercise (r=-0.652, p=0.021). A significant positive correlation was also found between IL-1ra and endurance at 1 hour post-exercise (r=0.643, p=0.033). As such, it would seem that IL-6 may potentially act to enhance nerve function while other cytokines such as IL-1ra may have negative effects and reduce NCV.

Regulation of Systemic Acquired Resistance through the Interaction of Arabidopsis thaliana Transcription factors TGAI and TGA2 with NPRI

Arabidopsis thaliana is an established model plant system for studying plantpathogen interactions. The knowledge garnered from examining the mechanism of induced disease resistance in this model system can be applied to eliminate the cost and danger associated with current means of crop protection. A specific defense pathway, known as systemic acquired resistance (SAR), involves whole plant protection from a wide variety of bacterial, viral and fungal pathogens and remains induced weeks to months after being triggered. The ability of Arabidopsis to mount SAR depends on the accumulation of salicylic acid (SA), the NPRI (non-expressor of pathogenesis related gene 1) protein and the expression of a subset of pathogenesis related (PR) genes. NPRI exerts its effect in this pathway through interaction with a closely related class of bZIP transcription factors known as TGA factors, which are named for their recognition of the cognate DNA motif TGACG. We have discovered that one of these transcription factors, TGA2, behaves as a repressor in unchallenged Arabidopsis and acts to repress NPRI-dependent activation of PRJ. TGA1, which bears moderate sequence similarity to TGA2, acts as a transcriptional activator in unchallenged Arabidopsis, however the significance of this activity is J unclear. Once SAR has been induced, TGAI and TGA2 interact with NPRI to form complexes that are capable of activating transcription. Curiously, although TGAI is capable of transactivating, the ability of the TGAI-NPRI complex to activate transcription results from a novel transactivation domain in NPRI. This transactivation domain, which depends on the oxidation of cysteines 521 and 529, is also responsible for the transactivation ability of the TGA2-NPRI complex. Although the exact mechanism preventing TGA2-NPRI interaction in unchallenged Arabidopsis is unclear, the regulation of TGAI-NPRI interaction is based on the redox status of cysteines 260 and 266 in TGAl. We determined that a glutaredoxin, which is an enzyme capable of regulating a protein's redox status, interacts with the reduced form of TGAI and this interaction results .in the glutathionylation of TGAI and a loss of interaction with NPRl. Taken together, these results expand our understanding of how TGA transcription factors and NPRI behave to regulate events and gene expression during SAR. Furthermore, the regulation of the behavior of both TGAI and NPRI by their redox status and the involvement of a glutaredoxin in modulating TGAI-NPRI interaction suggests the redox regulation of proteins is a general mechanism implemented in SAR.

Retrospective Exploration of the 3Rs Education Program: Systemic Facilitators and Barriers to Implementation

Individuals with intellectual disabilities (ID) have historically been denied basic rights and thus have been subjected to abuse. The 3Rs: Rights, Respect and Responsibility Human Rights Education Program was implemented and researched through a partnership with Community Living Welland Pelham and Brock University initially and then cascade training on the program was provided to five developmental service sector agencies from across the Niagara Region. This research evaluated the role of the 3Rs education program on the shift to a rights-based service agenda across those five agencies. Interviews were conducted with the Executive Director and Liaison staff from each of the agencies and a thematic analysis was used to describe factors that facilitated organizational changes and a cultural shift. Systemic barriers to the change were also explored. The results indicated that the 3Rs education program provides the catalyst necessary for the shift to a rights-based service agenda and that the resultant changes in practices now embedded in the organizations are reflective of a shift to a rights-based service agenda.

A research capacity strengthening project for infectious diseases in Honduras: experience and lessons learned

Background: In Honduras, research capacity strengthening (RCS) has not received sufficient attention, but an increase in research competencies would enable local scientists to advance knowledge and contribute to national priorities, including the Millennium Development Goals (MDGs). Objective: This project aimed at strengthening research capacity in infectious diseases in Honduras, focusing on the School of Microbiology of the National Autonomous University of Honduras (UNAH). The primary objective was the creation of a research-based graduate program for the continued training of researchers. Parallel objectives included institutional strengthening and the facilitation of partnerships and networks. Methods: Based on a multi-stakeholder consultation, an RCS workplan was designed and undertaken from 2007 to 2012. Due to unexpected adverse circumstances, the first 2 years were heavily dedicated to implementing the project's flagship, an MSc program in infectious and zoonotic diseases (MEIZ). In addition, infrastructure improvements and demand-driven continuing education opportunities were facilitated; biosafety and research ethics knowledge and practices were enhanced, and networks fostering collaborative work were created or expanded. Results: The project coincided with the peak of UNAH's radical administrative reform and an unprecedented constitutional crisis. Challenges notwithstanding, in September 2009, MEIZ admitted the first cohort of students, all of whom undertook MDG-related projects graduating successfully by 2012. Importantly, MEIZ has been helpful in expanding the School of Microbiology's traditional etiology-based, disciplinary model to infectious disease teaching and research. By fulfilling its objectives, the project contributed to a stronger research culture upholding safety and ethical values at the university. Conclusions: The resources and strategic vision afforded by the project enhanced UNAH's overall research capacity and its potential contribution to the MDGs. Furthermore, increased research activity and the ensuing improvement in performance indicators at the prime Honduran research institution invoke the need for a national research system in Honduras.

The Molecular Consequences of CK2-mediated Phosphorylation of the TGA2 Transcription Factor within Systemic Acquired Resistance of Arabidopsis thaliana

During infection, the model plant Arabidopsis thaliana is capable of activating long lasting defence responses both in tissue directly affected by the pathogen and in more distal tissue. Systemic acquired resistance (SAR) is a type of systemic defence response deployed against biotrophic pathogens resulting in altered plant gene expression and production of antimicrobial compounds. One such gene involved in plant defence is called pathogenesis-related 1 (PR1) and is under the control of several protein regulators. TGA II-clade transcription factors (namely TGA2) repress PR1 activity prior to infection by forming large oligomeric complexes effectively blocking gene transcription. After pathogen detection, these complexes are dispersed by a mechanism unknown until now and free TGA molecules interact with the non-expressor of pathogenesis-related gene 1 (NPR1) protein forming an activating complex enabling PR1 transcription. This study elucidates the TGA2 dissociation mechanism by introducing protein kinase CK2 into this process. This enzyme efficiently phosphorylates TGA2 resulting in two crucial events. Firstly, the DNA-binding ability of this transcription factor is completely abolished explaining how the large TGA2 complexes are quickly evicted from the PR1 promoter. Secondly, a portion of TGA2 molecules dissociate from the complexes after phosphorylation which likely makes them available for the formation of the TGA2-NPR1 activating complex. We also show that phosphorylation of a multiserine motif found within TGA2’s N terminus is responsible for the change of affinity to DNA, while modification of a single threonine in the leucine zipper domain seems to be responsible for deoligomerization. Despite the substantial changes caused by phosphorylation, TGA2 is still capable of interacting with NPR1 and these proteins together form a complex on DNA promoting PR1 transcription. Therefore, we propose a change in the current model of how PR1 is regulated by adding CK2 which targets TGA2 displacing it’s complexes from the promoter and providing solitary TGA2 molecules for assembly of the activating complex. Amino acid sequences of regions targeted by CK2 in Arabidopsis TGA2 are similar to those found in TGA2 homologs in rice and tobacco. Therefore, the molecular mechanism that we have identified may be conserved among various plants, including important crop species, adding to the significance of our findings.