Temperature- and host-dependent transcriptional responses in the entomopathogenic bacterium, Yersinia entomophaga MH96 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany Campus, New Zealand

Yersinia entomophaga MH96 is a virulent pathogenic bacterium that is infective towards a broad range of insects and is under development as a biopesticide. MH96 produces insecticidal toxin complex called Yen-TC that is secreted at temperatures of 25 °C and below and has been shown to be the primary virulence factor (VF) during per os challenge against the New Zealand grass grub, Costelytra giveni and other agricultural pests (Hurst et al., 2011a, 2019). New insights into the pathobiology of MH96 during insect infection were gained from the in vivo transcriptome, including identification of a core secreted weaponry of co-expressed/co-secreted VFs, including Yen-TC and other exoenzymes; however, many other diverse types of VFs, including toxins, effectors, fimbriae, secretion systems, efflux pumps, iron acquisition, stress response and metabolic adaptation were also identified as highly expressed under in vivo conditions. A small DNA-binding protein, Yen6, was shown to be under thermoregulation at the transcriptional level and host-dependent-regulation at the post-transcriptional level and contributed to virulence during intrahemocoelic infection of Galleria mellonella at 37 °C. The in vivo transcriptome of Δyen6 and in vitro DNA-binding specificity analysis provided evidence that Yen6 is a novel LytTR-containing regulator that activates a ribose uptake/metabolism gene cluster, rbsD-xylG-rbsC-xylF-rbsK-ccpA, and represses a fructose uptake/metabolism gene cluster, IIA-fruK-IIB and a gene for RNA-binding protein yhbY during infection at 37 °C. Another small DNA-binding protein, Yen7, was also implicated as a potential temperature-dependent activator of Yen-TC component genes and over-expression of yen7 resulted in restored secretion by MH96 at 37 °C; however, deletion of yen7 did not abrogate Yen-TC production. Experimental investigations into potential regulatory linkages between Yen6 and yen7 were undertaken, and evidence to date does not support Yen6 as transcriptional repressor of yen7. A 17.5 Kb unstable element within the genome of MH96 with linkages to Yen-TC and toxin secretion, motility and cell shape was identified. Overall the findings presented in this thesis represent the most detailed investigation of MH96 pathogenesis to date, reinforcing MH96 as one of the most highly entomopathogenic bacteria known to humankind; yet suggesting MH96 has possibly maintained at least one core thermoregulatory mechanism more typical of an opportunistic pathogen

The Host-Microbiota Axis in Chronic Wound Healing

Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management

Dissecting the Molecular Mechanisms of Motor Neurone Disease in Yeast and Worms

Superoxide dismutase 1 (SOD1) is an enzyme responsible for intracellular breakdown of toxic reactive oxygen species in prokaryotes and eukaryotes. Mutations in SOD1 are linked to the motor neurone disease Amyotrophic lateral sclerosis (ALS), with around 20% of familial (fALS) cases and, it is proposed, a significant proportion of sporadic cases caused by SOD1 mutations, however the mechanism of neuronal death remains unknown. Misfolding and aggregation of SOD1 leading to a ‘toxic gain of function’ is the most widely accepted mechanism, but further work is needed. To gain new insights into the underlying mechanism of cell toxicity, I am using two model systems: the yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans. One hypothesis being tested was proposed by Ghadge et al. (2006) who reported that truncated forms of SOD1 (both wild type and fALS–linked mutants) form toxic aggregates in spinal cords of chick embryos, hypothesising that proteolytic digestion of unstable mutant SOD1 creates these toxic fragments. My project aims to elucidate how fALS–linked SOD1 mutants and SOD1 truncations exert toxicity on S. cerevisiae and C. elegans. In S. cerevisiae, SOD1 truncations do not specifically localise to, or disrupt the functioning of, mitochondria nor reduce viability of yeast although cell growth is slowed. Conversely, a motor-defective phenotype is exhibited in transgenic fALS–linked SOD1 mutant (G85R) C. elegans accompanied by vulval defects, which lead to reduced lifespan. My findings suggest that amino acid supplementation of G85R C. elegans mutants improves their motor function and vulval defects, implicating a role of amino acid signalling in mutant SOD1-mediated toxicity

Communication Strategies Used During Organizational Change in a Health Care Organization

More than 4.9 million businesses exist in the United States, and leaders within these businesses have to acclimate to change. Clear and effective communication is vital to the success of an organization. According to scholars and health care leaders focusing on strategies to communicate change during organizational change is a critical aspect of sustainability and profitability. The conceptual framework of this study was communication theory. The purpose of this single case study was to explore successful strategies that some health care leaders used to communicate during organizational change in a health care organization in El Paso, Texas. The data collection process consisted of collecting data from semistructured interviews and organizational documents, and the analysis process included grouping key words and reconstructing data into themes. The 4 key themes that emerged from this process included building trust through organizational communication is critical during change, the use of technologies, as a tool for communication is key during change, 2-way communication needs to occur during organizational change, and communication about change is vital through comprehensive organizational meetings. Health care leaders provided insights on management and communication strategies and responsibilities leaders and employees go through during organizational change. The implications for positive social change include strategies to improve communication that could help health care leaders with their employees and their patients during organizational change, which could increase the profitability of the organization and potentially generate a more thriving and healthy community

Memory and Stereotypes for Lesbian/Gay Characters

Stereotype-consistency bias refers to the idea that people tend to remember stereotypical information about others better than non-stereotypical information (Fyock & Stangor, 1994). Limited research has examined how people may use stereotype-consistency bias when recalling information about LGBT characters in narratives (Bellezza & Bower, 1981; Clark & Woll, 1981; McGann & Goodwin, 2007; Snyder & Uranowitz, 1978). This line of research suggests that, instead of genuinely remembering stereotypical information better, participants tended to guess stereotypical answers to questions they do not know. In contrast to those studies, the experiment I conducted for this thesis suggests that heterosexual young adults tend to engage in stereotype inhibition, avoiding the use of gay and lesbian stereotypes. This may be due to a cohort effect, but future research is needed to determine the relevance of cognitive load and explicit homophobia. This information can be used for understanding how stereotyping occurs and developing interventions to reduce stereotype use

Another City is Possible: Mujeres de Maiz, Radical Indigenous Mestizaje and Activist Scholarship

This dissertation examines the political and creative practices of Mujeres de Maiz (MdM/Women of the Corn), an Indigenous mestiz@ led feminist of color visual and performing arts collective based in Los Angeles. Since their inception in 1997, Mujeres de Maiz has used politically and spiritually charged art that aims to challenge social injustices, revise dominant cultural representations and build meaningful communities across differences. The research was drawn from a dual method approach: textual analysis and participant observation ethnography with the MdM collective from 2009 to 2014 to examine how urban Indigenous mestiz@s represent themselves in textual and social spaces. Few studies have paid attention to constructions of Chicano indigeneity, and fewer still to feminist indigeneity, in the twenty-first century that exists outside of Chicano nationalist and "Chican@ indigenist" paradigms. In contrast, this work examines the ways women shape, transform and extend Chican@ indigeneity and express manifestations of a "radical indigenous mestizaje" through their artivism. This research seeks to propel Chican@ Studies towards making room for a transnational feminist, hemispheric and grounded approach to indigeneity by demonstrating that these approaches are already being constructed on the ground. The dissertation is comprised of case studies and models of activist scholarship

Strategies to Retain Employees in Clinical Laboratories

With 30 million citizens gaining access to U.S. healthcare through the Affordable Care Act by 2016, healthcare managers need preanalytic employees to ensure quality healthcare services can be provided. The purpose of this qualitative single case study explored strategies used to retain preanalytic employees. The target population consisted of 10 clinical laboratory managers in a single community-based clinical laboratory in the mid-southern United States, selected because of prior success in improved employee retention strategies. The conceptual framework grounding this study was the theory of work adjustment. Data triangulation occurred from using semistructured interviews and company documents. All interpretations from the data were subjected to member checking to ensure the trustworthiness of findings. Using the modified van Kaam method for data analysis, 3 themes included increasing employee training to enhance their roles as stakeholders, provide employee recognition and reward programs, and emphasizing to managers the importance of quality patient care. The application of the findings may contribute to social change by providing healthcare managers with new insights and strategies to retain preanalytic employees, reduce organizational costs associated with recruiting, replacing, and training of new employees, and potentially trained staff that provide quality healthcare services to community citizens

Physician understanding and application of surrogate decision-making laws in clinical practice

Background: Although state surrogate laws are the most common way surrogate decision makers are identified, no studies have been conducted to determine physician understanding of these laws or how these laws are utilized during clinical practice. The purpose of this study is to better understand how surrogate decision-making laws function in practice. Methods: A cross-sectional survey of 412 physicians working in Indiana hospitals was conducted between November 2014 and January 2015 to determine physicians' knowledge of Indiana's surrogate decision-making law and physicians' approaches to hypothetical cases using the law in clinical practice. Results: Fewer than half of physicians (48%) were able to correctly identify all legally allowable surrogate decision makers. Of those physicians who knew the law, nearly all of them (98%) indicated that they would violate the law during clinical practice by allowing nonlegal surrogates such as grandchildren to make medical decisions. Conclusions: A majority of physicians endorse relying on surrogates who have strong ties to the patient but are not legally allowable in Indiana. It is possible that these decisions reflect sound ethical reasoning even though they are illegal. Due to the narrow construction of some state surrogate decision laws, physicians may be placed in the position where they must either choose to follow medical ethical principles or the law. To alleviate these issues, state surrogate decision laws need to be amended to include a broader list of surrogates, such as extended family and close friends