Pathogenicity of nectriaceous fungi on avocado in Australia

Black root rot is a severe disease of young avocado trees in Australia causing black necrotic roots, tree stunting, and leaf drop prior to tree death. Nectriaceous fungi (Nectriaceae, Hypocreales), are commonly isolated from symptomatic roots. This research tested the pathogenicity of 19 isolates from Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis, and Ilyonectria, spp. collected from young avocado trees and other hosts. Glasshouse pathogenicity tests with ‘Reed’ avocado (Persea americana) seedlings confirmed that Calonectria ilicicola is a severe pathogen of avocado, causing stunting, wilting, and seedling death within 5 weeks of inoculation. Isolates of C. ilicicola from peanut, papaya, and custard apple were also shown to be aggressive pathogens of avocado, demonstrating a broad host range. An isolate of a Calonectria sp. from blueberry and avocado isolates of Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata, and D. anthuriicola caused significant root rot but not stunting within 5 to 9 weeks of inoculation. An isolate of an Ilyonectria sp. from grapevine closely related to Ilyonectria liriodendri, and avocado isolates of Cylindrocladiella pseudoinfestans, Gliocladiopsis peggii, and an Ilyonectria sp. were not pathogenic to avocado

Deoxynyboquinones as NQO1-targeted anticancer compounds and deoxynybomycins as potent and selective antibiotics

Cancer and antibiotic-resistant bacterial infections are currently two of the major health concerns facing the United States. Novel therapeutics capable of specifically targeting either cancer or resistant bacteria are greatly needed. Described herein are three separate efforts to address these needs. Described in Chapter 2 is the development of a targeted anticancer agent deoxynyboquinone (DNQ) which is specifically activated by the enzyme NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 is a 2-electron reductase that is known to be overexpressed in many solid tumors. Development of an anticancer quinone that is bioactivated by NQO1 has long been a goal of cancer therapy. Previously, several putative NQO1 substrates have been developed including mitomycin C, RH1, streptonigrin, and β-Lapachone (β-Lap). Recently the Hergenrother laboratory discovered the small molecule DNQ which has potent anticancer activity. Due to its quinone moiety and the fact that it causes reactive oxygen species (ROS) dependent cell death, we hypothesized that its activity was due to activation by NQO1. Described herein is a set of assays that was developed to determine the NQO1-dependence of anticancer compounds. Of the putative NQO1 substrates, only β-Lap and DNQ were found to be selectively activated by NQO1. Due to its excellent potency and pharmacokinetic profile, DNQ was explored further. Mechanistic evaluation of DNQ revealed that after reduction by NQO1, DNQ undergoes reduction-oxidation cycling which concurrently results in the formation of ROS. ROS causes extensive DNA damage that then activates poly(ADP-ribose) polymerase-1 dependent cell death. DNQ was found to be efficacious a murine model of lung cancer. Utilizing a modified version of the DNQ synthesis previously developed by the Hergenrother laboratory, derivatives were synthesized and evaluated. Several were found that have potent activity against a panel of breast and lung cancers along with improved solubility and toxicity profiles compared to DNQ. These derivatives are currently under investigation for in vivo activity. Described in Chapter 3 is the development of deoxynybomycin (DNM) as an antibiotic with potent activity against fluoroquinolone-resistant (FQR) bacteria. DNM is a natural product that has been shown previously to have antibiotic activity. Recently DNM was found to have potent activity against FQR Methicillin-resistant S. aureus (MRSA). This activity is due to the ability of DNM to inhibit the mutant DNA gyrase (specifically S84L gyrA) responsible for FQR. At the start of the studies described here, two main challenges to the further development of DNM existed: 1) Difficulty in attaining significant quantities of pure DNM for biological evaluation and 2) The poor solubility of DNM. The first issue was addressed by the development of a synthesis of DNM. A single reaction from a late stage intermediate of the DNQ synthesis allowed for the generation of DNM. The modular nature of the synthesis also allowed for the synthesis of a variety of derivatives some of which showed similar potency against FQR MRSA and greatly improved solubility. DNM and its derivative DNM-2 were tested against a variety of bacterial species to determine the activity profile for this class of compounds. The best activity was observed for FQR MRSA with S84L mutant of DNA gyrase and FQR VRE with S84I mutation. Less potent activity was observed for bacteria that commonly have other mutations such as S84F or S84Y. In vitro inhibition assays suggest that DNM is less potent against DNA gyrase with these mutations, but further studies need to be performed to confirm this. Additionally, DNM is inactive against Gram-negative bacteria likely due to its inability to traverse the outer membrane. Further studies to identify compounds active against Gram-negative bacteria are ongoing. Resistance to DNM was found to occur via regeneration of the WT DNA gyrase, thus re-sensitizing bacteria to FQs. This resistance cycling suggests that bacteria which develop resistance to DNM would be treatable. After determining that DNM and DNM-2 have good potency against FQR MRSA, studies evaluating their in vivo activity were performed. Initial pharmacokinetic analysis revealed that oral administration of DNM is not a useful administration route likely due to its poor solubility. However, DNM-2 has excellent oral absorption with area under the curve values which predict good in vivo efficacy. DNM-2 was used in further studies. Toxicity studies revealed no significant effects of DNM-2 on mice when treated at 50 mg/kg for ten consecutive days. Excitingly, DNM-2 was the first compound in the deoxynybomycin class to show in vivo activity, saving mice with FQR MRSA sepsis. Described in Chapter 4 is the analysis of the anticancer compound ersindole, an actiniophyllic acid analogue synthesized by the Martin laboratory. The anticancer activity of ersindole was discovered by the Hergenrother laboratory via a high throughput screen for compounds which induce breast cancer cell death. One of the most striking features of ersindole-induced cancer cell death is the shape of the dose response curve. Specifically, it has a very steep Hill slope and high Emax. These attributes reflect consistent and efficient induction of cancer cell death and suggest that ersindole is a very promising anticancer drug. Analysis of multiple cell lines and timepoints reveal that the steep Hill slope and high Emax of the ersindole dose response curve are general attributes of the compound. Previous mechanistic studies with ersindole suggested that it induced cancer cell death via induction of endoplasmic reticulum stress. This was further confirmed here via Western blot analysis and siRNA knockdown studies. Future efforts should focus on determining the molecular target of ersindole. Unfortunately, ersindole was found to induce hemolysis of red blood cells. A set of derivatives was investigated in an effort to find compounds that do not lyse red blood cells. Ersindole-9 was found to be nearly as potent as ersindole against a panel of cancer cell lines and to have a similarly shaped dose-response curve. Gratifyingly, ersindole-9 does not induce significant hemolysis. For this reason, ersindole-9 was studied in a murine model of breast cancer where it was found to have good efficacy. Evaluation of a second set of derivatives was then performed in order to find additional derivatives that are potent and do not induce hemolysis. Several leads were discovered. Further analysis of these compounds is needed to determine the best compound for future evaluation

In-situ aldehyde-modification of self-assembled acyl hydrazide hydrogels and dynamic component selection from complex aldehyde mixtures

Self-assembled hydrogels based on the industrially-relevant 1,3:2,4-dibenzylidene sorbitol framework functionalised with reactive acyl hydrazides (DBS-CONHNH2) peripheral groups react with aldehydes without disrupting the nanoscale gel network, adapting gel performance, and dynamically selecting specific aldehyde components from complex mixtures

Negotiating Health and Life: Syrian Refugees and The Politics of Access in Lebanon.

In the context of ongoing armed conflicts in Libya, Syria, Yemen, and Iraq, it is vital to foster nuanced understandings of the relationship between health, violence, and everyday life in the Middle East and North Africa. In this article, we explore how healthcare access interacts with humanitarian bureaucracy and refugees' daily experiences of exile. What are the stakes involved with accessing clinical services in humanitarian situations? How do local conditions structure access to healthcare? Building on the concept of “therapeutic geographies,” we argue for the integration of local socio-political context and situated knowledge into understandings of humanitarian healthcare systems. Using evidence gathered from participant observation among Syrian and Palestinian refugees in Lebanon, we demonstrate how procedures developed to facilitate care—such as refugee registration and insurance contracting—can interact with other factors to simultaneously prevent and/or disincentivize refugees' accessing healthcare services and expose them to structural violence. Drawing on two interconnected ethnographic encounters in a Palestinian refugee camp and in a Lebanese public hospital, we demonstrate how interactions surrounding the clinical encounter reveal the social, political, and logistical complexities of healthcare access. Moreover, rather than hospital visits representing discrete encounters with the Lebanese state, we contend that they reveal important moments in an ongoing process of negotiation and navigation within and through the constraints and uncertainties that shape refugee life. As a result, we advocate for the incorporation of situated forms of knowledge into humanitarian healthcare practices and the development of an understanding of healthcare access as nested in the larger experience of everyday refugee life

Clinical outcomes and cost-effectiveness of brief guided parent-delivered cognitive behavioural therapy and solution-focused brief therapy for treatment of childhood anxiety disorders: a randomised controlled trial

Background—Half of lifetime anxiety disorders emerge before 12 years of age, however access to evidence-based psychological therapies for affected children is poor. This Randomised Controlled Trial (RCT) compared the clinical outcome and cost-effectiveness of two brief psychological treatments for anxious children referred to routine child mental health settings. Methods—Children (5-12 years) referred to Primary Child and Mental Health Services across Oxfordshire, UK, for anxiety difficulties were randomly allocated (1:1) to brief Guided Parent-Delivered Cognitive Behavior Therapy (GPD-CBT) or Solution Focused Brief Therapy (SFBT). The primary outcome was Clinical Global Impressions of Improvement (CGI-I). Secondary outcomes were absence of primary anxiety diagnosis and all anxiety disorder diagnoses, self- and parent-reported anxiety symptoms and interference. Parents recorded patient level resource use. Quality Adjusted Life Years (QALYs) were derived from the CHU9D. Assessments were conducted pre-, post- (primary endpoint), and 6- months after treatment. Findings—136 patients were assigned to GPD-CBT (n=68) or SFBT (n=68). Analyses were conducted with the intent to treat population. No significant differences were observed on any clinical (CGI-I; Relative Risk (RR) = 1·01 (0·86, 1·19), p = 0·95) or economic (QALY mean difference = 0·006 (-0·009- 0·02), p = 0·42) outcome measure. However, the GPD-CBT treatment was associated with lower costs (mean difference: -£448; 95% CI: -£934, £37; p=0.070). Interpretation— There was no evidence of clinical superiority, however brief GPD-CBT is likely to be a cost-effective alternative to brief psychological treatment (SFBT) and could be considered as a first-line treatment for children with anxiety problems

Exploring How Australia's National Curriculum Supports the Aspirations of Aboriginal People

A culturally inclusive curriculum has long been considered beneficial to all students. The national Australian Curriculum set out to be so, including Aboriginal and Torres Strait Islander histories and cultures as a cross-curriculum priority. There is an assumption however that inclusion is an unproblematic good, and is a true representation of the 'reality' of Aboriginal and Torres Strait Islander Peoples' lived experiences and aspirations. Drawing on a Critical Discourse Analysis of the Australian Curriculum policy corpus and key informant interviews with members of an Aboriginal community, this dissertation explores how the aspirations of Aboriginal people are supported in dominant education discourses mobilised within the Australian Curriculum. The study identified a critical gap between the Australian Curriculum's positioning of Aboriginal knowledges, histories and cultures and the Aboriginal community's aspirations for their children's education. Within the Australian Curriculum policy corpus, 'Liberal Multicultural' and 'Inclusive' Discourses were dominant. Such discourses framed Aboriginal students as being vulnerable to marginalisation and in need of support to ensure equality in education. In contrast, community informants advocated for more critical discourses whereby Aboriginal students are seen as empowered, able to actively participate in mainstream society to engage in a process of community revitalisation. In drawing upon different and at times contradictory discourses to articulate their aspirations within a broader 'Community Revitalisation' Discourse, community members engaged in a creative act of bricolage in a highly contextually-dependent way

Risk factors for eating disorder symptoms at 12 years of age: A 6-year longitudinal cohort study

Eating disorders pose risks to health and wellbeing in young adolescents, but prospective studies of risk factors are scarce and this has impeded prevention efforts. This longitudinal study aimed to examine risk factors for eating disorder symptoms in a population-based birth cohort of young adolescents at 12 years. Participants from the Gateshead Millennium Study birth cohort (n = 516; 262 girls and 254 boys) completed self-report questionnaire measures of eating disorder symptoms and putative risk factors at age 7 years, 9 years and 12 years, including dietary restraint, depressive symptoms and body dissatisfaction. Body mass index (BMI) was also measured at each age. Within-time correlates of eating disorder symptoms at 12 years of age were greater body dissatisfaction for both sexes and, for girls only, higher depressive symptoms. For both sexes, higher eating disorder symptoms at 9 years old significantly predicted higher eating disorder symptoms at 12 years old. Dietary restraint at 7 years old predicted boys' eating disorder symptoms at age 12, but not girls'. Factors that did not predict eating disorder symptoms at 12 years of age were BMI (any age), girls’ dietary restraint at 7 years and body dissatisfaction at 7 and 9 years of age for both sexes. In this population-based study, different patterns of predictors and correlates of eating disorder symptoms were found for girls and boys. Body dissatisfaction, a purported risk factor for eating disorder symptoms in young adolescents, developed concurrently with eating disorder symptoms rather than preceding them. However, restraint at age 7 and eating disorder symptoms at age 9 years did predict 12-year eating disorder symptoms. Overall, our findings suggest that efforts to prevent disordered eating might beneficially focus on preadolescent populations

Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies