Enzymes with radical tendencies: the PFL family

The first glycyl radical in an enzyme was described 20 years ago and since then the family of glycyl radical enzymes (GREs) has expanded to include enzymes catalysing five chemically distinct reactions. The type enzymes of the family, anaerobic ribonucleotide reductase (RNRIII) and pyruvate formate lyase (PFL) had been studied long before it was known that they are GREs. Spectroscopic measurements on the radical and an observation that exposure to oxygen irreversibly inactivates the enzymes by cleavage of the protein proved that the radical is located on a particular glycine residue, close to the C-terminus of the protein. Both anaerobic RNRIII and PFL, are important for many anaerobic and facultative anaerobic bacteria as RNRIII is responsible for the synthesis of DNA precursors and PFL catalyses a key metabolic reaction in glycolysis. The crystal structures of both were solved in 1999 and they revealed that, although the enzymes do not share significant sequence identity, they share a similar structure - the radical site and residues necessary for catalysis are buried inside a ten stranded $\ualpha $/$\ubeta $-barrel. GREs are synthesised in an inactive form and are post-translationally activated by an activating enzyme which uses S-adenosyl methionine and an iron-sulphur cluster to generate the radical. One of the goals of this thesis work was to crystallise the activating enzyme of PFL. This task is challenging as, like GREs, the activating component is inactivated by oxygen. The experiments were therefore carried out in an oxygen free atmosphere. This is the first report of a crystalline GRE activating enzyme. Recently several new GREs have been characterised, all sharing sequence similarity to PFL but not to RNRIII. Also, the genome sequencing projects have identified many PFL-like GREs of unknown function, usually annotated as PFLs. In the present thesis I describe the grouping of these PFL family enzymes based on the sequence similarity and analyse the conservation patterns when compared to the structure of E. coli PFL. Based on this information an activation route is proposed. I also report a crystal structure of one of the PFL-like enzymes with unknown function, PFL2 from Archaeoglobus fulgidus. As A. fulgidus is a hyperthermophilic organism, possible mechanisms stabilising the structure are discussed. The organisation of an active site of PFL2 suggests that the enzyme may be a dehydratase. Keywords: glycyl radical, enzyme, pyruvate formate lyase, x-ray crystallography, bioinformatics

Inpatient experience of anorexia nervosa

Anorexia nervosa (AN) is an extremely serious mental illness, with a high mortality rate and many debilitating physical and psychological symptoms. While hospitalisation is sometimes required for patients with AN there remains no evidence base for “best practice’ inpatient treatment. With patients’ views recognised as critical to improving efficiency and outcomes, calls have been made for more qualitative research into inpatients’ experiences. In light of this the current paper utilised thematic analysis to examine 16 semi-structured interviews with inpatients diagnosed with AN, at a specialised eating disorders hospital unit. The study found an overarching theme of relationship ambivalence in connection with sub-themes of patients’ eating disorders, eating disorder co-patients, staff and treatment. Participants’ goals in relationship to their eating disorder and engagement in treatment shaped and were shaped by interactions with other inpatients with AN and staff. Clinical implications for this study and future research directions are discussed.

Thyroid Hormone Control of Cardiac Substrate Metabolism

Thyroid hormone (TH) plays an important role in maintaining a homeostasis in all the cells of our body. It also has significant cardiovascular effects, and abnormalities of its concentration can cause cardiovascular disease and even morbidity. Especially development of heart failure has been connected to low levels of thyroid hormone. A decrease in TH levels or TH-receptor binding adversely effects cardiac function. Although, this occurs in part through alterations in excitation-contraction and transport proteins, recent data from our laboratory indicate that TH also mediates changes in myocardial energy metabolism. Thyroid dysfunction may limit the heart s ability to shift substrate pathways and provide adequate energy supply during stress responses. Our goals of these studies were to determine substrate oxidation pattern in systemic and cardiac specific hypothyroidism at rest and at higher rates of oxygen demand. Additionally we investigated the TH mediated mechanisms in myocardial substrate selection and established the metabolic phenotype caused by a thyroid receptor dysfunction. We measured cardiac metabolism in an isolated heart model using 13Carbon isotopomer analyses with MR spectroscopy to determine function, oxygen consumption, fluxes and fractional contribution of acetyl-CoA to the citric acid cycle (CAC). Molecular pathways for changes in cardiac function and substrate shifts occurring during stress through thyroid receptor abnormalities were determined by protein analyses. Our results show that TH modifies substrate selection through nuclear-mediated and rapid posttranscriptional mechanisms. It modifies substrate selection differentially at rest and at higher rates of oxygen demand. Chronic TH deficiency depresses total CAC flux and selectively fatty acid flux, whereas acute TH supplementation decreases lactate oxidation. Insertion of a dominant negative thyroid receptor (Δ337T) alters metabolic phenotype and contractive efficiency in heart. The capability of the Δ337T heart to increase carbohydrate oxidation in response to stress seems to be limited. These studies provided a clearer understanding of the TH role in heart disease and shed light to identification of the molecular mechanisms that will facilitate in finding targets for heart failure prevention and treatment.

Evolutionary Genetics in the WIld : from Populations to Individuals

Predicting evolutionary outcomes and reconstructing past evolutionary transitions are among the main goals of evolutionary biology. Ultimately, understanding the mechanisms of evolutionary change will also provide answers to the timely question of whether and how organisms will adapt to changing environmental conditions. In this thesis, I have investigated the relative roles of natural selection, random genetic drift and genetic correlations in the evolution of complex traits at different levels of organisation from populations to individuals. I have shown that natural selection has been the driving force behind body shape divergence of marine and freshwater threespine stickleback (Gasterosteus aculeatus) populations, while genetic drift may have played a significant role in the more fine scale divergence among isolated freshwater populations. These results are concurrent with the patterns that have emerged in the published studies comparing the relative importance of natural selection and genetic drift as explanations for population divergence in different traits and taxa. I have also shown that body shape and armour divergence among threespine stickleback populations is likely to be biased by the patterns of genetic variation and covariation. Body shape and armour variation along the most likely direction of evolution the direction of maximum genetic variance reflects the general patterns of variation observed wild populations across the distribution range of the threespine stickleback. Conversely, it appears that genetic correlations between the sexes have not imposed significant constraints on the evolution of sexual dimorphism in threespine stickleback body shape and armour. I have demonstrated that the patterns of evolution seen in the wild can be experimentally recreated to tease out the effects of different selection agents in detail. In addition, I have shown how important it is to take into account the correlative nature of traits, when making interpretations about the effects of natural selection on individual traits. Overall, this thesis provides a demonstration of how considering the relative roles of different mechanism of evolutionary change at different levels of organisation can aid in an emergence of a comprehensive picture of how adaptive divergence in wild populations occurs.

Guest editorial: Urban nature for resilient and liveable cities

It is a transforming experience to imagine that in 50 years, our current built environment might look as foreign to our grandchildren as the computers of the 1960s look to us today. We can already see emerging attempts to create cities that are resilient and liveable in the face of physical stresses including population growth, increasing climate variability, resource shortages and pollution. The capacity for transforming every aspect of development towards resilience and liveability goals is profoundly exciting, from heating and cooling through to energy generation, water reticulation, food production, transportation, communication and recreational spaces...

Exploring the role of lean thinking in sustainable business practice

A large and growing body of literature has explored corporate environmental sustainability initiatives and their impacts locally, regionally and internationally. While the initiatives provide examples of environmental stewardship and cleaner production, a large proportion of the organisations considered in this literature have ‘sustainable practice’, ‘environmental stewardship’ or similar goals as add-ons to their core business strategy. Furthermore, there is limited evidence of organizations embracing and internalising sustainability principles throughout their activities, products or services. Many challenges and barriers impede outcomes as whole system design or holistic approach to address environmental issues, with some evidence to suggest that targeted initiatives could be useful in making progress. ‘Lean management’ and other lean thinking strategies are often put forward as part of such targeted approaches. Within this context, the authors have drawn on current literature to undertake a review of lean thinking practices and how these influence sustainable business practice, considering the balance of environmental and economic aspects of triple bottom line in sustainability. The review methodology comprised firstly identifying theoretical constructs to be studied, developing criteria for categorising the literature, evaluating the findings within each category and considering the implications of the findings for areas for future research. The evaluation revealed two main areas of consideration: - a) lean manufacturing tools and environmental performance, and; - b) integrated lean and green models and approaches. However the review highlighted the ad hoc use of lean thinking within corporate sustainability initiatives, and established a knowledge gap in the form of a system for being able to consider different categories of environmental impacts in different industries and choose best lean tools or models for a particular problem in a way to ensure holistic exploration. The findings included a specific typology of lean tools for different environmental impacts, drawing from multiple case studies. Within this research context, this paper presents the findings of the review; namely the emerging consensus on the relationships between lean thinking and sustainable business practice. The paper begins with an overview of the current literature regarding lean thinking and its documented role in sustainable business practice. The paper then includes an analysis of lean and green paradigms in different industries; and describes the typology of lean tools used to reduce specific environmental impacts and, integrated lean and green models and approaches. The paper intends to encourage industrial practitioners to consider the merits and potential risks with using specific lean tools to reduce context-specific environmental impacts. It also aims to highlight the potential for further investigation with regard to comparing different industries and conceptualising a generalizable system for ensuring lean thinking initiatives build towards sustainable business practice.

The Molecular Basis of Hydrolethalus Syndrome

Hydrolethalus syndrome (HLS) is a severe fetal malformation syndrome that is inherited by an autosomal recessive manner. HLS belongs to the Finnish disease heritage, an entity of rare diseases that are more prevalent in Finland than in other parts of the world. The phenotypic spectrum of the syndrome is wide and it is characterized by several developmental abnormalities, including hydrocephalus and absent midline structures in the brain, abnormal lobation of the lungs, polydactyly as well as micrognathia and other craniofacial anomalies. Polyhydramnios are relatively frequent during pregnancy. HLS can nowadays be effectively identified by ultrasound scan already at the end of the first trimester of pregnancy. One of the main goals in this study was to identify and characterize the gene defect underlying HLS. The defect was found from a previously unknown gene that was named HYLS1. Identification of the gene defect made it possible to confirm the HLS diagnosis genetically, an aspect that provides valuable information for the families in which a fetus is suspected to have HLS. Neuropathological findings of mutation confirmed HLS cases were described for the first time in detail in this study. Also, detailed general pathological findings were described. Since HYLS1 was an unknown gene with no relatives in the known gene families, many functional studies were performed in order to unravel the function of the gene and of the protein it codes for. Studies showed, for example, that the subcellular localization of the HYLS1 protein was different when the normal and the defective forms were compared. In addition, HYLS1 was shown to possess transactivation potential which was significantly diminished in the defective form. According to the results of this study it can be stated that HYLS1 most likely participates in transcriptional regulation and also in the regulation of cholesterol metabolism and that the function of HYLS1 is critical for normal fetal development.

Species-based and community-level approaches to conservation prioritization

One major reason for the global decline of biodiversity is habitat loss and fragmentation. Conservation areas can be designed to reduce biodiversity loss, but as resources are limited, conservation efforts need to be prioritized in order to achieve best possible outcomes. The field of systematic conservation planning developed as a response to opportunistic approaches to conservation that often resulted in biased representation of biological diversity. The last two decades have seen the development of increasingly sophisticated methods that account for information about biodiversity conservation goals (benefits), economical considerations (costs) and socio-political constraints. In this thesis I focus on two general topics related to systematic conservation planning. First, I address two aspects of the question about how biodiversity features should be valued. (i) I investigate the extremely important but often neglected issue of differential prioritization of species for conservation. Species prioritization can be based on various criteria, and is always goal-dependent, but can also be implemented in a scientifically more rigorous way than what is the usual practice. (ii) I introduce a novel framework for conservation prioritization, which is based on continuous benefit functions that convert increasing levels of biodiversity feature representation to increasing conservation value using the principle that more is better. Traditional target-based systematic conservation planning is a special case of this approach, in which a step function is used for the benefit function. We have further expanded the benefit function framework for area prioritization to address issues such as protected area size and habitat vulnerability. In the second part of the thesis I address the application of community level modelling strategies to conservation prioritization. One of the most serious issues in systematic conservation planning currently is not the deficiency of methodology for selection and design, but simply the lack of data. Community level modelling offers a surrogate strategy that makes conservation planning more feasible in data poor regions. We have reviewed the available community-level approaches to conservation planning. These range from simplistic classification techniques to sophisticated modelling and selection strategies. We have also developed a general and novel community level approach to conservation prioritization that significantly improves on methods that were available before. This thesis introduces further degrees of realism into conservation planning methodology. The benefit function -based conservation prioritization framework largely circumvents the problematic phase of target setting, and allowing for trade-offs between species representation provides a more flexible and hopefully more attractive approach to conservation practitioners. The community-level approach seems highly promising and should prove valuable for conservation planning especially in data poor regions. Future work should focus on integrating prioritization methods to deal with multiple aspects in combination influencing the prioritization process, and further testing and refining the community level strategies using real, large datasets.

Outer Membrane Protease/Adhesin PgtE of Salmonella enterica: Role in Salmonella-Host Interaction

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.

STEM education K-12: perspectives on integration

This commentary was stimulated by Yeping Li's first editorial (2014) citing one of the journal's goals as adding multidisciplinary perspectives to current studies of single disciplines comprising the focus of other journals. In this commentary I argue for a greater focus on STEM integration, with a more equitable representation of the four disciplines in studies purporting to advance STEM learning. The STEM acronym is often used in reference to just one of the disciplines, commonly science. Although the integration of STEM disciplines is increasingly advocated in the literature, studies that address multiple disciplines appear scant with mixed findings and inadequate directions for STEM advancement. Perspectives on how discipline integration can be achieved are varied, with reference to multidisciplinary, interdisciplinary, and transdisciplinary approaches adding to the debates. Such approaches include core concepts and skills being taught separately in each discipline but housed within a common theme; the introduction of closely linked concepts and skills from two or more disciplines with the aim of deepening understanding and skills; and the adoption of a transdisciplinary approach, where knowledge and skills from two or more disciplines are applied to real-world problems and projects with the aim of shaping the total learning experience. Research that targets STEM integration is an embryonic field with respect to advancing curriculum development and various student outcomes. For example, we still need more studies on how student learning outcomes arise not only from different forms of STEM integration but also from the particular disciplines that are being integrated. As noted in this commentary, it seems that mathematics learning benefits less than the other disciplines in programs claiming to focus on STEM integration. Factors contributing to this finding warrant more scrutiny. Likewise, learning outcomes for engineering within K-12 integrated STEM programs appear under-researched. This commentary advocates a greater focus on these two disciplines within integrated STEM education research. Drawing on recommendations from the literature, suggestions are offered for addressing the challenges of integrating multiple disciplines faced by the STEM community.

Juggling priorities: Balancing economic and social drivers to address the LLN needs of students in the VET sector

Economic success, and a commitment to the social benefits of inclusive training opportunities are important goals for public vocational education and training (VET). Currently, in Australia, VET policy is a shared responsibility between the Commonwealth and the States and Territories. Priorities for investment are juggled between: a) improving efficiency and responsiveness; and b) providing societal prosperity. Amid recent VET educational reforms and policy directives the authors of this paper undertook a pilot study examining language, literacy and numeracy support and inclusive teaching and learning practices in a Diploma of Nursing course. The data highlighted implications arising from new market driven education reforms. This article reports on identified factors that influenced inclusive learning opportunities, noticeably associated with two recent policy developments: the release of the FSK Foundation Skills Training Package (IBSA 2014); and the Queensland's Higher Skills Program Policy 2014-15.

Fuzzy waste load allocation model: a multiobjective approach

Fuzzy Waste Load Allocation Model (FWLAM), developed in an earlier study, derives the optimal fractional levels, for the base flow conditions, considering the goals of the Pollution Control Agency (PCA) and dischargers. The Modified Fuzzy Waste Load Allocation Model (MFWLAM) developed subsequently is a stochastic model and considers the moments (mean, variance and skewness) of water quality indicators, incorporating uncertainty due to randomness of input variables along with uncertainty due to imprecision. The risk of low water quality is reduced significantly by using this modified model, but inclusion of new constraints leads to a low value of acceptability level, A, interpreted as the maximized minimum satisfaction in the system. To improve this value, a new model, which is a combination Of FWLAM and MFWLAM, is presented, allowing for some violations in the constraints of MFWLAM. This combined model is a multiobjective optimization model having the objectives, maximization of acceptability level and minimization of violation of constraints. Fuzzy multiobjective programming, goal programming and fuzzy goal programming are used to find the solutions. For the optimization model, Probabilistic Global Search Lausanne (PGSL) is used as a nonlinear optimization tool. The methodology is applied to a case study of the Tunga-Bhadra river system in south India. The model results in a compromised solution of a higher value of acceptability level as compared to MFWLAM, with a satisfactory value of risk. Thus the goal of risk minimization is achieved with a comparatively better value of acceptability level.

Assessment of motivation to control alcohol use: The motivational thought frequency and state motivation scales for alcohol control

Introduction The Elaborated Intrusion Theory of Desire holds that desires for functional and dysfunctional goals share a common form. Both are embodied cognitive events, characterised by affective intensity and frequency. Accordingly, we developed scales to measure motivational cognitions for functional goals (Motivational Thought Frequency, MTF; State Motivation, SM), based on the existing Craving Experience Questionnaire (CEQ). When applied to increasing exercise, MTF and SM showed the same three-factor structure as the CEQ (Intensity, Imagery, Availability). The current study tested the internal structure and concurrent validity of the MTF and SM Scales when applied to control of alcohol consumption (MTF-A; SM-A). Methods Participants (N = 417) were adult tertiary students, staff or community members who had recently engaged in high-risk drinking or were currently trying to control alcohol consumption. They completed an online survey comprising the MTF-A, SM-A, Alcohol Use Disorders Identification Test (AUDIT), Readiness to Change Questionnaire (RCQ) and demographics. Results Confirmatory Factor Analysis gave acceptable fit for the MTF-A, but required the loss of one SM-A item, and was improved by intercorrelations of error terms. Higher scores were associated with more severe problems on the AUDIT and with higher Contemplation and Action scores on the RCQ. Conclusions The MTF-A and SM-A show potential as measures of motivation to control drinking. Future research will examine their predictive validity and sensitivity to change. The scales' application to both increasing functional and decreasing dysfunctional behaviours is consistent with EI Theory's contention that both goal types operate in similar ways.

A preliminary analysis of the impact of UN MDGs and RIO+20 on corporate social accountability practices

This chapter explores the impact of UN Millennium Development Goals (MDGs) and Rio + 20 in improving Corporate Social Responsibility (CSR) practices. While MDGs and Rio + 20 have suggested additive guidelines for improving CSR practices, they do not provide a strong legislative mandate. We find both MDGs and Rio + 20 have had limited cumulative effect on CSR practices and discourses within the corporate reports. UN bodies should bring a new policy and regulatory framework that addresses limitations in the principles espoused in the MDGs and Rio + 20. An independent monitoring system (a social compliance audit mechanism) can be mandated in an attempt to make incremental substantive change.

Business busts and U.S. auditors' interest in fraud detection: Evidence from the 20th century

We find evidence that U.S. auditors increased their attention to fraud detection during or immediately after the economic contractions of the 20th century, based on a content analysis of the 12 volumes of the 20th-century auditing reference series Montgomery’s Auditing. Contractions, however, do not seem to have affected auditors’ attention to the formal goal of fraud detection. The study suggests that auditors’ aversion to the heightened risks of fraud during economic downturns leads them to focus more on fraud detection at those times regardless of the particular guidance in formal audit standards. This study is the first to find some evidence of a recession-influenced difference between fraud detection practices and formal fraud detection goals.