Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

Mathematical modelling of chronic wound healing

Chronicwounds fail to proceed through an orderly process to produce anatomic and functional integrity and are a significant socioeconomic problem. There is much debate about the best way to treat these wounds. In this thesis we review earlier mathematical models of angiogenesis and wound healing. Many of these models assume a chemotactic response of endothelial cells, the primary cell type involved in angiogenesis. Modelling this chemotactic response leads to a system of advection-dominated partial differential equations and we review numerical methods to solve these equations and argue that the finite volume method with flux limiting is best-suited to these problems. One treatment of chronic wounds that is shrouded with controversy is hyperbaric oxygen therapy (HBOT). There is currently no conclusive data showing that HBOT can assist chronic wound healing, but there has been some clinical success. In this thesis we use several mathematical models of wound healing to investigate the use of hyperbaric oxygen therapy to assist the healing process - a novel threespecies model and a more complex six-species model. The second model accounts formore of the biological phenomena but does not lend itself tomathematical analysis. Bothmodels are then used tomake predictions about the efficacy of hyperbaric oxygen therapy and the optimal treatment protocol. Based on our modelling, we are able to make several predictions including that intermittent HBOT will assist chronic wound healing while normobaric oxygen is ineffective in treating such wounds, treatment should continue until healing is complete and finding the right protocol for an individual patient is crucial if HBOT is to be effective. Analysis of the models allows us to derive constraints for the range of HBOT protocols that will stimulate healing, which enables us to predict which patients are more likely to have a positive response to HBOT and thus has the potential to assist in improving both the success rate and thus the cost-effectiveness of this therapy.

Ocklawaha River restoration : the fate of the Rodman Reservoir

The Rodman Reservoir, an impoundment on the Ocklawaha River in north central Florida, is a last remnant of the Cross-Florida Barge Canal (CFBC). The canal, conceived in the 1820's, was designed by the U.S. Army Corps of Engineers (USACE) to shorten shipping lanes between the Fulf ports and the Atlantic coast. Opposition to CFBC by Florida's young environmental movement led to a half in construction of the CFBC in 1971, but decommissioning of the already-constructed Rodman dam and the reservoir behind it has been mired in controversy every since.

Georg Calixtus and the Humanist tradition

Georg Calixtus (1586–1656) was a Lutheran theologian, prominent in the German lands during the first half of the seventeenth century. Existing research focuses on Calixtus‘ contributions to religious and theological debates, particularly in regard to his role in the Syncretistic Controversy of the latter half of the seventeenth century, and in regard to his unique position as a Lutheran who aspired to reunion between the different Christian confessions. This thesis problematises this focus on Calixtus by theologians and ecclesiastical historians, and argues that the genesis and transmission of his ideas cannot be fully appreciated without considering his relationship with the broader intellectual milieu of early modern Europe. It does this by exploring Calixtus‘ interaction with the humanist tradition, in particular by reconsidering his relationship with Isaac Casaubon (1559–1614), and by exploring his work in light of intellectual movements that were taking place outside the Christian church. In so doing, this thesis argues that Calixtus made contributions to early modern thought that have been overlooked in the existing literature. It also becomes apparent that much research remains to be done to gain a more accurate picture of his place in the early modern intellectual landscape, and of his legacy to later generations of scholars.

Electron diffusion and back reaction in dye-sensitized solar cells : the effect of nonlinear recombination kinetics

The electron collection efficiency in dye-sensitized solar cells (DSCs) is usually related to the electron diffusion length, L = (Dτ)^1/2, where D is the diffusion coefficient of mobile electrons and τ is their lifetime, which is determined by electron transfer to the redox electrolyte. Analysis of incident photon-to-current efficiency (IPCE) spectra for front and rear illumination consistently gives smaller values of L than those derived from small amplitude methods. We show that the IPCE analysis is incorrect if recombination is not first-order in free electron concentration, and we demonstrate that the intensity dependence of the apparent L derived by first-order analysis of IPCE measurements and the voltage dependence of L derived from perturbation experiments can be fitted using the same reaction order, γ ≈ 0.8. The new analysis presented in this letter resolves the controversy over why L values derived from small amplitude methods are larger than those obtained from IPCE data.

Direct instruction in mathematics : issues for schools with high Indigenous enrolments : a literature review

Direct instruction, an approach that is becoming familiar to Queensland schools that have high Aboriginal and Torres Strait Islander populations, has been gaining substantial political and popular support in the United States of America [USA], England and Australia. Recent examples include the No Child Left Behind policy in the USA, the British National Numeracy Strategy and in Australia, Effective Third Wave Intervention Strategies. Direct instruction, stems directly from the model created in the 1960s under a Project Follow Through grant. It has been defined as a comprehensive system of education involving all aspects of instruction. Now in its third decade of influencing curriculum, instruction and research, direct instruction is also into its third decade of controversy because of its focus on explicit and highly directed instruction for learning. Characteristics of direct instruction are critiqued and discussed to identify implications for teaching and learning for Indigenous students.

Included by design : a case for regulation for accessible housing in Australia

Accessible housing is a scarce yet much needed commodity in Australia. A national agreement between industry and advocacy groups to a voluntary approach, called the Livable Design program, aims to provide access features in all new housing by 2020. Through a range of awareness raising initiatives, the program is anticipating increased supply by builders and increased demand by home-buyers. However the people who need accessible housing are the least likely and least able to buy it at the point of new sale and average homebuyers do not consider access features as a priority. This approach has not been successful overseas or in Australia in the past. Regulation with incentives supported by education and awareness has provided the best results, yet, regulation typically comes with controversy and resistance from the housing industry. A study is planned to identify how effective the Livable Design program is likely to be, what is likely to hinder it and why regulation is likely to be needed.

Functional heterogeneity as a two-dimensional concept : empirical evidence for new venture teams

Previous research on entrepreneurial teams has failed to settle the controversy over whether team heterogeneity helps or hinders new venture performance. Reconciling this inconsistency, this paper suggests a new conceptual approach to disentangle differential effects of team heterogeneity by modeling two separate heterogeneity dimensions, namely knowledge scope and knowledge disparity. Analyzing unique data on functional experiences of the members of 337 start-up teams, we find support for our contention of team heterogeneity as a two-dimensional concept. Results suggest that knowledge disparity negatively relates to both start-ups’ entrepreneurial and innovative performance. In contrast, we find knowledge scope to positively affect entrepreneurial performance, while it shows an inverse U-shaped relationship to innovative start-up performance.

Towards distributed citizen participation : lessons from WikiLeaks and the Queensland Floods

This paper examines the rapid and ad hoc development and interactions of participative citizen communities during acute events, using the examples of the 2011 floods in Queensland, Australia, and the global controversy surrounding Wikileaks and its spokesman, Julian Assange. The self-organising community responses to such events which can be observed in these cases bypass or leapfrog, at least temporarily, most organisational or administrative hurdles which may otherwise frustrate the establishment of online communities; they fast-track the processes of community development and structuration. By understanding them as a form of rapid prototyping, e-democracy initiatives can draw important lessons from observing the community activities around such acute events.

The use of Twitter hashtags in the formation of ad hoc publics

As the use of Twitter has become more commonplace throughout many nations, its role in political discussion has also increased. This has been evident in contexts ranging from general political discussion through local, state, and national elections (such as in the 2010 Australian elections) to protests and other activist mobilisation (for example in the current uprisings in Tunisia, Egypt, and Yemen, as well as in the controversy around Wikileaks). Research into the use of Twitter in such political contexts has also developed rapidly, aided by substantial advancements in quantitative and qualitative methodologies for capturing, processing, analysing, and visualising Twitter updates by large groups of users. Recent work has especially highlighted the role of the Twitter hashtag – a short keyword, prefixed with the hash symbol ‘#’ – as a means of coordinating a distributed discussion between more or less large groups of users, who do not need to be connected through existing ‘follower’ networks. Twitter hashtags – such as ‘#ausvotes’ for the 2010 Australian elections, ‘#londonriots’ for the coordination of information and political debates around the recent unrest in London, or ‘#wikileaks’ for the controversy around Wikileaks thus aid the formation of ad hoc publics around specific themes and topics. They emerge from within the Twitter community – sometimes as a result of pre-planning or quickly reached consensus, sometimes through protracted debate about what the appropriate hashtag for an event or topic should be (which may also lead to the formation of competing publics using different hashtags). Drawing on innovative methodologies for the study of Twitter content, this paper examines the use of hashtags in political debate in the context of a number of major case studies.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Comparing cost uplift in infrastructure delivery methods : a case based approach

In 2009 the Australian Federal and State governments are expected to have spent some AU$30 billion procuring infrastructure projects. For governments with finite resources but many competing projects, formal capital rationing is achieved through use of Business Cases. These Business cases articulate the merits of investing in particular projects along with the estimated costs and risks of each project. Despite the sheer size and impact of infrastructure projects, there is very little research in Australia, or internationally, on the performance of these projects against Business Case assumptions when the decision to invest is made. If such assumptions (particularly cost assumptions) are not met, then there is serious potential for the misallocation of Australia’s finite financial resources. This research addresses this important gap in the literature by using combined quantitative and qualitative research methods, to examine the actual performance of 14 major Australian government infrastructure projects. The research findings are controversial as they challenge widely held perceptions of the effectiveness of certain infrastructure delivery practices. Despite this controversy, the research has had a significant impact on the field and has been described as ‘outstanding’ and ‘definitive’ (Alliancing Association of Australasia), "one of the first of its kind" (Infrastructure Partnerships of Australia) and "making a critical difference to infrastructure procurement" (Victorian Department of Treasury). The implications for practice of the research have been profound and included the withdrawal by Government of various infrastructure procurement guidelines, the formulation of new infrastructure policies by several state governments and the preparation of new infrastructure guidelines that substantially reflect the research findings. Building on the practical research, a more rigorous academic investigation focussed on the comparative cost uplift of various project delivery strategies was submitted to Australia’s premier academic management conference, the Australian and New Zealand Academy of Management (ANZAM) Annual Conference. This paper has been accepted for the 2010 ANZAM National Conference following a process of double blind peer review with reviewers rating the paper’s overall contribution as "Excellent" and "Good".

Connecting Diversity : Paradoxes of Multicultural Australia

Commissioned by SBS, and published in March 2006, Connecting Diversity: Paradoxes of Multicultural Australia is a follow-up study to SBS’s 2002 report, Living Diversity: Australia’s Multicultural Future. The attitudes of many younger Australians from culturally diverse backgrounds reveal paradoxes about Australian multiculturalism today. This report sheds light on their views, experiences and expectations and the role of media in their lives. Younger, culturally and linguistically diverse Australians are often the subject of mediafanned controversy about disaffection, ‘ethnic gangs’ and cultural isolation. While these controversies tend to be localised – Cronulla, Inala or Bankstown – Connecting Diversity tells a national and quite different story. This research builds upon the findings of the 2002 report, Living Diversity: Australia’s Multicultural Future, which challenged common assumptions about contemporary multicultural Australia. In an era of fragmenting media and assumed political apathy, Connecting Diversity further examines many of the findings of the earlier study, with a new focus on younger people, cultural identity and media use. Connecting Diversity reveals individual experiences and often contradictory ideas about media and diversity in Australia. Disjunctions appear to exist between an individual’s experience and their thoughts about Australia’s national identity. Multiculturalism is valued for broadening the appreciation of difference, yet this support can coexist with concerns about perceived segregation, usually ‘elsewhere’ in Australia. Younger people tend to be more comfortable with cultural difference than previous generations and cite their own diverse network of friends as one of the reasons for this. Even so, some describe experiences of racism that engender a feeling of exclusion from ‘mainstream’ society. In their everyday lives, social relationships are navigated through regular and familiar connections on the one hand, and experiences and expressions of disconnection on the other. Racism and tolerance may be expressed almost simultaneously. These disconnections are often managed through ‘practical tolerance',allowing them to negotiate these apparent contradictions. The connections can be based simultaneously on such things as work, family,religion, friendships or location. The result is a multilayered sense of personal belonging and community connection. A large number of respondents in these focus groups expressed frustration at the failings of media, especially news and current affairs coverage, yet spoke enthusiastically about the accessibility and range of media compared to what was available to previous generations. In their many forms, media remain a key ingredient of self-identification among younger Australians of culturally diverse backgrounds who are especially cynical about media and disillusioned by their perceived inability to influence issues that are important to them. These findings reveal that although they may be cynical about media messages, these younger Australians are looking for connection through media and are seeking ways to participate in meaningful ways. This raises questions about the possibilities for media to empower younger people to play a part in genuine cultural democracy. By capturing the attitudes of Australians of culturally diverse backgrounds under the age of 40, Connecting Diversity: Paradoxes of Multicultural Australia provides an insight into social trends and the generational and cultural changes that are now shaping Australia.

China and the "Responsibility to Protect" (R2P) : the implications of the Libyan intervention

The emerging principle of a “responsibility to protect” (R2P) presents a direct challenge to China's traditional emphasis on the twin principles of non-intervention in the domestic affairs of other states and non-use of military force. This paper considers the evolution of China‟s relationship with R2P over the past ten years. In particular, it examines how China engaged with R2P during the recent Libyan crisis, and considers what impact this conflict may have first, on Chinese attitudes to R2P, and second, on the future development and implementation of the doctrine itself. This paper argues that China‟s decision to allow the passage of Security Council resolution 1973, authorising force in Libya, was shaped by an unusual set of pragmatic considerations, and should not be viewed as evidence of a dramatic shift in Chinese attitudes towards R2P. More broadly, controversy over the scope of NATO's military action in Libya has raised questions about R2P‟s legitimacy, which has contributed to a lack of timely international action in Syria and Yemen. In the short term at least, this post-Libya backlash against R2P is likely to constrain the Security Council‟s ability to respond decisively to other civilian protection situations.

Genetic evidence for the vital function of osterix in cementogenesis

To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of OSX and the formation of cellular cementum. We then generated 3.6 Col 1-OSX transgenic mice, which displayed accelerated cementum formation vs. WT controls. Importantly, the conditional deletion of OSX in the mesenchymal cells with two different Cre systems (the 2.3 kb Col 1 and an inducible CAG-CreER) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the OSX gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered SEM and resin-cast SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support 1) the mesenchymal origin of cellular cementum (from PDL progenitor cells); 2) the vital role of OSX in controlling the formation of cellular cementum; and 3) the limited remodeling of cellular cementum in adult mice.