Expression and function of osteopontin variants in HCV-related liver disease and hepatocellular carcinoma.

Osteopontin (OPN) is a highly secreted multi-functional sialoprotein that is widely expressed in tissues, blood and urine. It is involved in a number of normal physiological functions, but is also significantly elevated in a number of cancers. While OPN is significantly expressed in hepatocellular carcinoma (HCC) little is known as to its role and if it is expressed in the pre-cancerous hepatitis C virus (HCV) infected liver. In this thesis we show that OPN is expressed in the liver and in HCC as three variants, the full-length protein OPN-A and two splice variants OPN-B and OPN-C. Through production of stable Huh-7 cells expressing the OPN variants, we show for the first time that all variants increase proliferation of a range of cultured hepatoma cell lines in a paracrine manner through interactions with the cell surface OPN receptor CD44. Similarly, OPN-A (and to a lesser extent OPN-B and –C) accelerated Huh-7 derived tumor growth in a nude mouse model. We also show for the first time expression of all three OPN variants in the non-diseased liver as it was previously thought that splicing was a feature specific for tumor cells. Clinically, OPN is known to be highly expressed in HCC, however, its expression in chronic hepatitis C is not well documented. In this thesis we show that OPN mRNA expression is elevated in the HCV-infected liver with a trend towards increased expression as liver disease progresses. Consistent with an increase in mRNA, serum OPN levels were also increased in the HCV-infected liver although we could find no correlation with degree of liver disease. However, our sample size was small and this section of the thesis needs repeating with a larger HCV-infected patient cohort. Furthermore, we show that elevated OPN expression is not specific to the HCV-infected liver as OPN is also elevated in the HBV-infected and alcoholic liver suggesting that HCV does not drive OPN expression but is more likely as a result of the inflammatory process in the viral infected liver. Interestingly we also show that there is a shift of OPN expression from bile duct epithelial cells in the non-diseased liver to the hepatocyte in the HCV-infected liver which raises the question as to the role of OPN in hepatocyte transformation to facilitate the development of HCC. Our evaluation of serum OPN expression also suggests that OPN has potential as both a diagnostic and potentially prognostic biomarker for not only HCC (arising from HBV and HCV infections and alcohol abuse) but also the earlier stages of HCV-related liver disease. This work for the first time characterises the expression of all OPN variants in the liver including HCC and may be useful for identifying targeted OPN-based therapeutic approaches for HCC and other cancers. Furthermore it also suggests that monitoring OPN in chronic hepatitis C may be useful in monitoring liver disease progression and early detection of HCC.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Distinct DNA Damage Signaling in the Brain Distinguishes ATLD, NBS, and ATR-Seckel Syndrome

DNA double strand breaks create a situation of extreme stress under which a cell must either be capable of repairing the lesions in order to continue replication or succumb to death. Not surprisingly, deficiencies in DNA repair genes often lead to human diseases frequently associated with genomic instability, cancer proneness, and neuropathology. Neurological consequences of aberrant DNA repair mechanisms vary depending upon the affected gene and the pathway in which it operates. Ataxia-telangiectasia (A-T) is the prototypical disease associated with DNA double strand break (DSB) repair deficiency and is characterized by severe neural pathology. A-T results from homozygous mutations that inactivate the ataxia-telangiectasia mutated (ATM) gene, and typically presents in early childhood as cerebellar ataxia accompanied by ocular telangiectasias, elevated α-fetoprotein levels, and lymphoma predisposition compounded by radiosensitivity. The ATM protein is a large (~380 kDa) protein kinase within the PI3K family, which mediates cell cycle checkpoint activity and induces apoptosis through phosphorylation of copious substrates. It is thought to be through apoptotic induction of newly post-mitotic cells harboring lethal levels of DNA damage that Atm maintains integrity of the CNS, as it prevents incorporation of genomically compromised neurons into the mature nervous system. Hypomorphic mutations in the Mre11/Rad50/NBS1 complex (MRN complex), which is required for efficient ATM activity, result in distinct syndromes. Ataxia-telangiectasia-like disease (ATLD) results from truncating mutations in Mre11 and presents with neurodegeneration similar to that observed in A-T, but with longer latency and less severity. Mutations in the FHA and BRCT domains of NBS1 result in Nijmegen Breakage Syndrome (NBS), which is characterized by microcephaly without evidence of neurodegeneration. Thus far mutations in Rad50 have not been reported in humans. In order to better understand the roles of ATM and MRN signaling in maintaining physical and functional integrity of the CNS, mutant mice either null for Atm or carrying hypomorphic mutations in Mre11 or Nbs1 were subjected to either endogenous or exogenous forms of DNA damage and the signaling response was assessed in the brain. To understand the contrasting neuropathology resulting from Mre11 or Nbs1 hypomorphic mutations, we analyzed neural tissue from Mre11ATLD1/ATLD1 and Nbs1DB/DB mice after genotoxic stress. We found a pronounced resistance to DNA damage-induced apoptosis after ionizing radiation or DNA ligase IV (Lig4) loss in the Mre11ATLD1/ATLD1 nervous system that was associated with defective Atm activation and phosphorylation of its substrate p53. Apoptosis occurred normally in the Nbs1ΔB/ΔB brain. We also conditionally disrupted Lig4 throughout the nervous system using Nestin-cre (Lig4Nes-Cre), and while viable, these mice showed pronounced microcephaly and a prominent age-related accumulation of DNA damage throughout the brain. Either Atm-/- or Mre11ATLD1/ATLD1 genetic backgrounds, but not Nbs1ΔB/ΔB rescued Lig4Nes-Cre microcephaly. Thus, DNA damage signaling in the nervous system is different between ATLD and NBS, and likely explains their respective neuropathology. Ataxia-telangiectasia and Rad3-related (ATR) is a large serine/threonine kinase that is highly similar in structure to ATM and phosphorylates many overlapping substrates. Hypomorphic mutations in ATR have been associated with some cases of Seckel syndrome, a microcephalic condition. Despite the similarities between ATM and ATR, inactivation of either kinase has quite distinct consequences. Unlike ATM, ATR is essential for embryonic development, as Atr-/- embryos are lethal by E7.5. To better understand the differing roles of ATM and ATR in the nervous system, a conditional Atr mutant was crossed onto the Nestin-cre transgenic line. AtrloxP/loxP; Nestin-cre ( AtrNes-cre) animals displayed early post-natal lethality and severe neurogenesis defects, particularly striking in the cerebellum due to decreased proliferation and viability of granule neuron progenitors. Surprisingly, progenitors in the rhombic lip were largely unaffected in AtrNes-cre embryos whereas the granule cell precursors of the cerebellar external germinal layer (EGL) displayed DNA damage as early as E13.5 and p53-dependant apoptosis by E15.5. Between E15.5 and E16.5 p53 independent cell cycle arrest was inferred by decreased proliferation of the remaining granule cell precursors in the EGL. DNA damage was not observed in the rhombic lip until E16.5. The forebrain showed a similar pattern of acquired damage followed by apoptosis and decreased proliferation. The p53-null genetic background did not substantially rescue the AtrNes-cre phenotype despite blocking apoptosis. This data indicates that proliferation arrest is the major factor causing ATR-Seckel syndrome and that ATR does not significantly contribute to p53-induced apoptosis in proliferating neural progenitors. AtrNes-cre animals were crossed onto an Atm conditional background (AtmNes-cre) in order to assess how ATM and ATR cooperate in maintaining homeostasis in the brain. The AtmNes-cre background failed to alter the AtrNes-cre phenotype, underscoring the divergence of ATR and ATM function during embryogenesis, despite overlapping substrates between the kinases. In summary, this work provides us with important insight into the requirements for specific DNA damage signaling molecules in the nervous system. ATM and ATR appear to have distinct roles in the nervous system with ATM required for elimination of compromised early post-mitotic neurons, while ATR is critical for maintaining integrity and replicative capacity of progenitor populations. Based upon this study, there is no obvious cross-talk or overlap between Atm and Atr signaling in the nervous system as each plays a separate role in different cell populations. Furthermore, we have begun to tease out the existence of independent signaling roles between Mre11 and Nbs1 in regard to Atm activity. While Mre11 mutations associated with ATLD compromise Atm-dependant apoptosis, Nbs1 hypomorphism linked to NBS does not appear to negatively impact upon known Atm function in the nervous system. Overall these data provide new insights into the specific genetic requirements for DNA double strand break repair in the nervous system

You are no longer creative when you give up : Technical theatre’s creative sleight of hand

The Western Australian Academy of Performing Arts at Edith Cowan University contains vocational education training programmes including technical design courses with broad reach covering arts administration, stage management, stage lighting, sound design, set and costume design. In an unsettling problematic, teachers and students in the broadly themed Production and Design courses often find themselves isolated from the other creative disciplines or battle with the perception that their work is in fact not creative but entirely the technical implementation of ‘someone’s else’s vision’. This approach seems to dismiss the creative thinking required in the development and orchestration of the design and denies the complexity inherent in anything ‘technical’. This paper will address this disparity by drawing from the perceptions of a select number of current staff from Production and Design subjects. We understand that this is a very specific take on the subject from a small number of interested folk, in fact it is deliberately idiosyncratic and narrow in research scope, and in no way indicates the viewpoints of the Australian production and design community at large. Rather, we put forward a particular point of view, given at a particular time, in order to argue that there is merit in addressing what we see as a ‘hierarchy of value’ and seek further conversation about how we may find a way that the technical/mechanical and the creative are not considered as mutually exclusive. By doing so this would not only be a pedagogical shift, but a movement in cultural paradigm

You are no longer creative when you give up : Technical theatre’s creative sleight of hand

The Western Australian Academy of Performing Arts at Edith Cowan University contains vocational education training programmes including technical design courses with broad reach covering arts administration, stage management, stage lighting, sound design, set and costume design. In an unsettling problematic, teachers and students in the broadly themed Production and Design courses often find themselves isolated from the other creative disciplines or battle with the perception that their work is in fact not creative but entirely the technical implementation of ‘someone’s else’s vision’. This approach seems to dismiss the creative thinking required in the development and orchestration of the design and denies the complexity inherent in anything ‘technical’. This paper will address this disparity by drawing from the perceptions of a select number of current staff from Production and Design subjects. We understand that this is a very specific take on the subject from a small number of interested folk, in fact it is deliberately idiosyncratic and narrow in research scope, and in no way indicates the viewpoints of the Australian production and design community at large. Rather, we put forward a particular point of view, given at a particular time, in order to argue that there is merit in addressing what we see as a ‘hierarchy of value’ and seek further conversation about how we may find a way that the technical/mechanical and the creative are not considered as mutually exclusive. By doing so this would not only be a pedagogical shift, but a movement in cultural paradigm

Thermal Moonquake Identification and Localization from Apollo 17's Lunar Seismic Profiling Experiment

No abstract availabl

Could a Mid-Level Dental Provider Increase Access to Oral Health Care in Michigan?

According to a 2000 Surgeon General’s report, the United States faces an epidemic of unmet oral health needs, the result of both the high cost of care and geographic maldistribution of providers. This article assesses the extent of this unmet health care needs in Michigan, and examines one possible solution: the introduction of a mid-level dental provider (MDP) who could provide preventive and basic restorative care, under the supervision of a Michigan dentist. MDPs in various forms currently practice in over 50 countries including Canada and the U.K. The evidence suggests that a large and rigorous pilot of mid-level dental providers should be undertaken in Michigan, to inform policymakers about the structure’s potential for improving access to oral health care for vulnerable populations in the state

Simple Financial Economic Models of Fremont Maize Storage and an Assessment of External Threat

This paper presents a pair of models of the storage of maize. One is directly based on standard financial models of portfolio choice. Rather than optimally balancing a financial portfolio by choosing from a variety of financial instruments, our agents optimize holdings of maize by choosing from a variety of storage locations. Agents face a tradeoff between the effort of transporting maize to high elevation granaries versus the safety they offer from theft. The second model uses a multi-period framework to look at the costs and benefits of building a granary in the first place. We use our models to extract a perceived probability of maize theft by outsiders among the Fremont Indians that lived in Eastern Utah roughly 1000 - 700 years ago. We base our estimates on the caloric content of maize, the caloric cost of transporting it to granaries high above the valley floor where the maize was grown, and the costs of building and maintaining them. Our calculations show that a fairly low level of risk, on the order of 5% to 20%, could easily rationalize the use of cliffside granaries