A longitudinal assessment of chronic wound fluid to detect biochemical indicators of healing

Chronic venous leg ulcers are a detrimental health issue plaguing our society, resulting in long term pain, immobility and decreased quality of life for a large proportion of sufferers. The frequency of these chronic wounds has led current research to focus on the wound environment to provide important information regarding the prolonged, fluctuated or static healing patterns of these wounds. Disruption to the normal wound healing process results in release of multiple factors in the wound environment that could correlate to wound chronicity. These biochemical factors can often be detected through non-invasively sampling chronic wound fluid (CWF) from the site of injury. Of note, whilst there are numerous studies comparing acute and chronic wound fluids, there have not been any reports in the literature employing a longitudinal study in order to track biochemical changes in wound fluid as patients transition from a non-healing to healed state. Initially the objective of this study was to identify biochemical changes in CWF associated with wound healing using a proteomic approach. The proteomic approach incorporated a multi-dimensional liquid chromatography fractionation technique coupled with mass spectrometry (MS) to enable identification of proteins present in lower concentrations in CWF. Not surprisingly, many of the proteins identified in wound fluid were acute phase proteins normally expressed during the inflammatory phase of healing. However, the number of proteins positively identified by MS was quite low. This was attributed to the diverse range in concentration of protein species in CWF making it challenging to detect the diagnostically relevant low molecular weight proteins. In view of this, SELDI-TOF MS was also explored as a means to target low molecular weight proteins in sequential patient CWF samples during the course of healing. Unfortunately, the results generated did not yield any peaks of interest that were altered as wounds transitioned to a healed state. During the course of proteomic assessment of CWF, it became evident that a fraction of non-proteinaceous compounds strongly absorbed at 280 nm. Subsequent analyses confirmed that most of these compounds were in fact part of the purine catabolic pathway, possessing distinctive aromatic rings and which results in high absorbance at 254 nm. The accumulation of these purinogenic compounds in CWF suggests that the wound bed is poorly oxygenated resulting in a switch to anaerobic metabolism and consequently ATP breakdown. In addition, the presence of the terminal purine catabolite, uric acid (UA), indicates that the enzyme xanthine oxidoreductase (XOR) catalyses the reaction of hypoxanthine to xanthine and finally to UA. More importantly, the studies provide evidence for the first time of the exogenous presence of XOR in CWF. XOR is the only enzyme in humans capable of catalysing the production of UA in conjunction with a burst of the highly reactive superoxide radical and other oxidants like H2O2. Excessive release of these free radicals in the wound environment can cause cellular damage disrupting the normal wound healing process. In view of this, a sensitive and specific assay was established for monitoring low concentrations of these catabolites in CWF. This procedure involved combining high performance liquid chromatography (HPLC) with tandem mass spectrometry and multiple reaction monitoring (MRM). This application was selective, using specific MRM transitions and HPLC separations for each analyte, making it ideal for the detection and quantitation of purine catabolites in CWF. The results demonstrated that elevated levels of UA were detected in wound fluid obtained from patients with clinically worse ulcers. This suggests that XOR is active in the wound site generating significant amounts of reactive oxygen species (ROS). In addition, analysis of the amount of purine precursors in wound fluid revealed elevated levels of purine precursors in wound fluid from patients with less severe ulcers. Taken together, the results generated in this thesis suggest that monitoring changes of purine catabolites in CWF is likely to provide valuable information regarding the healing patterns of chronic venous leg ulcers. XOR catalysis of purine precursors not only provides a method for monitoring the onset, prognosis and progress of chronic venous leg ulcers, but also provides a potential therapeutic target by inhibiting XOR, thus blocking UA and ROS production. Targeting a combination of these purinogenic compounds and XOR could lead to the development of novel point of care diagnostic tests. Therefore, further investigation of these processes during wound healing will be worthwhile and may assist in elucidating the pathogenesis of this disease state, which in turn may lead to the development of new diagnostics and therapies that target these processes.

High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species

Red cap

The field of research is contemporary theatre practice with a community focus. In 2007, La Boite Theatre Company partnered with the Queensland Music Festival to produce an operatic representation of the 1964 Mt Isa industrial dispute, focussed on the charismatic figure of Pat Mackie. “Community theatre” is often criticised on grounds that the work aims only to satisfy community outcomes. This work explored whether a story from a specific location, which is very much an embedded story in the culture of the Mt Isa community, could be told in such a way as to appeal to, resonate with, and have relevance for, broader national and international audiences. To address this question required rigorous interrogation of both content and form. The play was researched through interviews with members of the Mt Isa community, political leadership at the time of the dispute, and participants of the dispute, including Pat Mackie himself. The production was then framed as an oratorio. Uniquely, the play had two back-to-back seasons; the first in Mount Isa (3 shows: 1500 people including a significant number of school children) and a 4-week season at the Roundhouse Theatre, Brisbane (over 5,000 attendances). In each location, a chorale was formed of community participants who, alongside the professional cast, performed the work. The production and its complementary exhibition had a significant local and national profile. The project was featured in The Australian newspaper’s Queensland Music Festival wrap-up as an exemplar of successful community engagement and creative adventure. Playlab Press has since published the script.

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones : a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeo-stasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, im-munohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the os-teocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogene-sis.

Victoria's “Day in Prison Program” : an evaluation and critique

The use of “Day in Prison” programs to deter young adult offenders is a concept which originated in the United States and was replicated in Australia during the late 1970s. After almost a decade of uncertainty this model of ‘crime prevention’ re-emerged in Victoria with the introduction of a pilot “Day in Prison” program. This article traces the development and operation of the Victorian experience and provides evaluation research findings which conclude that coercive, intimidatory and degrading aversion techniques should not be utilised by the criminal justice system for the purposes of individual deterrence.

Risk factors for Mycobacterium tuberculosis infection among children in Greenland

Objective To examine the risk factors for Mycobacterium tuberculosis infection (MTI) among Greenlandic children for the purpose of identifying those at highest risk of infection. Methods Between 2005 and 2007, 1797 Greenlandic schoolchildren in five different areas were tested for MTI with an interferon gamma release assay (IGRA) and a tuberculin skin test (TST). Parents or guardians were surveyed using a standardized self-administered questionnaire to obtain data on crowding in the household, parents’ educational level and the child’s health status. Demographic data for each child – i.e. parents’ place of birth, number of siblings, distance between siblings (next younger and next older), birth order and mother’s age when the child was born – were also extracted from a public registry. Logistic regression was used to check for associations between these variables and MTI, and all results were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). Children were considered to have MTI if they tested positive on both the IGRA assay and the TST. Findings The overall prevalence of MTI was 8.5% (152/1797). MTI was diagnosed in 26.7% of the children with a known TB contact, as opposed to 6.4% of the children without such contact. Overall, the MTI rate was higher among Inuit children (OR: 4.22; 95% CI: 1.55–11.5) and among children born less than one year after the birth of the next older sibling (OR: 2.48; 95% CI: 1.33–4.63). Self-reported TB contact modified the profile to include household crowding and low mother’s education. Children who had an older MTI-positive sibling were much more likely to test positive for MTI themselves (OR: 14.2; 95% CI: 5.75–35.0) than children without an infected older sibling. Conclusion Ethnicity, sibling relations, number of household residents and maternal level of education are factors associated with the risk of TB infection among children in Greenland. The strong household clustering of MTI suggests that family sources of exposure are important.

Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.

Aromatic l-amino acid decarboxylase : histochemical localization in rat kidney and lack of effect of dietary potassium or sodium loading on enzyme distribution

Utilizing a mono-specific antiserum produced in rabbits to hog kidney aromatic L-amino acid decarboxylase (AADC), the enzyme was localized in rat kidney by immunoperoxidase staining. AADC was located predominantly in the proximal convoluted tubules; there was also weak staining in the distal convoluted tubules and collecting ducts. An increase in dietary potassium or sodium intake produced no change in density or distribution of AADC staining in kidney. An assay of AADC enzyme activity showed no difference in cortex or medulla with chronic potassium loading. A change in distribution or activity of renal AADC does not explain the postulated dopaminergic modulation of renal function that occurs with potassium or sodium loading.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

Background Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. Methods The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Results Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. Conclusion Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.

Ageing of people with an intellectual disability: Effective training for frontline workers

While the attainment of late life represents a significant achievement for people with an intellectual disability, increased life expectancy has resulted in growing concerns about the extent to which disability service providers are ready to meet the changing needs of increasing numbers of older people and facilitate their ongoing social inclusion. Training of frontline disability staff is widely accepted as an effective strategy for increasing organisational capacity to contribute to improved quality of life for people with an intellectual disability. The study identifies training needs analyses and 'ready-to-deliver' training programs for frontline disability services staff working with adults with an intellectual disability who are ageing, assesses whether the training programs contribute to improved quality of life outcomes for service users, and makes recommendations for future research and development of training for disability services staff who work with older people with intellectual disability.

Properties of the bovine viral diarrhoea virus replicase in extracts of infected MDBK cells

An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially doublestranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5–10mM Mg2+ and although it was also active in 1–2mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.