Fibroblast nemosis in malignant progression of epithelial cells

Paracrine regulation between the components of the tumour microenvironment cancer cells, activated fibroblasts, immune and endothelial cells is under intense investigation. The signals between the different cell types are mediated by soluble factors, such as growth factors, proinflammatory cytokines and proteolytic enzymes. Nemosis is an experimental in vitro model of fibroblast activation, leading to increased production of such mediators. Nemotic activation of fibroblasts occurs as they are forced to cluster thereby forming a multicellular spheroid. The aim of the present studies was to elucidate the mechanisms underlying the nemotic response of cancer-associated fibroblasts (CAF) and the role of nemosis in paracrine regulation between activated fibroblasts and benign and malignant epithelial cells. The results presented in this thesis demonstrate that the nemotic response of CAFs and normal fibroblasts differs, and inter-individual variations exist between fibroblast populations. In co-culture experiments, fibroblasts increased colony formation of squamous cell carcinoma (SCC) cells, and CAFs further augmented this, highlighting the tumour-evolving properties of CAFs. Furthermore, fibroblast monolayers in those co-cultures started to cluster spontaneously. This kind of spontaneous nemosis response might take place also in vivo, although more direct evidence of this still needs to be obtained. The HaCaT skin carcinoma progression model was used to study the effects of benign and malignant keratinocytes on fibroblast nemosis. Benign HaCaT cells inhibited fibroblast nemosis, observed as inhibition of cyclooxygenase 2 (COX-2) induction in nemotic spheroids. In contrast, malignant HaCaTs further augmented the nemotic response by increasing expression of COX-2 and the growth factors hepatocyte growth factor / scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF), as well as causing a myofibroblastic differentiation of nemotic fibroblasts into fibroblasts resembling CAFs. On the other side of this reciprocal signalling, factors secreted into conditioned medium by the nemotic fibroblasts promoted proliferation and motility of the HaCaT cell lines. Notably, the nemotic fibroblast medium increased the expression of p63, a transcription factor linked to carcinogenesis, also in the highly metastatic HaCaT cells. These results emphasize the paracrine role of factors secreted by activated fibroblasts in driving tumour progression. We also investigated the epithelial-mesenchymal transition (EMT) of the HaCaT clones in response to transforming growth factor β (TGF-β), which is a well-characterized inducer of EMT. TGF-β caused growth arrest and loss of epithelial cell junctions in the HaCaT derivatives, but mesenchymal markers were not induced, suggesting a partial, but not complete EMT response. Inflammation induced by COX-2 has been proposed to be a key mechanism in EMT of benign cells. Corroborating this notion, COX-2 was induced only in benign, not in malignant HaCaT derivatives. Furthermore, in cells in which TGF-β caused COX-2 induction, migration was clearly augmented. The concept of treating cancer is changing from targeting solely the cancer cells to targeting the whole microenvironment. The results of this work emphasise the role of activated fibroblasts in cancer progression and that CAFs should also be taken into consideration in the treatment of cancer. The results from these studies suggests that nemosis could be used as a diagnostic tool to distinguish in vitro activated fibroblasts from tumour stroma and also in studying the paracrine signalling that is mediated to other cell types via soluble factors.

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.

A mixed-methods research approach to the review of competency standards for orthotist/prosthetists in Australia

Aim: The requirement for an allied health workforce is expanding as the global burden of disease increases internationally. To safely meet the demand for an expanded workforce of orthotist/prosthetists in Australia, competency based standards, which are up-to-date and evidence-based, are required. The aims of this study were to determine the minimum level for entry into the orthotic/prosthetic profession; to develop entry level competency standards for the profession; and to validate the developed entry-level competency standards within the profession nationally, using an evidence-based approach. Methods: A mixed-methods research design was applied, using a three-step sequential exploratory design, where step 1 involved collecting and analyzing qualitative data from two focus groups; step 2 involved exploratory instrument development and testing, developing the draft competency standards; and step 3 involved quantitative data collection and analysis – a Delphi survey. In stage 1 (steps 1 and 2), the two focus groups – an expert and a recent graduate group of Australian orthotist/prosthetists – were led by an experienced facilitator, to identify gaps in the current competency standards and then to outline a key purpose, and work roles and tasks for the profession. The resulting domains and activities of the first draft of the competency standards were synthesized using thematic analysis. In stage 2 (step 3), the draft-competency standards were circulated to a purposive sample of the membership of the Australian Orthotic Prosthetic Association, using three rounds of Delphi survey. A project reference group of orthotist/prosthetists reviewed the results of both stages. Results: In stage 1, the expert (n = 10) and the new graduate (n = 8) groups separately identified work roles and tasks, which formed the initial draft of the competency standards. Further drafts were refined and performance criteria added by the project reference group, resulting in the final draft-competency standards. In stage 2, the final draft-competency standards were circulated to 56 members (n = 44 final round) of the Association, who agreed on the key purpose, 6 domains, 18 activities, and 68 performance criteria of the final competency standards. Conclusion: This study outlines a rigorous and evidence-based mixed-methods approach for developing and endorsing professional competency standards, which is representative of the views of the profession of orthotist/prosthetists.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Mixed ligand complexes in vinyl polymerization-II: [NN-ethylenebis(salicylideneiminato)] (benzoylacetonato)cobalt(III) as an initiator

The polymerization of methyl methacrylate initiated by a mixed ligand complex. [NN-ethylenebis(salicylideneiminato)](benzoylacetonato)cobalt(III) has been studied in bulk and in benzene at 70° and 80°. The rate of polymerization is proportional to (concentration of the chelate)Image and the monomer exponent is close to 1.5. The activation energy and the kinetic and transfer constants are evaluated. A free radical mechanism has been proposed.

Molecular biology and functions of epilysin (MMP-28)

Epilysin (MMP-28) is the most recently identified member of the matrix metalloproteinase (MMP) family of extracellular proteases. Together these enzymes are capable of degrading almost all components of the extracellular matrix (ECM) and are thus involved in important biological processes such as development, wound healing and immune functions, but also in pathological processes such as tumor invasion, metastasis and arthritis. MMPs do not act solely by degrading the ECM. They also regulate cell behavior by releasing growth factors and biologically active peptides from the ECM, by modulating cell surface receptors and adhesion molecules and by regulating the activity of many important mediators in inflammatory pathways. The aim of this study was to define the unique role of epilysin within the MMP-family, to elucidate how and when it is expressed and how its catalytic activity is regulated. To gain information on its essential functions and substrates, the specific aim was to characterize how epilysin affects the phenotype of epithelial cells, where it is biologically expressed. During the course of the study we found that the epilysin promoter contains a well conserved GT-box that is essential for the basic expression of this gene. Transcription factors Sp1 and Sp3 bind this sequence and could hence regulate both the basic and cell type and differentiation stage specific expression of epilysin. We cloned mouse epilysin cDNA and found that epilysin is well conserved between human and mouse genomes and that epilysin is glycosylated and activated by furin. Similarly to in human tissues, epilysin is normally expressed in a number of mouse tissues. The expression pattern differs from most other MMPs, which are expressed only in response to injury or inflammation and in pathological processes like cancer. These findings implicate that epilysin could be involved in tissue homeostasis, perhaps fine-tuning the phenotype of epithelial cells according to signals from the ECM. In view of these results, it was unexpected to find that epilysin can induce a stable epithelial to mesenchymal transition (EMT) when overexpressed in epithelial lung carcinoma cells. Transforming growth factor b (TGF-b) was recognized as a crucial mediator of this process, which was characterized by the loss of E-cadherin mediated cell-cell adhesion, elevated expression of gelatinase B and MT1-MMP and increased cell migration and invasion into collagen I gels. We also observed that epilysin is bound to the surface of epithelial cells and that this interaction is lost upon cell transformation and is susceptible to degradation by membrane type-1-MMP (MT1-MMP). The wide expression of epilysin under physiological conditions implicates that its effects on epithelial cell phenotype in vivo are not as dramatic as seen in our in vitro cell system. Nevertheless, current results indicate a possible interaction between epilysin and TGF-b also under physiological circumstances, where epilysin activity may not induce EMT but, instead, trigger less permanent changes in TGF-b signaling and cell motility. Epilysin may thus play an important role in TGF-b regulated events such as wound healing and inflammation, processes where involvement of epilysin has been indicated.

Phase transitions in (NH4)2SO4 and (NH4)2SO4---K2SO4 mixed crystals as studied by EPR of CrO3- radicals

A study of the phase transitions in (NH4)2SO4 and (NH4)2SO4---K2SO4 mixed crystals by EPR of the CrO3- ion is reported. The results indicate a bilinear coupling of the order parameter with spontaneous polarization and a crossover from a discontinuous to a continuous nature of the phase transition in mixed crystals.

Species-site matching in mixed species plantations of native trees in tropical Australia

Mixed species plantations using native trees are increasingly being considered for sustainable timber production. Successful application of mixed species forestry systems requires knowledge of the potential spatial interaction between species in order to minimise the chance of dominance and suppression and to maximise wood production. Here, we examined species performances across 52 experimental plots of tree mixtures established on cleared rainforest land to analyse relationships between the growth of component species and climate and soil conditions. We derived site index (SI) equations for ten priority species to evaluate performance and site preferences. Variation in SI of focus species demonstrated that there are strong species-specific responses to climate and soil variables. The best predictor of tree growth for rainforest species Elaeocarpus grandis and Flindersia brayleyana was soil type, as trees grew significantly better on well-draining than on poorly drained soil profiles. Both E. grandis and Eucalyptus pellita showed strong growth response to variation in mean rain days per month. Our study generates understanding of the relative performance of species in mixed species plantations in the Wet Tropics of Australia and improves our ability to predict species growth compatibilities at potential planting sites within the region. Given appropriate species selections and plantation design, mixed plantations of high-value native timber species are capable of sustaining relatively high productivity at a range of sites up to age 10 years, and may offer a feasible approach for large-scale reforestation.

Quality of life after early enteral feeding versus standard care for proven or suspected advanced epithelial ovarian cancer: Results from a randomised trial

Background Malnutrition is common in patients with advanced epithelial ovarian cancer (EOC), and is associated with impaired quality of life (QoL), longer hospital stay and higher risk of treatment-related adverse events. This phase III multi-centre randomised clinical trial tested early enteral feeding versus standard care on postoperative QoL. Methods From 2009 to 2013, 109 patients requiring surgery for suspected advanced EOC, moderately to severely malnourished were enrolled at five sites across Queensland and randomised to intervention (n = 53) or control (n = 56) groups. Intervention involved intraoperative nasojejunal tube placement and enteral feeding until adequate oral intake could be maintained. Despite being randomised to intervention, 20 patients did not receive feeds (13 did not receive the feeding tube; 7 had it removed early). Control involved postoperative diet as tolerated. QoL was measured at baseline, 6 weeks postoperatively and 30 days after the third cycle of chemotherapy. The primary outcome measure was the difference in QoL between the intervention and the control group. Secondary endpoints included treatment-related adverse event occurrence, length of stay, postoperative services use, and nutritional status. Results Baseline characteristics were comparable between treatment groups. No significant difference in QoL was found between the groups at any time point. There was a trend towards better nutritional status in patients who received the intervention but the differences did not reach statistical significance except for the intention-to-treat analysis at 7 days postoperatively (11.8 intervention vs. 13.8 control, p 0.04). Conclusion Early enteral feeding did not significantly improve patients' QoL compared to standard of care but may improve nutritional status.

Prediction of breeding values for average fruit weight in mango using a multivariate individual mixed model

Mango is an important horticultural fruit crop and breeding is a key strategy to improve ongoing sustainability. Knowledge of breeding values of potential parents is important for maximising progress from breeding. This study successfully employed a mixed linear model methods incorporating a pedigree to predict breeding values for average fruit weight from highly unbalanced data for genotypes planted over three field trials and assessed over several harvest seasons. Average fruit weight was found to be under strong additive genetic control. There was high correlation between hybrids propagated as seedlings and hybrids propagated as scions grafted onto rootstocks. Estimates of additive genetic correlation among trials ranged from 0.69 to 0.88 with correlations among harvest seasons within trials greater than 0.96. These results suggest that progress from selection for broad adaptation can be achieved, particularly as no repeatable environmental factor that could be used to predict G x E could be identified. Predicted breeding values for 35 known cultivars are presented for use in ongoing breeding programs.

Circulating tumor cells: Isolation, expansion, characterization and translation to clinic

Circulating tumor cells (CTCs) are the seeds for cancer metastases development, which is responsible for >90% of cancer-related deaths. Accurate quantification of CTCs in human fluids could be an invaluable tool for understanding cancer prognosis, delivering personalized medicine to prevent metastasis and finding cancer therapy effectiveness. Although CTCs were first discovered more than 200 years ago, until now it has been a nightmare for clinical practitioners to capture and diagnose CTCs in clinical settings. Our society needs rapid, sensitive, and reliable assays to identify the CTCs from blood in order to help save millions of lives. Due to the phenotypic EMT transition, CTCs are undetected for more than one-third of metastatic breast cancer patients in clinics. To tackle the above challenges, the first volume in “Circulating Tumor Cells (CTCs): Detection Methods, Health Impact and Emerging Clinical Challenges discusses recent developments of different technologies, which have the capability to target and elucidate the phenotype heterogenity of CTCS. It contains seven chapters written by world leaders in this area, covering basic science to possible device design which can have beneficial applications in society. This book is unique in its design and content, providing an in-depth analysis to elucidate biological mechanisms of cancer disease progression, CTC detection challenges, possible health effects and the latest research on evolving technologies which have the capability to tackle the above challenges. It describes the broad range of coverage on understanding CTCs biology from early predictors of the metastatic spread of cancer, new promising technology for CTC separation and detection in clinical environment and monitoring therapy efficacy via finding the heterogeneous nature of CTCs. (Imprint: Nova Biomedical)

Development and testing of allometric equations for estimating above-ground biomass of mixed-species environmental plantings

To quantify the impact that planting indigenous trees and shrubs in mixed communities (environmental plantings) have on net sequestration of carbon and other environmental or commercial benefits, precise and non-biased estimates of biomass are required. Because these plantings consist of several species, estimation of their biomass through allometric relationships is a challenging task. We explored methods to accurately estimate biomass through harvesting 3139 trees and shrubs from 22 plantings, and collating similar datasets from earlier studies, in non-arid (>300mm rainfallyear-1) regions of southern and eastern Australia. Site-and-species specific allometric equations were developed, as were three types of generalised, multi-site, allometric equations based on categories of species and growth-habits: (i) species-specific, (ii) genus and growth-habit, and (iii) universal growth-habit irrespective of genus. Biomass was measured at plot level at eight contrasting sites to test the accuracy of prediction of tonnes dry matter of above-ground biomass per hectare using different classes of allometric equations. A finer-scale analysis tested performance of these at an individual-tree level across a wider range of sites. Although the percentage error in prediction could be high at a given site (up to 45%), it was relatively low (<11%) when generalised allometry-predictions of biomass was used to make regional- or estate-level estimates across a range of sites. Precision, and thus accuracy, increased slightly with the level of specificity of allometry. Inclusion of site-specific factors in generic equations increased efficiency of prediction of above-ground biomass by as much as 8%. Site-and-species-specific equations are the most accurate for site-based predictions. Generic allometric equations developed here, particularly the generic species-specific equations, can be confidently applied to provide regional- or estate-level estimates of above-ground biomass and carbon. © 2013 Elsevier B.V.