In vitro functional characterisation of IGF-I : VN-induced breast cancer progression

Members of the insulin-like growth factor (IGF) family have been shown to play critical roles in normal growth and development, as well as in tumour biology. The IGF system is complex and the biological effects of the IGFs are determined by their diverse interactions between many molecules, including their interactions with extracellular matrix (ECM) proteins. Recent studies have demonstrated that IGFs associate with the ECM protein vitronectin (VN) through IGF-binding proteins (IGFBP) and that this interaction modulates IGF-stimulated biological functions, namely cell migration and cell survival through the cooperative involvement of the type-I IGF receptor (IGF-1R) and VN-binding integrins. Since IGFs play important roles in the transformation and progression of breast cancer and VN has been found to be over-expressed at the leading edge of breast tumours, this project aimed to describe the effects of IGF-I:VN interactions on breast cell function. This was undertaken to dissect the molecular mechanisms underlying IGF-I:VN-induced responses and to design inhibitors to block the effects of such interactions. The studies described herein demonstrate that the increase in migration of MCF-7 breast cancer cells in response to the IGF-I:IGFBP-5:VN complex is accompanied by differential expression of genes known to be involved in migration, invasion and/or survival, including Tissue-factor (TF), Stratifin (SFN), Ephrin-B2, Sharp-2 and PAI-1. This „migration gene signature‟ was confirmed using real-time PCR analysis. Substitution of the native IGF-I within the IGF-I:IGFBP:VN complex with the IGF-I analogue, \[L24]\[A31]-IGF-I, which has a reduced affinity for the IGF-1R, failed to stimulate cell migration and interestingly, also failed to induce the differential gene expression. This supports the involvement of the IGF-1R in mediating these changes in gene expression. Furthermore, lentiviral shRNA-mediated stable knockdown of TF and SFN completely abrogated the increased cell migration induced by IGF-I:IGFBP:VN complexes in MCF-7 cells. Indeed, when these cells were grown in 3D Matrigel™ cultures a decrease in the overall size of the 3D spheroids in response to the IGF-I:IGFBP:VN complexes was observed compared to the parental MCF-7 cells. This suggests that TF and SFN have a role in complex-stimulated cell survival. Moreover, signalling studies performed on cells with the reduced expression of either TF or SFN had a decreased IGF-1R activation, suggesting the involvement of signalling pathways downstream of IGF-1R in TF- and/or SFN-mediated cell migration and cell survival. Taken together, these studies provide evidence for a common mechanism activated downstream of the IGF-1R that induces the expression of the „migration gene signature‟ in response to the IGF-I:IGFBP:VN complex that confers breast cancer cells the propensity to migrate and survive. Given the functional significance of the interdependence of ECM and growth factor (GF) interactions in stimulating processes key to breast cancer progression, this project aimed at developing strategies to prevent such growth factor:ECM interactions in an effort to inhibit the downstream functional effects. This may result in the reduction in the levels of ECM-bound IGF-I present in close proximity to the cells, thereby leading to a reduction in the stimulation of IGF-1R present on the cell surface. Indeed, the inhibition of IGF-I-mediated effects through the disruption of its association with ECM would not alter the physiological levels of IGF-I and potentially only exert effects in situations where abnormal over expression of ECM proteins are found; namely carcinomas and hyperproliferative diseases. In summary, this PhD project has identified novel, innovative and realistic strategies that can be used in vitro to inhibit the functions exerted by the IGF-I:IGFBP:VN multiprotein complexes critical for cancer progression, with a potential to be translated into in vivo investigations. Furthermore, TF and SFN were found to mediate IGF-I:IGFBP:VN-induced effects, thereby revealing their potential to be used as therapeutic targets or as predictive biomarkers for the efficacy of IGF-1R targeting therapies in breast cancer patients. In addition to its therapeutic and clinical scope, this PhD project has significantly contributed to the understanding of the role of the IGF system in breast tumour biology by providing valuable new information on the mechanistic events underpinning IGF-I:VN-mediated effects on breast cell functions. Furthermore, this is the first instance where favourable binding sites for IGF-II, IGFBP-3 and IGFBP-5 on VN have been identified. Taken together, this study has functionally characterised the interactions between IGF-I and VN and through innovative strategies has provided a platform for the development of novel therapies targeting these interactions and their downstream effects.

The role of psychological and physiological factors in decision making under risk and in a dilemma

We study the difference in the result of two different risk elicitation methods by linking estimates of risk attitudes to gender, age, personality traits, a decision in a dilemma situation, and physiological states measured by heart rate variability (HRV). Our results indicate that differences between the methods can partly be explained by gender, but not by personality traits. Furthermore, HRV is linked to risktaking in the experiment for at least one of the methods, indicating that more stressed individuals display more risk aversion. Finally, we and that risk attitudes are not predictive of the ability to decide in a dilemma, but personality traits are. Surprisingly, there is also no apparent relationship between the physiological state during the dilemma situation and the ability to make a decision.

Natural and engineered plasmin inhibitors : applications and design strategies

Plasmin is the primary enzyme responsible for dissolution of fibrin in the circulatory system. Plasminogen, the zymogen of plasmin is expressed ubiquitously in the human body [1], with the predominant source being the liver [2, 3]. Plasminogen is produced as an 810 amino acid protein with a 19 amino acid leader peptide, which is cleaved during secretion to produce the mature 791 amino acid one-chain zymogen. This is converted to plasmin by cleavage of the Arg561 - Val562 scissile bond [4], resulting in an active protease consisting of two disulfide linked chains. The amino-terminal heavy chain (residues Glu1-Arg561) is comprised of a plasminogen/apple/nematode (PAN) domain [5] and five kringle domains of approximately equal size [6] while the light chain (residues Val562-Asn791) contains a serine protease domain homologous to trypsin with a catalytic triad comprising His603, Asp646 and Ser741 [7]. Both plasmin and plasminogen occur in two forms, full length and a Lys77-Lys78 activated variant produced through self catalysis (Figure 1). The former exists in a tight conformation through binding of Lys50 and/or Lys62 to kringle domain 5 [8, 9] while Lys78-plasminogen assumes a more relaxed conformation rendering it more susceptible to plasmin conversion [10, 11].

Effect of recovery modality on 4-hour repeated treadmill running performance and changes in physiological variables

The purpose of this study was to compare the effectiveness of three different recovery modalities - active (ACT), passive (PAS) and contrast temperature water immersion (CTW) - on the performance of repeated treadmill running, lactate concentration and pH. Fourteen males performed two pairs of treadmill runs to exhaustion at 120% and 90% of peak running speed (PRS) over a 4-hour period. ACT, PAS or CTW was performed for 15-min after the first pair of treadmill runs. ACT consisted of running at 40% PRS, PAS consisted of standing stationary and CTW consisted of alternating between 60-s cold (10°C) and 120-s hot (42°C) water immersion. Run times were converted to time to cover set distance using critical power. Type of recovery modality did not have a significant effect on change in time to cover 400 m (Mean±SD; ACT 2.7±3.6 s, PAS 2.9±4.2 s, CTW 4.2±6.9 s), 1000 m (ACT 2.2±4.0 s, PAS 4.8±8.6 s, CTW 2.1±7.2 s) or 5000 m (ACT 1.4±29.0 s, PAS 16.7±58.5 s, CTW 11.7±33.0 s). Post exercise blood lactate concentration was lower in ACT and CTW compared with PAS. Participants reported an increased perception of recovery in the CTW compared with ACT and PAS. Blood pH was not significantly influenced by recovery modality. Data suggest both ACT and CTW reduce lactate accumulation after high intensity running, but high intensity treadmill running performance is returned to baseline 4-hours after the initial exercise bout regardless of the recovery strategy employed.

Histone deacetylase inhibitors target diabetes via chromatin remodeling or as chemical chaperones?

Globally, obesity and diabetes (particularly type 2 diabetes) represents a major challenge to world health. Despite decades of intense research efforts, the genetic basis involved in diabetes pathogenesis & conditions associated with obesity are still poorly understood. Recent advances have led to exciting new developments implicating epigenetics as an important mechanism underpinning diabetes and obesity related disease. One epigenetic mechanism known as the "histone code" describes the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as lysine acetyltransferases or KATs and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. Some of the known inhibitors of HDACs (HDACi) have also been shown to act as "chemical chaperones" to alleviate diabetic symptoms. In this review, we discuss the available evidence concerning the roles of HDACs in regulating chaperone function and how this may have implications in the management of diabetes. © 2009 Bentham Science Publishers Ltd.

Cultural inhibitors to hearing the voices of child sexual abuse by church personnel

It is increasingly recognised that Christian Churches and their Institutions have historically been sites where the voices of survivors of child sexual abuse (csa) by Church personnel have not been effectively heard. This paper draws on data from a research project which sought the voices of Church Leaders who were identified as being pro-active in addressing csa within their individual denominations. From this research several key inhibitors to hearing survivor’s voices, within Churches of Australia, were identified. These key inhibitors include the culture of Churches themselves, gendered ideologies, constructions of leadership and the deployment of forgiveness. The identification of such factors creates space to learn more effective strategies for hearing the voices of survivors both within Churches and their organisations and externally. This paper goes beyond considering these factors to report on a collaborative project initiated, between Survivors Australia and Dr Death. This project specifically targets the voices of Australian Survivors of csa by Church leaders. It is hoped that this project will not only achieve the primary objective of hearing and valuing the voices of survivors of csa by Church leaders, but will also provide impetus for the creation of alternative ways of managing complaints of csa by Church leaders in Australia. Such complaints processes will be increasingly survivor focussed and include the creation of spaces where the voices of survivors are valued.

Design of physiological control and magnetic levitation systems for a total artificial heart

Total Artificial Hearts are mechanical pumps which can be used to replace the failing natural heart. This novel study developed a means of controlling a new design of pump to reproduce physiological flow bringing closer the realisation of a practical artificial heart. Using a mathematical model of the device, an optimisation algorithm was used to determine the best configuration for the magnetic levitation system of the pump. The prototype device was constructed and tested in a mock circulation loop. A physiological controller was designed to replicate the Frank-Starling like balancing behaviour of the natural heart. The device and controller provided sufficient support for a human patient while also demonstrating good response to various physiological conditions and events. This novel work brings the design of a practical artificial heart closer to realisation.

Foot massage and physiological stress in people with dementia : a randomized controlled trial

Background There is increasing interest in using complementary and alternative treatments to manage behavioural and psychological symptoms of dementia such as agitation, aggression and depressed mood. Objective To compare the effect of foot massage (intervention) and quiet presence (control) on agitation and mood in people with dementia. Design A randomised controlled trial using a within-subjects, crossover design. Settings Five long-term care facilities in Brisbane, Australia. The primary outcome was the Cohen-Mansfield Agitation Inventory (CMAI) and the secondary outcome was the Observed Emotion Rating Scale (OERS). The screening and data collection research assistants, families, and care staff were blinded to participant allocation. Participants Participants of the study were 55 long-term care residents aged 74–103 years (mean age 86.5), with moderate to severe dementia and a history of agitated behaviour according to the Pittsburgh Agitation Scale. A computer-program randomised participants to 10-min foot massage (intervention) or quiet presence (control), every weekday for 3 weeks. Results A carry-over effect was identified in the data, and so the data was treated as a parallel groups RCT. The mean total CMAI increased in both groups (reflecting an increase in agitation) with this increase greater in the quiet presence group than the foot massage group (p=0.03). There was a trend towards a difference on OERS General Alertness, with a positive change in alertness for participants in the foot massage group (indicating reduced alertness) and a negative change for participants in the quiet presence group (indicating increased alertness) (F(1,51)=3.88, p=0.05, partial ή2=0.07). Conclusions The findings highlight the need for further research on the specific conditions under which massage might promote relaxation and improve mood for people with dementia. The unfamiliar research assistants and variations in usual activity may have contributed to the increase in agitation and this needs further research.

Physiological tolerance times while wearing explosive ordnance disposal protective clothing in simulated environmental extremes

Explosive ordnance disposal (EOD) technicians are required to wear protective clothing to protect themselves from the threat of overpressure, fragmentation, impact and heat. The engineering requirements to minimise these threats results in an extremely heavy and cumbersome clothing ensemble that increases the internal heat generation of the wearer, while the clothing’s thermal properties reduce heat dissipation. This study aimed to evaluate the heat strain encountered wearing EOD protective clothing in simulated environmental extremes across a range of differing work intensities. Eight healthy males [age 25±6 years (mean ± sd), height 180±7 cm, body mass 79±9 kg, V˙O2max 57±6 ml.kg−1.min−1] undertook nine trials while wearing an EOD9 suit (weighing 33.4 kg). The trials involved walking on a treadmill at 2.5, 4 and 5.5 km⋅h−1 at each of the following environmental conditions, 21, 30 and 37°C wet bulb globe temperature (WBGT) in a randomised controlled crossover design. The trials were ceased if the participants’ core temperature reached 39°C, if heart rate exceeded 90% of maximum, if walking time reached 60 minutes or due to fatigue/nausea. Tolerance times ranged from 10–60 minutes and were significantly reduced in the higher walking speeds and environmental conditions. In a total of 15 trials (21%) participants completed 60 minutes of walking; however, this was predominantly at the slower walking speeds in the 21°C WBGT environment. Of the remaining 57 trials, 50 were ceased, due to attainment of 90% maximal heart rate. These near maximal heart rates resulted in moderate-high levels of physiological strain in all trials, despite core temperature only reaching 39°C in one of the 72 trials.

Structures of μOconotoxins from Conusmarmoreus. Inhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons

The μO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxin-resistant (TTX-R) Na+ current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxin-sensitive Na+ currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the μO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small β-sheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone “loops” between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known δ- and ω-conotoxins, which along with the μO-conotoxins are members of the O superfamily. Loop 2 of ω-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels, and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels.

Synthesis of novel inhibitors blocking Wnt signaling downstream of β-Catenin

Large scale screening of libraries consisting of natural and small molecules led to the identification of many small molecule inhibitors repressing Wnt/β-Catenin signaling. However, targeted synthesis of novel Wnt pathway inhibitors has been rarely described. We developed a modular and expedient way to create the aromatic ring system with an aliphatic ring in between. Our synthesis opens up the possibility, in principle, to substitute all positions at the ring system with any desired substituent. Here, we tested five different haloquinone analogs carrying methoxy- and hydroxy-groups at different positions. Bona fide Wnt activity assays in cell culture and in Xenopus embryos revealed that two of these compounds act as potent inhibitors of aberrant activated Wnt/β-Catenin signaling.

Calcitonin receptor plays a physiological role to protect against hypercalcemia in mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]-induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

Physiological basis of salt stress tolerance in rice expressing the antiapoptotic gene SfIAP

Programmed cell death-associated genes, especially antiapoptosis-related genes have been reported to confer tolerance to a wide range of biotic and abiotic stresses in dicotyledonous plants such as tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.). This is the first time the antiapoptotic gene SfIAP was transformed into a monocotyledonous representative: rice (Oryza sativa L.). Transgenic rice strains expressing SfIAP were generated by the Agrobacterium-mediated transformation method and rice embryogenic calli, and assessed for their ability to confer tolerance to salt stress at both the seedling and reproductive stages using a combination of molecular, agronomical, physiological and biochemical techniques. The results show that plants expressing SfIAP have higher salt tolerance levels in comparison to the wild-type and vector controls. By preventing cell death at the onset of salt stress and maintaining the cell membrane’s integrity, SfIAP transgenic rice plants can retain plant water status, ion homeostasis, photosynthetic efficiency and growth to combat salinity successfully.

Contribution of individual, workplace, psychosocial and physiological factors to neck pain in female office workers

This study investigated the relative contribution of individual, workplace, psychosocial and physiological features associated with neck pain in female office workers towards developing appropriate intervention programs. Workers without disability (Neck Disability Index (NDI) score≤8, n=33); workers with neck pain and disability (NDI≥9/100, n=52) and 22 controls (women who did not work and without neck pain) participated in this study. Two logistic regression models were constructed to test the association between various measures in (1) workers with and without disability, and (2) workers without disability and controls. Measures included those found to be significantly associated with higher NDI in our previous studies: psychosocial domains; individual factors; task demands; quantitative sensory measures and measures of motor function. In the final model, higher score on negative affectivity scale (OR=4.47), greater activity in the neck flexors during cranio-cervical flexion (OR=1.44), cold hyperalgesia (OR=1.27) and longer duration of symptoms (OR=1.19) remained significantly associated with neck pain in workers. Workers without disability and controls could only be differentiated by greater muscle activity in the cervical flexors and extensors during a typing task. No psychosocial domains remained in either regression model. These results suggest that impairments in the sensory and motor system should be considered in any assessment of the office worker with neck pain and may have stronger influences on the presenting symptoms than workplace and psychosocial features.

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.