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.

Transforming perceptions and responses to student difference : the journey of seven beginning teachers

Drawing on critical social theory and transformative learning, this study investigates the sociocultural factors that enable a move away from the deficit model of learning. The multicase study examines beginning teachers’ perceptions of learning and teaching for students experiencing learning difficulties. The fluid conception of identity occupied by beginning teachers was explored as the participants challenged structural ideologies and their personal belief systems. The study argues that personal factors such as, engaging in critical reflection and beliefs, and school factors such as job security and school context influence how beginning teachers respond to diverse learners and position themselves as agentive.

A case study of pedagogical responses to internationalisation at a faith-based secondary school in Australia

This case study investigated pedagogical responses to internationalisation by a faith-based secondary school in Australia. Using social constructivism as the theoretical framework the study examined teaching and learning for culturally and linguistically diverse students. Data generated through questionnaires, focus groups, individual interviews and document archives were analysed and interpreted using thematic analysis. The findings showed that teachers believed themselves to be ill-equipped to teach international students. Their concerns centred on a lack of explicit pedagogical, cultural and linguistic knowledge to help the students acculturate and learn. Recommendations include the dissemination of school policies to teachers, intentional staff collaboration and professional development to address the teachers’ needs for internationalisation.

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.

The importance of individual difference : examining anthropomorphic tendency and responses to spokes-characters

Anthropomorphism is a cognitive bias, which occurs when individuals see human characteristics in a non-human agent, object or animal. Anthropomorphism is especially interesting to marketers, because once anthropomorphic bias has been triggered, it can lead to a greater feeling of connectedness to a non-human agent (Tam, Lee and Chao, 2013), the emulation of behaviours (Aggarwal and McGill, 2012) or greater attribution of brand personality and brand liking (Delbaere, McQuarrie and Phillips, 2011). Importantly, research now shows that levels of this tendency vary between individuals (Waytz, Cacioppo and Epley, 2010), but research to date has failed to focus on how anthropomorphic tendency influences individual responses to marketing communications messages. Spokes-characters present an ideal context through which to examine this gap, given that they function as personified brands, designed to trigger consumer anthropomorphic tendency. Further, little is understood about how spokes-characters operate and which consumers will prefer them to their human counterparts. Like anthropomorphic research, much empirical work to date has focused on design and outcomes, examining the sender’s encoding process and the feedback generated, but ignoring the individual decoding process that is so important to understanding individual differences and message effectiveness. The current research employs three experiments using an online survey with stimulus exposure to show that anthropomorphic tendency, personality similarity and spokes-character type all have relevance to the understanding of this complex relationship. Study one and two indicate that while a human spokesperson is still preferred by many, higher levels of anthropomorphic tendency increase likeability of cartoon spokes characters. Study three highlights the importance of personality similarity, which further increases likability. Additional analyses provide key findings concerning the nature of anthropomorphic tendency as an individual difference and trait. This research contributes to a greater understanding of anthropomorphism theory and fills existing gaps in the consumer psychology and marketing communications literature.

Are two threats worse than one? The effects of face race and emotional expression on fear conditioning

Facial cues of racial outgroup or anger mediate fear learning that is resistant to extinction. Whether this resistance is potentiated if fear is conditioned to angry, other race faces has not been established. Two groups of Caucasian participants were conditioned with two happy and two angry face conditional stimuli (CSs). During acquisition, one happy and one angry face were paired with an aversive unconditional stimulus whereas the second happy and angry faces were presented alone. CS face race (Caucasian, African American) was varied between groups. During habituation, electrodermal responses were larger to angry faces regardless of race and declined less to other race faces. Extinction was immediate for Caucasian happy faces, delayed for angry faces regardless of race, and slowest for happy racial outgroup faces. Combining the facial cues of other race and anger does not enhance resistance to extinction of fear.

Packed red blood cells suppress myeloid dendritic cell and monocyte inflammatory responses in a whole blood model of transfusion

Purpose/Objective: The basis for poor outcomes in some patients post transfusion remains largely unknown. Despite leukodepletion, there is still evidence of immunomodulatory effects of transfusion that require further study. In addition, there is evidence that the age of blood components transfused significantly affects patient outcomes. Myeloid dendritic cell (DC) and monocyte immune function were studied utilising an in vitro whole blood model of transfusion. Materials and methods: Freshly collected (‘recipient’) whole blood was cultured with ABO compatible leukodepleted PRBC at 25% blood replacement-volume (6hrs). PRBC were assayed at [Day (D) 2, 14, 28and 42 (date-of expiry)]. In parallel, LPS or Zymosan (Zy) were added to mimic infection. Recipients were maintained for the duration of the time course (2 recipients, 4 PRBC units, n = 8).Recipient DC and monocyte intracellular cytokines and chemokines (IL-6, IL-10, IL-12,TNF-a, IL-1a, IL-8, IP-10, MIP-1a, MIP-1b, MCP-1) were measured using flow cytometry. Changes in immune response were calculated by comparison to a parallel no transfusion control (Wilcoxin matched pairs). Influence of storage age was calculated using ANOVA. Results: Significant suppression of DC and monocyte inflammatory responses were evident. DC and monocyte production of IL-1a was reduced following exposure to PRBC regardless of storage age (P < 0.05 at all time points). Storage independent PRBC mediated suppression of DC and monocyte IL-1a was also evident in cultures costimulated with Zy. In cultures co-stimulated with either LPS or Zy, significant suppression of DC and monocyte TNF-a and IL-6 was also evident. PRBC storage attenuated monocyte TNF-a production when co-cultured with LPS (P < 0.01 ANOVA). DC and monocyte production of MIP-1a was significantly reduced following exposure to PRBC (DC: P < 0.05 at D2, 28, 42; Monocyte P < 0.05 all time points). In cultures co-stimulated with LPS and zymosan, a similar suppression of MIP-1a production was also evident, and production of both DC and monocyte MIP-1b and IP-10 were also significantly reduced. Conclusions: The complexity of the transfusion context was reflected in the whole blood approach utilised. Significant suppression of these key DC and monocyte immune responses may contribute to patient outcomes, such as increased risk of infection and longer hospital stay, following blood transfusion.

Bridging the biology of the ovine TRALI model – human inflammatory cell responses to TRALI inducing supernatants

Background Transfusion-related acute lung injury (TRALI) is a serious and potentially fatal consequence of transfusion. A two-event TRALI model demonstrated date-of-expiry - day (D) 5 platelet (PLT) and D42 packed red blood cell (PRBC) supernatants (SN) induced TRALI in LPS-treated sheep. We have adapted a whole blood transfusion culture model as an investigative bridge between the ovine TRALI model human responses to transfusion. Methods A whole blood transfusion model was adapted to replicate the ovine model - specifically +/- 0.23μg/mL LPS as the first event and 10% SN volume (transfusion) as the second event. Four pooled SN from blood products, previously used in the TRALI ovine model, were investigated: D1-PLT, D5-PLT, D1-PRBC, and D42-PRBC. Fresh human whole blood (recipient) was mixed with combinations of LPS and BP-SN stimuli and incubated in vitro for 6 hrs. Addition of golgi plug enabled measurement of monocyte cytokine production (IL-6, IL-8, IL-10, IL-12, TNF-α, IL-1α, CXCL-5, IP-10, MIP-1α, MCP-1) using multi-colour flow cytometry. Responses for 6 recipients were assessed. Results In the presence of LPS, D42-PRBC-SN significantly increased monocyte IL-6 (P=0.031), IL-8 (P=0.016) and IL-1α (P=0.008) production compared to D1-PRBC-SN. This response to D42-PRBC-SN was LPS-dependent, and was not evident in non-LPSstimulated controls. This response was also specific to D42-PRBC-SN, as similar changes were not evident for the D5-PLT-SN, compared to the D1-PLT-SN, regardless of the presence of LPS. D5-PLT-SN significantly increased IL-12 production (P=0.024) compared to D1-PLT-SN. This response was again LPS-dependent. Conclusions These data demonstrate a novel two-event mechanism of monocyte inflammatory response that was dependent upon both the presence of date-of-expiry blood product SN and LPS. Further, these results demonstrate different cytokines responses induced by date-of-expiry PLT-SN and PRBC-SN. These data are consistent with the evidence from the ovine TRALI model, and enhancing its relevance to transfusion related changes in humans.

Low formalin concentrations induce fine-tuned responses that are sex and age-dependent : a developmental study

The formalin test is increasingly applied as a model of inflammatory pain using high formalin concentrations (5–15%). However, little is known about the effects of low formalin concentrations on related behavioural responses. To examine this, rat pups were subjected to various concentrations of formalin at four developmental stages: 7, 13, 22, and 82 days of age. At postnatal day (PND) 7, sex differences in flinching but not licking responses were observed with 0.5% formalin evoking higher flinching in males than in females. A dose response was evident in that 0.5% formalin also produced higher licking responses compared to 0.3% or 0.4% formalin. At PND 13, a concentration of 0.8% formalin evoked a biphasic response. At PND 22, a concentration of 1.1% evoked higher flinching and licking responses during the late phase (10–30 min) in both males and females. During the early phase (0–5 min), 1.1% evoked higher licking responses compared to 0.9% or 1% formalin. 1.1% formalin produced a biphasic response that was not evident with 0.9 or 1%. At PND 82, rats displayed a biphasic pattern in response to three formalin concentrations (1.25%, 1.75% and 2.25%) with the presence of an interphase for both 1.75% and 2.25% but not for 1.25%. These data suggest that low formalin concentrations induce fine-tuned responses that are not apparent with the high formalin concentration commonly used in the formalin test. These data also show that the developing nociceptive system is very sensitive to subtle changes in formalin concentrations.

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state

Sex-based comparisons of myofibrillar protein synthesis after resistance exercise in the fed state. J Appl Physiol 112: 1805-1813, 2012. First published March 1, 2012; doi:10.1152/japplphysiol.00170.2012.- We made sex-based comparisons of rates of myofibrillar protein synthesis (MPS) and anabolic signaling after a single bout of high-intensity resistance exercise. Eight men (20 ± 10 yr, BMI = 24.3 ± 2.4) and eight women (22 ± 1.8 yr, BMI = 23.0 ± 1.9) underwent primed constant infusions of L-[ring-13C6]phenylalanine on consecutive days with serial muscle biopsies. Biopsies were taken from the vastus lateralis at rest and 1, 3, 5, 24, 26, and 28 h after exercise. Twenty-five grams of whey protein was ingested immediately and 26 h after exercise. We also measured exercise-induced serum testosterone because it is purported to contribute to increases in myofibrillar protein synthesis (MPS) postexercise and its absence has been hypothesized to attenuate adaptative responses to resistance exercise in women. The exercise-induced area under the testosterone curve was 45-fold greater in men than women in the early (1 h) recovery period following exercise (P < 0.001). MPS was elevated similarly in men and women (2.3- and 2.7-fold, respectively) 1-5 h postexercise and after protein ingestion following 24 h recovery. Phosphorylation of mTORSer2448 was elevated to a greater extent in men than women acutely after exercise (P = 0.003), whereas increased phosphorylation of p70S6K1Thr389 was not different between sexes. Androgen receptor content was greater in men (main effect for sex, P = 0.049). Atrogin-1 mRNA abundance was decreased after 5 h recovery in both men and women (P < 0.001), and MuRF-1 expression was elevated in men after protein ingestion following 24 h recovery (P = 0.003). These results demonstrate minor sex-based differences in signaling responses and no difference in the MPS response to resistance exercise in the fed state. Interestingly, our data demonstrate that exerciseinduced increases in MPS are dissociated from postexercise testosteronemia and that stimulation of MPS occurs effectively with low systemic testosterone concentrations in women.

Rapid aminoacidemia enhances myofibrillar protein synthesis and anabolic intramuscular signaling responses after resistance exercise

Background: Ingestion of whey or casein yields divergent patterns of aminoacidemia that influence whole-body and skeletal muscle myofibrillar protein synthesis (MPS) after exercise. Direct comparisons of the effects of contrasting absorption rates exhibited by these proteins are confounded by their differing amino acid contents. Objective: Our objective was to determine the effect of divergent aminoacidemia by manipulating ingestion patterns of whey protein alone on MPS and anabolic signaling after resistance exercise. Design: In separate trials, 8 healthy men consumed whey protein either as a single bolus (BOLUS; 25-g dose) or as repeated, small, "pulsed" drinks (PULSE; ten 2.5-g drinks every 20 min) to mimic a more slowly digested protein. MPS and phosphorylation of signaling proteins involved in protein synthesis were measured at rest and after resistance exercise. Results: BOLUS increased blood essential amino acid (EAA) concentrations above those of PULSE (162% compared with 53%, P < 0.001) 60 min after exercise, whereas PULSE resulted in a smaller but sustained increase in aminoacidemia that remained elevated above BOLUS amounts later (180-220 min after exercise, P < 0.05). Despite an identical net area under the EAA curve, MPS was elevated to a greater extent after BOLUS than after PULSE early (1-3 h: 95% compared with 42%) and later (3-5 h: 193% compared with 121%) (both P < 0.05). There were greater changes in the phosphorylation of the Akt-mammalian target of rapamycin pathway after BOLUS than after PULSE. Conclusions: Rapid aminoacidemia in the postexercise period enhances MPS and anabolic signaling to a greater extent than an identical amount of protein fed in small pulses that mimic a more slowly digested protein. A pronounced peak aminoacidemia after exercise enhances protein synthesis.

Effect of high-frequency resistance exercise on adaptive responses in skeletal muscle

PURPOSE: Regulation of skeletal muscle mass is highly dependent on contractile loading. The purpose of this study was to examine changes in growth factor and inflammatory pathways following high-frequency resistance training. METHODS: Using a novel design in which male Sprague-Dawley rats undertook a "stacked" resistance training protocol designed to generate a summation of transient exercise-induced signaling responses (four bouts of three sets × 10 repetitions of squat exercise, separated by 3 h of recovery), we determined the effects of high training frequency on signaling pathways and transcriptional activity regulating muscle mass. RESULTS: The stacked training regimen resulted in acute suppression of insulin-like growth factor 1 mRNA abundance (P < 0.05) and Akt phosphorylation (P < 0.05), an effect that persisted 48 h after the final training bout. Conversely, stacked training elicited a coordinated increase in the expression of tumor necrosis factor alpha, inhibitor kappa B kinase alpha/beta activity (P < 0.05), and p38 mitogen-activated protein kinase phosphorylation (P < 0.05) at 3 h after each training bout. In addition, the stacked series of resistance exercise bouts induced an increase in p70 S6 kinase phosphorylation 3 h after bouts ×3 and ×4, independent of the phosphorylation state of Akt. CONCLUSIONS: Our results indicate that high resistance training frequency extends the transient activation of inflammatory signaling cascades, concomitant with persistent suppression of key mediators of anabolic responses. We provide novel insights into the effects of the timing of exercise-induced overload and recovery on signal transduction pathways and transcriptional activity regulating skeletal muscle mass in vivo.

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.

Behavioral evaluation of eight rat lines selected for high and low anxiety-related responses

Anxiety traits can be stable and permanent characteristics of an individual across time that is less susceptible of influences by a particular situation. One way to study trait anxiety in an experimental context is through the use of rat lines, selected according to contrasting phenotypes of fear and anxiety. It is not clear whether the behavioral differences between two contrasting rat lines in one given anxiety test are also present in others paradigms of state anxiety. Here, we examine the extent to which multiple anxiety traits generalize across selected animal lines originally selected for a single anxiety trait. We review the behavioral results available in the literature of eight rat genetic models of trait anxiety - namely Maudsley Reactive and Non-reactive rats, Floripa H and L rats, Tsukuba High and Low Emotional rats, High and Low Anxiety-related rats, High and Low Ultrasonic Vocalization rats, Roman High and Low Avoidance rats, Syracuse High and Low Avoidance rats, and Carioca High and Low Conditioned Freezing rats - across 11 behavioral paradigms of innate anxiety or aversive learning frequently used in the experimental setting. We observed both convergence and divergence of behavioral responses in these selected lines across the 11 paradigms. We find that predisposition for specific anxiety traits will usually be generalized to other anxiety provoking stimuli. However this generalization is not observed across all genetic models indicating some unique trait and state interactions. Genetic models of enhanced-anxiety related responses are beginning to help define how anxiety can manifest differently depending on the underlying traits and the current environmentally induced state.

How can Australia sustain middle school education?

Middle school is a crucial area of education where adolescents experiencing physiological and psychological changes, and require expert guidance. As more research evidence is provided about adolescent learning, teachers are considered pivotal to adolescents’ educational development. Reform measures need to be targeted at the inservice and preservice teacher levels. This quantitative study employs a 40-item, five part Likert scale survey to understand preservice teachers’ (n = 142) perceptions of their confidence to teach in a middle school at the conclusion of their tertiary education. The survey instrument was developed from the literature, with connections to the Queensland College of Teachers' professional standards. Results indicated that they perceived themselves as capable of creating a positive classroom environment with seven items greater than 80%, except with behaviour management (< 80% for two items), and they considered their pedagogical knowledge to be adequate (i.e., 7 out of 8 items > 84%). Items associated with implementing a middle school curriculum had varied responses (e.g., implementing literacy and numeracy were 74%, while implementing learning with real world connections was 91%). This information may assist coursework designers. For example, if a significant percentages of preservice teachers indicate that they believe they were not well prepared for assessment and reporting at the middle school level, then course designers can target these areas more effectively.