Composite electrospun scaffolds for engineering tubular bone grafts

In this study, poly (e-caprolactone) [PCL] and its collagen composite blend (PCL=Col) were fabricated to scaffolds using electrospinning method. Incorporated collagen was present on the surface of the fibers, and it modulated the attachment and proliferation of pig bone marrow mesenchymal cells (pBMMCs). Osteogenic differentiation markers were more pronounced when these cells were cultured on PCL=Col fibrous meshes, as determined by immunohistochemistry for collagen type I, osteopontin, and osteocalcin. Matrix mineralization was observed only on osteogenically induced PCL=Col constructs. Long bone analogs were created by wrapping osteogenic cell sheets around the PCL=Col meshes to form hollow cylindrical cell-scaffold constructs. Culturing these constructs under dynamic conditions enhanced bone-like tissue formation and mechanical strength.We conclude that electrospun PCL=Col mesh is a promising material for bone engineering applications. Its combination with osteogenic cell sheets offers a novel and promising strategy for engineering of tubular bone analogs.

Modelling the effects of bone fragment contact in fracture healing

The fracture healing process is modulated by the mechanical environment created by imposed loads and motion between the bone fragments. Contact between the fragments obviously results in a significantly different stress and strain environment to a uniform fracture gap containing only soft tissue (e.g. haematoma). The assumption of the latter in existing computational models of the healing process will hence exaggerate the inter-fragmentary strain in many clinically-relevant cases. To address this issue, we introduce the concept of a contact zone that represents a variable degree of contact between cortices by the relative proportions of bone and soft tissue present. This is introduced as an initial condition in a two-dimensional iterative finite element model of a healing tibial fracture, in which material properties are defined by the volume fractions of each tissue present. The algorithm governing the formation of cartilage and bone in the fracture callus uses fuzzy logic rules based on strain energy density resulting from axial compression. The model predicts that increasing the degree of initial bone contact reduces the amount of callus formed (periosteal callus thickness 3.1mm without contact, down to 0.5mm with 10% bone in contact zone). This is consistent with the greater effective stiffness in the contact zone and hence, a smaller inter-fragmentary strain. These results demonstrate that the contact zone strategy reasonably simulates the differences in the healing sequence resulting from the closeness of reduction.

Multi-level order, friction and contradiction : the evolution of Australian film industry policy

Cultural policy studies have previously highlighted the importance of multiple logics, friction and contradiction in cultural policy. Recent developments in institutional theory provide a framework for analysing change in cultural policy which explores movement between these multiple and sometimes contradictory logics. This paper analyses the role of friction in the evolution of Australian film industry policy and in particular the tension between competing logics regarding nationalism, commercialism and the state. The paper is suggestive of the relevance of institutional theory as a framework for understanding cultural policy evolution.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.

P2 purinergic receptor mRNA in rat and human sinoatrial node and other heart regions.

It is known that adenosine 5'-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X(1-7) and P2Y(1-14) receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X(5) was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y(1), P2Y(2), and P2Y(14) mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y(2) and P2Y(14) levels were highest, respectively. We extended these studies to investigate P2X(4) receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X(4) receptor mRNA was upregulated by 93% in SAN (P < 0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X(4)-mediated effects might be modulated in heart failure. mRNA for P2X(4-7) and P2Y(1,2,4,6,12-14), but not P2X(2,3) and P2Y(11), was detected in human right atrium and SAN. In addition, mRNA for P2X(1) was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X(4) and P2X(7) mRNA was the highest for P2X receptors. P2Y(1) and P2Y(2) mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y(1), P2Y(2), and P2Y(14) were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility.

Mathematical models for describing the shape of the in vitro unstretched human crystalline lens

We developed orthogonal least-squares techniques for fitting crystalline lens shapes, and used the bootstrap method to determine uncertainties associated with the estimated vertex radii of curvature and asphericities of five different models. Three existing models were investigated including one that uses two separate conics for the anterior and posterior surfaces, and two whole lens models based on a modulated hyperbolic cosine function and on a generalized conic function. Two new models were proposed including one that uses two interdependent conics and a polynomial based whole lens model. The models were used to describe the in vitro shape for a data set of twenty human lenses with ages 7–82 years. The two-conic-surface model (7 mm zone diameter) and the interdependent surfaces model had significantly lower merit functions than the other three models for the data set, indicating that most likely they can describe human lens shape over a wide age range better than the other models (although with the two-conic-surfaces model being unable to describe the lens equatorial region). Considerable differences were found between some models regarding estimates of radii of curvature and surface asphericities. The hyperbolic cosine model and the new polynomial based whole lens model had the best precision in determining the radii of curvature and surface asphericities across the five considered models. Most models found significant increase in anterior, but not posterior, radius of curvature with age. Most models found a wide scatter of asphericities, but with the asphericities usually being positive and not significantly related to age. As the interdependent surfaces model had lower merit function than three whole lens models, there is further scope to develop an accurate model of the complete shape of human lenses of all ages. The results highlight the continued difficulty in selecting an appropriate model for the crystalline lens shape.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Different approaches to reduce shaft voltage in AC generators

This paper presents several shaft voltage reduction techniques for doubly-fed induction generators in wind turbine applications. These techniques includes: pulse width modulated voltage without zero vectors, multi-level inverters with proper PWM strategy, better generator design to minimize effective capacitive couplings in shaft voltage, active common-mode filter, reducing dc-link voltage and increasing modulation index. These methods have been verified with mathematical analysis and simulations.

Heparan sulfate mediates the proliferation and differentiation of rat mesenchymal stem cells

The growth and differentiation of mesenchymal stem cells is controlled by various growth factors, the activities of which can be modulated by heparan sulfates. We have previously underscored the necessity of sulfated glycosaminoglycans for the FGF-2-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of heparan sulfate to cultures of primary rat MSCs stimulates their proliferation leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation and osteogenic differentiation of rMSCs when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. Furthermore, we show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth resulting in the cells being unable to reach the critical density necessary to induce differentiation. Interestingly, blocking FGFR1 signaling in post-confluent osteogenic cultures significantly increased calcium deposition. Taken together our data clearly suggests that FGFR1 signaling plays an important role during osteogenic differentiation, firstly by stimulating cell growth that is closely followed by an inhibitory affect once the cells have reached confluence. It also underlines the importance of HS as a co-receptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.

A comparison of different methods to determine diffusion length in dye-sensitized solar cells

A new steady state method for determination of the electron diffusion length in dye-sensitized solar cells (DSCs) is described and illustrated with data obtained using cells containing three different types of electrolyte. The method is based on using near-IR absorbance methods to establish pairs of illumination intensity for which the total number of trapped electrons is the same at open circuit (where all electrons are lost by interfacial electron transfer) as at short circuit (where the majority of electrons are collected at the contact). Electron diffusion length values obtained by this method are compared with values derived by intensity modulated methods and by impedance measurements under illumination. The results indicate that the values of electron diffusion length derived from the steady state measurements are consistently lower than the values obtained by the non steady-state methods. For all three electrolytes used in the study, the electron diffusion length was sufficiently high to guarantee electron collection efficiencies greater than 90%. Measurement of the trap distributions by near-IR absorption confirmed earlier observations of much higher electron trap densities for electrolytes containing Li+ ions. It is suggested that the electron trap distributions may not be intrinsic properties of the TiO2 nanoparticles, but may be associated with electron-ion interactions.

The politics of literacy in David Almond and Dave McKean’s "The Savage" (2008)

Competency in language and literacy are central to contemporary debates about education in Anglophone nations around the world. This paper suggests that such debates are informing not just educational policy but children’s literature itself as can be seen in Almond and McKean’s The Savage. This hybrid text combines prose and graphic narrative and narration in order to tell the story of Blue, a young British boy negotiating his identity in the aftermath of his father's death. While foregrounding a narrative of ideal masculinity, The Savage enacts and privileges a formal and thematic ideal of literacy as index of individual agency and development. Almond and McKean produce a politicised understanding of language and literacy that simultaneously positions The Savage in a textual tradition of socio-culturally disenfranchised youth, and intervenes in that tradition to (perhaps ironically) affirm the very conditions previously critiqued by that very tradition. Where earlier authors such as Barry Hines sought to challenge normative accounts of language and literacy in order to indict educational policy and praxes, Almond and McKean work to naturalise the very logics of education and agency by which their protagonist has been disenfranchised. In doing so, The Savage exemplifies current approaches to education which claim to value social and cultural diversity while imposing national standardised testing predicated on assumptions about the legitimacy of uniform standards and definitions of literacy.

Animal Control - What constitutes a reliable cue to stop animal movement ?

Controlling free-ranging livestock requires low-stress cues to alter animal behaviour. Recently modulated sound and electric shock were demonstrated to be effective in controlling free-ranging cattle. In this study the behaviour of 60, 300 kg Belmont Red heifers were observed for behavioural changes when presented cues designed to impede their movement through an alley. The heifers were given an overnight drylot shrink off feed but not drinking water prior to being tested. Individual cattle were allowed to move down a 6.5 m wide alley towards a pen of peers and feed located 71 m from their point of release. Each animal was allowed to move through the alley unimpeded five times to establish a basal behavioural pattern. Animals were then randomly assigned to treatments consisting of sound plus shock, vibration plus shock, a visual cue plus shock, shock by itself and a control. The time each animal required to reach the pen of peers and feed was recorded. If the animal was prevented from reaching the pen of peers and feed by not penetrating through the cue barrier at set points along the alley for at least 60 sec the test was stopped and the animal was returned to peers located behind the release pen. Cues and shock were manually applied from a laptop while animals were observed from a 3.5 m tower located outside the alley. Electric shock, sound, vibration and Global Position System (GPS) hardware were housed in a neck collar. Results and implications will be discussed.

The regulation of food intake in humans

Knowledge of the regulation of food intake is crucial to an understanding of body weight and obesity. Strictly speaking, we should refer to the control of food intake whose expression is modulated in the interests of the regulation of body weight. Food intake is controlled, body weight is regulated. However, this semantic distinction only serves to emphasize the importance of food intake. Traditionally food intake has been researched within the homeostatic approach to physiological systems pioneered by Claude Bernard, Walter Cannon and others; and because feeding is a form of behaviour, it forms part of what Curt Richter referred to as the behavioural regulation of body weight (or behavioural homeostasis). This approach views food intake as the vehicle for energy supply whose expression is modulated by a metabolic drive generated in response to a requirement for energy. The idea was that eating behaviour is stimulated and inhibited by internal signalling systems (for the drive and suppression of eating respectively) in order to regulate the internal environment (energy stores, tissue needs).

Performance analysis of adaptive lattice filters for FM signals and alpha-stable processes

The performance of an adaptive filter may be studied through the behaviour of the optimal and adaptive coefficients in a given environment. This thesis investigates the performance of finite impulse response adaptive lattice filters for two classes of input signals: (a) frequency modulated signals with polynomial phases of order p in complex Gaussian white noise (as nonstationary signals), and (b) the impulsive autoregressive processes with alpha-stable distributions (as non-Gaussian signals). Initially, an overview is given for linear prediction and adaptive filtering. The convergence and tracking properties of the stochastic gradient algorithms are discussed for stationary and nonstationary input signals. It is explained that the stochastic gradient lattice algorithm has many advantages over the least-mean square algorithm. Some of these advantages are having a modular structure, easy-guaranteed stability, less sensitivity to the eigenvalue spread of the input autocorrelation matrix, and easy quantization of filter coefficients (normally called reflection coefficients). We then characterize the performance of the stochastic gradient lattice algorithm for the frequency modulated signals through the optimal and adaptive lattice reflection coefficients. This is a difficult task due to the nonlinear dependence of the adaptive reflection coefficients on the preceding stages and the input signal. To ease the derivations, we assume that reflection coefficients of each stage are independent of the inputs to that stage. Then the optimal lattice filter is derived for the frequency modulated signals. This is performed by computing the optimal values of residual errors, reflection coefficients, and recovery errors. Next, we show the tracking behaviour of adaptive reflection coefficients for frequency modulated signals. This is carried out by computing the tracking model of these coefficients for the stochastic gradient lattice algorithm in average. The second-order convergence of the adaptive coefficients is investigated by modeling the theoretical asymptotic variance of the gradient noise at each stage. The accuracy of the analytical results is verified by computer simulations. Using the previous analytical results, we show a new property, the polynomial order reducing property of adaptive lattice filters. This property may be used to reduce the order of the polynomial phase of input frequency modulated signals. Considering two examples, we show how this property may be used in processing frequency modulated signals. In the first example, a detection procedure in carried out on a frequency modulated signal with a second-order polynomial phase in complex Gaussian white noise. We showed that using this technique a better probability of detection is obtained for the reduced-order phase signals compared to that of the traditional energy detector. Also, it is empirically shown that the distribution of the gradient noise in the first adaptive reflection coefficients approximates the Gaussian law. In the second example, the instantaneous frequency of the same observed signal is estimated. We show that by using this technique a lower mean square error is achieved for the estimated frequencies at high signal-to-noise ratios in comparison to that of the adaptive line enhancer. The performance of adaptive lattice filters is then investigated for the second type of input signals, i.e., impulsive autoregressive processes with alpha-stable distributions . The concept of alpha-stable distributions is first introduced. We discuss that the stochastic gradient algorithm which performs desirable results for finite variance input signals (like frequency modulated signals in noise) does not perform a fast convergence for infinite variance stable processes (due to using the minimum mean-square error criterion). To deal with such problems, the concept of minimum dispersion criterion, fractional lower order moments, and recently-developed algorithms for stable processes are introduced. We then study the possibility of using the lattice structure for impulsive stable processes. Accordingly, two new algorithms including the least-mean P-norm lattice algorithm and its normalized version are proposed for lattice filters based on the fractional lower order moments. Simulation results show that using the proposed algorithms, faster convergence speeds are achieved for parameters estimation of autoregressive stable processes with low to moderate degrees of impulsiveness in comparison to many other algorithms. Also, we discuss the effect of impulsiveness of stable processes on generating some misalignment between the estimated parameters and the true values. Due to the infinite variance of stable processes, the performance of the proposed algorithms is only investigated using extensive computer simulations.