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.

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly 3-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 mg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing microcomputed tomography (mCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non- BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by mCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, microcompression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects. Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.

Simultaneous kinetic spectrophotometric determination of norfloxacin and rifampicin in pharmaceutical formulation and human urine samples by use of chemometrics approaches

A kinetic spectrophotometric method with aid of chemometrics is proposed for the simultaneous determination of norfloxacin and rifampicin in mixtures. The proposed method was applied for the simultaneous determination of these two compounds in pharmaceutical formulation and human urine samples, and the results obtained are similar to those obtained by high performance liquid chromatography.

Vibrational spectroscopic studies of laboratory scale polymer melt processing : application to a thermoplastic polyurethane nanocomposite

A laboratory scale twin screw extruder has been interfaced with a near infrared (NIR) spectrometer via a fibre optic link so that NIR spectra can be collected continuously during the small scale experimental melt state processing of polymeric materials. This system can be used to investigate melt state processes such as reactive extrusion, in real time, in order to explore the kinetics and mechanism of the reaction. A further advantage of the system is that it has the capability to measure apparent viscosity simultaneously which gives important additional information about molecular weight changes and polymer degradation during processing. The system was used to study the melt processing of a nanocomposite consisting of a thermoplastic polyurethane and an organically modified layered silicate.

A psychosocial approach to understanding young Australians' mobile phone behaviour

This thesis by publication contributes to our knowledge of psychological factors underlying a modern day phenomenon, young people’s mobile phone behaviour. Specifically, the thesis reports a PhD program of research which adopted a social psychological approach to explore mobile phone behaviour among young Australians aged between 15 and 24 years. A particular focus of the research program was to explore both the cognitive and behavioural aspects of young people’s mobile phone behaviour which for the purposes of this thesis is defined as mobile phone involvement. The research program comprised three separate stages which were developmental in nature, in that, the findings of each stage of the research program informed the next. The overarching goal of the program of research was to improve our understanding of the psychosocial factors influencing young people’s mobile phone behaviour. To achieve this overall goal, there were a number of aims to the research program which reflect the developmental nature of this thesis. Given the limited research into the mobile phone behaviour in Australia, the first two aims of the research program were to explore patterns of mobile phone behaviour among Australian youth and explore the social psychological factors relating to their mobile phone behaviour. Following this exploration, the research program sought to develop a measure which captures the cognitive and behavioural aspects of mobile phone behaviour. Finally, the research program aimed to examine and differentiate the psychosocial predictors of young people’s frequency of mobile phone use and their level of involvement with their mobile phone. Both qualitative and quantitative methodologies were used throughout the program of research. Five papers prepared during the three stages of the research program form the bulk of this thesis. The first stage of the research program was a qualitative investigation of young people’s mobile phone behaviour. Thirty-two young Australians participated in a series of focus groups in which they discussed their mobile phone behaviour. Thematic data analysis explored patterns of mobile phone behaviour among young people, developed an understanding of psychological factors influencing their use of mobile phones, and identified that symptoms of addiction were emerging in young people’s mobile phone behaviour. Two papers (Papers 1 and 2) emanated from this first stage of the research program. Paper 1 explored patterns of mobile phone behaviour and revealed that mobile phones were perceived as being highly beneficial to young people’s lives, with the ability to remain in constant contact with others being particularly valued. The paper also identified that symptoms of behavioural addiction including withdrawal, cognitive and behavioural salience, and loss of control, emerged in participants’ descriptions of their mobile phone behaviour. Paper 2 explored how young people’s need to belong and their social identity (two constructs previously unexplored in the context of mobile phone behaviour) related to their mobile phone behaviour. It was revealed that young people use their mobile phones to facilitate social attachments. Additionally, friends and peers influenced young people’s mobile phone behaviour; for example, their choice of mobile phone carrier and their most frequent type of mobile phone use. These papers laid the foundation for the further investigation of addictive patterns of behaviour and the role of social psychological factors on young people’s mobile behaviour throughout the research program. Stage 2 of the research program focussed on developing a new parsimonious measure of mobile phone behaviour, the Mobile Phone Involvement Questionnaire (MPIQ), which captured the cognitive and behavioural aspects of mobile phone use. Additionally, the stage included a preliminary exploration of factors influencing young people’s mobile phone behaviour. Participants (N = 946) completed a questionnaire which included a pool of items assessing symptoms of behavioural addiction, the uses and gratifications relating to mobile phone use, and self-identity and validation from others in the context of mobile phone behaviour. Two papers (Papers 3 & 4) emanated from the second stage of the research program. Paper 3 provided an important link between the qualitative and quantitative components of the research program. Qualitative data from Stage 1 indicated the reasons young people use their mobile phones and identified addictive characteristics present in young people’s mobile phone behaviour. Results of the quantitative study conducted in Stage 2 of the research program revealed the uses and gratifications relating to young people’s mobile phone behaviour and the effect of these gratifications on young people’s frequency of mobile phone use and three indicators of addiction, withdrawal, salience, and loss of control. Three major uses and gratifications: self (such as feeling good or as a fashion item), social (such as contacting friends), and security (such as use in an emergency) were found to underlie much of young people’s mobile phone behaviour. Self and social gratifications predicted young people’s frequency of mobile phone use and the three indicators of addiction but security gratifications did not. These results provided an important foundation for the inclusion of more specific psychosocial predictors in the later stages of the research program. Paper 4 reported the development of the mobile phone involvement questionnaire and a preliminary exploration of the effect of self-identity and validation from others on young people’s mobile phone behaviour. The MPIQ assessed a unitary construct and was a reliable measure amongst this cohort. Results found that self-identity influenced the frequency of young people’s use whereas self-identity and validation from others influenced their level of mobile phone involvement. These findings provided an important indication that, in addition to self factors, other people have a strong influence on young people’s involvement with their mobile phone and that mobile phone involvement is conceptually different to frequency of mobile phone use. Stage 3 of the research program empirically examined the psychosocial predictors of young people’s mobile behaviour and one paper, Paper 5, emanated from this stage. Young people (N = 292) from throughout Australia completed an online survey assessing the role of self-identity, ingroup norm, the need to belong, and self-esteem on their frequency of mobile phone use and their mobile phone involvement. Self-identity was the only psychosocial predictor of young people’s frequency of mobile phone use. In contrast, self-identity, ingroup norm, and need to belong all influenced young people’s level of involvement with their mobile phone. Additionally, the effect of self-esteem on young people’s mobile phone involvement was mediated by their need to belong. These results indicate that young people who perceive their mobile phone to be an integral part of their self-identity, who perceive that mobile phone is common amongst friends and peers, and who have a strong need for attachment to others, in some cases driven by a desire to enhance their self-esteem, are most likely to become highly involved with their mobile phones. Overall, this PhD program of research has provided an important contribution to our understanding of young Australians’ mobile phone behaviour. Results of the program have broadened our knowledge of factors influencing mobile phone behaviour beyond the approaches used in previous research. The use of various social psychological theories combined with a behavioural addiction framework provided a novel examination of young people’s mobile behaviour. In particular, the development of a new measure of mobile phone behaviour in the research program facilitated the differentiation of the psychosocial factors influencing frequency of young people’s mobile phone behaviour and their level of involvement with their mobile phone. Results of the research program indicate the important role that mobile phone behaviour plays in young people’s social development and also signals the characteristics of those people who may become highly involved with their mobile phone. Future research could build on this thesis by exploring whether mobile phones are affecting traditional social psychological processes and whether the results in this research program are generalisable to other cohorts and other communication technologies.

Layered titanate nanofibers as efficient adsorbents for removal of toxic radioactive and heavy metal ions from water

Titanate nanofibers with two formulas, Na2Ti3O7 and Na1.5H0.5Ti3O7, respectively, exhibit ideal properties for removal of radioactive and heavy metal ions in wastewater, such as Sr2+ , Ba2+ (as substitute of 226Ra2+), and Pb2+ ions. These nanofibers can be fabricated readily by a reaction between titania and caustic soda and have structures in which TiO6 octahedra join each other to form layers with negative charges; the sodium cations exist within the interlayer regions and are exchangeable. They can selectively adsorb the bivalent radioactive ions and heavy metal ions from water through ion exchange process. More importantly, such sorption finally induces considerable deformation of the layer structure, resulting in permanent entrapment of the toxic bivalent cations in the fibers so that the toxic ions can be safely deposited. This study highlights that nanoparticles of inorganic ion exchangers with layered structure are potential materials for efficient removal of the toxic ions from contaminated water.