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.

Complementarity and the uncertainty principle as aesthetic principles : the practice and performance of The Physics Project

Using the generative processes developed over two stages of creative development and the performance of The Physics Project at the Loft at the Creative Industries Precinct at the Queensland University of Technology (QUT) from 5th – 8th April 2006 as a case study, this exegesis considers how the principles of contemporary physics can be reframed as aesthetic principles in the creation of contemporary performance. The Physics Project is an original performance work that melds live performance, video and web-casting and overlaps an exploration of personal identity with the physics of space, time, light and complementarity. It considers the acts of translation between the language of physics and the language of contemporary performance that occur via process and form. This exegesis also examines the devices in contemporary performance making and contemporary performance that extend the reach of the performance, including the integration of the live and the mediated and the use of metanarratives.

Choice of image features for depth-axis control in image based visual servo control

This paper is concerned with choosing image features for image based visual servo control and how this choice influences the closed-loop dynamics of the system. In prior work, image features tend to be chosen on the basis of image processing simplicity and noise sensitivity. In this paper we show that the choice of feature directly influences the closed-loop dynamics in task-space. We focus on the depth axis control of a visual servo system and compare analytically various approaches that have been reported recently in the literature. The theoretical predictions are verified by experiment.

Defending the sanctity of life principle : a reply to John Keown

This article is a response to Professor Keown’s criticism of my paper “Finding a Way Through the Ethical and Legal Maze: Withdrawal of Medical Treatment and Euthanasia” (2005) 13 (3) Medical Law Review 357. The article takes up and responds to a number of criticisms raised by Keown in an attempt to further the debate concerning the moral and legal status of withdrawing life-sustaining measures, its distinction from euthanasia, and the implications of the lawfulness of withdrawal for the principle of the sanctity of life.

The late discovery of adoptive and donor insemination offspring status : ethical implications for conceptual understandings of the ‘best interests of the child’ principle

Late discovery is a term used to describe the experience of discovering the truth of one’s genetic origins as an adult. Following discovery, late discoverers face a lack of recognition and acknowledgment of their concerns from family, friends, community and institutions. They experience pain, anger, loss, grief and frustration. This presentation shares the findings of the first qualitative study of both late discovery of adoptive and donor insemination offspring (heterosexual couple use only) experiences. It is also the first study of late discovery experiences undertaken from an ethical perspective. While this study recruited new participants, it also included an ethical re-analysis of existing late discovery accounts across both practices. The findings of this study (a) draws links between past adoption and current donor insemination (heterosexual couple only) practices, (b) reveals that late discoverers are demanding acknowledgment and recognition of the particularity of their experiences, and (c) offers insights into conceptual understandings of the ‘best interests of the child’ principle. These insights derive from the lived experiences of those whose biological and social worlds have been sundered and secrecy and denial of difference used to conceal this. It suggests that acknowledging the equal moral status of the child may be useful in strengthening conceptual understandings of the ‘best interests of the child’ principle. This equal moral status involves ensuring that personal autonomy and the ability to exercise free will is protected; that the integrity of the relationships of trust expected and demanded between parent/s and children is defended and supported; and that equal access to normative socio-cultural practices, that is; non-fictionalised birth certificates and open records, is guaranteed.

Metacognitive narrative psychotherapy for people diagnosed with schizophrenia : an outline of a principle-based treatment manual

Schizophrenia is often characterised by diminished self-experience. This article describes the development and principles of a manual for a psychotherapeutic treatment model that aims to enhance self-experience in people diagnosed with schizophrenia. Metacognitive Narrative Psychotherapy draws upon dialogical theory of self and the work of Lysaker and colleagues, in conjunction with narrative principles of therapy as operationalised by Vromans. To date, no manual for a metacognitive narrative approach to the treatment of schizophrenia exists. After a brief description of narrative understandings of schizophrenia, the development of the manual is described. Five general phases of treatment are outlined: (1) developing a therapeutic relationship; (2) eliciting narratives; (3) enhancing metacognitive capacity; (4) enriching narratives, and; (5) living enriched narratives. Proscribed practices are also described. Examples of therapeutic interventions and dialogue are provided to further explain the application of interventions in-session. The manual has been piloted in a study investigating the effectiveness of Metacognitive Narrative Psychotherapy in the treatment of people diagnosed with schizophrenia spectrum disorders.

The Human Fertilisation and Embryology Act 2008 : restrictions on the creation of “saviour siblings” and the relevance of the harm principle

This paper provides an overview of the regulatory developments in the UK which impact on the use of in vitro fertilization (IVF) and embryo screening techniques for the creation of “saviour siblings.” Prior to the changes implemented under the Human Fertilisation and Embryology Act 2008, this specific use of IVF was not addressed by the legislative framework and regulated only by way of policy issued by the Human Fertilisation and Embryology Authority (HFEA). Following the implementation of the statutory reforms, a number of restrictive conditions are now imposed on the face of the legislation. This paper considers whether there is any justification for restricting access to IVF and pre-implantation tissue typing for the creation of “saviour siblings.” The analysis is undertaken by examining the normative factors that have guided the development of the UK regulatory approach prior to the 2008 legislative reforms. The approach adopted in relation to the “saviour sibling” issue is compared to more general HFEA policy, which has prioritized the notion of reproductive choice and determined that restrictions on access are only justified on the basis of harm considerations.