Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

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.

Preventing the selection of “deaf embryos” under the Human Fertilisation and Embryology Act 2008 : problematizing disability?

Section 14(4) of the Human Fertilisation and Embryology Act 2008 imposes – within the general licensing conditions listed in the Human Fertilisation and Embryology Act 1990 – a prohibition to prevent the selection and implantation of embryos for the purpose of creating a child who will be born with a “serious disability.” This article offers a perspective that demonstrates the problematic nature of the consultation, review, and legislative reform process surrounding s 14(4). The term “serious disability” is not defined within the legislation, but we highlight the fact that s 14(4) was passed with the case of selecting deaf children in mind. We consider some of the literature on the topic of disability and deafness, which, we think, casts some doubt on the view that deafness is a “serious disability.” The main position we advance is that the lack of serious engagement with alternative viewpoints during the legislative process was unsatisfactory. We argue that the contested nature of deafness necessitates a more robust consultation process and a clearer explanation and defence of the normative position that underpins s 14(4).

Editorial: The Human Fertilisation and Embryology Act 2008

This editorial first describes the workshop out of which the present special issue arose. The editors then identify the need for a multidisciplinary collection examining the Human Fertilisation and Embryology Act 2008 from both legal and political perspectives, including the consultation process, campaigning and parliamentary debates leading to its passage, and the concluded legislation and its effects. The editorial provides an overview of the legislative reform process, key legislative changes, and the various contributions to the special issue. Cross-cutting themes include the value of a qualitative, discourse-based approach to research in this area; the need to understand the 2008 Act in historical context; unforeseen practical implications of the legislative provisions; and silences and missed opportunities in the legislation. Finally, a postscript covers the changing landscape of hybrid embryo research since the passage of the Act, and the uncertain future of the Human Fertilisation and Embryology Authority at the time of writing.

Load bearing improves the limits of agreement for repeated in vivo measures of the speed of sound in human Achilles tendon

Axial acoustic wave propagation has been widely used in evaluating the mechanical properties of human bone in vivo. However, application of this technique to monitor soft tissues, such as tendon, has received comparatively little scientific attention. Laboratory-based research has established that axial acoustic wave transmission is not only related to the physical properties of equine tendon but is also proportional to tensile load to which it is exposed (Miles et al., 1996; Pourcelot et al., 2005). The reproducibility of the technique for in vivo measurements in human tendon, however, has not been established. The aim of this study was to evaluate the limits of agreement for repeated measures of the speed of sound (SoS) in human Achilles tendon in vivo. Methods: A custom built ultrasound device, consisting of an A-mode 1MHz emitter and two regularly spaced receivers, was used to measure the SoS in the mid-portion of the Achilles tendon in ten healthy males and ten females (mean age: 33.8 years, range 23-56 yrs; height: 1.73±0.08 m; weight: 68.4±15.3 kg). The emitter and receivers were held at fixed positions by a polyethylene frame and maintained in close contact with the skin overlying the tendon by means of elasticated straps. Repeated SoS measurements were taken with the subject prone (non-weightbearing and relaxed Achilles tendon) and during quiet bipedal and unipedal stance. In each instance, the device was detached and repositioned prior to measurement. Results: Limits of agreement for repeated SoS measures during non-weightbearing and bipedal and unipedal stance were ±53, ±28 and ±21 m/s, respectively. The average SoS in the non-weightbearing Achilles tendon was 1804±198 m/s. There was a significant increase in the average SoS during bilateral (2122±135 m/s) (P < 0.05) and unilateral (2221±79 m/s) stance (P < 0.05). Conclusions: Repeated SoS measures in human Achilles tendon were more reliable during stance than under non-weightbearing conditions. These findings are consistent with previous research in equine tendon in which lower variability in SoS was observed with increasing tensile load (Crevier-Denoix et al, 2009). Since the limits of agreement for Achilles tendon SoS are nearly 5% of the changes previously observed during walking and therapeutic heel raise exercises, acoustic wave transmission provides a promising new non-invasive method for determining tendon properties during sports and rehabilitation related activities.

Computer experiment – a case study for modelling and simulation of manufacturing systems

Deterministic computer simulation of physical experiments is now a common technique in science and engineering. Often, physical experiments are too time consuming, expensive or impossible to conduct. Complex computer models or codes, rather than physical experiments lead to the study of computer experiments, which are used to investigate many scientific phenomena. A computer experiment consists of a number of runs of the computer code with different input choices. The Design and Analysis of Computer Experiments is a rapidly growing technique in statistical experimental design. This paper aims to discuss some practical issues when designing a computer simulation and/or experiments for manufacturing systems. A case study approach is reviewed and presented.

Stockholm Syndrome : architecture, politics and the human body

The exchange between the body and architecture walks a fine line between violence and pleasure. It is through the body that the subject engages with the architectural act, not via thought or reason, but through action. The materiality of architecture is the often the catalyst for some intense association; the wall that defines gender or class, the double bolted door that incarcerates, the enclosed privacy of the bedroom to the love affair. Architecture is the physical manifestation of Lefebvre’s inscribed space. It enacts the social and political systems through bodily occupation. Architecture, when tested by the occupation of bodies, anchors ideology in both space and time. The architect’s script can be powerful when rehearsed honestly to the building’s intentions and just as beautiful when rebuked by the act of protest or unfaithful occupation. This research examines this fine line of violence and pleasure in architecture through performance, in the work of Bryony Lavin’s play Stockholm and Revolving Door by Allora & Calzadilla as part of the recent Kaldor Public Art Projects exhibition 13 Rooms in Sydney. The research is underpinned by the work of Architect and theorist, Bernard Tschumi in his two essays, Violence of Architecture and The Pleasure of Architecture. Studying architecture through the lens of performance shifts the focus of examination from pure thought to the body; because architecture is occupied through the body and not the mind.

The rural school experiment : creating a Queensland yeoman

Using historical narrative and extensive archival research, this thesis portrays the story of the twentieth century Queensland Rural Schools. The initiative started at Nambour Primary School in 1917, and extended over the next four decades to encompass thirty primary schools that functioned as centralized institutions training children in agricultural science, domestic science, and manual trade training. The Rural Schools formed the foundation of a systemised approach to agricultural education intended to facilitate the State’s closer settlement ideology. The purpose of the Rural Schools was to mitigate urbanisation, circumvent foreign incursion and increase Queensland’s productivity by turning boys into farmers, or the tradesmen required to support them, and girls into the homemakers that these farmers needed as wives and mothers for the next generation. Effectively Queensland took rural boys and girls and created a new yeomanry to aid the State’s development.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

Introduction. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. Methods. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone based scaffold plus 0.54µg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. Results. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. Conclusions. The results of this study demonstrate that rhBMP-2 plus PCL-based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.

Delivery of dimethyloxallyl glycine in mesoporous bioactive glass scaffolds to improve angiogenesis and osteogenesis of human bone marrow stromal cells

Development of hypoxia-mimicking bone tissue engineering scaffolds is of great importance in stimulating angiogenesis for bone regeneration. Dimethyloxallyl glycine (DMOG) is a cell-permeable, competitive inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression. The aim of this study was to develop hypoxia-mimicking scaffolds by delivering DMOG in mesoporous bioactive glass (MBG) scaffolds and to investigate whether the delivery of DMOG could induce a hypoxic microenvironment for human bone marrow stromal cells (hBMSC). MBG scaffolds with varied mesoporous structures (e.g. surface area and mesopore volume) were prepared by controlling the contents of mesopore-template agent. The composition, large-pore microstructure and mesoporous properties of MBG scaffolds were characterized. The effect of mesoporous properties on the loading and release of DMOG in MBG scaffolds was investigated. The effects of DMOG delivery on the cell morphology, cell viability, HIF-1α stabilization, vascular endothelial growth factor (VEGF) secretion and bone-related gene expression (alkaline phosphatase, ALP; osteocalcin, OCN; and osteopontin, OPN) of hBMSC in MBG scaffolds were systematically investigated. The results showed that the loading and release of DMOG in MBG scaffolds can be efficiently controlled by regulating their mesoporous properties via the addition of different contents of mesopore-template agent. DMOG delivery in MBG scaffolds had no cytotoxic effect on the viability of hBMSC. DMOG delivery significantly induced HIF-1α stabilization, VEGF secretion and bone-related gene expression of hBMSC in MBG scaffolds in which DMOG counteracted the effect of HIF-PH and stabilized HIF-1α expression under normoxic condition. Furthermore, it was found that MBG scaffolds with slow DMOG release significantly enhanced the expression of bone-related genes more than those with instant DMOG release. The results suggest that the controllable delivery of DMOG in MBG scaffolds can mimic a hypoxic microenvironment, which not only improves the angiogenic capacity of hBMSC, but also enhances their osteogenic differentiation.

Evolutionary genetics and human assisted movement of a globally invasive pest (Russian wheat aphid : Diuraphis noxia)

This PhD study has examined the population genetics of the Russian wheat aphid (RWA, Diuraphis noxia), one of the world’s most invasive agricultural pests, throughout its native and introduced global range. Firstly, this study investigated the geographic distribution of genetic diversity within and among RWA populations in western China. Analysis of mitochondrial data from 18 sites provided evidence for the long-term existence and expansion of RWAs in western China. The results refute the hypothesis that RWA is an exotic species only present in China since 1975. The estimated date of RWA expansion throughout western China coincides with the debut of wheat domestication and cultivation practices in western Asia in the Holocene. It is concluded that western China represents the limit of the far eastern native range of this species. Analysis of microsatellite data indicated high contemporary gene flow among northern populations in western China, while clear geographic isolation between northern and southern populations was identified across the Tianshan mountain range and extensive desert regions. Secondly, this study analyzed the worldwide pathway of invasion using both microsatellite and endosymbiont genetic data. Individual RWAs were obtained from native populations in Central Asia and the Middle East and invasive populations in Africa and the Americas. Results indicated two pathways of RWA invasion from 1) Syria in the Middle East to North Africa and 2) Turkey to South Africa, Mexico and then North and South America. Very little clone diversity was identified among invasive populations suggesting that a limited founder event occurred together with predominantly asexual reproduction and rapid population expansion. The most likely explanation for the rapid spread (within two years) from South Africa to the New World is by human movement, probably as a result of the transfer of wheat breeding material. Furthermore, the mitochondrial data revealed the presence of a universal haplotype and it is proposed that this haplotype is representative of a wheat associated super-clone that has gained dominance worldwide as a result of the widespread planting of domesticated wheat. Finally, this study examined salivary gland gene diversity to determine whether a functional basis for RWA invasiveness could be identified. Peroxidase DNA sequence data were obtained for a selection of worldwide RWA samples. Results demonstrated that most native populations were polymorphic while invasive populations were monomorphic, supporting previous conclusions relating to demographic founder effects in invasive populations. Purifying selection most likely explains the existence of a universal allele present in Middle Eastern populations, while balancing selection was evident in East Asian populations. Selection acting on the peroxidase gene may provide an allele-dependent advantage linked to the successful establishment of RWAs on wheat, and ultimately their invasion potential. In conclusion, this study is the most comprehensive molecular genetic investigation of RWA population genetics undertaken to date and provides significant insights into the source and pathway of global invasion and the potential existence of a wheat-adapted genotype that has colonised major wheat growing countries worldwide except for Australia. This research has major biosecurity implications for Australia’s grain industry.

Investigating epidermogenesis in a human skin equivalent model

Skin is the largest, and arguably, the most important organ of the body. It is a complex and multi-dimensional tissue, thus making it essentially impossible to fully model in vitro in conventional 2-dimensional culture systems. In view of this, rodents or pigs are utilised to study wound healing therapeutics or to investigate the biological effects of treatments on skin. However, there are many differences between the wound healing processes in rodents compared to humans (contraction vs. re-epithelialisation) and there are also ethical issues associated with animal testing for scientific research. Therefore, the development of skin equivalent (HSE) models from surgical discard human skin has become an important area of research. The studies in this thesis compare, for the first time, native human skin and the epidermogenesis process in a HSE model. The HSE was reported to be a comparable model for human skin in terms of expression and localisation of key epidermal cell markers. This validated HSE model was utilised to study the potential wound healing therapeutic, hyperbaric oxygen (HBO) therapy. There is a significant body of evidence suggesting that lack of cutaneous oxygen results in and potentiates the chronic, non-healing wound environment. Although the evidence is anecdotal, HBO therapy has displayed positive effects on re-oxygenation of chronic wounds and the clinical outcomes suggest that HBO treatment may be beneficial. Therefore, the HSE was subjected to a daily clinical HBO regime and assessed in terms of keratinocyte migration, proliferation, differentiation and epidermal thickening. HBO treatment was observed to increase epidermal thickness, in particular stratum corneum thickening, but it did not alter the expression or localisation of standard epidermal cell markers. In order to elucidate the mechanistic changes occurring in response to HBO treatment in the HSE model, gene microarrays were performed, followed by qRT-PCR of select genes which were differentially regulated in response to HBO treatment. The biological diversity of the HSEs created from individual skin donors, however, overrode the differences in gene expression between treatment groups. Network analysis of functional changes in the HSE model revealed general trends consistent with normal skin growth and maturation. As a more robust and longer term study of these molecular changes, protein localisation and expression was investigated in sections from the HSEs undergoing epidermogenesis in response to HBO treatment. These proteins were CDCP1, Metallothionein, Kallikrein (KLK) 1 and KLK7 and early growth response 1. While the protein expression within the HSE models exposed to HBO treatment were not consistent in all HSEs derived from all skin donors, this is the first study to detect and compare both KLK1 and CDCP1 protein expression in both a HSE model and native human skin. Furthermore, this is the first study to provide such an in depth analysis of the effect of HBO treatment on a HSE model. The data presented in this thesis, demonstrates high levels of variation between individuals and their response to HBO treatment, consistent with the clinical variation that is currently observed.

Diurnal variation of ocular biometrics under natural and defocused conditions

It is well known that a broad range of ocular anatomical and physiological parameters undergo significant diurnal variation. However, the natural diurnal variations that occur in the length of the human eye (axial length) and their underlying causes have been less well studied. Improvements in optical methods for the measurement of ocular biometrics now allow more precise and comprehensive measurements of axial length to be performed than has previously been possible. Research from animal models also suggests a link between diurnal axial length variations and longer term myopic eye growth, and that retinal image defocus can disrupt these diurnal rhythms in axial length. This research programme has examined the diurnal variations in axial length in young normal eyes, the contributing components and the influence of optical stimuli on these changes. In the first experiment, the normal pattern and consistency of the diurnal variations in axial length were examined at 10 different times (5 measurements each day, at ~ 3-hour intervals from ~ 9 am to ~ 9 pm) over 2 consecutive days on 30 young adult subjects (15 myopes, 15 emmetropes). Additionally, variations in a range of other ocular biometric measurements such as choroidal thickness, intraocular pressure, and other ocular biometrics were also explored as potential factors that may be associated with the observed variations in axial length. To investigate the potential influence of refractive error on diurnal axial length variations, the differences in the magnitude and pattern of diurnal variations in axial length between the myopic and emmetropic subjects were examined. Axial length underwent significant diurnal variation that was consistently observed over the 2 consecutive days of measurements, with the longest axial length typically occurring during the day, and the shortest at night. Significant diurnal variations were also observed in choroidal thickness, IOP and other ocular biometrics (such as central corneal thickness, anterior chamber depth and vitreous chamber depth) of the eye. Diurnal variations in vitreous chamber depth, IOP (positive associations) and choroidal thickness (negative association) were all significantly correlated with the diurnal changes in axial length. Choroidal thickness was found to fluctuate approximately in antiphase to the axial length changes, with the average timing of the longest axial length coinciding with the thinnest choroid and vice versa. There were no significant differences in the ocular diurnal variations associated with refractive error. Given that the diurnal changes in axial length could be associated with the changes in the eye’s optical quality, whether the optical quality of the eye also undergoes diurnal variation in the same cohort of young adult myopes and emmetropes over 2 consecutive days was also examined. Significant diurnal variations were observed only in the best sphere refraction (power vector M) and in the spherical aberration of the eye over two consecutive days of testing. The changes in the eyes lower and higher order ocular optics were not significantly associated with the diurnal variations in axial length and the other measured ocular biometric parameters. No significant differences were observed in the magnitude and timing of diurnal variations in lower-order and higher-order optics associated with refractive error. Since the small natural fluctuations in the eye’s optical quality did not appear to be sufficient to influence the natural diurnal fluctuations in ocular biometric parameters, in the next experiment, the influence of monocular myopic defocus (+1.50 DS) upon the normal diurnal variations in axial length and choroidal thickness of young adult emmetropic human subjects (n=13) imposed over a 12 hour period was examined. A series of axial length and choroidal thickness measurements (collected at ~3 hourly intervals, with the first measurement at ~9 am and the final measurement at ~9 pm) were obtained over three consecutive days. The natural diurnal rhythms (Day 1, no defocus), diurnal rhythms with monocular myopic defocus (Day 2, +1.50 DS spectacle lens over the right eye), and the recovery from any defocus induced changes (Day 3, no defocus) were examined. Significant diurnal variations over the course of the day were observed in both axial length and choroidal thickness on each of the three measurement days. The introduction of monocular myopic defocus led to significant reductions in the mean amplitude of diurnal change, and phase shifts in the peak timing of the diurnal rhythms in axial length and choroidal thickness. These defocus induced changes were found to be transient in nature and returned to normal the day following removal of the defocus. To further investigate the influence of optical stimuli on human diurnal rhythms, in the final experiment, the influence of monocular hyperopic defocus on the normal diurnal rhythms in axial length and choroidal thickness was examined in young adult emmetropic subjects (n=15). Similar to the previous experiment, the natural diurnal rhythms (Day 1, no defocus), diurnal rhythms with monocular hyperopic defocus (Day 2, -2.00 DS spectacle lens over the right eye), and the recovery from any defocus induced changes (Day 3, no defocus) were examined over three consecutive days. Both axial length and choroidal thickness underwent significant diurnal variations on each of the three days. The introduction of monocular hyperopic defocus resulted in a significant increase in the amplitude of diurnal change, but no change in the peak timing of diurnal rhythms in both parameters. The ocular changes associated with hyperopic defocus returned to normal, the day following removal of the defocus. This research has shown that axial length undergoes significant diurnal variation in young adult human eyes, and has shown that the natural diurnal variations in choroidal thickness and IOP are significantly associated, and may underlie these diurnal fluctuations in axial length. This work also demonstrated for the first time that exposing young human eyes to monocular myopic and hyperopic defocus leads to a significant disruption in the normal diurnal rhythms of axial length and choroidal thickness. These changes in axial length with defocus may reflect underlying mechanisms in the human eye that are involved in the regulation of longer term eye growth.

The role of human capital in economic growth revisited

Benhabib and Spiegel (1994) examine the role of human capital in the development process empirically using a theory-driven specification rather than the standard production function approach. While they find evidence of a positive impact of human capital on income growth, their result is not robust to the inclusion of inequality as an additional covariate. Using an alternate dataset and different measures of inequality, we find robust support for the hypothesis that human capital matters even when we account for the adverse effect of income inequality on growth.