Improving safety and establishing episomal maintenance of Adeno-associated viral vectors

Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems in basic and pre-clinical research and have been employed in more than 160 clinical trials. AAV vectors are commonly produced in producer cell lines like HEK293 by co-transfection with a so-called vector plasmid and one (in this work) or two so-called helper plasmids. The vector plasmid contains the transgene cassette of interest (TEC) flanked by AAV’s inverted terminal repeats (ITRs) which serve as packaging signals, whereas the helper plasmid provides the required AAV and helper virus functions in trans. A pivotal aspect of AAV vectorology is the manufacturing of AAV vectors free from impurities arising during the production process. These impurities include AAV vector preparations that contain capsids containing prokaryotic sequences, e.g. antibiotic resistance genes originating from the producer plasmids. In the first part of the thesis we aimed at improving the safety of AAV vectors. As we found that encapsidated prokaryotic sequences (using the ampicillin resistance gene as indicator) cannot be re-moved by standard purification methods we investigated whether the producer plasmids could be replaced by Minicircles (MCs). MCs are circular DNA constructs which contain no functional or coding prokaryotic sequences; they only consist of the TEC and a short sequence required for production and purification. MC counterparts of a vector plasmid encoding for enhanced green fluorescent (eGFP) protein and a helper plasmid encoding for AAV serotype 2 (AAV2) and helper Adenovirus (Ad) genes were designed and produced by PlasmidFactory (Bielefeld, Germany). Using all four possible combinations of plasmid and MCs, single-stranded AAV2 vectors (ssAAV) and self-complementary AAV vectors (scAAV) were produced and characterized for vector quantity, quality and functionality. The analyses showed that plasmids can be replaced by MCs without decreasing the efficiency of vector production and vector quality. MC-derived scAAV vector preparations even exceeded plasmid-derived preparations, as they displayed up to 30-fold improved transduction efficiencies. Using MCs as tools, we found that the vector plasmid is the main source of encapsidated prokaryotic sequences. Remarkably, we found that plasmid-derived scAAV vector preparations contained a much higher relative amount of prokaryotic sequences (up to 26.1 %, relative to TEC) compared to ssAAV vector preparations (up to 2.9 %). By replacing both plasmids by MCs the amount of functional prokaryotic sequences could be decreased to below the limit of quantification. Additional analyses for DNA impurities other than prokaryotic sequences showed that scAAV vectors generally contained a higher amount of non-vector DNA (e.g. adenoviral sequences) than ssAAV vectors. For both, ssAAV and scAAV vector preparations, MC-derived vectors tended to contain lower amounts of foreign DNA. None of the vectors tested could be shown to induce immunogenicity. In summary we could demonstrate that the quality of AAV vector preparations could be significantly improved by replacing producer plasmids by MCs. Upon transduction of a target tissue, AAV vector genomes predominantly remain in an episomal state, as duplex DNA circles or concatemers. These episomal forms mediate long-term transgene expression in terminally differentiated cells, but are lost in proliferating cells due to cell division. Therefore, in the second part of the thesis, in cooperation with Claudia Hagedorn and Hans J. Lipps (University Witten/Herdecke) an AAV vector genome was equipped with an autonomous replication element (Scaffold/matrix attachment region (S/MAR)). AAV-S/MAR encoding for eGFP and a blasticidin resistance gene and a control vector with the same TEC but lacking the S/MAR element (AAV-ΔS/MAR) were produced and transduced into highly proliferative HeLa cells. Antibiotic pressure was employed to select for cells stably maintaining the vector genome. AAV-S/MAR transduced cells yielded a higher number of colonies than AAV-ΔS/MAR-transduced cells. Colonies derived from each vector transduction were picked and cultured further. They remained eGFP-positive (up to 70 days, maximum cultivation period) even in the absence of antibiotic selection pressure. Interestingly, the mitotic stability of both AAV-S/MAR and control vector AAV-ΔS/MAR was found to be a result of episomal maintenance of the vector genome. This finding indicates that, under specific conditions such as the mild selection pressure we employed, “common” AAV vectors persist episomally. Thus, the S/MAR element increases the establishment frequency of stable episomes, but is not a prerequisite.

Above ground biomass functions with vegetation indices for multiple use systems of two evergreen oaks

Remote sensing is a promising approach for above ground biomass estimation, as forest parameters can be obtained indirectly. The analysis in space and time is quite straight forward due to the flexibility of the method to determine forest crown parameters with remote sensing. It can be used to evaluate and monitoring for example the development of a forest area in time and the impact of disturbances, such as silvicultural practices or deforestation. The vegetation indices, which condense data in a quantitative numeric manner, have been used to estimate several forest parameters, such as the volume, basal area and above ground biomass. The objective of this study was the development of allometric functions to estimate above ground biomass using vegetation indices as independent variables. The vegetation indices used were the Normalized Difference Vegetation Index (NDVI), Enhanced Vegetation Index (EVI), Simple Ratio (SR) and Soil-Adjusted Vegetation Index (SAVI). QuickBird satellite data, with 0.70 m of spatial resolution, was orthorectified, geometrically and atmospheric corrected, and the digital number were converted to top of atmosphere reflectance (ToA). Forest inventory data and published allometric functions at tree level were used to estimate above ground biomass per plot. Linear functions were fitted for the monospecies and multispecies stands of two evergreen oaks (Quercus suber and Quercus rotundifolia) in multiple use systems, montados. The allometric above ground biomass functions were fitted considering the mean and the median of each vegetation index per grid as independent variable. Species composition as a dummy variable was also considered as an independent variable. The linear functions with better performance are those with mean NDVI or mean SR as independent variable. Noteworthy is that the two better functions for monospecies cork oak stands have median NDVI or median SR as independent variable. When species composition dummy variables are included in the function (with stepwise regression) the best model has median NDVI as independent variable. The vegetation indices with the worse model performance were EVI and SAVI.

Building Information Modelling for FM at Sydney Opera House

“SOH see significant benefit in digitising its drawings and operation and maintenance manuals. Since SOH do not currently have digital models of the Opera House structure or other components, there is an opportunity for this national case study to promote the application of Digital Facility Modelling using standardized Building Information Models (BIM)”. The digital modelling element of this project examined the potential of building information models for Facility Management focusing on the following areas: • The re-usability of building information for FM purposes • BIM as an Integrated information model for facility management • Extendibility of the BIM to cope with business specific requirements • Commercial facility management software using standardised building information models • The ability to add (organisation specific) intelligence to the model • A roadmap for SOH to adopt BIM for FM The project has established that BIM – building information modelling - is an appropriate and potentially beneficial technology for the storage of integrated building, maintenance and management data for SOH. Based on the attributes of a BIM, several advantages can be envisioned: consistency in the data, intelligence in the model, multiple representations, source of information for intelligent programs and intelligent queries. The IFC – open building exchange standard – specification provides comprehensive support for asset and facility management functions, and offers new management, collaboration and procurement relationships based on sharing of intelligent building data. The major advantages of using an open standard are: information can be read and manipulated by any compliant software, reduced user “lock in” to proprietary solutions, third party software can be the “best of breed” to suit the process and scope at hand, standardised BIM solutions consider the wider implications of information exchange outside the scope of any particular vendor, information can be archived as ASCII files for archival purposes, and data quality can be enhanced as the now single source of users’ information has improved accuracy, correctness, currency, completeness and relevance. SOH current building standards have been successfully drafted for a BIM environment and are confidently expected to be fully developed when BIM is adopted operationally by SOH. There have been remarkably few technical difficulties in converting the House’s existing conventions and standards to the new model based environment. This demonstrates that the IFC model represents world practice for building data representation and management (see Sydney Opera House – FM Exemplar Project Report Number 2005-001-C-3, Open Specification for BIM: Sydney Opera House Case Study). Availability of FM applications based on BIM is in its infancy but focussed systems are already in operation internationally and show excellent prospects for implementation systems at SOH. In addition to the generic benefits of standardised BIM described above, the following FM specific advantages can be expected from this new integrated facilities management environment: faster and more effective processes, controlled whole life costs and environmental data, better customer service, common operational picture for current and strategic planning, visual decision-making and a total ownership cost model. Tests with partial BIM data – provided by several of SOH’s current consultants – show that the creation of a SOH complete model is realistic, but subject to resolution of compliance and detailed functional support by participating software applications. The showcase has demonstrated successfully that IFC based exchange is possible with several common BIM based applications through the creation of a new partial model of the building. Data exchanged has been geometrically accurate (the SOH building structure represents some of the most complex building elements) and supports rich information describing the types of objects, with their properties and relationships.

Getting creative in health care

Health care accounts for a substantial and growing share of national expenditures, and Australia’s health-care system faces some unprecedented pressures. This paper examines the contribution of creative expertise and services to Australian health care. They are found to be making a range of contributions to the development and delivery of health-care goods and services, the initial training and ongoing professionalism of doctors and nurses, and the effective functioning of health-care buildings. Creative activities within health-care services are also undertaken by medical professionals and patients. Key functions that creative activities address are innovation and service delivery in information management and analysis, and making complex information comprehensible or more useful, assisting communication and reducing psycho-social and distance-mediated barriers, and improving the efficiency and effectiveness of services.

A criminological analysis : using real-time monitoring to gather data on online predators

The Internet presents a constantly evolving frontier for criminology and policing, especially in relation to online predators – paedophiles operating within the Internet for safer access to children, child pornography and networking opportunities with other online predators. The goals of this qualitative study are to undertake behavioural research – identify personality types and archetypes of online predators and compare and contrast them with behavioural profiles and other psychological research on offline paedophiles and sex offenders. It is also an endeavour to gather intelligence on the technological utilisation of online predators and conduct observational research on the social structures of online predator communities. These goals were achieved through the covert monitoring and logging of public activity within four Internet Relay Chat(rooms) (IRC) themed around child sexual abuse and which were located on the Undernet network. Five days of monitoring was conducted on these four chatrooms between Wednesday 1 to Sunday 5 April 2009; this raw data was collated and analysed. The analysis identified four personality types – the gentleman predator, the sadist, the businessman and the pretender – and eight archetypes consisting of the groomers, dealers, negotiators, roleplayers, networkers, chat requestors, posters and travellers. The characteristics and traits of these personality types and archetypes, which were extracted from the literature dealing with offline paedophiles and sex offenders, are detailed and contrasted against the online sexual predators identified within the chatrooms, revealing many similarities and interesting differences particularly with the businessman and pretender personality types. These personality types and archetypes were illustrated by selecting users who displayed the appropriate characteristics and tracking them through the four chatrooms, revealing intelligence data on the use of proxies servers – especially via the Tor software – and other security strategies such as Undernet’s host masking service. Name and age changes, which is used as a potential sexual grooming tactic was also revealed through the use of Analyst’s Notebook software and information on ISP information revealed the likelihood that many online predators were not using any safety mechanism and relying on the anonymity of the Internet. The activities of these online predators were analysed, especially in regards to child sexual grooming and the ‘posting’ of child pornography, which revealed a few of the methods in which online predators utilised new Internet technologies to sexually groom and abuse children – using technologies such as instant messengers, webcams and microphones – as well as store and disseminate illegal materials on image sharing websites and peer-to-peer software such as Gigatribe. Analysis of the social structures of the chatrooms was also carried out and the community functions and characteristics of each chatroom explored. The findings of this research have indicated several opportunities for further research. As a result of this research, recommendations are given on policy, prevention and response strategies with regards to online predators.

Adaptive Bayesian compound designs for dose finding studies

We consider the problem of how to efficiently and safely design dose finding studies. Both current and novel utility functions are explored using Bayesian adaptive design methodology for the estimation of a maximum tolerated dose (MTD). In particular, we explore widely adopted approaches such as the continual reassessment method and minimizing the variance of the estimate of an MTD. New utility functions are constructed in the Bayesian framework and are evaluated against current approaches. To reduce computing time, importance sampling is implemented to re-weight posterior samples thus avoiding the need to draw samples using Markov chain Monte Carlo techniques. Further, as such studies are generally first-in-man, the safety of patients is paramount. We therefore explore methods for the incorporation of safety considerations into utility functions to ensure that only safe and well-predicted doses are administered. The amalgamation of Bayesian methodology, adaptive design and compound utility functions is termed adaptive Bayesian compound design (ABCD). The performance of this amalgamation of methodology is investigated via the simulation of dose finding studies. The paper concludes with a discussion of results and extensions that could be included into our approach.

Functional role of cell surface CUB domain-containing protein 1 in tumour cell dissemination

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time- and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2–mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2–mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.

The influence of cultural selection on strategic communication

Strategic communication is held to be a key process by which organisations respond to environmental uncertainty. In the received view articulated in the literatures of organisational communication and public relations, strategic communication results from collaborative efforts by organisational members to create shared understanding about environmental uncertainty and, as a result of this collective understanding, formulate appropriate communication responses. In this study, I explore how such collaborative efforts towards the development of strategic communication are derived from, and bounded by, culturally shared values and assumptions. Study of the influences of an organisation‟s culture on the formulation of strategic communication is a fundamental conceptual challenge for public relations and, to date, a largely unaddressed area of research. This thesis responds to this challenge by describing a key property of organisational culture – the action of cultural selection (Durham, 1992). I integrate this property of cultural selection to extend and refine the descriptive range of Weick‟s (1969, 1979) classic sociocultural model of organizing. From this integration I propose a new model, the Cultural Selection of Strategic Communication (CSSC). Underpinning the CSSC model is the central proposition that because of the action of cultural selection during organizing processes, the inherently conservative properties of an organisation‟s culture constrain development of effective strategic communication in ways that may be unrelated to the outcomes of “environmental scanning” and other monitoring functions heralded by the public relations literature as central to organisational adaptation. Thus, by examining the development of strategic communication, I describe a central conservative influence on the social ecology of organisations. This research also responds to Butschi and Steyn‟s (2006) call for the development of theory focusing on strategic communication as well as Grunig (2006) and Sriramesh‟s (2007) call for research to further understand the role of culture in public relations practice. In keeping with the explorative and descriptive goals of this study, I employ organisational ethnography to examine the influence of cultural selection on the development of strategic communication. In this methodological approach, I use the technique of progressive contextualisation to compare data from two related but distinct cultural settings. This approach provides a range of descriptive opportunities to permit a deeper understanding of the work of cultural selection. Findings of this study propose that culture, operating as a system of shared and socially transmitted social knowledge, acts through the property of cultural selection to influence decision making, and decrease conceptual variation within a group. The findings support the view that strategic communication, as a cultural product derived from the influence of cultural selection, is an essential feature to understand the social ecology of an organisation.

Raman spectroscopy of the multi-anion mineral schlossmacherite (H3O,Ca)Al3(AsO4,PO4,SO4)2(OH)6

The mineral schlossmacherite (H3O,Ca)Al3(AsO4,PO4,SO4)2(OH)6 , a multi-cation-multi-anion mineral of the beudantite mineral subgroup has been characterised by Raman spectroscopy. The mineral and related minerals functions as a heavy metal collector and is often amorphous or poorly crystalline, such that XRD identification is difficult. The Raman spectra are dominated by an intense band at 864 cm-1, assigned to the symmetric stretching mode of the AsO43- anion. Raman bands at 809 and 819 cm-1 are assigned to the antisymmetric stretching mode of AsO43- . The sulphate anion is characterised by bands at 1000 cm-1 (ν1), and at 1031, 1082 and 1139 cm-1 (ν3). Two sets of bands in the OH stretching region are observed: firstly between 2800 and 3000 cm-1 with bands observed at 2850, 2868, 2918 cm-1 and secondly between 3300 and 3600 with bands observed at 3363, 3382, 3410, 3449 and 3537 cm-1. These bands enabled the calculation of hydrogen bond distances and show a wide range of H-bond distances.

Using process models to support design of airport terminals

The complex design process of airport terminal needs to support a wide range of changes in operational facilities for both usual and unusual/emergency events. Process model describes how activities within a process are connected and also states logical information flow of the various activities. The traditional design process overlooks the necessity of information flow from the process model to the actual building design, which needs to be considered as a integral part of building design. The current research introduced a generic method to obtain design related information from process model to incorporate with the design process. Appropriate integration of the process model prior to the design process uncovers the relationship exist between spaces and their relevant functions, which could be missed in the traditional design approach. The current paper examines the available Business Process Model (BPM) and generates modified Business Process Model(mBPM) of check-in facilities of Brisbane International airport. The information adopted from mBPM then transform into possible physical layout utilizing graph theory.

Teachers as advocates for widening participation

In England and Australia, higher education institutions (HEIs) are expected to widen participation (WP) in higher education (HE) to enhance social justice and improve individual and national economic returns. Furthermore, HEIs are the major providers of initial and in-service teacher education. This article surveys international literature to explore ways in which teacher education programmes could and do contribute to preparing teachers to advocate for WP, including drawing on learning from WP research that demonstrates the value of current HE students engaging young people in schools and colleges to support them in seriously considering progressing to HE. We conclude that teachers and pre-service teachers are well placed to be advocates for WP. In the majority of higher education institutions, however, WP and teacher education functions are not working collaboratively to embed advocacy for WP into teacher education programmes.

Quantile regression without the curse of unsmoothness

We consider quantile regression models and investigate the induced smoothing method for obtaining the covariance matrix of the regression parameter estimates. We show that the difference between the smoothed and unsmoothed estimating functions in quantile regression is negligible. The detailed and simple computational algorithms for calculating the asymptotic covariance are provided. Intensive simulation studies indicate that the proposed method performs very well. We also illustrate the algorithm by analyzing the rainfall–runoff data from Murray Upland, Australia.

Pornography as entertainment

For the majority of its producers and consumers, pornography functions as entertainment rather than art. This paper draws on my recent work mapping out entertainment as an area of study (for the new Entertainment Industries programme at Queensland University of Technology) to explore what it means for this object of study to treat it as adult entertainment. Entertainment is audience-centred culture. It is commonly based around characters and story. It encourages seriality, and is unafraid of adaptation. Its dominant mode is fun, its favourite narrative resolution the happy ending. It commonly encourages audience activity and its aesthetics are organized around fast-moving, vulgar spectacle. Its primary purpose is to create an emotional response. In this article I test mainstream pornography against each of these characteristics as a way of mapping out the shape of pornography as it functions in its everyday form, and explain the advantages of such an approach.

Tocotrienol as potential anticancer agent

Vitamin E is composed of two structurally similar compounds: tocopherols (TPs) and tocotrienols (T3). Despite being overshadowed by TP over the past few decades, T3 is now considered to be a promising anticancer agent due to its potent effects against a wide range of cancers. A growing body of evidence suggests that in addition to its antioxidative and pro-apoptotic functions, T3 possesses a number of anticancer properties that make it superior to TP. These include the inhibition of epithelial-to-mesenchymal transitions, the suppression of vascular endothelial growth factor tumor angiogenic pathway and the induction of antitumor immunity. More recently, T3, but not TP, has been shown to have chemosensitization and anti-cancer stem cell effects, further demonstrating the potential of T3 as an effective anticancer therapeutic agent. With most of the previous clinical studies on TP producing disappointing results, research has now focused on testing T3 as the next generation vitamin E for chemoprevention and cancer treatment. This review will summarize recent developments in the understanding of the anticancer effects of T3. We will also discuss current progress in clinical trials involving T3 as an adjuvant to conventional cancer therapy.

Process based synthesis to evaluate design flexibility in airport terminal layout

Flexible design concept is a relatively new trend in airport terminal design which is believed to facilitate the ever changing needs of a terminal. Current architectural design processes become more complex every day because of the introduction of new building technologies where the concept of flexible airport terminal would apparently make the design process even more complex. Previous studies have demonstrated that ever growing aviation industry requires airport terminals to be planned, designed and constructed in such a way that should allow flexibility in design process. In order to adopt the philosophy of ‘design for flexibility’ architects need to address a wide range of differing needs. An appropriate integration of the process models, prior to the airport terminal design process, is expected to uncover the relationships that exist between spatial layout and their corresponding functions. The current paper seeks to develop a way of sharing space adjacency related information obtained from the Business Process Models (BPM) to assist in defining flexible airport terminal layouts. Critical design parameters are briefly investigated at this stage of research whilst reviewing the available design alternatives and an evaluation framework is proposed in the current paper. Information obtained from various design layouts should assist in identifying and defining flexible design matrices allowing architects to interpret and to apply those throughout the lifecycle of the terminal building.