Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.

Novel ciliate genetic code variants including the reassignment of all three stop codons to sense codons in Condylostoma magnum

mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3' end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state.mRNA translation in many ciliates utilizes variant genetic codes where stop codons are reassigned to specify amino acids. To characterize the repertoire of ciliate genetic codes, we analyzed ciliate transcriptomes from marine environments. Using codon substitution frequencies in ciliate protein-coding genes and their orthologs, we inferred the genetic codes of 24 ciliate species. Nine did not match genetic code tables currently assigned by NCBI. Surprisingly, we identified a novel genetic code where all three standard stop codons (TAA, TAG, and TGA) specify amino acids in Condylostoma magnum. We provide evidence suggesting that the functions of these codons in C. magnum depend on their location within mRNA. They are decoded as amino acids at internal positions, but specify translation termination when in close proximity to an mRNA 3' end. The frequency of stop codons in protein coding sequences of closely related Climacostomum virens suggests that it may represent a transitory state.

Caractérisation des variants R100 et D191 de la protéine de dégradation de l’hème ChuS

ChuS est une protéine provenant d’une bactérie pathogène de l’humain qui a la capacité de lier et de dégrader l’hème de l’hôte pour en libérer le fer nécessaire à la croissance bactérienne. Deux résidus potentiellement importants du site actif, l’arginine 100 (R100) et l’acide aspartique 191 (D191) ont été investigués afin de mieux comprendre leur rôle dans la catalyse par ChuS. Nos résultats révèlent que le résidu D191 n’est pas essentiel à l’activité catalytique, mais qu’il est important pour la stabilité du complexe entre l’enzyme et l’un de ses substrats (hème) alors que le résidu R100 est absolument essentiel à l’activité catalytique. Nos études cinétiques et spectroscopiques détaillées fournissent des informations qui permettent de mieux comprendre le mécanisme catalytique de ChuS. Elles aident ainsi à obtenir une meilleure compréhension des voies d’acquisition de l’hème comme source de fer chez les bactéries pathogènes et à définir de nouvelles cibles thérapeutiques.

Estudio molecular en dos hermanas afectadas por ictiosis congénita autosómica recesiva : descripción de una nueva mutación causal en TGM1

Se describe la variante homocigota c.320-2A>G de TGM1 en dos hermanas con ictiosis congénita autosómica recesiva. El clonaje de los transcritos generados por esta variante permitió identificar tres mecanismos moleculares de splicing alternativos.

Comparison of sequence variants in the PDK4 and COX4I2 genes between racing and cutting lines of quarter horses and associations with the speed index.

Different selection objectives within the Quarter Horse breed led to the formation of groups with distinct skills, including the racing and cutting lines. With a smaller population size in Brazil, but of great economic representativeness, the racing line is characterized by animals that can reach high speeds over short distances and within a short period of time. The cutting line is destined for functional tests, exploring skills such as agility and obedience. Although the athletic performance of horses is likely to be influenced by a large number of genes, few genetic variants have so far been related to this trait and this was done exclusively in Thoroughbreds, including the g.38973231G>A singlenucleotide polymorphism in the PDK4 gene and the g.22684390C>T single-nucleotide polymorphism in the COX4I2 gene. The results of the present study demonstrate the presence of polymorphic PDK4 and COX4I2 genes in Quarter Horses. The analysis of 296 racing animals and 68 cutting animals revealed significant differences in allele and genotype frequencies between the two lines. The same was not observed when these frequencies were compared between extreme racing performance phenotypes. There were also no significant associations between alleles of the two polymorphisms and the speed index. These results suggest that the alleles of the PDK4 and COX4I2 genes, which are related to better racecourse performance in Thoroughbreds, are probably associated with beneficial adaptations in aerobic metabolism and therefore play secondary roles in sprint racing performance in Quarter Horses, which is mainly anaerobic.

MSTN, CKM, and DMRT3 gene variants in different lines of quarter horses.

and creatine kinase muscle (CKM) (g.22999655C>A) genes have been associated with optimum racing distance and muscle development and racing performance in Thoroughbred horses, respectively. Considering that, since its formation, the Quarter Horse breed has received important genetic influence from the English breed, the genes cited become important candidates for athletic performance in the racing line of the American breed. An SNP in the equine doublesex and mab-3-related transcription factor 3 (DMRT3) gene (g.22999655C>A) has been described, which is responsible for the gait phenotype in homozygous individuals. Using a sample of 296 Quarter Horses of the racing line and 68 animals of the cutting line, the objective of this study was to compare the frequencies of the three SNPs cited above between a random subsample of animals of the cutting line (n ¼ 20) and animals with extreme phenotypes for racing performance (n ¼ 20 per extreme phenotype). The MSTN SNP showed practically no variation, with the observation of only one heterozygous animal (CT) in the cutting line, suggesting that this gene has been under great selective pressure within the racing segment. The CKM gene variant studied was found to be polymorphic, but no significant associates were observed between its alleles and the different lines or groups. Two animals carrying the CA heterozygous DMRT3 genotype were identified in the group with poor racing performance and one in the cutting line, indicating that this variant can be a limiting factor for the development of greater speeds.

Development of a Rep-inducible, BBTV-based expression system in banana

Banana bunchy top is regarded as the most important viral disease of banana, causing significant yield losses worldwide. The disease is caused by Banana bunchy top virus (BBTV), which is a circular ssDNA virus belonging to the genus Babuvirus in the family Nanoviridae. There are currently few effective control strategies for this and other ssDNA viruses. “In Plant Activation” (InPAct) is a novel technology being developed at QUT for ssDNA virus-activated suicide gene expression. The technology exploits the rolling circle replication mechanism of ssDNA viruses and is based on a unique “split” gene design such that suicide gene expression is only activated in the presence of the viral Rep. This PhD project aimed to develop a BBTV-based InPAct system as a suicide gene strategy to control BBTV. The BBTV-based InPAct vector design requires a BBTV intergenic region (IR) to be embedded within an intron in the gene expression cassette. To ensure that the BBTV IR would not interfere with intron splicing, a TEST vector was initially generated that contained the entire BBTV IR embedded within an intron in a β-glucuronidase (GUS) expression vector. Transient GUS assays in banana embryogenic cell suspensions indicated that cryptic intron splice sites were present within the IR. Transcript analysis revealed two cryptic intron splice sites in the Domain III sequence of the CR-M within the IR. Removal of the CR-M from the TEST vector resulted in an enhancement of GUS expression suggesting that the cryptic intron splice sites had been removed. An InPAct GUS vector was subsequently generated that contained the modified BBTV IR, with the CR-M (minus Domain III) repositioned within the InPAct cassette. Using transient histochemical and fluorometric GUS assays in banana embryogenic cells, the InPAct GUS vector was shown to be activated in the presence of the BBTV Rep. However, the presence of both BBTV Rep and Clink was shown to have a deleterious effect on GUS expression suggesting that these proteins were cytotoxic at the levels expressed. Analysis of replication of the InPAct vectors by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector through the nicking/ligation activity of BBTV Rep. However, Rep-mediated episomal replicons, indicative of rolling circle replication of the released circularised cassettes, were not observed. The inability of the InPAct cassette to be replicated was further investigated. To examine whether the absence of Domain III of the CR-M was responsible, a suite of modified BBTV-based InPAct GUS vectors was constructed that contained the CR-M with the inclusion of Domain III, the CR-M with the inclusion of Domain III and additional upstream IR sequence, or no CR-M. Analysis of replication by Southern hybridisation revealed that neither the presence of Domain III, nor the entire CR-M, had an effect on replication levels. Since the InPAct cassette was significantly larger than the native BBTV genomic components (approximately 1 kb), the effect of InPAct cassette size on replication was also investigated. A suite of size variant BBTV-based vectors was constructed that increased the size of a replication competent cassette to 1.1 kbp through to 2.1 kbp.. Analysis of replication by Southern hybridisation revealed that an increase in vector size above approximately 1.5 - 1.7 kbp resulted in a decrease in replication. Following the demonstration of Rep-mediated release, circularisation and expression from the InPAct GUS vector, an InPAct vector was generated in which the uidA reporter gene was replaced with the ribonuclease-encoding suicide gene, barnase. Initially, a TEST vector was generated to assess the cytotoxicity of Barnase on banana cells. Although transient assays revealed a Barnase-induced cytotoxic effect in banana cells, the expression levels were sub-optimal. An InPAct BARNASE vector was generated and tested for BBTV Rep-activated Barnase expression using transient assays in banana embryogenic cells. High levels of background expression from the InPAct BARNASE vector made it difficult to accurately assess Rep-activated Barnase expression. Analysis of replication by Southern hybridisation revealed low levels of InPAct cassette-based episomal DNA released from the vector but no Rep-mediated episomal replicons indicative of rolling circle replication of the released circularised cassettes were again observed. Despite the inability of the InPAct vectors to replicate to enable high level gene expression, the InPAct BARNASE vector was assessed in planta for BBTV Rep-mediated activation of Barnase expression. Eleven lines of transgenic InPAct BARNASE banana plants were generated by Agrobacterium-mediated transformation and were challenged with viruliferous Pentalonia nigronervosa. At least one clonal plant in each line developed bunchy top symptoms and infection was confirmed by PCR. No localised lesions were observed on any plants, nor was there any localised GUS expression in the one InPAct GUS line challenged with viruliferous aphids. The results presented in this thesis are the first study towards the development of a BBTV-based InPAct system as a Rep-activatable suicide gene expression system to control BBTV. Although further optimisation of the vectors is necessary, the preliminary results suggest that this approach has the potential to be an effective control strategy for BBTV. The use of iterons within the InPAct vectors that are recognised by Reps from different ssDNA plant viruses may provide a broad-spectrum resistance strategy against multiple ssDNA plant viruses. Further, this technology holds great promise as a platform technology for the molecular farming of high-value proteins in vitro or in vivo through expression of the ssDNA virus Rep protein.

Strongly Secure Certificateless Key Agreement

We introduce a formal model for certificateless authenticated key exchange (CL-AKE) protocols. Contrary to what might be expected, we show that the natural combination of an ID-based AKE protocol with a public key based AKE protocol cannot provide strong security. We provide the first one-round CL-AKE scheme proven secure in the random oracle model. We introduce two variants of the Diffie-Hellman trapdoor the introduced by \cite{DBLP:conf/eurocrypt/CashKS08}. The proposed key agreement scheme is secure as long as each party has at least one uncompromised secret. Thus, our scheme is secure even if the key generation centre learns the ephemeral secrets of both parties.

Process configuration in YAWL

This chapter describes how the YAWL meta-model was extended to support the definition of variation points. These variation points can be used to describe different variants of a YAWL process model in a unified, configurable model. The model can then be configured to suit the needs of specific settings, e.g. for a new organization of project.

Calculation of habitable dwelling surfaces : an empirical analysis for the agglomerations of Munich and Sydney

In many countries the calculation of habitable dwelling surface is characterised by a chaotic variety of calculation variants hardly comprehensible for the end user - sometimes not even reproducible for the expert. Therefore dossiers were analysed on the basis of a random choice in order to determine the method according to which the habitable dwelling surface was measured and to find out wether customers can scrutinize the calculations. The paper compares Sydney and Munich, where in both cases property prices are situated at the high end of the market

Influence of UGT1A9 intronic I399C4T polymorphism on SN-38 glucuronidation in Asian cancer patients

Genetic polymorphisms in hepatically expressed UGT1A1 and UGT1A9 contribute to the interindividual variability i-n irinotecan disposition and toxicity. We screened UGT1A1 (UGT1A1*60, g.−3140G>A, UGT1A1*28 and UGT1A1*6) and UGT1A9 (g.−118(T)9>10 and I399C>T) genes for polymorphic variants in the promoter and coding regions, and the genotypic effect of UGT1A9 I399C>T polymorphism on irinotecan disposition in Asian cancer patients was investigated. Blood samples were collected from 45 patients after administration of irinotecan as a 90 min intravenous infusion of 375 mg/m2 once in every 3 weeks. Genotypic–phenotypic correlates showed that cancer patients heterozygous or homozygous for the I399C>T allele had approximately 2-fold lower systemic exposure to SN-38 (P<0.05) and a trend towards a higher relative extent of glucuronidation (REG) of SN-38 (P>0.05). UGT1A1–1A9 diplotype analysis showed that patients harbouring the H1/H2 (TG6GT10T/GG6GT9C) diplotype had 2.4-fold lower systemic exposure to SN-38 glucuronide (SN-38G) compared with patients harbouring the H1/H5 (TG6GT10T/GG6GT10C) diplotype (P=0.025). In conclusion, this in vivo study supports the in vitro findings of Girard et al. and suggests that the UGT1A9 I399C>T variant may be an important glucuronidating allele affecting the pharmacokinetics of SN-38 and SN-38G in Asian cancer patients receiving irinotecan chemotherapy.

Variation of the Stiles-Crawford effect with accommodation and myopia

Background: Mechanical forces either due to accommodation or myopia may stretch the retina and/or cause shear between the retina and choroid. This can be investigated by making use of the Stiles-Crawford effect (SCE), which is the phenomenon of light changing in apparent brightness as it enters through different positions in the pupil. The SCE can be measured by psychophysical and objective techniques, with the SCE parameters being directionality (rate of change across the pupil), and orientation (the location of peak sensitivity in the pupil). Aims: 1. To study the changes in foveal SCE with accommodation in emmetropes and myopes using a subjective (psychophysical) technique. 2. To develop and evaluate a quick objective technique of measuring the SCE using the multifocal electroretinogram. Methods: The SCE was measured in 6 young emmetropes and 6 young myopes for up to 8 D accommodation stimulus with a psychophysical technique and its variants. An objective technique using the multifocal electroretinogram was developed and evaluated with 5 emmetropes. Results: Using the psychophysical technique, the SCE directionality increased by similar amounts in both emmetropes and myopes as accommodation increased, with an increase of 15-20% with 6 D of accommodation. However, there were no significant orientation changes. Additional measurements showed that most of the change in the directionality was probably an artefact of optical factors such as higher-order aberrations and accommodative lag rather a true effect of accommodation. The multifocal technique demonstrated the presence of the SCE, but results were noisy and too variable to detect any changes in SCE directionality or orientation with accommodation. Conclusion: There is little true change in the SCE with accommodation responses up to 6 D in either emmetropes or myopes, although it is possible that substantial changes might occur at very high accommodation levels. The objective technique using the multifocal electroretinogram was quicker and less demanding for the subjects than the psychophysical technique, but as implemented in this thesis, it is not a reliable method of measuring the SCE.

Campylobacter jejuni and campylobacter coli genotyping by high-resolution melting analysis of a flaA fragment

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaAHRManalysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Latent variable models in statistical genetics

Understanding the complexities that are involved in the genetics of multifactorial diseases is still a monumental task. In addition to environmental factors that can influence the risk of disease, there is also a number of other complicating factors. Genetic variants associated with age of disease onset may be different from those variants associated with overall risk of disease, and variants may be located in positions that are not consistent with the traditional protein coding genetic paradigm. Latent Variable Models are well suited for the analysis of genetic data. A latent variable is one that we do not directly observe, but which is believed to exist or is included for computational or analytic convenience in a model. This thesis presents a mixture of methodological developments utilising latent variables, and results from case studies in genetic epidemiology and comparative genomics. Epidemiological studies have identified a number of environmental risk factors for appendicitis, but the disease aetiology of this oft thought useless vestige remains largely a mystery. The effects of smoking on other gastrointestinal disorders are well documented, and in light of this, the thesis investigates the association between smoking and appendicitis through the use of latent variables. By utilising data from a large Australian twin study questionnaire as both cohort and case-control, evidence is found for the association between tobacco smoking and appendicitis. Twin and family studies have also found evidence for the role of heredity in the risk of appendicitis. Results from previous studies are extended here to estimate the heritability of age-at-onset and account for the eect of smoking. This thesis presents a novel approach for performing a genome-wide variance components linkage analysis on transformed residuals from a Cox regression. This method finds evidence for a dierent subset of genes responsible for variation in age at onset than those associated with overall risk of appendicitis. Motivated by increasing evidence of functional activity in regions of the genome once thought of as evolutionary graveyards, this thesis develops a generalisation to the Bayesian multiple changepoint model on aligned DNA sequences for more than two species. This sensitive technique is applied to evaluating the distributions of evolutionary rates, with the finding that they are much more complex than previously apparent. We show strong evidence for at least 9 well-resolved evolutionary rate classes in an alignment of four Drosophila species and at least 7 classes in an alignment of four mammals, including human. A pattern of enrichment and depletion of genic regions in the profiled segments suggests they are functionally significant, and most likely consist of various functional classes. Furthermore, a method of incorporating alignment characteristics representative of function such as GC content and type of mutation into the segmentation model is developed within this thesis. Evidence of fine-structured segmental variation is presented.

How environmental and organizational complexity affects opportunity recognition and exploitation in development projects

Principal Topic: Project structures are often created by entrepreneurs and large corporate organizations to develop new products. Since new product development projects (NPDP) are more often situated within a larger organization, intrapreneurship or corporate entrepreneurship plays an important role in bringing these projects to fruition. Since NPDP often involves the development of a new product using immature technology, we describe development of an immature technology. The Joint Strike Fighter (JSF) F-35 aircraft is being developed by the U.S. Department of Defense and eight allied nations. In 2001 Lockheed Martin won a $19 billion contract to develop an affordable, stealthy and supersonic all-weather strike fighter designed to replace a wide range of aging fighter aircraft. In this research we define a complex project as one that demonstrates a number of sources of uncertainty to a degree, or level of severity, that makes it extremely difficult to predict project outcomes, to control or manage project (Remington & Zolin, Forthcoming). Project complexity has been conceptualized by Remington and Pollock (2007) in terms of four major sources of complexity; temporal, directional, structural and technological complexity (See Figure 1). Temporal complexity exists when projects experience significant environmental change outside the direct influence or control of the project. The Global Economic Crisis of 2008 - 2009 is a good example of the type of environmental change that can make a project complex as, for example in the JSF project, where project managers attempt to respond to changes in interest rates, international currency exchange rates and commodity prices etc. Directional complexity exists in a project where stakeholders' goals are unclear or undefined, where progress is hindered by unknown political agendas, or where stakeholders disagree or misunderstand project goals. In the JSF project all the services and all non countries have to agree to the specifications of the three variants of the aircraft; Conventional Take Off and Landing (CTOL), Short Take Off/Vertical Landing (STOVL) and the Carrier Variant (CV). Because the Navy requires a plane that can take off and land on an aircraft carrier, that required a special variant of the aircraft design, adding complexity to the project. Technical complexity occurs in a project using technology that is immature or where design characteristics are unknown or untried. Developing a plane that can take off on a very short runway and land vertically created may highly interdependent technological challenges to correctly locate, direct and balance the lift fans, modulate the airflow and provide equivalent amount of thrust from the downward vectored rear exhaust to lift the aircraft and at the same time control engine temperatures. These technological challenges make costing and scheduling equally challenging. Structural complexity in a project comes from the sheer numbers of elements such as the number of people, teams or organizations involved, ambiguity regarding the elements, and the massive degree of interconnectedness between them. While Lockheed Martin is the prime contractor, they are assisted in major aspects of the JSF development by Northrop Grumman, BAE Systems, Pratt & Whitney and GE/Rolls-Royce Fighter Engineer Team and innumerable subcontractors. In addition to identifying opportunities to achieve project goals, complex projects also need to identify and exploit opportunities to increase agility in response to changing stakeholder demands or to reduce project risks. Complexity Leadership Theory contends that in complex environments adaptive and enabling leadership are needed (Uhl-Bien, Marion and McKelvey, 2007). Adaptive leadership facilitates creativity, learning and adaptability, while enabling leadership handles the conflicts that inevitably arise between adaptive leadership and traditional administrative leadership (Uhl-Bien and Marion, 2007). Hence, adaptive leadership involves the recognition and opportunities to adapt, while and enabling leadership involves the exploitation of these opportunities. Our research questions revolve around the type or source of complexity and its relationship to opportunity recognition and exploitation. For example, is it only external environmental complexity that creates the need for the entrepreneurial behaviours, such as opportunity recognition and opportunity exploitation? Do the internal dimensions of project complexity, such as technological and structural complexity, also create the need for opportunity recognition and opportunity exploitation? The Kropp, Zolin and Lindsay model (2009) describes a relationship between entrepreneurial orientation (EO), opportunity recognition (OR), and opportunity exploitation (OX) in complex projects, with environmental and organizational contextual variables as moderators. We extend their model by defining the affects of external complexity and internal complexity on OR and OX. ---------- Methodology/Key Propositions: When the environment complex EO is more likely to result in OR because project members will be actively looking for solutions to problems created by environmental change. But in projects that are technologically or structurally complex project leaders and members may try to make the minimum changes possible to reduce the risk of creating new problems due to delays or schedule changes. In projects with environmental or technological complexity project leaders who encourage the innovativeness dimension of EO will increase OR in complex projects. But projects with technical or structural complexity innovativeness will not necessarily result in the recognition and exploitation of opportunities due to the over-riding importance of maintaining stability in the highly intricate and interconnected project structure. We propose that in projects with environmental complexity creating the need for change and innovation project leaders, who are willing to accept and manage risk, are more likely to identify opportunities to increase project effectiveness and efficiency. In contrast in projects with internal complexity a much higher willingness to accept risk will be necessary to trigger opportunity recognition. In structurally complex projects we predict it will be less likely to find a relationship between risk taking and OP. When the environment is complex, and a project has autonomy, they will be motivated to execute opportunities to improve the project's performance. In contrast, when the project has high internal complexity, they will be more cautious in execution. When a project experiences high competitive aggressiveness and their environment is complex, project leaders will be motivated to execute opportunities to improve the project's performance. In contrast, when the project has high internal complexity, they will be more cautious in execution. This paper reports the first stage of a three year study into the behaviours of managers, leaders and team members of complex projects. We conduct a qualitative study involving a Group Discussion with experienced project leaders. The objective is to determine how leaders of large and potentially complex projects perceive that external and internal complexity will influence the affects of EO on OR. ---------- Results and Implications: These results will help identify and distinguish the impact of external and internal complexity on entrepreneurial behaviours in NPDP. Project managers will be better able to quickly decide how and when to respond to changes in the environment and internal project events.