Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

A comprehensive investigation into the hydrodynamic and capture/retention performance of a gross pollutant trap

A novel and comprehensive testing approach to examine the performance of gross pollutant traps (GPTs) was developed. A proprietary GPT with internal screens for capturing gross pollutants—organic matter and anthropogenic litter—was used as a case study. This work is the first investigation of its kind and provides valuable practical information for the design, selection and operation of GPTs and also the management of street waste in an urban environment. It used a combination of physical and theoretical models to examine in detail the hydrodynamic and capture/retention characteristics of the GPT. The results showed that the GPT operated efficiently until at least 68% of the screens were blocked, particularly at high flow rates. At lower flow rates, the high capture/retention performance trend was reversed. It was also found that a raised inlet GPT offered a better capture/retention performance. This finding indicates that cleaning operations could be more effectively planned in conjunction with the deterioration in GPT’s capture/retention performance.

Turbulent velocity and suspended sediment concentration measurements in an urban environment of the Brisbane River Flood Plain at Gardens Point on 12-13 January 2011

The flood flow in urbanised areas constitutes a major hazard to the population and infrastructure as seen during the summer 2010-2011 floods in Queensland (Australia). Flood flows in urban environments have been studied relatively recently, although no study considered the impact of turbulence in the flow. During the 12-13 January 2011 flood of the Brisbane River, some turbulence measurements were conducted in an inundated urban environment in Gardens Point Road next to Brisbane's central business district (CBD) at relatively high frequency (50 Hz). The properties of the sediment flood deposits were characterised and the acoustic Doppler velocimeter unit was calibrated to obtain both instantaneous velocity components and suspended sediment concentration in the same sampling volume with the same temporal resolution. While the flow motion in Gardens Point Road was subcritical, the water elevations and velocities fluctuated with a distinctive period between 50 and 80 s. The low frequency fluctuations were linked with some local topographic effects: i.e, some local choke induced by an upstream constriction between stairwells caused some slow oscillations with a period close to the natural sloshing period of the car park. The instantaneous velocity data were analysed using a triple decomposition, and the same triple decomposition was applied to the water depth, velocity flux, suspended sediment concentration and suspended sediment flux data. The velocity fluctuation data showed a large energy component in the slow fluctuation range. For the first two tests at z = 0.35 m, the turbulence data suggested some isotropy. At z = 0.083 m, on the other hand, the findings indicated some flow anisotropy. The suspended sediment concentration (SSC) data presented a general trend with increasing SSC for decreasing water depth. During a test (T4), some long -period oscillations were observed with a period about 18 minutes. The cause of these oscillations remains unknown to the authors. The last test (T5) took place in very shallow waters and high suspended sediment concentrations. It is suggested that the flow in the car park was disconnected from the main channel. Overall the flow conditions at the sampling sites corresponded to a specific momentum between 0.2 to 0.4 m2 which would be near the upper end of the scale for safe evacuation of individuals in flooded areas. But the authors do not believe the evacuation of individuals in Gardens Point Road would have been safe because of the intense water surges and flow turbulence. More generally any criterion for safe evacuation solely based upon the flow velocity, water depth or specific momentum cannot account for the hazards caused by the flow turbulence, water depth fluctuations and water surges.

Turbulence characteristics of a small subtropical estuary during and after some moderate rainfall

In natural estuaries, scalar diffusion and dispersion are driven by turbulence. In the present study, detailed turbulence measurements were conducted in a small subtropical estuary with semi-diurnal tides under neap tide conditions. Three acoustic Doppler velocimeters were installed mid-estuary at fixed locations close together. The units were sampled simultaneously and continuously at relatively high frequency for 50 h. The results illustrated the influence of tidal forcing in the small estuary, although low frequency longitudinal velocity oscillations were observed and believed to be induced by external resonance. The boundary shear stress data implied that the turbulent shear in the lower flow region was one order of magnitude larger than the boundary shear itself. The observation differed from turbulence data in a laboratory channel, but a key feature of natural estuary flow was the significant three dimensional effects associated with strong secondary currents including transverse shear events. The velocity covariances and triple correlations, as well as the backscatter intensity and covariances, were calculated for the entire field study. The covariances of the longitudinal velocity component showed some tidal trend, while the covariances of the transverse horizontal velocity component exhibited trends that reflected changes in secondary current patterns between ebb and flood tides. The triple correlation data tended to show some differences between ebb and flood tides. The acoustic backscatter intensity data were characterised by large fluctuations during the entire study, with dimensionless fluctuation intensity I0b =Ib between 0.46 and 0.54. An unusual feature of the field study was some moderate rainfall prior to and during the first part of the sampling period. Visual observations showed some surface scars and marked channels, while some mini transient fronts were observed.

Turbulent measurements in a small subtropical estuary with semidiurnal tides

Since predictions of scalar dispersion in small estuaries can rarely be predicted accurately, new field measurements were conducted continuously at relatively high frequency for up to 50 h (per investigation) in a small subtropical estuary with semidiurnal tides. The bulk flow parameters varied in time with periods comparable to tidal cycles and other large-scale processes. The turbulence properties depended upon the instantaneous local flow properties. They were little affected by the flow history, but their structure and temporal variability were influenced by a variety of parameters including the tidal conditions and bathymetry. A striking feature of the data sets was the large fluctuations in all turbulence characteristics during the tidal cycle, and basic differences between neap and spring tide turbulence.

Turbulence measurements in a small subtropical estuary under king tide conditions

In natural waterways and estuaries, the understanding of turbulent mixing is critical to the knowledge of sediment transport, stormwater runoff during flood events, and release of nutrient-rich wastewater into ecosystems. In the present study, some field measurements were conducted in a small subtropical estuary with micro-tidal range and semi-diurnal tides during king tide conditions: i. e., the tidal range was the largest for both 2009 and 2010. The turbulent velocity measurements were performed continuously at high-frequency (50Hz) for 60 h. Two acoustic Doppler velocimeters (ADVs) were sampled simultaneously in the middle estuarine zone, and a third ADV was deployed in the upper estuary for 12 h only. The results provided an unique characterisation of the turbulence in both middle and upper estuarine zones under the king tide conditions. The present observations showed some marked differences between king tide and neap tide conditions. During the king tide conditions, the tidal forcing was the dominant water exchange and circulation mechanism in the estuary. In contrast, the long-term oscillations linked with internal and external resonance played a major role in the turbulent mixing during neap tides. The data set showed further that the upper estuarine zone was drastically less affected by the spring tide range: the flow motion remained slow, but the turbulent velocity data were affected by the propagation of a transient front during the very early flood tide motion at the sampling site. © 2012 Springer Science+Business Media B.V.

Turbulence and suspended sediment measurements in an urban environment during the Brisbane River flood of January 2011

In urbanised areas, the flood flows constitute a hazard to populations and infrastructure as illustrated during major floods in 2011. During the 2011 Brisbane River flood, some turbulent velocity data were collected using acoustic Doppler velocimetry in an inundated street. The field deployment showed some unusual features of flood flow in the urban environment. That is, the water elevations and velocities fluctuated with distinctive periods between 50 and 100 s linked with some local topographic effects. The instantaneous velocity data were analysed using a triple decomposition. The velocity fluctuations included a large energy component in the slow fluctuation range, while the turbulent motion components were much smaller. The suspended sediment data showed some significant longitudinal flux. Altogether the results highlighted that the triple decomposition approach originally developed for period flows is well suited to complicated flows in an inundated urban environment.

Suspended sediment properties and suspended sediment flux estimates in an inundated urban environment during a major flood event

During a major flood event, the inundation of urban environments leads to some complicated flow motion most often associated with significant sediment fluxes. In the present study, a series of field measurements were conducted in an inundated section of the City of Brisbane (Australia) about the peak of a major flood in January 2011. Some experiments were performed to use ADV backscatter amplitude as a surrogate estimate of the suspended sediment concentration (SSC) during the flood event. The flood water deposit samples were predominantly silty material with a median particle size about 25 μm and they exhibited a non-Newtonian behavior under rheological testing. In the inundated urban environment during the flood, estimates of suspended sediment concentration presented a general trend with increasing SSC for decreasing water depth. The suspended sediment flux data showed some substantial sediment flux amplitudes consistent with the murky appearance of floodwaters. Altogether the results highlighted the large suspended sediment loads and fluctuations in the inundated urban setting associated possibly with a non-Newtonian behavior. During the receding flood, some unusual long-period oscillations were observed (periods about 18 min), although the cause of these oscillations remains unknown. The field deployment was conducted in challenging conditions highlighting a number of practical issues during a natural disaster.

Turbulent mixing and sediment processes in peri-urban estuariesin South-East Queensland (Australia)

An estuary is formed at the mouth of a river where the tides meet a freshwater flow and it may be classified as a function of the salinity distribution and density stratification. An overview of the broad characteristics of the estuaries of South-East Queensland(Australia) is presented herein, where the small peri-urban estuaries may provide an useful indicator of potential changes which might occur in larger systems with growing urbanisation. Small peri-urban estuaries exhibits many key hydrological features and associated with ecosystem types of larger estuaries, albeit at smaller scales, often with a greater extent of urban development as a proportion of catchment area. We explore the potential for some smaller peri-urban estuaries to be used as natural laboratories to gain some much needed information on the estuarine processes, although any dynamics similarity is presently limited by critical absence of in-depth physical investigation in larger estuarine systems. The absence of the detailed turbulence and sedimentary data hampers the understanding and modelling of the estuarine zones. The interactions between the various stake holders are likely to define the vision for the future of South-East Queensland's peri-urban estuaries. This will require a solid understanding of the bio-physical function and capacity of the peri-urban estuaries. Based upon the knowledge gap, it is recommended that an adaptive trial and error approach be adopted for the future of investigation and management strategies.

A case study of interactions between flood flow and buildings in an urban environment : Gardens Point during the 12-13 January 2011 flood of the Brisbane River (Australia)

Flood flows in inundated urban environment constitute a natural hazard. During the 12- 13 January 2011 flood of the Brisbane River, detailed water elevation, velocity and suspended sediment data were recorded in an inundated street at the peak of the flood. The field observations highlighted a number of unusual flow interactions with the urban surroundings. These included some slow fluctuations in water elevations and velocity with distinctive periods between 50 and 100 s caused by some local topographic effect (choking), superposed with some fast turbulent fluctuations. The suspended sediment data highlighted some significant suspended sediment loads in the inundated zone.

Turbulence in an inundated urban environment during a major flood : implications in terms of people evacuation and sediment deposition

Floods through inundated urban environments constitute a hazard to the population and infrastructure. A series of field measurements were performed in an inundated section of the City of Brisbane (Australia) during a major flood in January 2011. Using an acoustic Doppler velocimeter (ADV), detailed velocity and suspended sediment concentration measurements were conducted about the peak of the flood. The results are discussed with a focus on the safety of individuals in floodwaters and the sediment deposition during the flood recession. The force of the floodwaters in Gardens Point Road was deemed unsafe for individual evacuation. A comparison with past laboratory results suggested that previous recommendations could be inappropriate and unsafe in real flood flows.

New criterion for the stability of a human body in floodwaters

The authors must be congratulated for their original and important study. The flooding of urbanised areas constitutes a hazard to the population and infrastructure. Floods through inundated urban environments have been studied only recently and few considered the potential impact of flowing waters on pedestrians...

Turbulence in an inundated urban environment during a major flood : implications in terms of people evacuation and sediment deposition

Floods through inundated urban environments constitute a hazard to the population and infrastructure. A series of field measurements were performed in an inundated section of the City of Brisbane (Australia) during a major flood in January 2011. Using an acoustic Doppler velocimeter (ADV), detailed velocity and suspended sediment concentration measurements were conducted about the peak of the flood. The results are discussed with a focus on the safety of individuals in floodwaters and the sediment deposition during the flood recession. The force of the floodwaters in Gardens Point Road was deemed unsafe for individual evacuation. A comparison with past laboratory results suggested that previous recommendations could be inappropriate and unsafe in real flood flows.

Turbulence measurements in a shallow tidal estuary : analysis based on triple decomposition

In an estuary, mixing and dispersion are the result of the combination of large scale advection and small scale turbulence which are both complex to estimate. A field study was conducted in a small sub-tropical estuary in which high frequency (50 Hz) turbulent data were recorded continuously for about 48 hours. A triple decomposition technique was introduced to isolate the contributions of tides, resonance and turbulence in the flow field. A striking feature of the data set was the slow fluctuations which exhibited large amplitudes up to 50% the tidal amplitude under neap tide conditions. The triple decomposition technique allowed a characterisation of broader temporal scales of high frequency fluctuation data sampled during a number of full tidal cycles.

High-resolution GNSS-tracked drifter for studying surface dispersion in shallow water

The use of GNSS tracked Lagrangian drifters allows more realistic quantification of fluid motion and dispersion coefficients than Eulerian techniques because such drifters are analogues of particles that are relevant to flow field characterisation and pollutant dispersion. Using the fast growing Real Time Kinematic (RTK) positioning technique derived from Global Satellite Navigation Systems (GNSS), drifters are developed for high frequency (10 Hz) sampling with position estimates to centimetre accuracy. The drifters are designed with small size and less direct wind drag to follow the sub-surface flow which characterizes dispersion in shallow waters. An analysis of position error from stationary observation indicates that the drifter can efficiently resolve motion up to 1 Hz. The result of the field deployments of the drifter in conjunction with acoustic Eulerian devices shows higher estimate of the drifter streamwise velocities. Single particle statistical analysis of field deployments in a shallow estuarine zone yielded dispersion coefficients estimate comparable to those of dye tracer studies. The drifters capture the tidal elevation during field studies in a tidal estuary.