Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

The role of the synthetic enzyme GAD65 in the control of neuronal γ-aminobutyric acid release

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of γ-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65−/− mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K+-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65−/− mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65−/− mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.

Control parameters in the batchwise production of synthetic resins

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Application of computer control to the production of synthetic resins

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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.

Novel synthetic bio-mimic polymers for potential cell delivery therapy

Cell-based therapy is one of the major potential therapeutic strategies for cardiovascular, neuronal and degenerative diseases in recent years. Synthetic biodegradable polymers have been utilized increasingly in pharmaceutical, medical and biomedical engineering. Control of the interaction of living cells and biomaterials surfaces is one of the major goals in the design and development of new polymeric biomaterials in tissue engineering. The aims of this study is to develop a novel bio-mimic polymeric materials which will facilitate the delivery cells, control cell bioactivities and enhance the focal integration of graft cells with host tissues.

Vibrational spectroscopy of synthetic stercorite H(NH4)Na(PO4)•4H2O – a comparison with the natural cave mineral

In order to mimic the chemical reactions in cave systems, the analogue of the mineral stercorite H(NH4)Na(PO4)•4H2O has been synthesised. X-ray diffraction of the stercorite analogue matches the stercorite reference pattern. A comparison is made with the vibrational spectra of synthetic stercorite analogue and the natural Cave mineral. The mineral in nature is formed by the reaction of bat guano chemicals on calcite substrates. A single Raman band at 920 cm-1 (Cave) and 922 cm-1 (synthesised) defines the presence of hydrogen phosphate in the mineral. In the synthetic stercorite analogue, additional bands are observed and are attributed to the dihydrogen and phosphate anions. The vibrational spectra of synthetic stercorite only partly match that of the natural stercorite. It is suggested that natural stercorite is more pure than that of synthesised stercorite. Antisymmetric stretching bands are observed in the infrared spectrum at 1052, 1097, 1135 and 1173 cm-1. Raman spectroscopy shows the stercorite mineral is based upon the hydrogen phosphate anion and not the phosphate anion. Raman and infrared bands are found and assigned to PO43-, H2O, OH and NH stretching vibrations. Raman spectroscopy shows the synthetic analogue is similar to the natural mineral. A mechanism for the formation of stercorite is provided.

Stability of hydrotalcites formed from Bayer refinery environmental control processes

Bauxite refinery residues (red mud) are derived from the Bayer process by the digestion of crushed bauxite in concentrated sodium hydroxide at elevated temperatures and pressures. This slurry residue, if untreated, is unsuitable for discharge directly into the environment and is usually stored in tailing dams. The liquid portion has the potential for discharge, but requires pre-treatment before this can occur. The seawater neutralisation treatment facilitates a significant reduction in pH and dissolved metal concentrations, through the precipitation of hydrotalcite-like compounds and some other Mg, Ca, and Al hydroxide and carbonate minerals. The hydrotalcite-like compounds, precipitated during seawater neutralisation, also remove a range of transition metals, oxy-anions and other anionic species through a combination of intercalation and adsorption reactions: smaller anions are intercalated into the hydrotalcite matrix, while larger molecules are adsorbed on the particle surfaces. A phenomenon known as ‘reversion’ can occur if the seawater neutralisation process is not properly controlled. Reversion causes an increase in the pH and dissolved impurity levels of the neutralised effluent, rendering it unsuitable for discharge. It is believed that slow dissolution of components of the red mud residue and compounds formed during the neutralisation process are responsible for reversion. This investigation looked at characterising natural hydrotalcite (Mg6Al2(OH)16(CO3)∙4H2O) and ‘Bayer’ hydrotalcite (synthesised using the seawater neutralisation process) using a variety of techniques including X-ray diffraction, infrared and Raman spectroscopy, and thermogravimetric analysis. This investigation showed that Bayer hydrotalcite is comprised of a mixture of 3:1 and 4:1 hydrotalcite structures and exhibited similar chemical characteristic to the 4:1 synthetic hydrotalcite. Hydrotalcite formed from the seawater neutralisation of Bauxite refinery residues has been found not to cause reversion. Other components in red mud were investigated to determine the cause of reversion and this investigation found three components that contributed to reversion: 1) tricalcium aluminate, 2) hydrocalumite and 3) calcium hydroxide. Increasing the amount of magnesium in the neutralisation process has been found to be successful in reducing reversion.

Vibrational spectroscopy of synthetic archerite (K,NH4)H2PO4 : and in comparison with the natural cave mineral

In order to mimic the formation of archerite in cave minerals, the mineral analogue has been synthesised. The cave mineral is formed by the reaction of the chemicals in bat guano with calcite substrates. X-ray diffraction proves that the synthesised archerite analogue was pure. The vibrational spectra of the synthesised mineral are compared with that of the natural cave mineral. Raman and infrared bands are assigned to H2PO4-, OH and NH stretching and bending vibrations. The Raman band at 917 cm-1 is assigned to the HOP stretching vibration of the H2PO4- units. Bands in the 1200 to 1800 cm-1 region are associated with NH4+ bending modes. Vibrational spectroscopy enables the molecular structure of archerite to be analysed.

Fast and robust stereo matching algorithms for mining automation

The mining environment, being complex, irregular and time varying, presents a challenging prospect for stereo vision. For this application, speed, reliability, and the ability to produce a dense depth map are of foremost importance. This paper evaluates a number of matching techniques for possible use in a stereo vision sensor for mining automation applications. Area-based techniques have been investigated because they have the potential to yield dense maps, are amenable to fast hardware implementation, and are suited to textured scenes. In addition, two non-parametric transforms, namely, the rank and census, have been investigated. Matching algorithms using these transforms were found to have a number of clear advantages, including reliability in the presence of radiometric distortion, low computational complexity, and amenability to hardware implementation.

A framework for a dense matching algorithm : Results using matching metrics and order statistic filters

An algorithm for computing dense correspondences between images of a stereo pair or image sequence is presented. The algorithm can make use of both standard matching metrics and the rank and census filters, two filters based on order statistics which have been applied to the image matching problem. Their advantages include robustness to radiometric distortion and amenability to hardware implementation. Results obtained using both real stereo pairs and a synthetic stereo pair with ground truth were compared. The rank and census filters were shown to significantly improve performance in the case of radiometric distortion. In all cases, the results obtained were comparable to, if not better than, those obtained using standard matching metrics. Furthermore, the rank and census have the additional advantage that their computational overhead is less than these metrics. For all techniques tested, the difference between the results obtained for the synthetic stereo pair, and the ground truth results was small.

Order statistic filters for image matching

The rank and census are two filters based on order statistics which have been applied to the image matching problem for stereo pairs. Advantages of these filters include their robustness to radiometric distortion and small amounts of random noise, and their amenability to hardware implementation. In this paper, a new matching algorithm is presented, which provides an overall framework for matching, and is used to compare the rank and census techniques with standard matching metrics. The algorithm was tested using both real stereo pairs and a synthetic pair with ground truth. The rank and census filters were shown to significantly improve performance in the case of radiometric distortion. In all cases, the results obtained were comparable to, if not better than, those obtained using standard matching metrics. Furthermore, the rank and census have the additional advantage that their computational overhead is less than these metrics. For all techniques tested, the difference between the results obtained for the synthetic stereo pair, and the ground truth results was small.