Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / Nature has developed a generous number of peptides carrying out various essential functions in all living organisms. Human body produces peptides as signaling molecules, such as hormones, to transmit messages from cell to cell and regulate metabolic homeostasis. Microbes synthesize peptides as antibiotics to inhibit the growth of other microorganisms. These peptides display an exceeding diversity of amino acid composition, peptide sequence, secondary structure and post-translational modification. Inspired by nature, researchers have developed peptides as a unique modality of therapeutics, combining the best attributes of small-molecule drugs and protein-based biopharmaceuticals. This work has sought to explore the potential of peptides as synthetic receptors, binders of protein and probes for bacteria detection. The research started from a foldable cyclic peptide scaffold, prolinomycin, a proline-rich analogue of valinomycin. The peptide can chelate a potassium ion folding into a drum like structure, which provides a platform to display and preoganize functional side chains for target binding. We first investigated its folding behavior under physiological conditions. We demonstrate that the metal-assisted folding of the prolinomycin scaffold tolerates various side chain mutations. The stability of the structure can be improved by introducing crosslinking moieties. Based on this scaffold, we rationally designed synthetic receptors of various amines by utilizing iminoboronate chemistry with acetylphenyl boronic acid (APBA). Furthermore, I pursued phage display, a powerful technique to develop high affinity peptide binders of protein targets. Proteins are the most appealing targets for drug development and disease biomarkers discovery. We chose sortase A (SrtA) as a model target protein to screen for potent peptide binders. A peptide inhibitor of sortase A with single-digit micromolar affinity was identified from a cyclic peptide library displayed by phage. In addition, from the chemically modified phage display peptide library presenting APBA motifs, peptide binders with specificity and micromolar affinity towards SrtA were discovered. Instead of binding to the active site, the peptide could recognize the surface of the protein. Additionally, to further expand the chemical space of phage display, I constructed a phage display peptide library presenting N-terminal cysteine (NCys) which can undergo site-specific chemical modifications. Two pieces of chemistry were applied, including thiazolidino boronate (Tzb) mediated acylation reaction of NCys and 2-cyanobenzothiazole (CBT)-NCys condensation. The site-specific dual modifications on NCys and internal Cys of phage-encoded peptides were achieved. Furthermore, a strategy to N, S-doubly label NCys via an alternative pathway of CBT condensation was reported, which presents a significant addition to the toolbox for site-specific protein modifications. Finally, by functionalizing graphene field effect transistors (G-FET) with peptide probes, we developed the first selective, electrical detection of the pathogenic bacterial species Staphylococcus aureus and antibiotic resistant Acinetobacter baumannii on a single platform. Overall, peptides provide enormous opportunities for therapeutics development. Research herein demonstrated principles of peptide design for specific molecular recognition. Novel chemistry strategies have been developed to expand the molecular diversity of peptide libraries. We believed that the advances in peptide design and screening would promote peptide-based drug discovery. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

One major challenge in synthetic biology is how to design genetic circuits with predictable behaviors in various biological contexts. There are two limitations to addressing this challenge in mammalian cells. First, models that can predict circuit behaviors accurately in bacteria cells cannot be directly translated to mammalian cells. Second, upon interconnection, the behavior of a module, the building block of a circuit, may be different from its behavior in a standalone setting. In this thesis, I present a bottom-up modeling framework that can be used to predict circuit behaviors in transiently transfected mammalian cells (TTMC). The first part of the framework is based on a novel bin-dependent ODE model that can describe the behavior of modules in TTMC accurately. The second part of the framework rests upon a method of modular composition that allows model-based design of circuits. The efficacies of the bin-dependent model and the method of modular composition are validated via experimental data. The effects of retroactivity, a loading effect that arises from modular composition, on circuit behaviors are also investigated.

PhD,School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand / Tissue specific regulation of the progesterone receptor is central to female health. The synthetic steroid, RU486 is a partial progesterone and oestrogen receptor antagonist, functioning to actively silence progesterone receptor gene associated transcription. For this reason, it has been used both as a contraceptive and quite controversially as an abortive agent. In this study, both cellular and gene specific effects of RU486 were investigated in a rat model of early pregnancy, this including the key phases of the plasma membrane transformation, the window of receptivity and early implantation. As all of these stages are hormonally regulated by progesterone and oestrogen, the focus here was to elucidate the mechanisms of action of a single dose of RU486, used as a postcoital contraceptive, at day 3.0 of pregnancy, to successfully prevent implantation of a viable blastocyst and subsequent pregnancy. In association with the cellular preparation of uterine epithelial cells for implantation, selected molecular targets and events were investigated at a protein and gene expression level, both prior to and after RU486 treatment, to assess the effects of either a deficit or excessive expression of these gene products on uterine preparation and eventual implantation. Factors here included the progesterone receptor, markers of apoptosis (Bax and Bcl2), mediators of angiogenesis (VEGF, bFGF and PDGF) and biomarkers of endometrial implantation (LIF, Calcitonin and Muc-1). Together, an ultrastructural and light microscopy analysis showed RU486 to morphologically alter the uterine endometrial cells and to disrupt the plasma membrane transformation of early pregnancy, predisposing these cells towards apoptosis. In association with this, progesterone receptor gene and protein expression was ubiquitously decreased throughout pregnancy. With regards to the implantation process of early pregnancy, the luminal epithelial cells undergo apoptosis to allow the hatching blastocyst to penetrate and implant within the uterine wall. This is partially mediated by the ratio of the expression of the apoptotic factors Bax and Bcl-2. Surprisingly here, RU486 caused an overall anti-apoptotic environment, despite previously observed high levels of apoptotic activity. This indicates that factors other than Bax and Bcl-2 influence the RU486-induced apoptosis. A crucial event of early pregnancy is the establishment of an adequate blood supply to sustain and nourish the implanting blastocyst. There was a decided reduction in the angiogenic response of early pregnancy, as a direct consequence of RU486 treatment; the normally high levels of VEGF and bFGF during early pregnancy, were markedly decreased at all three days of pregnancy. This was reflected in the lack of increased vascularisation as normally signalled by the indicator dye, Pontamine Sky blue. In contrast to the overall increase in VEGF and bFGF at the time of blastocyst implantation during early pregnancy, increased PDGF expression was localised to the implantation sites, strongly suggesting a role for this angiogenic factor in endothelial cell proliferation. v The endometrial biomarkers are indicative of implantation, their expression patterns varying around the phase of implantation. These markers are essential to implantation, as when LIF and Calcitonin are deregulated and Muc-1 persists on the apical surface of the endometrium, implantation fails. These events are precisely what occur following RU486 treatment. In summary, the overall effects of RU486 in the rat model of early pregnancy, when used as a postcoital contraceptive, indicate highly effective inhibition of progesterone and oestrogen effects on the endometrium, mediated by their receptors. More specifically, the structural and molecular events mirror those described in ovariectomised animal models, suggesting a hormonally under-stimulated endometrium. Clearly from the present study, the precise priming of the endometrium in preparation for blastocyst implantation is severely impaired by RU486 through a number of signalling pathways, thus predisposing the uterus to pregnancy failure.

Thesis advisor: Eranthie Weerapana / Thesis advisor: Jianmin Gao / Proteins are composed of the 20 naturally occurring amino acids and are further modified by a variety of post-translational modifications (PTMS). Naturally occurring amino acids are diverse in structure and function. Catalytic amino acids, or nucleophilic amino acids, are of particular interest because of their contribution to chemical transformations in the cell. Synthetic covalent modification is a means to further functionalize or diversify proteins. These modifications, or enhancements, allow for improved understanding of protein structure, function and activity. For instance, isotope labeling of amino acid side chains in NMR studies enable investigators to study protein dynamics upon substrate or ligand binding. Fluorescence labeling is particularly useful to investigate protein cellular localization. Covalent modification is a useful tool to investigate the relative level of activity for protein known to be regulated by PTMs. An important feature of covalent modification reactions is site specificity, as this dictates the location, number of modifications, and protein targets. Tyrosine is of particular interest because it is both nucleophilic and aromatic. These characteristics contribute to the existence of tyrosine residues in both the protein surface and hydrophobic cores. Tyrosine is incorporated into proteins at a relatively low frequency. Unlike lysine, which is ubiquitous on protein surfaces, the low number of potential sites for general tyrosine modifications makes it an attractive site for surface bioconjugation modifications. A low number of surface modifications is less likely to perturb native protein function. Bioconjugation reactions give access to functionalizing the surface of proteins with moieties such as fluorophores, PEG, peptides, or drugs. Tyrosine is an attractive target for modifications because it is found in the active sites of a variety of enzymes such as sialidases, glutathione-S transferases, corticosteroid 11-beta-dehydrogensase, DNA topoisomerase, and ferredoxin-NADP+ reductase. Provided here is a survey of the known non-selective and selective synthetic chemical modification reactions for tyrosine. To investigate nucleophilic amino acids, Activity Based Protein Profiling (ABPP) may be implemented to investigate the role of these residues. ABPP utilizes small molecule covalent probes as a tool to selectively target enzymes in their active state. To investigate a protein of interest (POI) (or class of proteins) by ABPP, it is necessary to use a small molecule covalent probe that selectively reacts with the POI over other proteins within the proteome. Due to this requirement, it is necessary to expand the current ABPP probe toolbox to increase the coverage of what proteins in the proteome may be studied. Inspired by findings in the literature, our lab sought to explore the utility of various aryl halides for implementation in ABPP probes to overcome this limitation. This study revealed dichlorotriazine as a biologically relevant and reactive electrophile. A focus was placed on a dichlorotriazine containing probe library (LAS1-LAS20). LAS17 was discovered to be a potent and selective inhibitor of human glutathione S-transferase pi (GSTP1). Further studies revealed GSTP1 as a novel therapeutic target for the treatment of triple negative breast cancer. Other studies revealed several members of the dichlorotriazine library were found to covalently modify purified recombinant human aldolase A (ALDOA) in the presence of a complex cellular background. Additionally, LAS9 was identified as an inhibitor of ALDOA retro aldol condensation activity in vitro. Lastly, the final chapter highlights two collaborations in which tandem mass spectrometry experiments aid in the characterization of experimental data. In the first collaboration, a quantitative cysteine reactivity profiling method was used to characterize the selectivity of a cysteine reactive covalent NRF2-inducing small molecule, MIND4-17. In the second collaboration, analysis of tryptic mass spectrometry data enabled high resolution characterization of peptide sequencing for superfolder green fluorescent protein (sfGFP) expressed from observed internal nonsense suppression. Identification of the misincorporated amino acid facilitated the elucidation of the cross-talk mechanism. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Nucleic acids store genetic information in the sequence of nucleobases. Through duplex formation and template directed synthesis, the information stored in nucleic acids determines their three-dimensional structure and function. Nucleic acids are essential molecules for biological processes and have been used in nanotechnology. Modified nucleic acids have been synthesised that still form duplexes and can be tolerated by enzymes, suggesting that it is possible to construct a synthetic system comparable to nucleic acids, orthogonal to nucleic acids. This thesis describes the synthesis of a new class of synthetic information molecule, characterisation of the duplex forming properties, and attempts at templated oligomerisation reactions. The new synthetic information molecule is based on the phenylacetylene oligomer framework developed by Moore and co-workers. Recognition was achieved via a base-pair that is made from a single point high affinity H-bond, with phenol as the H-bond donor (D) and phosphine oxide as the H-bond acceptor (A). The Sonogashira coupling was used to construct the phenylacetylene oligomer backbone. The AA, DD and AD 2-mers were synthesised and complementary 2-mers showed cooperative duplex formation. No intramolecular H-bonding due to folding was observed in the AD mixed 2-mer. Longer oligomers were synthesised using a method of oligomerisation and chromatographic separation by reverse-phase preparatory HPLC. Homo-oligomers up to the 7-mer were isolated and binding studies between complementary all donor, all acceptor homo-oligomers showed increasing duplex stability with each additional recognition unit in the oligomer chain. Oligomers containing both acceptor and donor recognition modules in the same chain were synthesised and NMR dilution studies were used to investigate their ability to fold. Preliminary experiments were carried out to evaluate the ability of these information molecules to template oligomerisation reactions, but when reactions were carried out at concentrations low enough for a significant template effect, no coupling reactions were observed.

About 74 million of the world population needs assistive leg devices on daily basis on account of some form of disability. Although the standard wheelchairs perform well on level ground terrains but they prove ineffective on soft ground and in crossing large obstacles. For dealing with the advance challenges of navigating the human environment a biped walker seems to a more suitable choice. The research presented here is focused on building and actuating a two-joint leg structure that is an integral part of ongoing leg-chair project in Popovic Labs. The project provides a proof of concept that how the synthetic hydro muscles (also developed by Popovic Labs) can be used for the control of large artificial limb joints. Here we discuss the designing, testing and corresponding refining of electronics design, actuation and control of the bi-joint hydro muscle actuated leg structure .We will also elaborate on the requirements, design, problems and refinement of some of the important mechanical components like Coupler/Decoupler and Force Multipliers.

In heterogeneous catalysis, the phase of a catalytic agent, which is responsible for reducing the activation energy of a reaction, is different from the phase of its reactants or substrates. Often, soluble catalysts are tightly associated with an inert carrier in order to artificially alter their phase. Applying this concept to biocatalysis yields a system in which enzyme molecules are immobilized on a solid support. This often serves to stabilize the enzyme, as well as enhance the recyclability of the enzyme since it is no longer soluble. In this dissertation, two methods of enzyme immobilization are evaluated: adsorption to a solid surface and whole-cell biocatalysis. The latter is then engineered for improved kinetics and functional activity using principles of synthetic biology. Adsorption of a protein to a solid surface is driven by the same thermodynamic factors that are responsible for the folding of a protein. Hydrophobic interactions, ionic interactions, covalent bonding, and weak forces all contribute to minimizing the free energy of a protein, which defines its secondary, tertiary, and quaternary structures. Upon introduction to a surface, these different forces rearrange across the surface of the substrate to minimize the free energy of the system. Many factors influence this behavior, including particle curvature, material properties of the surface, and the stability of the protein. In the preexisting body of work, much of the research performed regarding the effects of thermal stability on adsorption were performed using mutant proteins whose structures were intentionally altered for a range of stabilities. In Chapter 2, we evaluate the effects of thermal stability on adsorption behavior using naturally evolved enzymes from the AKR superfamily, namely AdhD and hAR. These enzymes were selected for their structural homology, but vastly different thermal stabilities. Using these proteins, we demonstrate that the previously held theories of thermostable protein adsorption behavior are not entirely applicable to naturally evolved proteins that are not artificially stabilized. We also propose a modification to the classic 4-state adsorption/desorption model by introducing new pathways and protein states based on our experiments. In addition to physisorption, whole-cell biocatalysis was explored as an enzyme immobilization platform. In general, this can be accomplished by cytosolic expression, periplasmic expression, or surface display. After weighing these options, we chose periplasmic expression in E. coli for our biocatalysts. As for the catalytic component, we selected carbonic anhydrase (CA), a class of Zn+2-binding metalloenzymes that are capable of catalyzing the reversible hydration of CO2. This enzyme was selected for the breadth of applications it can be used for, as well as its ubiquity in nature and extremely fast kinetics. Two isoforms were selected (Cab and Cam) for their respective benefits and were periplasmically expressed using 2 different leader peptides, which we discuss in Chapter 3. The enzyme loading in the periplasm, kinetics, thermal stability, and functional activity are all reported for the resulting whole-cell biocatalysts. We also describe a new method for the measurement of the operational stability of CA-based biocatalysts. Modifications to the whole-cell biocatalysts are described in Chapter 4 and Chapter 5. In Chapter 4, we demonstrate that expression of a viral envelope protein enhances the permeability of the outer membranes of E. coli cells. We characterize this improvement by measuring small-molecule permeance, whole-cell kinetics, and functional activity of the modified biocatalysts. We also quantify this enhancement by applying concepts of porous chemical catalysts to our whole-cells. In doing so, we show improvements in parameters such as the effectiveness factor, Thiele modulus, diffusivity, and permeability. Finally, in Chapter 5 we show enhancement of the functional activity of the whole-cell biocatalysts by displaying small peptides on the outer surfaces of the cells. The modified cells are shown to enhance precipitation of calcium carbonate, a common end product of carbon mineralization. Improved solid formation rates are also reported and possible explanations for these effects are discussed. Overall, this dissertation explores immobilization of enzymes to create heterogeneous biocatalysts. First, the effects of immobilization on enzyme structure, stability, and activity are shown for two different immobilization techniques: adsorption to a solid surface and periplasmic expression in E. coli cells. After characterization, engineering of the whole-cell biocatalysts for improved properties is presented. Finally, future directions for this work are discussed which would advance our understanding of heterogeneous biocatalysts, as well as enhance their utility.

Glucocorticoids - cortisol and corticosterone - are steroid hormones synthesised in the adrenal gland that are important mediators of the stress response. Glucocorticoids are also vital in development to aid in organ maturation. Endogenous glucocorticoid levels rapidly rise before birth in all mammals to promote fetal organ maturation. Because preterm birth occurs before this natural rise in glucocorticoid levels, pregnant women at risk of preterm delivery are administered synthetic glucocorticoids to mature the fetal lung and aid neonatal survival. Mice that globally lack the glucocorticoid receptor (GR) die at birth, attributed to lung immaturity. Effects on tissues other than the lung remain less well characterised. Previous work has shown endogenous glucocorticoid action is also essential to mature the mouse fetal heart. Mice globally lacking GR have small, functionally and structurally immature hearts. Mice with tissue-specific deletion of GR in cardiomyocytes and vascular smooth muscle cells (SMGRKO mice; generated using Sm22α-Cre) also have an increased risk of death around the time of birth, suggesting that glucocorticoid maturation of the cardiovascular system is important for neonatal survival. GR expression within the fetal mouse heart initiates at E10.5 but GR in the myocardium is not activated and localised to the nucleus until E15.5. This suggests that mice can respond to glucocorticoid from E10.5. Here, it was hypothesised that antenatal glucocorticoid exposure, prior to the increase in endogenous glucocorticoid levels, would advance fetal heart maturation and this will depend on cardiovascular GR. To investigate the effects of antenatal glucocorticoid treatment on fetal heart maturation in mid-gestation and identify effects mediated by GR, mice with a conditional deletion of GR in cardiomyocytes and vascular smooth muscle cells were studied (SMGRKO mice). Pregnant mice received dexamethasone (dex) in the drinking water from E12.5-E15.5. Levels of Fkbp5 mRNA (a marker of glucocorticoid action) were unchanged between control and SMGRKO mice at E15.5 or following dex treatment. This suggested a lack of response to dex treatment. However, liquid chromatography mass spectrometry measurement confirmed the presence of dex and its active metabolite 6- hydroxydexamethasone (6OHDex) in livers of E15.5 fetuses from dex treated dams (fetal: Dex 0.46 ± 0.1 ng/g, 6OHDex 13.6 ± 0.35 ng/g; dam: Dex 7.96 ± 3.65 ng/g, 6OHDex 4.75 ± 1.2 ng/g). Livers of fetuses exposed to dex had lower levels of the naturally occurring active glucocorticoid, corticosterone, compared to vehicle treated fetuses. This suggests HPA axis suppression in dex exposed fetuses. Maternal liver showed no significant difference in corticosterone levels between dex and vehicle treated mice, suggesting that whilst dex suppressed the HPA axis in fetuses, it did not in the dams. To determine any persistent effects of early antenatal dex treatment on fetal heart, a later time point in gestation, E17.5, was also assessed. At E17.5, 2-days following cessation of dex treatment, dex and its metabolites were undetectable in the fetal and maternal liver. However, corticosterone levels remained reduced in fetal liver at E17.5 in dex exposed animals (vehicle treated: 4.31 ± 0.47 ng/g, Dex treated: 1.72 ± 0.42 ng/g, p < 0.01), whilst levels in the dam liver did not differ from vehicle treated controls. This suggests prolonged HPA axis suppression following dex treatment, which reduced the natural late-gestation rise in glucocorticoids required for fetal organ maturation. To determine whether early antenatal dex treatment could advance fetal heart function, Doppler imaging with a Vevo 770 high frequency ultrasound imager was used. Isovolumetric contraction time, isovolumetric relaxation time and ejection time of the left ventricle were unaltered by dex treatment. However, at E15.5 the mitral deceleration index (MDI), a measure of diastolic function that takes into account loading conditions, was 1.5 fold lower in vehicle treated SMGRKO mice than control (Cre-) littermates (p < 0.05). This reduction in SMGRKO mice suggests glucocorticoids are required within the fetal cardiomyocytes and/or vascular smooth muscle cells to mature the diastolic function of the fetal heart. Dex exposure had no effect on MDI in SMGRKO fetuses, but reduced the MDI by 1.5 fold in control mice to similar levels as in SMGRKO mice (p < 0.05). RNA analysis revealed a trend (p=0.09) for reduced levels of Nr3c1 mRNA (encoding GR) in hearts of E15.5 control (Cre-) fetuses following dex treatment. Although this requires confirmation at the level of GR protein, this finding together with the lack of induction of the GR target, Fkbp5, suggests dex may cause glucocorticoid resistance through down-regulation of GR. At E17.5, 2-days following cessation of dex there were no changes in systolic parameters and the reduction in MDI found at E15.5, following dex, had normalised. Litter size was reduced (close to a 50% reduction) at E17.5 in dex treated mice. This was similar between SMGRKO and control fetuses. The cause of death was not established, but potentially could be due to the reduction in the natural rise in glucocorticoids at E17.5, previously shown to be important for fetal heart maturation. It is therefore possible that mice with more immature hearts may die before reaching E17.5. RNA analysis was undertaken to determine any mechanistic alterations following dex treatment, which could support fetal heart functional alterations found at E15.5. In contrast to expectation, dex also decreased expression of mRNA encoding the calcium handling proteins SERCA2a, NCX1, and CaV1.2 in E15.5 fetal mouse hearts in both control and SMGRKO mice (p < 0.05), compared with the respective vehicle treated mice. These proteins had previously shown to be induced by glucocorticoid action in cardiomyocytes. However, the similar down-regulation in both genotypes indicates this effect is not dependent on GR in cardiomyocytes. Lowered SERCA2a activity following dex treatment could contribute to the changes in MDI observed in control mice. Similarly, Scnn1a and Kcnj12 mRNA levels, previously found to be induced by glucocorticoids in cardiomyocytes, were down-regulated in the E15.5 fetal heart in vivo following dex. Collectively, these data are consistent with glucocorticoid resistance or down-regulation of glucocorticoid action in E15.5 fetal hearts following dex administration. Mutations in KCNJ12 are associated with long QT syndrome, which is characterised by a delayed repolarisation of the heart following each contraction. An altered relaxation of the fetal heart found in control mice following dex could therefore be due to a prolongation of the cardiac action potential, particularly with a delayed repolarisation, because of lower Kcnj12 expression. At E17.5, there were no significant differences in expression of calcium handling genes or ion channel mRNAs between genotypes or following earlier dex exposure. Thus, effects of dex on mRNA expression level may not persist, which could account for the lack of functional changes observed 2-days following cessation of treatment. Because effects seen in vivo with dex treatment were contrary to those predicted, and to further investigate the effect of dex upon calcium content, an in vitro model of primary fetal E15.5 cardiomyocytes was used. Cardiomyocytes were treated with dex for 24 hours and effects on membrane potential voltage changes and calcium transients measured. Following dex, isolated fetal cardiomyocytes showed an elongated repolarisation phase of the action potential (untreated: 120.45 ± 13.81 ms, Dex: 142.34 ± 12.97 ms, p < 0.01), and duration of calcium transients (untreated: 103.31 ± 13.78 ms, Dex: 120.43 ± 23.36 ms, p < 0.05). This assessment of fetal cardiomyocytes was preliminary work to aid in the understanding of mechanisms of fetal heart functional alterations associated with glucocorticoid regulation. The results suggest glucocorticoids may be important in regulating calcium levels. In summary, dex treatment in mice from E12.5-E15.5 did not advance fetal heart maturation. It reduced litter size at E17.5, irrespective of whether GR was expressed in cardiomyocytes or not. The normal late-gestation increase in endogenous glucocorticoid levels in the fetus was reduced by dex, even after treatment finished. / The suppression of corticosterone levels following antenatal dex may reduce maturation of the heart at E15.5 and could be responsible for the reduction in litter size. Downregulation of GR in the fetal heart, may be a mechanism that results in glucocorticoid resistance following antenatal dex treatment, which could explain the lack of beneficial effects of antenatal dex upon fetal heart maturation in these experiments in mice.

Toxicity of organic solvents to microbial hosts is a major consideration in the economical production of biofuels such as ethanol and especially butanol, with low product concentrations leading to high recovery costs. The key to rational engineering of solvent tolerant microorganisms for such processes lies in obtaining appropriate tolerance genes (modules) suited for different compounds. In this project, a synthetic biology approach was adopted to generate a library of standardised BioBrick parts involved in different stress responses. Using a multiple-assay approach, including a bioluminescence assay, these stress response genes were tested individually and in combination to determine their effects on survival in ethanol, nbutanol, acetone and fermentation inhibitors produced by biomass pre-treatment. A set of tolerance modules was obtained for ethanol and n-butanol. Proof-of-concept tests suggested that ethanol and n-butanol toxicity was mainly due to damage to membrane, cellular proteins and DNA possibly by oxidative stress. No synergistic interactions were observed from a combination of different tolerance genes. Further tests carried out using enzyme and fluorescence-based assays to elucidate the effect of n-butanol on the cell envelope showed that the solvent released lipopolysaccharides from the outer membrane of E. coli and also caused both outer and inner membranes to be leaky. Very high n-butanol concentrations resulted in an altered cell shape and bleb formation suggesting an impairment in cell division and peptidoglycan biosynthesis. The cell membrane was modified by cis-trans isomerisation of unsaturated fatty acids in the phospholipids resulting in a reduction of membrane leakage which in effect, increased n-butanol tolerance in E. coli. In conclusion, results from this research suggest that strategies to protect the membrane and cellular proteins should be included in rational engineering of n-butanol tolerant bacteria.

The effect of stoichiometry on the pyrolytic decomposition, oxidation and ignition behaviour of synthetic violarite and pentlandite has been established. These minerals, of general formula (Fe,Ni)(subscript)3S(subscript)4 and (Fe,Ni)(subscript)9S(subscript)8 respectively, may vary considerably in Fe:Ni ratio. Pentlandite can also show some variation in metal:sulfur ratio. A series of samples, ranging in stoichiometry from Fe(subscript)0.96Ni(subscript)1.97S(subscript)4 to Fe(subscript)0.20Ni(subscript)2.72S(subscript)4 and Fe(subscript)5.80Ni(subscript)3.15S(subscript)8 to Fe(subscript)3.40Ni(subscript)5.55S(subscript)8, were synthesised and characterised using wet chemical analysis, electron probe micro-analysis (EPMA), scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer, Emmett and Teller (BET) surface area analysis.The thermal behaviour of these sulfides was examined using simultaneous Thermogravimetry-Differential Thermal Analysis (TG-DTA) at different heating rates and in different atmospheres. Partially reacted samples were collected at various temperatures and analysed using XRD, EPMA, SEM, optical microscopy (OM), and Fourier transform infrared (FTIR) spectroscopy. The endmembers of the violarite and pentlandite series were examined in detail to determine the effect of stoichiometry on the reaction mechanism. In this study the reaction mechanism refers to the sequence of reactions occurring during pyrolytic decomposition or oxidation of the sulfide minerals. Samples were sieved into four particle size fractions, 125-90, 90-63, 63-45 and 45-20 gm, to determine the effect of particle size on the reaction mechanism.When violarite was heated in an inert atmosphere at 10 degrees celsius min(subscript)-1, it initially decomposed to a monosulfide solid solution (mss), (Fe,Ni)(subscript)1-xS, and vaesite, (Ni,Fe)S(subscript)2, indicated by a ++ / sharp endothermic peak in the DTA trace. The decomposition temperature was found to be linearly dependent on the iron:nickel ratio, decreasing from 495 degrees celsius to 450 degrees celsius as the iron:nickel ratio decreased from 0.49 to 0.07. This was followed by a broader endothermic peak coinciding with a rapid mass loss, which was associated with the decomposition of vaesite to mss with the loss of sulfur. Between 615-805 degrees celsius the mss was converted to a high temperature form of heazlewoodite, (Fe,Ni)(subscript)3+/-S(subscript)2 melted incongruently at 835 degrees celsius and 805 degrees celsius for Fe(subscript)0.96Ni(subscript)1.97S(subscript)4 and Fe(subscript)0.20Ni(subscript)2.72S(subscript)4 respectively, with further loss of sulfur vapour forming a central sulfide liquid of general formula (Fe,Ni)(subscript)1+xS.Under similar experimental conditions, pentlandite pyrolytically decomposed forming mss and heazlewoodite with no associated loss of sulfur. The decomposition temperature decreased as the iron:nickel ratio deviated from the ideal value of 1:1. A maximum decomposition temperature of 610 degrees celsius was found at an iron:nickel ratio of 1.00, decreasing to 580 degrees celsius at a ratio of 1.84 and 0.61. Sulfur was evolved slowly at temperatures in excess of 760 degrees celsius as mss was converted to heazlewoodite, indicated by a gradual mass loss. The heazlewoodite then melted incongruently in excess of 840 degrees celsius indicated by a sharp endothermic peak, resulting in a further loss of sulfur.The oxidation of violarite and pentlandite was investigated at a heating rate of 10 degrees celsius min(subscript)-1 in an air atmosphere. The oxidation of violarite was initiated by decomposition to mss resulting in a rapid mass loss associated with the evolution of sulfur vapour, and an exothermic peak due to the gas phase oxidation of ++ / the sulfur. The iron sulfide component of the mss was then preferentially oxidised to iron(II) sulfate between 485-575 degrees celsius, upon which the sulfate decomposed and the remaining iron sulfide was preferentially oxidised to hematite. The mss core was then converted to (Fe,Ni)(subscript)3+/-xS(subscript)2 between 635-715 degrees celsius, resulting in the loss of further sulfur which was oxidised. The sulfide core, which consisted of predominantly Ni(subscript)3+/-xS(subscript)2 with a minor amount of iron still remaining in solid solution, incongruently melted at a constant temperature of 795 degrees celsius regardless of the initial stoichiometry of the violarite sample. This was followed by the rapid oxidation of the liquid sulfide resulting in a sharp exothermic peak in the DTA trace.For pentlandite, the TG-DTA curve exhibited an initial mass gain commencing at approximately 400 degrees celsius, which was attributed to the preferential oxidation of iron. Evidence from SEM indicated that iron migrated towards the oxygen interface, where it was oxidised to hematite. During this process the metal: sulfur ratio decreased and pentlandite was converted to mss. The iron sulfide component of the mss phase was then preferentially oxidised to hematite as indicated by a major exotherm, which occurred in the temperature range 575-665 degrees celsius, forming an oxide product layer around a nickel sulfide core. The oxidation of the remaining nickel sulfide followed the same reaction sequence to that of violarite.By increasing the heating rate to 40 degrees celsius min(subscript)-1, and carrying out the oxidation in pure oxygen, the tendency of the sulfides to ignite was established. Ignition was characterised by a highly exothermic reaction which coincided with a rapid mass loss over a short time period. Overheating of the samples above the programmed furnace ++ / temperature was also observed. Violarite exhibited ignition behaviour while pentlandite did not.Both sulfides were subjected to shock heating conditions (heating rate = 1500 - 5000 degrees celsius min(subscript)-1, oxygen atmosphere) using isothermal thermogravimetry (TG). This method produces heating rates analogous to those which are experienced in the reaction shaft of an industrial flash smelter. The effect of stoichiometry on ignition temperature and extent of oxidation for the entire series of synthetic violarites and pentlandites was determined. Partially ignited and ignited products were collected from isothermal TG experiments and were examined by OM, SEM and EPMA to establish the ignition mechanism.Both violarite and pentlandite ignited using the isothermal TG technique. A clear relationship was found between the stoichiometry of violarite and pentlandite and the ignition temperature, with an increase in the iron:nickel ratio causing a decrease in the ignition temperature. The ignition temperature also decreased as the size of the particles decreased.The extent of oxidation increased as the iron:nickel ratio increased, and also increased as the particle size decreased.