Chloroethenes such as tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE) and vinyl chloride (VC), are major contaminants in subsurface systems threatening water quality and human health. Under anaerobic conditions, PCE and TCE can be reductively dechlorinated to ethene. Recent findings indicate that members of the Dehalococcoides group are responsible for ethene formation at chloroethene-contaminated sites. Dehalococcoides species exhibit diverse dechlorination activities, but share highly similar 16S rRNA genes. Hence, additional gene targets that go beyond the 16S rRNA gene are needed to reliably detect and quantify Dehalococcoides populations involved in high rate chloroethene detoxification at contaminated sites. Dehalococcoides sp. strain BAV1 couples growth to reductive dechlorination of VC to ethene. To shed light on the genes involved in reductive dechlorination in strain BAV1, degenerate primers targeting reductive dehalogenase (RDase) genes of Dehalococcoides were designed using available sequence information. PCR amplification with these primers yielded seven putative RDase genes with genomic DNA from strain BAV1 as template. Transcription analysis identified one RDase gene possibly involved in VC dechlorination, which was named bvcA. The bvcA gene was not present in Dehalococcoides strains that failed to couple growth with reductive dechlorination of VC (i.e., Dehalococcoides isolates CBDB1, FL2 and 195). Primers specific for bvcA detected this gene in several, but not all, Dehalococcoides-containing, ethene-producing mixed cultures. Apparently, the bvcA-targeted primers do not capture the diversity of VC RDase genes. Nevertheless, a relevant target was identified, and bvcA-targeted primers are commercially applied to monitor Dehalococcoides sp. strain BAV1 and related organisms at contaminated sites undergoing bioremediation treatment. Additional RDase genes were identified in Dehalococcoides sp. strain FL2, and expression analysis was performed when FL2 was grown with cis-DCE and TCE as electron acceptors. Multiple RDase genes were transcribed with each electron acceptor. This work identified novel process-specific target genes that are useful for site assessment and bioremediation monitoring at chloroethene-contaminated sites. In particular, bvcA emerged as a relevant target for monitoring the critical detoxification step from VC, to ethene. Additionally, the RDase genes retrieved in this work form a basis for further exploration of the specific functions and regulation mechanisms involved in reductive dechlorination processes.

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This dissertation examines the applicability of the iron-based degradative solidification/stabilization (DS/S-Fe(II)) to various chlorinated aliphatic hydrocarbons (CAHs) that are common chemicals of concern at contaminated sites. The research focuses on the transformation of 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloro-ethane (1,1,2,2-TetCA) and 1,2-dichloroehtane (1,2-DCA) by Fe(II) in cement slurries. It also investigates the degradation of 1,1,1-TCA by a mixture of Fe(II), cement and three iron-bearing phyllosilicates. Transformation of 1,1,1-TCA and 1,1,2,2-TetCA by Fe(II) in 10% cement slurries was characterized using batch reactors. Dechlorination kinetics of 1,1,1-TCA and TCE* (TCE that was produced by transformation of 1,1,2,2-TetCA) was strongly dependent on Fe(II) dose, pH and initial target organic concentration. Degradation of target organics in DS/S-Fe(II) process was generally described by a pseudo-first-order rate law. However, saturation relationships between the rate constants and Fe(II) dose or between the initial degradation rates and target organic concentration were observed. These behaviors were properly described by a modified Langmuir-Hinshelwood kinetic model. This supports the working hypothesis of this research that reductive dechlorination of chlorinated ethanes occurs on the surface of active solids formed in mixtures of Fe(II) and cement. Transformation products for 1,1,1-TCA and 1,1,2,2-TetCA in mixtures of Fe(II) and cement were identified. The major product of the degradation of 1,1,1-TCA was 1,1-DCA, which indicates that the reaction followed a hydrogenolysis pathway. However, a small amount of ethane was also observed. TCE* was rapidly produced by degradation of 1,1,2,2-TetCA and is expected to undergo β-elimination to produce acetylene. Dechlorination of 1,1,1-TCA in suspension of Fe(II), cement and three soil minerals (biotite, vermiculite, montmorillonite) was characterized using batch reactors. A first-order rate model was generally used to describe the dechlorination kinetics of 1,1,1-TCA in this heterogeneous system. The rate constants for 1,1,1-TCA in mixtures of Fe(II), cement and soil minerals were influenced by soil mineral types, Fe(II) dose and the mass ratio of cement to soil mineral. It was demonstrated that structural Fe(II) and surface-bound Fe(II) in the soil minerals affect dechlorination kinetics and the effects vary with mineral types. Furthermore, it suggests that the reductant formed from Fe(II) and cement hydration components is also effective in systems that include soil minerals.

This dissertation examines the applicability of the iron-based degradative solidification/stabilization (DS/S-Fe(II)) to various chlorinated aliphatic hydrocarbons (CAHs) that are common chemicals of concern at contaminated sites. The research focuses on the transformation of 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloro-ethane (1,1,2,2-TetCA) and 1,2-dichloroehtane (1,2-DCA) by Fe(II) in cement slurries. It also investigates the degradation of 1,1,1-TCA by a mixture of Fe(II), cement and three iron-bearing phyllosilicates. Transformation of 1,1,1-TCA and 1,1,2,2-TetCA by Fe(II) in 10% cement slurries was characterized using batch reactors. Dechlorination kinetics of 1,1,1-TCA and TCE* (TCE that was produced by transformation of 1,1,2,2-TetCA) was strongly dependent on Fe(II) dose, pH and initial target organic concentration. Degradation of target organics in DS/S-Fe(II) process was generally described by a pseudo-first-order rate law. However, saturation relationships between the rate constants and Fe(II) dose or between the initial degradation rates and target organic concentration were observed. These behaviors were properly described by a modified Langmuir-Hinshelwood kinetic model. This supports the working hypothesis of this research that reductive dechlorination of chlorinated ethanes occurs on the surface of active solids formed in mixtures of Fe(II) and cement. Transformation products for 1,1,1-TCA and 1,1,2,2-TetCA in mixtures of Fe(II) and cement were identified. The major product of the degradation of 1,1,1-TCA was 1,1-DCA, which indicates that the reaction followed a hydrogenolysis pathway. However, a small amount of ethane was also observed. TCE* was rapidly produced by degradation of 1,1,2,2-TetCA and is expected to undergo β-elimination to produce acetylene. Dechlorination of 1,1,1-TCA in suspension of Fe(II), cement and three soil minerals (biotite, vermiculite, montmorillonite) was characterized using batch reactors. A first-order rate model was generally used to describe the dechlorination kinetics of 1,1,1-TCA in this heterogeneous system. The rate constants for 1,1,1-TCA in mixtures of Fe(II), cement and soil minerals were influenced by soil mineral types, Fe(II) dose and the mass ratio of cement to soil mineral. It was demonstrated that structural Fe(II) and surface-bound Fe(II) in the soil minerals affect dechlorination kinetics and the effects vary with mineral types. Furthermore, it suggests that the reductant formed from Fe(II) and cement hydration components is also effective in systems that include soil minerals.

The industrial solvent trichloroethylene (TCE) is among the most ubiquitous chlorinated compounds found in groundwater pollution. TCE in environment can be removed by physical, chemical and biological procedures. The objective of this pilot-scale study was to apply an enhanced in situ bioremediation technology to remediate TCE-contaminated groundwater. Both aerobic and anaerobic remedial systems were evaluated at a TCE-spill site located in southern Taiwan. In the aerobic test zone, the effectiveness of air, nutrient, and sugarcane molasses injection to enhance the aerobic cometabolism on TCE degradation was evaluated. In the anaerobic test zone, the effectiveness of nutrient and sugarcane molasses injection to enhance the anaerobic reductive dechlorination on TCE degradation was also evaluated. Polymerase chain reaction was applied to analyze the gene variation in TCE-microbial degraders during the treatment process. Results from this study indicate that the aerobic TCE-degraders (type ¢º methanotrophs) and the gene of degradation enzymes (toluene monooxygenase, toluene dioxygenase, particulate methane monooxygenase) were detected after the treatment process in the aerobic test zone. Moreover, TCE concentration dropped from approximately 0.1 mg/L to below 0.05 mg/L in the aerobic test zone after six months of treatment. In the anaerobic treatment zone, Dehalococcoides (anaerobic TCE-degrader) and the gene of degradation enzyme (vcrA) were detected and a significant drop of TCE concentration was also observed. Results reveal that both the aerobic cometabolism and anaerobic dechlorination are feasible and applicable technologies to clean up TCE contaminated aquifers.

Modern readings of the Tractatus focus on the concept of nonsense instead of the meta-physics presented in the book. In fact a resolute reading sees the body of the system as mere nonsense and calls it a “ladder that should be thrown away”[1]. Other readings have seen the Tractatus as conveying knowledge-how[2] and some have seen the work as to be illustrating limitation of doctrinal systems such as the Mosaic Law[3]. In this paper it is argued that the Tractatus can be read as leading to a reductio ad absurdum read in its context. A reductive reading is presented that interprets the purpose of the book as conveying a method that can be used to defeat skepticism. The concept of nonsense as developed in the Tractatus is crucial for this understanding. It turns out that you can in fact discard the meta-physics of the book since when you have defeated skepticism you have come to a solid place. There is a way to determine the truth-value of all propositions about the world. Thus a resolute reading of the Tractatus is confirmed, when it comes to the meta-physical content, but the book still conveys knowledge how. [1] Cora Diamond, “Throwing away the ladder”, Philosophy, Volume 63 (243), pp. 5-27, 1998. [2] Adrian W. Moore and Peter Sullivan, “Ineffability and Nonsense”, Aristotelian Society Supplementary, Volume 77, pp. 169-223, 2003. [3] Michael Kremer, “The Purpose of Tractarian Nonsense”, Noûs, Volume 35, pp. 39-73, 2001.

Modern organic chemistry strives to achieve rapid molecular complexity from simple achiral substrates. One method by which this may be achieved is with enolate formation followed by attack on an electrophile which can generate one, two or even more new stereocentres in one step. However regioselective generation of an enolate in the presence of several enolisable sites has always proved problematical. A partial answer to this problem has been provided by the development of the reductive aldol reaction. The first part of this thesis is concerned with describing a highly diastereoselective Co(II)-catalysed reductive aldol reaction between α,β-unsaturated amides and ketones. The reaction proceeds using substoichiometric quantities of cobalt(II) in the presence of a stoichiometric quantity of the reductant diethylzinc. Using both N,Ndimethyl and morpholine amides, the reactions are tolerant of substituted aromatic ketones as well as aliphatic ketones. The reaction also proceeded well when the β-carbon was substituted with both aromatic and aliphatic groups resulting in improved diastereoselection. The racemic work is followed by the development of an asymmetric version of the reaction using oxazolidinone chiral auxiliaries that impart high levels of diastereofacial selectivity. The reaction was found to proceed with a variety of aromatic ketones and once again, substitution of the β-carbon resulted in improved diastereoselectivity. Finally work on formal homo aldol cyclisations using substoichiometric quantities of Ni(II) also in the presence of a stoichiometric quantity of diethylzinc is described. This work aims to develop methodology that involves double cyclisations with the formation of up to five contiguous stereocentres. Although unsuccessful, useful conclusions for future work were made as well as the serendipitous discovery of a apparent base catalysed alternative cyclisation pathway that successfully generated two new rings and four contiguous stereocentres.

Conventional cancer chemotherapy often lacks specificity and is consequently associated with significant normal tissue toxicities. Molecular chemotherapy offers the potential to target the activation of inert prodrugs by utilising tumour-specific catalytic enzymes to restrict cytotoxicity to neoplastic tissues. Appropriate expression of therapeutic enzymes can be achieved by exploiting the genetic distinctions that exist between tumour and normal tissues through the use of tissue- or disease-specific promoters. Alternatively, the unique physiological differences that arise in solid tumour masses as a consequence of the abnormal vascular architecture might also be exploited to achieve therapeutic selectivity. The most conspicuous of these differences is the presence of areas of low oxygen tension (hypoxia) arising through both diffusion and perfusion-limited oxygen availability. Hypoxia is an important cause of radioresistance and is a independent prognostic indicator for local recurrence, metastatic spread and overall survival. Evidence also implicates hypoxia in chemotherapeutic resistance and genetic instability, as well as the progression of and selection for an aggressive neoplastic phenotype. Attempts to eliminate tumour hypoxia have met with some success, but the opportunity to utilise thisphenomenonfor therapeutic gain, through the exploitation of the unique reductive tissue environment have lead to the development of hypoxic-specific cytotoxins. These bioreductive prodrugs rely. on the natural complement of tumour enzymes to catalyse their activation under low tissue oxygen tensions. Levels of these reductases are potentially heterogeneous and are often down-regulated in the neoplastic state. The artificial reintroduction of high levels of reductive enzyme expression may be of significant therapeutic value, particularly if expression is restricted to the hypoxic tissue enviroment in which the prodrugs will be activated. This might be achieved through the utilisation of the specific cisacting sequences that are responsive to hypoxia-regulated transcription factors. A diverse spectrum of genes are known to be induced as a consequence of oxygen deprivation, being involved in systemic oxygen supply, vascular tone, neovascularisation, iron homeostasis, glucose metabolism, drug detoxification and protein chaperoning. The details of the cis-acting sequences and transcription factors that mediate this oxygen-sensitive gene control are beginning to emerge. This provides the opportunity to exploit these defined sequences to regulate therapeutic genes in a hypoxia-responsive manner. This thesis describes the evaluation of three potential prodruglenzyme paradigms that may have application in this context. Further, the potential of hypoxia-response-elements to specifically regulate heterologous genes in response to low oxygen tension is described. The application of such an oxygen-regulated gene-directed enzyme/prodrug therapy to solid tumours may provide chemotherapeutic specificity aimed at a clinically important tumour subpopulation.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the reductive catabolism of the pyrimidine bases uracil and thymine. The clinical relevance of this enzyme is illustrated in individuals presenting with the inherited metabolic disorder thymine uraciluria. This syndrome is characterised by high plasma concentrations of thymine and uracil, and may result in clinical features including mental retardation and dysmorphia. DPD is also clinically relevant in the metabolism and subsequent inactivation of the chemotherapeutic agent 5- fluououracil (5FU). DPD activity has been shown to be highly variable in populations of healthy volunteers and cancer patients, but the mechanisms of regulation of DPD activity are as yet poorly understood. The extent of this variation may determine the efficacy or the severity of the side effects of this treatment. The aim of this research was to evaluate DPD in terms of mRNA expression, protein expression, and activity in a variety of normal and tumour tissues in an attempt to gain an insight into the regulation of DPD. Protein expression and catalytic activity were measured using the well-characterised techniques of Western blotting, and the HPLC separation of 5FU metabolites respectively. However, the method evaluating DPD mRNA expression needed to be developed and validated. After the appraisal of various mRNA detection and quantitation methodologies, competitive polymerase chain reaction (cPCR) was selected as the most suitable method for evaluating DPD transcription in these studies. The RNA samples are reverse transcribed into cDNA which then undergoes PCR amplification in the presence of known amounts of a synthetic template ('competitor') and competes for PCR primers with the target of interest. In each PCR reaction different quantities of target and competitor PCR product will be of both PCR products the concentration of the target template in the cDNA sample can be determined. Competitive PCR was demonstrated to be a highly sensitive and specific method for quantitating DPD mRNA expression, and could be used for tissues with both high and low levels of DPD (liver colon respectively). The technique was also found to be highly reproducible and reliable and was deemed to be suitable for use in further studies. To gain an understanding of the regulation of DPD in colorectal tumour, and the effect it may have upon the activity of 5FU in a specific location, the expression/activity profile of DPD was assessed in colorectal tumour, matched normal colorectal tissue, colorectal metastases to liver, and matched normnal liver. DPD activity, mRNA, and protein levels were all significantly higher in the normal liver than colon, and in the normal liver compared to liver metastases. In the colorectal tissues, mRNA levels were significantly lower in the colorectal tumour than normal colonic mucosa, however no significant difference could be determined between tissues for DPD protein and activity. A good relationship was determined between DPD activity and protein expression in colorectal tumour tissue (rs=0.61, p=0.01), whereas a weaker relationship was determined between DPD mRNA and activity for all colorectal tumour, metastases, and normal tissues (0.43, p 0.1). DPD activity has been detected in most tissues tested to date but appears to be tissue specific with higher levels observed in liver and peripheral blood mononuclear cells than other tissues. In these studies, DPD mRNA, protein, and activity were all found to be higher in the human liver tissue than normal colon.