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The mechanism of the Ley–Griffith alcohol oxidation has been elucidated using time-resolved spectroscopic methods., The Ley–Griffith reaction is utilized extensively in the selective oxidation of alcohols to aldehydes or ketones. The central catalyst is commercially available tetra-n-propylammonium perruthenate (TPAP, n-Pr4N[RuO4]) which is used in combination with the co-oxidant N-methylmorpholine N-oxide (NMO). Although this reaction has been employed for more than 30 years, the mechanism remains unknown. Herein we report a comprehensive study of the oxidation of diphenylmethanol using the Ley–Griffith reagents to show that the rate determining step involves a single alcohol molecule, which is oxidised by a single perruthenate anion; NMO does not appear in rate law. A key finding of this study is that when pure n-Pr4N[RuO4] is employed in anhydrous solvent, alcohol oxidation initially proceeds very slowly. After this induction period, water produced by alcohol oxidation leads to partial formation of insoluble RuO2, which dramatically accelerates catalysis via a heterogeneous process. This is particularly relevant in a synthetic context where catalyst degradation is usually problematic. In this case a small amount of n-Pr4N[RuO4] must decompose to RuO2 to facilitate catalysis.

Congenital CMV is the current leading infectious cause of congenital abnormalities in the developed world, affecting 0.3-2% of newborns. Of these, 10% are symptomatic at birth, while a further 10% of these develop later sequelae. Clinical manifestations vary between miscarriage or stillbirth, and cytomegalic inclusion disease of the newborn. Despite the significance of CMV as a congenital disease in humans, there remains little understanding of the factors determining the pathogenesis of congenital CMV infection from the infected mother to fetus. Altered cytokine profiles in compartments of pregnancy have been shown to be indicative of various disease states, such as viral or bacterial infection and pre-eclampsia, as well as preterm labour. However, such profiles have never been established in midtrimester amniotic fluid or localised in term placentae with CMV infection. This thesis examines the hypothesis that there are changes in pro-inflammatory and chemotactic cytokine levels that accompany congenital CMV infection in specific cells and compartments of pregnancy, and that these changes lead to fetal damage. Baseline amniotic fluid levels of 27 cytokines were established in 100 pregnant women at 14-16 weeks gestation using a fluorescent bead-based multiplex immunoassay system. These were also compared with 33 matched maternal sera sampled at amniocentesis. Uninfected status was established with negative multiplex PCR (mPCR) results for 17 known congenital infectious agents. Corresponding maternal serology results also showed no evidence of recent or active infection. Maternal serum concentrations were positively correlated with amniotic fluid levels for MIP-1β (r = 0.39, p = 0.027). IL-1ra amniotic fluid levels were found to increase with advancing maternal age (r = 0.210, p = 0.036). Mean IL-5 levels were significantly higher in amniotic fluids from pregnancies with male fetuses than those with female fetuses (p = 0.036). Normal amniotic fluid concentrations for five cytokines (IL-6, IL-8, IP-10, MCP-1, IL-1ra) were found to be significantly elevated over maternal serum concentrations in matched pairs (p < 0.05). Concentrations of 12 cytokines (eotaxin, IFN-γ, IL-9, IL-12, IL-15, IL-17, MIP-1α, MIP-1β, RANTES, TNF-α, VEGF, PDGF bb) were significantly elevated in maternal serum compared to paired amniotic fluid at midtrimester (p < 0.05). Cytokine profiles in midtrimester amniotic fluid were examined in CMV-infected pregnancies. Amniotic fluid samples were divided into four groups for cytokine comparison, based on mPCR results and maternal serology: Group I (maternal primary CMV infection) – CMV IgM-positive, CMV PCR-positive, with documented maternal CMV seroconversion during pregnancy (n = 8); Group II – CMV IgM/IgG-positive, mPCR-negative (n = 8); Group III – IgM-positive for agents other than CMV, mPCR-negative (n = 11); Group IV (uninfected) – IgM-negative for all viruses, mPCR-negative (n = 100). Matched maternal serum samples were divided into two groups for analysis: Group I (n = 12) and Group IV (n = 33). This was the first reporting of elevation of the cytokines, IL-1ra, IL-10, IL-15, IL-17 and TNF-α; the chemokines, IP 10, MCP-1 and MIP-1β; and the growth factors, GM-CSF, PDGF bb; and the reduction of the cytokine IL-4, in midtrimester amniotic fluid from pregnant women with primary CMV infection compared with amniotic fluid from healthy uninfected women (p < 0.0083). IP-10 (p < 0.001) and MCP-1 (p = 0.038) levels in maternal serum from CMV-infected women were elevated at midtrimester compared with the uninfected group. These findings demonstrate an association between elevated inflammatory cytokine levels and midtrimester amniotic fluid and maternal serum from CMV-infected pregnancies. A subset of cytokines was examined in amniotic membranes, umbilical cord and placental disk tissue in situ, as well as in extravillous trophoblast and fibroblast cell lines in vitro. In immunohistochemical studies, an increased presence of pro-inflammatory IL-6 and IP-10, and reduced presence of anti-inflammatory IL-1ra with CMV infection in the amniotic membranes and umbilical cord in situ was observed. Examination of cytokine dynamics in extravillous cytotrophoblasts in vitro demonstrated that initial cytokine production and release after CMV infection was independent of complete viral replication, and that relative levels of cytokine release after CMV infection may be strain- and cell- dependent. The differential expression of candidate CMV genes between extravillous cytotrophoblasts and lung fibroblast cell lines was observed in microarray experiments. Six CMV genes were found to be expressed at higher levels in extravillous cytotrophoblast cells compared to in fibroblast cells, while 16 genes were found to be lower, suggesting that CMV gene expression profiles vary amongst the numerous cell types in a CMV-infected placenta. Four known (UL36, US28) or potential (UL28, UL5) immunomodulatory genes were differentially expressed in fibroblasts over extravillous trophoblast cells, suggesting a differential role of these CMV-encoded gene products between placental cell types in the pathogenesis of the virus across the fetomaternal barrier. In conclusion, this thesis contributes to the understanding of the influence of cytokines in the pathogenesis of congenital CMV infection. The association of elevated levels of pro-inflammatory cytokines and CMV infection in amniotic fluid, placental tissues and placental cells suggests that the promotion of an inflammatory environment at key points of interaction between fetal and maternal compartments of pregnancy is a potential mechanism for the dissemination of CMV infection across the fetomaternal barrier. Differential expression of CMV replication and immunomodulatory genes between different cell types of the placenta may be a further contributing factor in the pathogenesis of congenital CMV infection.