No description available.

No description available.

No description available.

No description available.

The bertha armyworm (Mamestra configurata) is a major pest of canola and other oilseed crops. A promising control agent for this species is the baculovirus Mamestra configurata nucleopolyhedrovirus (MacoNPV). Baculoviruses are insect-specific viruses. Infections initiate in the host midgut following ingestion of virus particles called occlusion bodies. For a productive infection to occur, the occlusion bodies must dissolve to release the infectious occlusion-derived virions. These virions must pass through the peritrophic matrix, a protein-chitin meshwork that lines the midgut of most insects and provides protection against abrasion and pathogen invasion. The mechanism by which the baculovirus virions transit the peritrophic matrix is unknown. Following the initial infection of midgut cells, a second virion phenotype, the budded virus, is released from infected cells and establishes a systemic infection within the insect. The 11K group of genes, which are conserved among baculovirus species and other insect-infecting viruses, encode proteins with a predicted chitin-binding domain. The degree of conservation of these genes among insect-infecting viruses suggests that they may play a role in insect infectivity. It is possible that the gene products could be involved in an interaction between the baculovirus occlusion-derived virions and the peritrophic matrix or the chitin-secreting cells of the midgut epithelium, and therefore may be involved in initial oral infectivity. The two 11K genes from MacoNPV (ORF 118 and ORF 164), and their homologues in a second species of baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV [ORF145 and ORF150]) were expressed in a baculovirus expression system. The ability of the proteins, Maco118, Maco164, Ac145, and Ac150, to bind to chitin was assessed in vitro using chitin-coated beads. Each of the four proteins binds to chitin, and hydrophobic interactions mediate the binding. Other binding mechanisms are likely involved, but were not determined in this project. To determine the function of these proteins, a series of gene knockout and repair constructs was produced for AcMNPV ORF 145 and ORF 150 using an established bacmid system. An analysis of the knockout and repair constructs using quantitative real-time polymerase chain reaction showed that deletion of either ORF 145 or ORF 150 had no effect on the rate of budded virus production or viral DNA replication. Oral and injection bioassays were performed in Trichoplusia ni larvae to determine if there were differences in infectivity between the knockout, repair, and wild type constructs. Injection assays, in which budded virus from each construct was injected directly into the insect haemocoel, therefore bypassing the midgut and peritrophic matrix, indicated that there was no statistical difference in infectivity between the knockout, repair, and wild type constructs at a dose of 15 TCID50 U per larva. Oral bioassays, in which larvae were fed occlusion bodies from each virus construct, indicated that there was no statistical difference in mortality rates between the knockout, repair, and wild type constructs. The results from this study indicate that although the baculovirus 11K genes are highly conserved among baculovirus species, and the 11K gene products from MacoNPV and AcMNPV interact with chitin, they are not required for oral infectivity in T. ni larvae, and likely serve another function in the baculovirus infection cycle.

The hydrogenase enzyme is an effective replacement for the expensive platinum catalysts used in hydrogen fuel cells today. However, many enzymes themselves are found in extreme environments and are inactive under standard conditions, but current active site models have a much larger over-potential for H+ reduction than the actual enzyme. Most research today involves the improvement of these synthetic models in an attempt to lower reduction potential, increase reaction kinetics, or improve catalytic activity. Research focuses on the synthesis of active site models with a carbon chain bridgehead linker of varying length. Synthesis of these molecules is achieved by the reaction of a dithiol with triiron dodecacarbonyl under an inert atmosphere to avoid the formation of by-products. Dithiols with four or more carbon atoms must first be converted to cyclic disulfides before the reaction with the iron The hydrogenase enzyme is an effective replacement for the expensive platinum catalysts used in hydrogen fuel cells today. However, many enzymes themselves are found in extreme environments and are inactive under standard conditions, but current active site models have a much larger over-potential for H+ reduction than the actual enzyme. Most research today involves the improvement of these synthetic models in an attempt to lower reduction potential, increase reaction kinetics, or improve catalytic activity. Research focuses on the synthesis of active site models with a carbon chain bridgehead linker of varying length. Synthesis of these molecules is achieved by the reaction of a dithiol with triiron dodecacarbonyl under an inert atmosphere to avoid the formation of by-products. Dithiols with four or more carbon atoms must first be converted to cyclic disulfides before the reaction with the iron dodecacarbonyl. This prevents the formation of an unwanted side product. Both butyl- and pentyldithiolatohexacarbonyldiiron model complexes have been characterized by IR, NMR, and X-ray spectroscopy. Active site models can also feature two unlinked sulfur atoms. These models have two conformational isomers that depend on the spatial location of the R-group bonded to each sulfur atom. This research also focuses on the synthesis of unlinked active site models with a variety of R-groups, and a temperature controlled NMR study of the isomeration reaction to determine the reaction rate. / Review of literature -- Synthesis of [FeFe]-hydrogenase active site mimics with bridged sulfur atoms -- Preliminary kinetics study of [FeFe]-hydrogenase active site mimics. / Department of Chemistry

No description available.

MeCP2 was initially identified as an abundant protein in the brain, with an affinity for methylated DNA in vitro. Interestingly, both deficiency and excess of the protein leads to severe neurological problems, such as Rett syndrome, which is the result of mutations in the MECP2 gene. Subsequent transfection experiments showed that MeCP2 can recruit corepressor complexes and inhibit gene expression in vivo. MeCP2 was therefore thought to repress specific gene targets and the aetiology of Rett syndrome was proposed to result from aberrant gene expression in the MeCP2-deficient brain. Although gene expression is perturbed in the Mecp2-null mouse brain, few specific targets have been verified and alternative hypotheses for MeCP2 function have been put forward. Previous binding studies have also failed to clearly identify MeCP2 targets. To shed light on these matters, a novel technique was generated to isolate neuronal and glial nuclei and established that the amount of MeCP2 is unexpectedly high in neurons, with an abundance approaching that of the histone octamer. Chromatin immunoprecipitation experiments on mature mouse brain showed widespread binding of MeCP2, consistent with its high abundance, tracking the methyl-CpG density of the genome. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. The mutant brain also shows elevated transcription of repetitive elements, which are distributed throughout the mouse genome. Based on this data, we propose that MeCP2 binds genome wide and suppresses spurious transcription through binding in a DNA methylation dependent manner.

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl isomerase enzyme (PPIs) that catalyze the cis-trans inter-conversion of peptidyl-prolyl bonds in proteins. This non-covalent post-translational modification is a reversible mechanism to modulate protein structure and function. PPIs have been implicated in a wide variety of processes from protein folding to signal transduction. Despite these enzymes being ubiquitous, the substrates and functions of most PPIs have yet to be described. FKBP25 is a nuclear FKBP that has been shown to associate with transcription factors and chromatin modifying enzymes, however its functions and substrates remain largely unresolved. FKBP25 is the human ortholog of S. cerevisiae Fpr4, which has been shown to regulate the chromatin landscape by two distinct mechanisms: 1. Acting as a histone chaperone at ribosomal DNA, and 2. Isomerizing histone prolines. Based on these observations, I hypothesized FKBP25 regulates chromatin and/or ribosome biogenesis through isomerization of histone prolines and a discrete collection of substrate proteins. While small molecule inhibitors exist for FKBPs, applying them to dissect the specific function(s) of any given FKBP is confounded by the fact that multiple FKBPs are found in each organism, and several are inhibited by these molecules. In Chapter 2, I biochemically and structurally characterize a set of FKBP25 loss-of-function mutants, yielding a toolset capable of distinguishing between catalytic and non-catalytic functions. These reagents provide the tools necessary to analyze potential substrates of FKBP25 identified in my research going forward. In Chapter 3, I present the first unbiased proteomic screen of FKBP25 associated proteins and show that it interacts with a large number of ribosomal proteins, ribosomal processing factors and a smaller subset of chromatin proteins. I focus on the interaction between FKBP25 and nucleolin, a multi-functional nucleolar protein, and show that FKBP25 interacts with nucleolin and the pre-60s ribosomal subunit in an RNA dependent fashion. In Chapter 4, I gain insight into the role of FKBP25 in ribosome biology, and demonstratex that FKBP25 regulates RNA binding activity of nucleolin, however this does not appear to involve cis-trans prolyl isomerization. Collectively, my work establishes FKBP25 as the first human FKBP to be implicated in the maturation of the pre-60S ribosomal subunit in the nucleus. My data supports a model whereby FKBP25 associates with the assembling large ribosomal subunit, where it is likely to chaperone protein-RNA interactions. / Graduate

Bisphosphonates (BPs) are stable analogues of pyrophosphate widely used for the treatment of bone diseases characterised by increased bone resorption. Studies over the years have shown that the pharmacological potencies of BPs are dependent both on their binding affinities for bone mineral and on their inhibitory actions on osteoclasts. In addition, potential effects on other cell types present locally in the environment of skeletal tissues have been reported. The present study systematically evaluated the relative mineral-binding affinities of individual BPs of clinically relevance in mixtures of these compounds and the changes with elution pH by using column chromatography with ceramic hydroxyapatite and fluoroapatite combined with mass spectrometric identification and quantitation of the individual BPs. The results indicate that pH has a profound effect on the ionisation of the phosphonate and R2 functional groups, with BPs having greater affinities at lower pH as shown by increased retention times. Moreover, two other approaches, namely using Langmuir adsorption isotherms and competition assays based on fluorescent BP, have been developed to assess the mineral-binding capacities and dissociation constants of BPs. These results suggest that there are substantial differences among BPs in their binding to hydroxyapatite. From the cellular aspect of my study, I present evidence for the anti-apoptotic effects of BPs in osteocytes and osteoblasts. However, the study of prosurvival signalling pathways involved in these cells needs to be optimised. The work described in this thesis provides novel insights into the physiological and biological mechanisms of BP action. My project has provided a better knowledge of the physicochemical properties of BPs, which are highly relevant to their differential distributions within bone, their biological potencies, and their durations of action. Additionally, the cell culture studies may provide new information on the cellular effects of BPs on osteocytes and osteoblasts.