Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalyti

Evolution of entanglement within classical light states

We investigate the evolution of quantum correlations over the lifetime of a multi-photon state. Measurements reveal time-dependent oscillations of the entanglement fidelity for photon pairs created by a single semiconductor quantum dot. The oscillations are attributed to the phase acquired in the intermediate, non-degenerate, exciton-photon state and are consistent with simulations. We conclude that emission of photon pairs by a typical quantum dot with finite polarisation splitting is in fact entangled in a time-evolving state, and not classically correlated as previously regarded

Spliceosomal intron and spliceosome evolution in Giardia lamblia and other diplomonads

Spliceosomal introns interrupt protein coding genes in all characterized eukaryotic nuclear genomes and are removed by a large RNA-protein complex termed the spliceosome. Diplomonads are diverse unicellular eukaryotes that display compact genomes with few spliceosomal introns. My thesis objectives were to explore spliceosomal intron and spliceosome diversity as well as RNA processing mechanisms in the diplomonads Giardia lamblia and Spironucleus spp. Surprisingly, G. lamblia was found to contain a proportionally large number of fragmented spliceosomal introns that are spliced in trans from separate pre-mRNA molecules. Next, both evolutionarily divergent and conventional spliceosomal small nuclear RNAs were identified in G. lamblia and Spironucleus spp. and an RNA 3ʹ end motif was determined to be involved in processing of both non-coding RNAs and trans-introns in G. lamblia. These findings shed light on spliceosome and spliceosomal intron evolution in eukaryotes undergoing severe genomic reduction and potentially complete loss of their spliceosomal introns.University of Lethbridge (SGS Graduate Fellowship), Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery Grant and Alexander Graham Bell Canada Graduate Scholarships)

Wavelet Signatures and Diagnostics for the Assessment of ICU Agitation-Sedation Protocols

The use of quantitative modelling to enhance understanding of the agitation-sedation (A-S) system and the provision of an A-S simulation platform are key tools in this area of patient critical care. A suite of wavelet techniques and metrics based on the discrete wavelet transform (DWT) are developed in this chapter which are shown to successfully establish the validity of deterministic agitation-sedation (A-S) models against empirical (recorded) dynamic A-S infusion profiles. The DWT approach is shown to provide robust performance metrics of A-S control and also yield excellent visual assessment tools. This approach is generalisable to any study which investigates the similarity or closeness of bivariate time series of, say, a large number of units (patients, households etc) and of disparate lengths and of possibly extremely long length. This work demonstrates the value of the DWT for assessing ICU agitation-sedation deterministic models, and suggests new wavelet based diagnostics by which to assess the A-S models

Prediction of intramammary infection status across the dry period from lifetime cow records

The dry period is very important for mammary gland health, with the aim not only to cure existing intramammary infections (IMI) but also to prevent new IMI. Although it is known that the dry period is an important time for optimizing udder health, the probability that individual cows will succumb to a new IMI or, if infected, will fail to cure an IMI is not well established. The aim of this study was to investigate whether lifetime cow data, available through routine on-farm milk recording, could be used to predict changes in IMI status across the dry period for individual cows that were (1) deemed high somatic cell count (SCC; >199,000 cells/mL) or (2) low SCC (<200,000 cells/mL) at the last test day before drying off. Milk recording data collected between September 1994 and July 2014 from 114 herds in the United Kingdom were used. Two 2-level random effects models were built and both cure and new IMI were used as outcome variables in separate models. Cows with a smaller proportion of test days with a high SCC in the lactation before drying off, a smaller proportion of test days recording a high SCC in the lactation before the current lactation, of lower parity, producing less milk before drying off, of lower days in milk at drying off, and of lower SCC just before drying off were more likely to cure across the dry period. Dry period length had no effect on the likelihood of cure. Individual cows with a smaller proportion of test days recording a high SCC in the lactation before the current, of lower parity, of lower milk production at drying off, and fewer days in milk at drying off were less likely to develop a new IMI. Dry period length was found to have no effect on the probability of new IMI. Model predictions showed that a high level of discrimination was possible between cows with a high and low risk of both cures and new infections across the dry period

Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch

Re-assessing the infection strategies of the entomopathogenic nematode Steinernema feltiae (Rhabditidae; Steinernematidae)

Previous studies have indicated that between 60 and 80% of a population of entomopathogenic nematodes do not infect their insect hosts at any one period in time. Two hypotheses explain this behaviour: the first that there is a subpopulation of non-infectious nematodes and the second that the non-infectious group is created by inhibitory cues derived from infected insects. Through an experimental approach with the Galleria mellonella-Steinernema feltiae system we show that both mechanisms operate together. When conditions for infection were optimized, the sum of individual infection behaviours was similar to the number infecting as a population, implying observed infection rates are driven by intrinsic mechanisms. In addition, there was evidence that an infected host released a chemical cue into the environment which inhibited subsequent levels of infection. This degree of inhibition was independent of the number of infecting nematodes. Both these mechanisms are dynamic, so the observed proportion of infectious nematodes depended heavily on the time of exposure. The implications of these findings for both the design of laboratory trials and the use of entomopathogenic nematodes in biological control are discussed

Evaluating the efficacy of entomopathogenic nematodes for the biological control of crop pests: A nonequilibrium approach

The efficacy of entomopathogenic nematodes for biological control is assessed using deterministic models. Typically, the examination of such models involves stability analyses to determine the long‐term persistence of control. However, in agricultural systems, control is often needed within a single season. Hence, the transient dynamics of the systems were assessed under specific, short‐term control scenarios using stage‐structured models. Analyses suggest that preemptive application may be the optimum strategy if nematode mortality rates are low; applying before pest invasion can result in greater control than applying afterward. In addition, repeated applications will suppress a pest, providing the application rate exceeds a threshold. However, the period between applications affects control success, so the economic injury level of the crop and the life history of the pest should be evaluated before deciding the strategy. In all scenarios, the most important parameter influencing control is the transmission rate. These findings are applicable to more traditional biological control agents (e.g., microparasites and parasitoids), and we recommend the approach adopted here when considering their practical use. It is concluded that it is essential to consider the specific crop and pest characteristics and the definition of control success before selecting the appropriate control strategy