Talk and silence: instantiations and articulations

This paper considers the desire for unity, reconciliation and consensus underpinning three models of talking – namely, 'the meeting', 'the dyadic love relationship', and 'the psychoanalytic session'. We highlight the three domains’ shared intellectual and historical heritage wherein talk is seen as a mode of achieving unity (of the group, of the dyad, or of the self) and conversely 'silence' is seen as pathology. Through looking at the role of silence in the works of Lacan, Joyce, and Beckett, we then examine how conversations with a collective, an Other, the self, etc. can all be enriched by ambivalence, antagonism and, in particular, silence. In contrast to the conventional understanding, silence is not the 'end' of understanding, but rather a new beginning. From this perspective, silence can be the basis upon which we can begin to imagine a principled relationship with the Other.

Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.

The synthesis and biological evaluation of lanostane and cholestane-type natural products

The main objective of this thesis is to outline the synthetic chemistry involved in the preparation of a range of novel lanostane and cholestane derivatives, and subsequent investigation into their biological activity in cancer cells. The biological results obtained throughout the project have driven the strategic synthesis of new compounds, in an effort to optimise the anti cancer potential of lanostane and cholestane derivatives. The first chapter begins with an overview of steroidal compounds and details a literature review of the natural sources of these moieties, as well as their biosynthesis and reported synthetic derivatives. The biological activity of interesting natural and synthetic analogues is also discussed. In addition, an insight into some currently prescribed pharmaceutical compounds, with functional groups relevant to this project, is presented. The second chapter discusses the methods employed for the synthesis of these novel lanostane and cholestane derivatives, and comprises three main sections. Firstly, various oxidation products of lanosterol are synthesised, mainly via epoxidations of the C-8,9 and C- 24,25 alkenes, and also allylic oxidations at these positions. Secondly, amine derivatives of lanosterol are formed by cleaving the lanostane side chain, thereby yielding a new cholestane nucleus, and performing several reductive aminations on the resulting key aldehyde intermediates. Various amines such as piperidine, morpholine, diethylamine and aniline are employed in the reductive amination reactions to yield novel cholestane steroids with amine side chains. Finally, starting from stigmasterol and proceeding with the same methodology of cleaving the steroidal side chain and subsequently performing reductive aminations, novel cholestane derivatives of the biologically active amines are synthesised. The cytotoxicity of these compounds against CaCo-2 and U937 cell lines is presented in terms of percentage viability of cells, IC50 value and apoptosis. The MTT assay is used to determine the percentage viability of cells, and the IC50 data is generated from the MTT results. Apoptosis is measured in terms of fold increase relative to a carrier control. In summary, the compounds formed are discussed in terms of chemical synthesis, spectroscopic interpretation and biological activity. The main reaction pathways involved in the chemistry within this project are various oxidations and reductive amination. The final chapter is a detailed account of the full experimental procedures for the compounds synthesised during this work, including characterisation using spectroscopic and analytical data.

A novel activation mechanism for Clostridial bacteriophage endolysins

Bacteriophage-encoded endolysins are produced at the end of the phage lytic cycle for the degradation of the host bacterial cell. Endolysins offer the potential as alternatives to antibiotics as biocontrol agents or therapeutics. The lytic mechanisms of three bacteriophage endolysins that target Clostridium species living under different conditions were investigated. For these endolysins a trigger and release mechanism is proposed for their activation. During host lysis, holin lesion formation suddenly permeabilises the membrane which exposes the cytosol-sequestered endolysins to a sudden environmental shock. This shock is suggested to trigger a conformational switch of the endolysins between two distinct dimer states. The switch between dimer states is proposed to activate a novel autocleavage mechanism that cleaves the linker connecting the N-terminal catalytic domain and the C-terminal domain to release the catalytic domain for more efficient digestion of the bacterial cell wall. Crystal structures of cleaved fragments of CD27L and CTP1L were previously obtained. In these structures cleavage occurs at the stem of the linker connected to the C-terminal domain. Despite a sequence identity of only 22% between 81 residues of the C-terminal domains of CD27L and CTP1L, they represent a novel fold that is identified in a number of different lysins. Within the crystal structures the two distinct dimerization modes are represented: the elongated head‐on dimer and the side-by‐side dimer. Introducing mutations that inhibit either of the dimerization states caused a decrease in the efficiency of both the autocleavage mechanism and the lytic activity of the endolysins. The two dimer states were validated for the full-length endolysins in solution by using right angle light scattering, small angle X‐ray scattering and cross-linking experiments. Overall, the data represents a new type of regulation governed by the C-terminal domains that is used to activate these endolysins once they enter the bacterial cell wall.