The role of synchronization in digital communications using chaos - Part II: Chaotic modulation and chaotic synchronization.

For pt. I see ibid., vol. 44, p. 927-36 (1997). In a digital communications system, data are transmitted from one location to another by mapping bit sequences to symbols, and symbols to sample functions of analog waveforms. The analog waveform passes through a bandlimited (possibly time-varying) analog channel, where the signal is distorted and noise is added. In a conventional system the analog sample functions sent through the channel are weighted sums of one or more sinusoids; in a chaotic communications system the sample functions are segments of chaotic waveforms. At the receiver, the symbol may be recovered by means of coherent detection, where all possible sample functions are known, or by noncoherent detection, where one or more characteristics of the sample functions are estimated. In a coherent receiver, synchronization is the most commonly used technique for recovering the sample functions from the received waveform. These sample functions are then used as reference signals for a correlator. Synchronization-based coherent receivers have advantages over noncoherent receivers in terms of noise performance, bandwidth efficiency (in narrow-band systems) and/or data rate (in chaotic systems). These advantages are lost if synchronization cannot be maintained, for example, under poor propagation conditions. In these circumstances, communication without synchronization may be preferable. The theory of conventional telecommunications is extended to chaotic communications, chaotic modulation techniques and receiver configurations are surveyed, and chaotic synchronization schemes are described

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.

Identification of RNA editing in the human exome and development of DARNED DatabaseSF

RNA editing is a biological phenomena that alters nascent RNA transcripts by insertion, deletion and/or substitution of one or a few nucleotides. It is ubiquitous in all kingdoms of life and in viruses. The predominant editing event in organisms with a developed central nervous system is Adenosine to Inosine deamination. Inosine is recognized as Guanosine by the translational machinery and reverse-transcriptase. In primates, RNA editing occurs frequently in transcripts from repetitive regions of the genome. In humans, more than 500,000 editing instances have been identified, by applying computational pipelines on available ESTs and high-throughput sequencing data, and by using chemical methods. However, the functions of only a small number of cases have been studied thoroughly. RNA editing instances have been found to have roles in peptide variants synthesis by non-synonymous codon substitutions, transcript variants by alterations in splicing sites and gene silencing by miRNAs sequence modifications. We established the Database of RNA EDiting (DARNED) to accommo-date the reference genomic coordinates of substitution editing in human, mouse and fly transcripts from published literatures, with additional information on edited genomic coordinates collected from various databases e.g. UCSC, NCBI. DARNED contains mostly Adenosine to Inosine editing and allows searches based on genomic region, gene ID, and user provided sequence. The Database is accessible at http://darned.ucc.ie RNA editing instances in coding region are likely to result in recoding in protein synthesis. This encouraged me to focus my research on the occurrences of RNA editing specific CDS and non-Alu exonic regions. By applying various filters on discrepancies between available ESTs and their corresponding reference genomic sequences, putative RNA editing candidates were identified. High-throughput sequencing was used to validate these candidates. All predicted coordinates appeared to be either SNPs or unedited.

Molecular genetics of the imprinted gene - SPROUTY3 (SPRY3)

Sprouty proteins are key regulators of cell growth and branching morphogenesis during development. Human SPRY3 which maps to the pseudoautosomal region 2, undergoes random X-inactivation in females and preferential Y-inactivation in males, behaving as though genetically X-linked. Spry3 is widely expressed in neuronal tissues, being found at high levels in the cerebellum and particularly in the Purkinje cells which, notably, are deficient in the autistic brain. Spry3 is also highly expressed in other ganglia in adults including retinal ganglion cells, dorsal root ganglion and superior cervical ganglion. SPRY3 enhancer can drive SPRY3 expression in the lung airway, which is consistent with a role in branching morphogenesis and the function of the original Drosophila Spry gene, which is critical for lung morphogenesis, providing a possible explanation for an observed anatomic abnormality in the autistic lung airway. In the human and mouse, the SPRY3 core promoter contains an AG-rich repeat and we found evidence of coexpression, promoter binding and regulation of SPRY3 expression by transcription factors EGR1, ZNF263 and PAX6. Spry3 over-expression in mouse superior cervical ganglion cells inhibits axon branching and Spry3 knockdown in those cells increases axon branching, consistent with known functions of other Sprouty proteins. Novel SPRY3 upstream transcripts that I characterised originate from three start sites in the X-linked F8A3 – TMLHE gene region, which is recently implicated in autism causation. Arising from these findings, I propose that the lung airway abnormality and low levels of blood carnitine found in autism suggest that deregulation of SPRY3 may underpin a subset of autism cases.

Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.

Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.

Totality of experience: sound, music and mythology in Gus Van Sant’s “Death Quartet”

Reflecting on Gus Van Sant’s films Gerry (2003) Elephant (2004) and Last Days (2005), the director’s long-term sound-designer Leslie Shatz observed that “You have to get into the totality of the experience and not just the dialogue”. Shatz’s comment expresses something fundamental about the experimental approach to cinema and to soundscapes undertaken by Van Sant in these three films, unofficially known as the “Death Trilogy”. This thesis contends that Van Sant makes deliberate aesthetic choices which do indicate a distinctly “auteurist” leaning. However, I also argue that intertextual elements, prior knowledge, and audience participation in meaningmaking enhance the experience of, and reveal the nuances in, the soundtracks themselves. This thesis aims to contribute to a growing body of work within filmmusic scholarship concerned with resisting a traditional bias in the field: that film music should be understood as a means of characterisation and as emotional signifier. The films of the “Death Quartet”, which includes Paranoid Park (2007), I believe, offer fertile ground on which to explore these new approaches. It is my contention that these films deconstruct the traditional approach to soundtracking and the relationship between soundtrack and character, and that only an approach sensitive to the aesthetic and philosophical functions of music and sound can adequately acknowledge their unique cinematic qualities.