Healthcare-acquired infections (HAIs) cost the National Health Service (NHS) in England in excess of £1 billion per year. One of the main HAIs is caused by the endospore-forming bacterium Clostridium difficile. The most common cause of healthcare-acquired diarrhoea in the developed world, C. difficile was responsible for around 850 deaths in England and Wales in 2011. To help reduce the spread of the HAI-causing bacteria, terminal disinfection of isolation rooms and wards using hydrogen peroxide vapour is actively promoted. The key advantages of hydrogen peroxide vapour are its high oxidation potential which has been reported to inactivate bacteria, fungi and spores. An additional advantage of hydrogen peroxide vapour is that it is relatively environmentally friendly, breaking down into oxygen and water. Investigation into bacterial inactivation kinetics was undertaken at controlled, steady concentrations of hydrogen peroxide vapour in the range of 10 ppm to 90 ppm. An exposure chamber was designed whereby the bacterial spores could be exposed to constant concentrations of hydrogen peroxide for various exposure times. Bacterial spores (1-log10 to 8-log10 cfu) were filter deposited onto membranes to achieve an even layer for consistent exposure of the hydrogen peroxide vapour to the spores. Bacillus subtilis is often used for method development in bacterial studies; advantages are it has been shown to be highly resistant to hydrogen peroxide vapour and is not a human pathogen. Following the method development, different strains of C. difficile (ribotypes 014, 027, 103 and 220) were exposed to identify differences in resistance. Inactivation models (Chick-Watson, Series-Event, Weibull and Baranyi) were used to fit the data generated using the environmental chamber. Decimal reduction values (D-values) were calculated from the models for comparative studies regarding the inactivation achieved for the different bacteria and different hydrogen peroxide concentrations. The findings from this thesis revealed the Weibull model provides the best fit for most of the data. An initial shoulder period was identified for B. subtilis which was absent for C. difficile inactivation by hydrogen peroxide vapour; B. subtilis is therefore more resistant to hydrogen peroxide disinfection than C. difficile. Typical D-values for B. subtilis and C. difficile when exposed to hydrogen peroxide vapour at a concentration of 90 ppm were 140 and 1 min, respectively. C. difficile inactivation data were used to develop a model to estimate the log reduction that could be achieved during an inactivation cycle based on the concentration-time integral ( ). This model could be used to estimate the log reduction of commercially available hydrogen peroxide decontamination systems; these release a fixed amount of hydrogen peroxide into the room resulting in a peak concentration before decomposition to oxygen and water. Releasing the hydrogen peroxide into the room in this manner results in spatial and temporal variation; this could result in differences in bacterial inactivation in different areas within the room. Using the aforementioned regression model, the inactivation achieved at all locations within the room could be predicted, which could be used to optimise the current hydrogen peroxide decontamination cycles.

Η πολυλειτουργική πρωτεΐνη core του ιού της ηπατίτιδας C (HCV) εμπλέκεται στην ανάπτυξη ηπατοκυτταρικού καρκινώματος (HCC) που προκαλείται από τον ιό της ηπατίτιδας C, αλλά ο μηχανισμός με τον οποίο συμβαίνει αυτό δεν είναι κατανοητός. Η ενεργοποίηση του μονοπατιού Wnt/ β-κατενίνη, παίζει ένα σημαντικό ρόλο στην ανάπτυξη ηπατοκυττταρικού καρκίνου, και τροποποιείται από την πρωτεΐνη core του ιού της ηπατίτιδας C. Ο ιός της ηπατίτιδας C χαρακτηρίζεται από εκτεταμένη γενετική ποικιλομορφία και διαφορετικά κλινικά δείγματα διαφέρουν όσον αφορά την μολυσματικότητα τους και την παθογένεια που προκαλούν. Σκοπός αυτής της μελέτης είναι να καθοριστεί ο ρόλος της γενετικής ποικιλομορφίας της πρωτεΐνης HCV core στην ενεργοποίηση του μονοπατιού Wnt/ β-κατενίνη και να μελετηθεί ο μοριακός μηχανισμός με τον οποίο η πρωτεΐνη HCV core ρυθμίζει την ενεργοποίηση αυτή. Η ενεργότητα του μονοπατιού Wnt/β-κατενίνη μελετήθηκε σε HEK 293T και Huh 7.5 κυτταρικές σειρές που εκφράζουν παροδικά τις πρωτεΐνες core των γενοτύπων 1a, 1b, 3a, 4a, 4f και από ένα μοναδικό δείγμα του γενοτύπου 1a που προέρχεται από έναν ασθενή από την Καμπότζη (1aCam). Μελέτες βασισμένες στη μέτρηση ενεργότητας της λουσιφεράσης, Western blot ανάλυση και qPCR, χρησιμοποιήθηκαν για την μέτρηση των επιπέδων έκφρασης γονιδίων και πρωτεϊνών. Βρέθηκε ότι η HCV core πρωτεΐνη ρυθμίζει θετικά την μεσολαβούμενη από τη β-κατενίνη Tcf-εξαρτώμενη ενεργότητα της λουσιφεράσης σε ένα γενοτυπο-εξαρτώμενο τρόπο. Σε συμφωνά με τα αποτελέσματα αυτά βρέθηκε ότι η πρωτεΐνη HCV core σταθεροποίει τα επίπεδα της β-κατενίνης, τόσο σε παροδικά μετασχηματισμένα κύτταρα, όσο και σε κύτταρα που μολύνονται με βακουλοϊούς που εκφράσουν τις πρωτεΐνες core των υποτύπων 4a και 4f. Τέλος, βρέθηκε ότι η πρωτεΐνη HCV core συμβάλει στην θετική ρύθμιση των γονιδίων c-myc, αξίνης και Tbx3, τα οποία είναι καθοδικά γονίδια στόχοι του μονοπατιού Wnt/β-κατενίνη και εμπλέκονται στην ανάπτυξη ηπατοκυτταρικού καρκινώματος. Συμπερασματικά, οι πρωτεΐνες HCV core διαφορετικών γενοτύπων του ιού ρυθμίζουν διαφορετικά το μονοπάτι σηματοδότησης Wnt/β-κατενίνη και η διαφορετική αυτή ρύθμιση μπορεί να σχετίζεται με ικανότητα των διαφόρων γενοτύπων του ιού της ηπατίτιδας C να επάγουν την ανάπτυξη ηπατοκυτταρικού καρκινώματος. / The multifunctional HCV core protein is implicated in the development of hepatocellular carcinoma (HCC) caused by HCV infection, but the underlying mechanism is not fully understood. Activation of the Wnt/ β-catenin pathway plays a major role in HCC and is modulated by the HCV core protein. HCV is characterized by extensive genetic diversity and different clinical isolates do vary in their infectivity and pathogenesis mainly due to variations in the structure/function relationships of individual viral proteins. The aim of this study is to determine the possible influence of genetic variability in HCV core protein in enhancing the Wnt/ β-catenin signaling activity and to elucidate the molecular mechanisms by which HCV core modulates activation of β-catenin. The Wnt/β-catenin activity was investigated in transiently transfected HEK 293T and Huh 7.5 cell lines transiently expressing HCV core proteins from HCV genotypes 1a, 1b, 4a, 4f and from a unique isolate of genotype 1a obtained from a Cambodian patient (1aCam). Luciferase-based reporter assay, Western blot, and qPCR, were used to measure gene and protein expression levels. We found that, HCV core protein upregulates β-catenin-mediated Tcf-dependent luciferase activity in a genotype specific manner. Consistent to these findings, HCV core stabilizes β-catenin levels. Finally, we showed that HCV core contributes to the upregulation of Tbx3 gene expression, a downstream target gene of Wnt/ β-catenin pathway contributing to HCC development. In conclusion, HCV core protein from different genotypes appears to differentially regulate the Wnt/β-catenin signaling pathway and this finding may contribute to different potential of HCV genotypes to induce HCC.

ITC/USA 2011 Conference Proceedings / The Forty-Seventh Annual International Telemetering Conference and Technical Exhibition / October 24-27, 2011 / Bally's Las Vegas, Las Vegas, Nevada / Currently most of the missile telemetry systems operate in the lower S-Band frequency range (2200-2290 MHz). Due to Federal Government plans to repurpose this frequency spectrum to commercial entities, missile telemetry systems will have to migrate to operate in the lower C-Band range (4400-4940 MHz) as well. This move in the operational frequency requires the upgrading of not only the Range ground receiving equipment, but the airborne transmitting units as well. This paper will detail the efforts required to augment a missile telemetry unit from operating in S-Band to C-Band.

The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines. TLR3, TLR4 and retinoic acid-inducible gene 1 (RIG-1)/melanoma differentiation associated gene 5 (MDA-5) activation by double stranded (ds) RNA mimic polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) and synthetic ds-B-DNA respectively, mediated IFN-beta as well as TNF-alpha and IL-8 synthesis in monocyte-derived DCs. Using the pharmacological inhibitor of conventional PKCs (cPKCs), Gö6976, we demonstrated that this family of kinases was involved in TLR3, TLR4 and RIG-1/ MDA-5 signaling pathways leading to the production of IFN-beta but not of TNF-alpha and IL-8. Further analysis with the use of specific kinase inactive cPKC isoforms and siRNA targeted to PKCalpha, we established that PKCalpha was the isoform involved in the TLR3 signaling pathway. In the case of TLR3, we show that PKCalphaexerts its effect downstream of TRIF and TBK1. Moreover, we show that inactivation of PKCalpha specifically inhibits the activation of IRF-3 and not that of NF-kB. Through biochemical analysis, we assessed the contribution of PKCalpha in the critical events of IRF-3 activation: a) phosphorylation b) homodimerization c) nuclear translocation d) DNA-binding and e) recruitment of creb-binding protein (CBP). We conclude that inhibition of cPKCs severely hinders the association of IRF-3 with CBP. Overall, these data revealed the critical role of cPKCs in TLR-dependent and -independent pathways leading to IFN-beta synthesis. The selective targeting of IRF-3 by cPKCs prompted us to study the possible implications of cPKCs in the transcriptional control of IL-12 family members, some of which are regulated by IRF3. Indeed, recent studies have emerged demonstrating the essential role of IRF-3 in IL-12p35 and IL-27p28 gene expression (1;2). Likewise, we investigated the role of cPKCs in the regulation of LPS- and poly(I:C)-induced expression of IL-12(p40/p35), IL-23(p40/p19) and IL-27(p28/EBI3) in monocyte-derived DCs. Treatment of monocyte-derived DCs with Gö6976 down-regulated LPS- and poly(I:C)-induced IL-12 and IL-27 synthesis while it did not alter IL-23 production. Next, we showed that impaired IL-12 and IL-27 synthesis was due to repressed IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4 whereas IL-23p19 and IL-27EBI3 gene expression were not modified. Reporter gene assays demonstrated that cPKCs are involved in LPS- and poly(I:C)-induced IL-12p35 and IL-27p28 promoter activity. Finally, experiments in bone marrow-derived DCs from IRF-3-/- and wild type mice showed that IL-23 synthesis does not require IRF-3 activation. We conclude that cPKCs through the control of IRF-3 activity are critically involved in the regulation of IL-12 and IL-27 synthesis downstream of TLR3 and TLR4 while they do not participate in IRF-3-independent IL-23 synthesis. On whole, we demonstrated a novel function for cPKCs in the regulation of IRF-3 and IRF-3 dependent gene expression, specifically IFN-beta, IL-12 and IL-27. In light of the important and divergent roles of IFN-beta and IL-12 family of cytokines on the development of T helper (Th) Th1, Th2, Th17-mediated immune responses, cPKCs represent a potential target for therapeutic immunomodulation. This modulation needs to be carefully administered due to the complex interplay of the IL-12 family members in immunity.

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The nematode <i>Caenorhabditis elegans</i> is a small invertebrate whose nervous system, general anatomy, and normal development are all (comparatively) extremely simple and reproducible, and have all been well characterised. This dissertation describes work based on two different approaches to the study of the control of neural development in <i>C. elgans</i>. In the first part the course of neural outgrowth in the region of the ventral nerve cord was followed from electron microscope reconstructions of a series of fixed embryos. Following this, neurons whose processes grew out early were removed by laser ablation of their parent cells and the effect on subsequent nerve outgrowth was assayed by electron microscope reconstruction. The first process to grow along the ventral cord is that of AVG, and its presence is required for the normal, highly asymmetrical structure of the cord. Two more examples of dependency on particular nerve processes for correct guidance can be deduced from experiments in which cells at the back of the animal were removed. The observations of normal development and the ablation experiments can in some cases be related to defects seen in <i>uncoordinated</i> mutants with defective nerve process organisation. The second approach was to establish and analyse a computer data base containing all the synaptic connectivity data obtained by White et al. (1986), who reconstructed at an electron microscope level the entire central nervous system regions of two <i>C. elegans</i> specimens. Since the circuitry is highly reproducible, comparisons of connections between equivalent pairs of cells can be used to infer properties of synapse formation. Overall, the <i>C. elegans</i> circuitry is anatomically highly directional, and what little chemical synaptic feedback that is seen is mostly part of reciprocal synaptic connections. There is also evidence for physical organisation of the nerve processes in subbundles of the main process tract in the central nervous system.

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