Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori

Trophic role of small cetaceans and seals in Irish waters

Understanding the role of marine mammals in specific ecosystems and their interactions with fisheries involves, inter alia, an understanding of their diet and dietary requirements. In this thesis, the foraging ecology of seven marine mammal species that regularly occur in Irish waters was investigated by reconstructing diet using hard parts from digestive tracts and scats. Of the species examined, two (striped and Atlantic white-sided dolphin) can be considered offshore species or species inhabiting neritic waters, while five others usually inhabit more coastal areas (white-beaked dolphin, harbour porpoise, harbour seal and grey seal); the last species studied was the bottlenose dolphin whose population structure is more complex, with coastal and offshore populations. A total of 13,028 prey items from at least 81 different species (62 fish species, 14 cephalopods, four crustaceans, and a tunicate) were identified. 28% of the fish species were identified using bones other than otoliths, highlighting the importance of using all identifiable structures to reconstruct diet. Individually, each species of marine mammal presented a high diversity of prey taxa, but the locally abundant Trisopterus spp. were found to be the most important prey item for all species, indicating that Trisopterus spp. is probably a key species in understanding the role of these predators in Irish waters. In the coastal marine mammals, other Gadiformes species (haddock, pollack, saithe, whiting) also contributed substantially to the diet; in contrast, in pelagic or less coastal marine mammals, prey was largely comprised of planktivorous fish, such as Atlantic mackerel, horse mackerel, blue whiting, and mesopelagic prey. Striped dolphins and Atlantic white-sided dolphins are offshore small cetaceans foraging in neritic waters. Differences between the diet of striped dolphins collected in drift nets targeting tuna and stranded on Irish coasts showed a complex foraging behaviour; the diet information shows that although this dolphin forages mainly in oceanic waters it may occasionally forage on the continental shelf, feeding on available prey. The Atlantic white-sided dolphin diet showed that this species prefers to feed over the continental edge, where planktivorous fish are abundant. Some resource partitioning was found in bottlenose dolphins in Irish waters consistent with previous genetic and stable isotope analysis studies. Bottlenose dolphins in Irish waters appears to be generalist feeders consuming more than 30 prey species, however most of the diet comprised a few locally abundant species, especially gadoid fish including haddock/pollack/saithe group and Trisopterus spp., but the contribution of Atlantic hake, conger eels and the pelagic planktivorous horse mackerel were also important. Stomach content information suggests that three different feeding behaviours might occur in bottlenose dolphin populations in Irish waters; firstly a coastal behaviour, with animals feeding on prey that mainly inhabit areas close to the coast; secondly an offshore behaviour where dolphins feed on offshore species such as squid or mesopelagic fish; and a third more complex behaviour that involves movements over the continental shelf and close to the shelf edge. The other three coastal marine mammal species (harbour porpoise, harbour seal and grey seal) were found to be feeding on similar prey and competition for food resources among these sympatric species might occur. Both species of seals were found to have a high overlap (more than 80%) in their diet composition, but while grey seals feed on large fish (>110mm), harbour seals feed mostly on smaller fish (<110mm), suggesting some spatial segregation in foraging. Harbour porpoises and grey seals are potentially competing for the same food resource but some differences in prey species were found and some habitat partitioning might occur. Direct interaction (by catch) between dolphins and fisheries was detected in all species. Most of the prey found in the stomach contents from both stranded and by catch dolphins were smaller sizes than those targeted by commercial fisheries. In fact, the total annual food consumption of the species studied was found to be very small (225,160 tonnes) in comparison to fishery landings for the same area (~2 million tonnes). However, marine mammal species might be indirectly interacting with fisheries, removing forage fish. Incorporating the dietary information obtained from the four coastal species, an ECOPATH food web model was established for the Irish Sea, based on data from 2004. Five trophic levels were found, with bottlenose dolphins and grey and harbour seals occurring at the highest trophic level. A comparison with a previous model based on 1973 data suggests that while the overall Irish Sea ecosystem appears to be “maturing”, some indices indicate that the 2004 fishery was less efficient and was targeting fish at higher trophic levels than in 1973, which is reflected in the mean trophic level of the catch. Depletion or substantial decrease of some of the Irish Sea fish stocks has resulted in a significant decline in landings in this area. The integration of diet information in mass-balance models to construct ecosystem food-webs will help to understand the trophic role of these apex predators within the ecosystem.

Manipulation of magnetic anisotropy in nanostructures

Of late, the magnetic properties of micro/nano-structures have attracted intense research interest both fundamentally and technologically particularly to address the question that how the manipulation in the different layers of nanostructures, geometry of a patterned structure can affect the overall magnetic properties, while generating novel applications such as in magnetic sensors, storage devices, integrated inductive components and spintronic devices. Depending on the applications, materials with high, medium or low magnetic anisotropy and their possible manipulation are required. The most dramatic manifestation in this respect is the chance to manipulate the magnetic anisotropy over the intrinsic preferential direction of the magnetization, which can open up more functionality particularly for device applications. Types of magnetic anisotropies of different nanostructured materials and their manipulation techniques are investigated in this work. Detail experimental methods for the quantitative determination of magnetic anisotropy in nanomodulated Ni45Fe55 thin film are studied. Magnetic field induced in-plane rotations within the nanomodulated Ni45Fe55 continuous films revealed various rotational symmetries of magnetic anisotropy due to dipolar interactions showing a crossover from lower to higher fold of symmetry as a function of modulation geometry. In a second approach, the control of exchange anisotropy at ferromagnetic (FM) – aniferomagnetic (AFM) interface in multifferoic nanocomposite materials, where two different phase/types of materials were simultaneously synthesized, was investigated. The third part of this work was to study the electroplated thin films of metal alloy nanocomposite for enhanced exchange anisotropy. In this work a unique observation of an anti-clock wise as well as a clock wise hysteresis loop formation in the Ni,Fe solid solution with very low coercivity and large positive exchange anisotropy/exchange bias have been investigated. Hence, controllable positive and negative exchange anisotropy has been observed for the first time which has high potential applications such as in MRAM devices.

Contemporary pilgrimage in Ireland: a cultural geographic perspective

Using the lenses of contemporary cultural geography, this research develops an understanding of pilgrimage as a relational and reciprocal process that co-produces self and world. Drawing on the phenomenology of Merleau-Ponty, I argue that through the performances and experiences of contemporary pilgrimage in Ireland, participants and locations emerge as pilgrims and pilgrimage places. This approach unites different strands of cultural geography, including the mobilities field, more-than representational concerns, discussions of embodiment and practice, emotional and affective geographies, and religious and spiritual geographies. I explore how pilgrimage is an active process in which self and world, belief and practice, and the numinous and material entwine and merge. An autoethnographic methodology is enacted as an embodied, intersubjective, and reflexive research approach that incorporates the motivations, experiences, and beliefs of research participants, alongside my own descriptions and reflections. The methodology is focused on encountering and documenting pilgrimage practices as they occur in place and relating these embodied spatial practices to the accounts of pilgrims. The insights generated by engagements with research participants and through my own pilgrimages, offer new appreciations of the enduring appeal of pilgrimage in Ireland as a religious-spiritual and cultural activity that allows people to express personal intentions, to develop their faith, and to seek numinous encounters. Through the pilgrimages at Lough Derg, Croagh Patrick, and three holy wells, I produce a layered account of the empirical circumstances of the practices. The presentation of these places and events is enhanced by the use of evocative images and audiovisual recordings. By centring my study on the practices and experiences of different pilgrimages in present-day Ireland, and critically deploying strands of cultural geography and pilgrimage studies, this research produces new qualitative understandings of pilgrimage and contributes to discussions concerned with the relationships between self and world.

Redox biology of myeloid leukaemias

Internal tandem duplication of FMS-like receptor tyrosine kinase (FLT3-ITD) has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (dsbs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Herein, we observe that the majority of H2O2 in FLT3-ITD-expressing MV4-11 cells colocalises to the endoplasmic reticulum (ER). Furthermore, ER localisation of ROS in MV4-11 cells corresponds to the localisation of p22phox, a small membrane-bound subunit of NOX complex. Furthermore, we show that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, possess higher steady protein levels of p22phox than their wild type FLT3 (FLT3-WT)-expressing counterparts. Moreover, the inhibition of FLT3-ITD, using various FLT3 tyrosine kinase inhibitors, uniformly results in a posttranslational downregulation of p22phox. We also show that depletion of NOX2 and NOX4 and p22phox, but not NOX1 proteins causes a reduction in endogenous H2O2 levels. We show that genomic instability induced by FLT3-ITD leads to an increase in nuclear levels of H2O2. The presence of H2O2 in the nucleus is largely reduced by inhibition of FLT3-ITD or NOX. Furthermore, similar results are also observed following siRNA knockdowns of p22phox or NOX4. We demonstrate that 32D cells transfected with FLT3-ITD have a higher level of DNA damage than 32D cells transfected with FLT3-WT. Additionally, inhibition of FLT3-ITD, p22phox and NOX knockdowns decrease the number of DNA dsbs. In summary, this study presents a novel mechanism of genomic instability generation in FLT3-ITD-expressing AML cells, whereby FLT3-ITD activates NOX complexes by stabilising p22phox. This in turn leads to elevated generation of ROS and DNA damage in these cells.

Micro and nanostructured impedance sensors for biological and biomedical applications

This thesis work covered the fabrication and characterisation of impedance sensors for biological applications aiming in particular to the cytotoxicity monitoring of cultured cells exposed to different kind of chemical compounds and drugs and to the identification of different types of biological tissue (fat, muscles, nerves) using a sensor fabricated on the tip of a commercially available needle during peripheral nerve block procedures. Gold impedance electrodes have been successfully fabricated for impedance measurement on cells cultured on the electrode surface which was modified with the fabrication of gold nanopillars. These nanostructures have a height of 60nm or 100nm and they have highly ordered layout as they are fabricated through the e-beam technique. The fabrication of the threedimensional structures on the interdigitated electrodes was supposed to improve the sensitivity of the ECIS (electric cell-substrate impedance sensing) measurement while monitoring the cytotoxicity effects of two different drugs (Antrodia Camphorata extract and Nicotine) on three different cell lines (HeLa, A549 and BALBc 3T3) cultured on the impedance devices and change the morphology of the cells growing on the nanostructured electrodes. The fabrication of the nanostructures was achieved combining techniques like UV lithography, metal lift-off, evaporation and e-beam lithography techniques. The electrodes were packaged using a pressure sensitive, medical grade adhesive double-sided tape. The electrodes were then characterised with the aid of AFM and SEM imaging which confirmed the success of the fabrication processes showing the nanopillars fabricated with the right layout and dimensions figures. The introduction of nanopillars on the impedance electrodes, however, did not improve much the sensitivity of the assay with the exception of tests carried out with Nicotine. HeLa and A549 cells appeared to grow in a different way on the two surfaces, while no differences where noticed on the BALBc 3T3 cells. Impedance measurements obtained with the dead cells on the negative control electrodes or the test electrodes with the drugs can be compared to those done on the electrodes containing just media in the tested volume (as no cells are attached and cover the electrode surface). The impedance figures recorded using these electrodes were between 1.5kΩ and 2.5 kΩ, while the figures recorded on confluent cell layers range between 4kΩ and 5.5kΩ with peaks of almost 7 kΩ if there was more than one layer of cells growing on each other. There was then a very clear separation between the values of living cell compared to the dead ones which was almost 2.5 - 3kΩ. In this way it was very easy to determine whether the drugs affected the cells normal life cycle on not. However, little or no differences were noticed in the impedance analysis carried out on the two different kinds of electrodes using cultured cells. An increase of sensitivity was noticed only in a couple of experiments carried out on A549 cells growing on the nanostructured electrodes and exposed to different concentration of a solution containing Nicotine. More experiments to achieve a higher number of statistical evidences will be needed to prove these findings with an absolute confidence. The smart needle project aimed to reduce the limitations of the Electrical Nerve Stimulation (ENS) and the Ultra Sound Guided peripheral nerve block techniques giving the clinicians an additional tool for performing correctly the peripheral nerve block. Bioimpedance, as measured at the needle tip, provides additional information on needle tip location, thereby facilitating detection of intraneural needle placement. Using the needle as a precision instrument and guidance tool may provide additional information as to needle tip location and enhance safety in regional anaesthesia. In the time analysis, with the frequency fixed at 10kHz and the samples kept at 12°C, the approximate range for muscle bioimpedance was 203 – 616 Ω, the approximate bioimpedance range for fat was 5.02 - 17.8 kΩ and the approximate range for connective tissue was 790 Ω – 1.55 kΩ. While when the samples were heated at 37°C and measured again at 10kHz, the approximate bioimpedance range for muscle was 100-175Ω. The approximate bioimpedance range of fat was 627 Ω - 3.2 kΩ and the range for connective tissue was 221-540Ω. In the experiments done on the fresh slaughtered lamb carcass, replicating a scenario close to the real application, the impedance values recorded for fat were around 17 kΩ, for muscle and lean tissue around 1.3 kΩ while the nervous structures had an impedance value of 2.9 kΩ. With the data collected during this research, it was possible to conclude that measurements of bioimpedance at the needle tip location can give valuable information to the clinicians performing a peripheral nerve block procedure as the separation (in terms of impedance figures) was very marked between the different type of tissues. It is then feasible to use an impedance electrode fabricated on the needle tip to differentiate several tissues from the nerve tissue. Currently, several different methods are being studied to fabricate an impedance electrode on the surface of a commercially available needle used for the peripheral nerve block procedure.

Neonatal EEG classification using atomic decomposition

The electroencephalogram (EEG) is an important noninvasive tool used in the neonatal intensive care unit (NICU) for the neurologic evaluation of the sick newborn infant. It provides an excellent assessment of at-risk newborns and formulates a prognosis for long-term neurologic outcome.The automated analysis of neonatal EEG data in the NICU can provide valuable information to the clinician facilitating medical intervention. The aim of this thesis is to develop a system for automatic classification of neonatal EEG which can be mainly divided into two parts: (1) classification of neonatal EEG seizure from nonseizure, and (2) classifying neonatal background EEG into several grades based on the severity of the injury using atomic decomposition. Atomic decomposition techniques use redundant time-frequency dictionaries for sparse signal representations or approximations. The first novel contribution of this thesis is the development of a novel time-frequency dictionary coherent with the neonatal EEG seizure states. This dictionary was able to track the time-varying nature of the EEG signal. It was shown that by using atomic decomposition and the proposed novel dictionary, the neonatal EEG transition from nonseizure to seizure states could be detected efficiently. The second novel contribution of this thesis is the development of a neonatal seizure detection algorithm using several time-frequency features from the proposed novel dictionary. It was shown that the time-frequency features obtained from the atoms in the novel dictionary improved the seizure detection accuracy when compared to that obtained from the raw EEG signal. With the assistance of a supervised multiclass SVM classifier and several timefrequency features, several methods to automatically grade EEG were explored. In summary, the novel techniques proposed in this thesis contribute to the application of advanced signal processing techniques for automatic assessment of neonatal EEG recordings.

3D vanadium oxide inverse opal growth by electrodeposition

Three-dimensional vanadium pentoxide (V2O5) material architectures in the form of inverse opals (IOs) were fabricated using a simple electrodeposition process into artificial opal templates on stainless steel foil using an aqueous solution of VOSO4.χH2O with added ethanol. The direct deposition of V2O5 IOs was compared with V2O5 planar electrodeposition and confirms a similar progressive nucleation and growth mechanism. An in-depth examination of the chemical and morphological nature of the IO material was performed using X-ray crystallography, X-ray photoelectron spectroscopy, Raman scattering and scanning/transmission electron microscopy. Electrodeposition is demonstrated to be a function of the interstitial void fraction of the artificial opal and ionic diffusivity that leads to high quality, phase pure V2O5 inverse opals is not adversely affected by diffusion pathway tortuosity. Methods to alleviate electrodeposited overlayer formation on the artificial opal templates for the fabrication of the porous 3D structures are also demonstrated. Such a 3D material is ideally suited as a cathode for lithium ion batteries, electrochromic devices, sensors and for applications requiring high surface area electrochemically active metal oxides.

Solo and Chamber Music of Pulitzer Prize-Winning Composers

The Pulitzer Prize in Music, established in 1943, is one of America's most prestigious awards. It has been awarded to fifty-three composers for a "distinguished musical composition of significant dimension by an American that has had its first performance in the United States during the year." Composers who have won the Pulitzer Prize are considered to be at the pinnacle of their creativity and have provided the musical world with classical music compositions worthy of future notice. By tracing the history of Pulitzer Prize-winning composers and their compositions, researchers and musicians enhance their understanding of the historical evolution of American music, and its impact on American culture. Although the clarinet music of some of these composers is rarely performed today, their names will be forever linked to the Pulitzer, and because of that, their compositions will enjoy a certain sense of immortality. Of the fifty-four composers who have won the award, forty-seven have written for the clarinet in a solo or chamber music setting (five or less instruments). Just as each Pulitzer Prize-winning composition is a snapshot of the state of American music at that time, these works trace the history of American clarinet musical development, and therefore, they are valuable additions to the clarinet repertoire and worthy of performance. This dissertation project consists of two recitals featuring the solo and chamber clarinet music of sixteen Pulitzer Prize-winning composers, extended program notes containing information on each composer's life, their music, the Pulitzer Prize-winning composition and the recital selection, and a complete list of all Pulitzer Prize-winning composers and their solo and chamber clarinet music. Featured Composers Dominick Argento, To Be Sung Upon the Water Leslie Bassett, Soliloquies William Bolcom, Little Suite of Four Dances Aaron Copland, As it Fell Upon a Day John Corigliano, Soliloquy Norman Dello Joio, Concertante Morton Gould, Benny's Gig Charles Ives, Largo Douglas Moore, Quintet for Clarinet and Strings George Perle, Three Sonatas Quincy Porter, Quintet for Clarinet and Strings Mel Powell, Clarinade Shulamit Ran, Private Game Joseph Schwantner, Entropy Leo Sowerby, Sonata Ernst Toch, Adagio elegiaco

Probing the ultimate limits of plasmonic enhancement.

Metals support surface plasmons at optical wavelengths and have the ability to localize light to subwavelength regions. The field enhancements that occur in these regions set the ultimate limitations on a wide range of nonlinear and quantum optical phenomena. We found that the dominant limiting factor is not the resistive loss of the metal, but rather the intrinsic nonlocality of its dielectric response. A semiclassical model of the electronic response of a metal places strict bounds on the ultimate field enhancement. To demonstrate the accuracy of this model, we studied optical scattering from gold nanoparticles spaced a few angstroms from a gold film. The bounds derived from the models and experiments impose limitations on all nanophotonic systems.

Characterization and analysis of the exogenous application of selected phytohormones on C. reinhardtii metabolism

Gemstone Team Genes to Fuels

Seroprotection against tetanus in patients of an an emergency department in Belgium and evaluation of a bedside immunotest (Tetanos Quick Stick R). A prospective observational study

info:eu-repo/semantics/published

Evaluating the effectiveness of Z: The claims made about CICS and where we go from here

There have been few genuine success stories about industrial use of formal methods. Perhaps the best known and most celebrated is the use of Z by IBM (in collaboration with Oxford University's Programming Research Group) during the development of CICS/ESA (version 3.1). This work was rewarded with the prestigious Queen's Award for Technological Achievement in 1992 and is especially notable for two reasons: 1) because it is a commercial, rather than safety- or security-critical, system and 2) because the claims made about the effectiveness of Z are quantitative as well as qualitative. The most widely publicized claims are: less than half the normal number of customer-reported errors and a 9% savings in the total development costs of the release. This paper provides an independent assessment of the effectiveness of using Z on CICS based on the set of public domain documents. Using this evidence, we believe that the case study was important and valuable, but that the quantitative claims have not been substantiated. The intellectual arguments and rationale for formal methods are attractive, but their widespread commercial use is ultimately dependent upon more convincing quantitative demonstrations of effectiveness. Despite the pioneering efforts of IBM and PRG, there is still a need for rigorous, measurement-based case studies to assess when and how the methods are most effective. We describe how future similar case studies could be improved so that the results are more rigorous and conclusive.

Paying the price for loss of light

Considers the Court of Appeal ruling in Forsyth-Grant v Allen on the principles to be applied in assessing damages for the loss of light. Outlines the method for calculating damages for loss of right to light, the type of amenity which will be included in calculations for loss of amenity and the process applied in this case for assessing damages arising from the loss of profits that would have been made by the owner of the right to light if they had negotiated to relax the covenant, with reference to case law. Notes the limits to damages available for hypothetical loss and the difference between this award of profits and an account of profits.