Biological wastes as plant growth substrate amendments

Globally, agriculture is being intensified with mechanization and increased use of synthetic fertilizers and pesticides. There has been a scaling up of production to satisfy the demands of supermarket distribution. Problems associated with intensification of production, trade globalisation and a larger market demand for greater volumes of fresh produce, include consumers' concern about pesticide residues and leaching of nutrients and pesticides into the environment, as well as increases in the transmission of human food-poisoning pathogens on raw vegetables and in fruit juices. The first part of this research was concerned with the evaluation of a biological control strategy for soil-borne pathogens, these are difficult to eliminate and the chemicals of which the most effective fumigants e.g. methyl bromide, are being withdrawn form use. Chitin-containing crustaceans shellfish waste was investigated as a selective growth substrate amendment in the field, in glasshouse and in storage trials against Sclerotinia disease of Helianthus tuberosus, Phytophthora fragariae disease of Fragaria vesca and Fusarium disease of Dianthus. Results showed that addition to shellfish waste stimulated substrate microbial populations and lytic activity and induced plant defense proteins, namely chitinases and cellulases. Protective effects were seen in all crop models but the results indicate that further trials are required to confirm long-term efficacy. The second part of the research investigated the persistence of enteric bacteria in raw salad vegetables using model food poisoning isolates. In clinical investigations plants are sampled for bacterial contamination but no attempt is made to differentiate between epiphytes and endophytes. Results here indicate that the mode isolates persist endophytically thereby escaping conventional chlorine washes and they may also induce host defenses, which results in their suppression and in negative results in conventional plate count screening. Finally a discussion of criteria that should be considered for a HACCP plan for safe raw salad vegetable production is presented.

The application of aryne chemistry and use of α-diazo carbonyl derivatives in indazole synthesis with biological evaluation

This thesis outlines the synthetic chemistry involved in the preparation of a range of novel indazole compounds and details the subsequent investigation into their potential as biologically active agents. The synthetic route utilised in this research to form the indazole structure was the [3+2] dipolar cycloaddition of diazo carbonyl compounds with reactive aryne intermediates generated in situ. The preparation of further novel indazole derivatives containing different functional groups and substituents was performed by synthesising alternative 1,3- dipole and dipolarophile analogues and provided additionally diverse compounds. Further derivatisation of the indazole product was made possible by deacylation and alkylation methods. Transformation reactions were performed on alkenecontaining ester side chains to provide novel epoxide, aldehyde and tertiary amine derivatives. The first chapter is a review of the literature beginning with a short overview on the structure, reactivity and common synthetic routes to diazo carbonyl derivatives. More attention is given to the use of diazo compounds as 1,3-dipoles in cycloaddition reactions or where the diazo group is incorporated into the final product. A review of the interesting background, structure and reactivity of aryne intermediates is also presented. In addition, some common syntheses of indazole compounds are presented as well as a brief discussion on the importance of indazole compounds as therapeutic agents. The second chapter discusses the synthetic routes employed towards the synthesis of the range of indazoles. Initially, the syntheses of the diazo carbonyl and aryne precursors are described. Next, the synthetic methods to prepare the indazole compounds are provided followed by discussion on derivatisation of the indazole compounds including N-deacylation, N-benzylation and ester side-chain transformation of some alkene-containing indazoles. A series of novel indazole derivatives were submitted for anti-cancer screening at the U.S National Cancer Institute (NCI). A number of these derivatives were identified as hit compounds, with excellent growth inhibition. The results obtained from biological evaluation from the NCI are provided with further results pending from the Community for Open Antimicrobial Drug Discovery. The third chapter details the full experimental procedures, including spectroscopic and analytical data for all the compounds prepared during this research.

Design, synthesis and evaluation of novel ellipticine derivatives and analogues as anti-cancer agents

This thesis describes work carried out on the synthesis of novel 5- and 11-substituted ellipticines and derivatives of the ellipticine analogues, isoellipticine and deazaellipticine, followed by investigation of their potential as anti-cancer agents. Preparation of the key 5- and 11-substituted ellipticine targets involved the development of regiospecific, sequential alkylation reactions with alkenyllithium and Grignard reagents. Investigation of these novel reactions resulted in a new route towards 5-substituted ellipticines via Grignard reaction with vinylmagnesium bromide. These novel 5-vinylellipticine derivatives were further functionalised in an ozonolysis reaction, followed by oxidation to give a range of novel 5-substituted ellipticines. Less success was encountered in the 11-substituted ellipticine series, however preparation of these derivatives using a previously published route was accomplished, and the resulting 11-formylellipticine was further derivatised to give a panel of novel 9- and 11-substituted ellipticines, incorporating amide, carboxylate, imine and amine functionality. The successful route towards 5-substituted ellipticines was applied to the preparation of a range of novel 11-substituted isoellipticines and 6-substituted deazaellipticines, the first time substantial synthesis has been undertaken with these analogues. In addition to this, the first preparation of isoellipticinium salts is described, and a panel of novel isoellipticinium, 7 formylisoellipticinium and 7-hydroxyisoellipticinium salts were synthesised in good yields. Biological evaluation of a panel of 43 novel ellipticine, isoellipticine and deazaellipticine derivatives was accomplished with a topoisomerase II decatenation assay and submission to the NCI 60-cell line screen. Four novel isoellipticine topoisomerase II inhibitors were identified from the decatenation assay, with strong activity at 10 μM. In addition to this, NCI screening identified five highly cytotoxic ellipticine and isoellipticine compounds with remarkable selectivity profiles for different cancer types. These novel lead compounds represent new templates for further research and synthesis.

Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

Synthesis and evaluation of novel azaindolocarbazole derivatives as cancer chemotherapeutics

This thesis details the design and implementation of novel chemical routes towards a series of highly propitious 7-azaindolyl derivatives of the indolocarbazole (ICZ) and bisindolylmaleimide (BIM) families, with subsequent evaluation for use as cancer chemotherapeutic agents. A robust synthetic strategy was devised to allow the introduction of a 7-azaindolyl moiety into our molecular template. This approach allowed access to a wide range of β-keto ester and β-keto nitrile intermediates. Critical analysis identified F-ring modulation as a major theme towards the advancement of ICZ and BIM derivatives in drug therapy. Thus, the employment of cyclocondensation methodology furnished a number of novel aminopyrazole, isoxazolone, pyrazolone and pyrimidinone analogues, considerably widening the scope of the prevalent maleimide functionality. Photochemical cyclisation provided for the first reported aza ICZ containing a six-membered F-ring. Another method towards achieving the aza ICZ core involved use of a Perkin-type condensation approach, with chemical elaboration of the headgroup instigated post-aromatisation. Subsequent use of a modified Lossen rearrangement allowed access to further analogues containing a six-membered F-ring. Extensive screening of the novel aza ICZ and BIM derivatives was carried out against the NCI-60 cancer cell array, with nine prospective candidates selected for continued biological evaluation. From these assays, a number of compounds were shown to inhibit cancer cell growth at concentrations of below 10 nM. Indeed, the most active aza ICZ tested is currently under assessment by the Biological Evaluation Committee of the NCI due to excellent antiproliferative activity demonstrated across the panel of cell lines, with a mean GI50 of 34 nM, a mean total growth inhibition (TGI) of 4.6 μM and a mean cytotoxicity (LC50) of 63.1 μM. Correlation to known topoisomerase I (topo I) inhibitors was revealed by COMPARE analysis, and subsequent topo I-mediated DNA cleavage assays showed inhibitory activity below 1 μM for several derivatives.

Synthesis and reactivity of α-diazo-β-keto sulfoxides: scope and synthetic potential

The research described in this thesis focuses on the design and synthesis of stable α-diazosulfoxides and investigation of their reactivity under a variety of conditions (transition-metal catalysis, thermal, photochemical and microwave) with a particular emphasis on the synthesis of novel heterocyclic compounds with potential biological activity. The exclusive reaction pathway for these α-diazosulfoxides was found to be hetero-Wolff rearrangement to give α-oxosulfine intermediates. In the first chapter, a literature review of sulfines is presented, including a discussion of naturally occurring sulfines, and an overview of the synthesis and reactivity of sulfines. The potential of sulfines in organic synthesis and recent developments in particular are highlighted. The second chapter discusses the synthesis and reactivity of α-diazosulfoxides, building on earlier results in this research group. The synthesis of lactone-based α-diazosulfoxides and, for the first time, ketone-based benzofused and monocyclic α-diazosulfoxides is described. The reactivity of these α-diazosulfoxides is then explored under a variety of conditions, such as transition-metal catalysis, photochemical and microwave, generating labile α-oxosulfine intermediates, which are trapped using amines and dienes, in addition to the spontaneous reaction pathways which occur with α-oxosulfines in the absence of a trap. A new reaction pathway was explored with the lactone based α-oxosulfines, involving reaction with amines to generate novel 3-aminofuran-2(5H)-ones via carbophilic attack, in very good yields. The reactivity of ketone-based α-diazosulfoxides was explored for the first time, and once again, pseudo-Wolff rearrangement to the α-oxosulfines was the exclusive reaction pathway observed. The intermediacy of the α-oxosulfines was confirmed by trapping as cycloadducts, with the stereochemical features dependant on the reaction conditions. In the absence of a diene trap, a number of reaction fates from the α-oxosulfines were observed, including complete sulfinyl extrusion to give indanones, sulfur extrusion to give indanediones, and, to a lesser extent, dimerisation. The indanediones were characterised by trapping as quinoxalines, to enable full characterisation. One of the overriding outcomes of this thesis was the provision of new insights into the behaviour of α-oxosulfines with different transition metal catalysts, and under thermal, microwave and photolysis conditions. A series of 3-aminofuran-2(5H)-ones and benzofused dihydro-2H-thiopyran S-oxides were submitted for anticancer screening at the U.S. National Cancer Institute. A number of these derivatives were identified as hit compounds, with excellent cell growth inhibition. One 3-aminofuran-2(5H)-one derivative has been chosen for further screening. The third chapter details the full experimental procedures, including spectroscopic and analytical data for the compounds prepared during this research. The data for the crystal structures are contained in the attached CD.

An examination of the underlying trait structure of psychopathy: applications for screening and measurement

This thesis seeks to clarify the faceted organisation of psychopathy with a view to developing a comprehensive protocol for the assessment of core psychopathic personality traits. The framework developed will, as best as possible, be free of sample bias. The Self-and Informant-report Deviant Personality Screen (DPS) is introduced and a series of empirical studies are conducted to examine the psychometric properties and construct validity of these measures in general and offender populations. Findings from these studies provide strong support for the utility of the DPS scales for the appraisal of psychopathy across diverse population samples. In addition to this, the utility of cognitive based performance measures for the assessment of emotional deficits in psychopathy is evaluated. Results from this study suggest limited correspondence between these measurement techniques and self-report psychopathy measures. Finally, research conducted on offenders suggests that information obtained from DPS reports may be useful within a broad framework of risk assessment. Further empirical and theoretical implications of the research are discussed.

Synthesis of iminophosphine and phosphinoiminol cyclometallated Pt (II) and Pt (IV) chloro complexes and studies into their biological and photophysical properties

This thesis focuses on the synthesis and analysis of novel chloride based platinum complexes derived from iminophosphine and phosphinoamide ligands, along with studies on their reactivity towards substitution and oxidation reactions. Also explored here are the potential applications of these complexes for biological and luminescent purposes. Chapter one provides an extensive overview of platinum coordination chemistry with examples of various mixed donor ligands along with the history of platinum anticancer therapy. It also looks at metals in medicine, both for biological functions as well as for therapeutic purposes and gives a background to some other applications for platinum complexes. Chapter two outlines the design and synthetic strategies employed for the development of novel platinum (II) chloride complexes from iminophosphine and phosphinoamide ligands. Also reported is the cyclometallation of these complexes to form stable tridentate mixed donor platinum (II) compounds. In Chapter three the development of a direct method for displacing a chloride from a platinum metal centre with a desired phosphine is reported. Numerous methods for successful oxidation of the platinum (II) complexes will also be explored, leading to novel platinum (IV) complexes being reported here also. The importance of stabilisation of the displaced anion, chloride, by the solvent system will also be discussed in this chapter. Chapter four investigates the reactivity of the platinum (II) complexes towards two different biomolecules to form novel platinum bio-adducts. The potential application of the platinum (II) cyclometallates as chemotherapeutics will also be explored here using in-vitro cancer cell testing. Finally, luminescence studies are also reported here for the ligands and platinum complexes reported in chapter two and three to investigate potential applications in this field also. Chapter five provides a final conclusion and an overall summary of the entire project as well as identifying key areas for future work.

Characterisation of the microbiota of traditional fermented beverages and screening these and other populations for novel antimicrobial producers and gene clusters

To screen for novel ribosomally synthesised antimicrobials, in-silico genome mining was performed on all publically available fully sequenced bacterial genomes. 49 novel type 1 lantibiotic clusters were identified from a number of species, genera and phyla not usually associated with lantibiotic production, and indicates high prevalence. A crucial step towards the commercialisation of fermented beverages is the characterisation of the microbial content. To achieve this goal, we applied next-generation sequencing techniques to analyse the bacterial and yeast populations of the organic, symbiotically-fermented beverages kefir, water kefir and kombucha. A number of minor components were revealed, many of which had not previously been associated with these beverages. The dominant microorganism in each of the water kefir grains and fermentates was Zymomonas, an ethanol-producing bacterium that had not previously been detected on such a scale. These studies represent the most accurate description of these populations to date, and should aid in future starter design and in determining which species are responsible for specific attributes of the beverages. Finally, high-throughput robotics was applied to screen for the presence of antimicrobial producers associated with these beverages. This revealed a low frequency of bacteriocin production amongst the bacterial isolates, with only lactococcins A, B and LcnN of lactococcin M being identified. However, a proteinaceous antimicrobial produced by the yeast Dekkera bruxellensis, isolated from kombucha, was found to be active against Lactobacillus bulgaricus. This peptide was patially purified.

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.

Environmental and biological characteristics of lagoons on the southwest coast of Ireland

Coastal lagoons are defined as shallow coastal water bodies partially separated from the adjacent sea by a restrictive barrier. Coastal lagoons are protected under Annex I of the European Habitats Directive (92/43/EEC). Lagoons are also considered to be “transitional water bodies” and are therefore included in the “register of protected areas” under the Water Framework Directive (2000/60/EC). Consequently, EU member states are required to establish monitoring plans and to regularly report on lagoon condition and conservation status. Irish lagoons are considered relatively rare and unusual because of their North Atlantic, macrotidal location on high energy coastlines and have received little attention. This work aimed to assess the physicochemical and ecological status of three lagoons, Cuskinny, Farranamanagh and Toormore, on the southwest coast of Ireland. Baseline salinity, nutrient and biological conditions were determined in order to provide reference conditions to detect perturbations, and to inform future maintenance of ecosystem health. Accumulation of organic matter is an increasing pressure in coastal lagoon habitats worldwide, often compounding existing eutrophication problems. This research also aimed to investigate the in situ decomposition process in a lagoon habitat together with exploring the associated invertebrate assemblages. Re-classification of the lagoons, under the guidelines of the Venice system for the classifications of marine waters according to salinity, was completed by taking spatial and temporal changes in salinity regimes into consideration. Based on the results of this study, Cuskinny, Farranamanagh and Toormore lagoons are now classified as mesohaline (5 ppt – 18 ppt), oligohaline (0.5 ppt – 5 ppt) and polyhaline (18 ppt – 30 ppt), respectively. Varying vertical, longitudinal and transverse salinity patterns were observed in the three lagoons. Strong correlations between salinity and cumulative rainfall highlighted the important role of precipitation in controlling the lagoon environment. Maximum effect of precipitation on the salinity of the lagoon was observed between four and fourteen days later depending on catchment area geology, indicating the uniqueness of each lagoon system. Seasonal nutrient patterns were evident in the lagoons. Nutrient concentrations were found to be reflective of the catchment area and the magnitude of the freshwater inflow. Assessment based on the Redfield molar ratio indicated a trend towards phosphorus, rather than nitrogen, limitation in Irish lagoons. Investigation of the decomposition process in Cuskinny Lagoon revealed that greatest biomass loss occurred in the winter season. Lowest biomass loss occurred in spring, possibly due to the high density of invertebrates feeding on the thick microbial layer rather than the decomposing litter. It has been reported that the decomposition of plant biomass is highest in the preferential distribution area of the plant species; however, no similar trend was observed in this study with the most active zones of decomposition varying spatially throughout the seasons. Macroinvertebrate analysis revealed low species diversity but high abundance, indicating the dominance of a small number of species. Invertebrate assemblages within the lagoon varied significantly from communities in the adjacent freshwater or marine environments. Although carried out in coastal lagoons on the southwest coast of Ireland, it is envisaged that the overall findings of this study have relevance throughout the entire island of Ireland and possibly to many North Atlantic coastal lagoon ecosystems elsewhere.

Experiences of the foster care process in the Republic of Ireland: retrospective accounts from biological children of foster carers

This thesis examines the experiences of the biological children of foster carers. In particular it explores their experiences in relation to inclusion, consultation and decision-making. The study also examines the support and training needs of birth children in foster families. Using a qualitative methodology in-depth, semi-structured interviews were conducted with fifteen birth children of foster carers aged between 18 and 30 years. The research findings show that for the majority of birth children, fostering was overall a positive experience which helped them develop into individuals who were caring and nonjudgemental. However, from the data collected in this study, it is clear that fostering also brings a range of challenges for birth children in foster families, such as managing feelings of loss, grief, jealousy and guilt when foster children leave. Birth children are reluctant to discuss these issues with their parents and often did not approach fostering social workers as they did not have a meaningful relationship in order to discuss their concerns. The findings also demonstrate that birth children undertake a lot of emotional work in supporting their parents, birth siblings and foster siblings. Despite the important role played by birth children in the fostering process, this contribution often goes unrecognised and unacknowledged by fostering professionals and agencies with birth children not included or consulted about foster care decisions that affect them. It is argued here that birth children are viewed by foster care professionals and agencies from a deficit based perspective. However, this study contends that it is not just foster parents who are involved in the foster care process, but the entire foster family. The findings of this study show that birth children are competent social actors capable of making valuable contributions to foster care decisions that affect their lives and that of their family.

Synthesis and evaluation of novel quinolines and quinazolinediones as potential anti-cancer agents

This thesis outlines the design and effectuation of novel chemical routes towards a nascent class of functionalised quinoline-5,8-diones and the expansion of a series of contemporary quinazolinediones towards an innovative family of pyridinoquinazolinetetrone derivatives. This fragment based approach is envisaged to lead to advancements in the three scaffolds, expanding the SAR pool of both quinolines and quinazolinediones with subsequent evaluation of chemotherapeutic potential as well as furnishing a new class of tricycle for biological investigation. Development of novel quinoline-5,8-diones is provided for by expanding on existing methodology. Using a variety of nucleophiles on a critical intermediate, a broad range of novel compounds was afforded allowing chemotherapeutic potential to be assessed, while also serving as intermediates for accomplishing novel pyridinoquinazolinetetrone congeners. In order to incorporate functionality into our quinazolinedione template, an efficient synthetic strategy was constructed which provided a robust route to effectuate a highly derivatised pyrimidinedione ring. As derivatisation of this template is unreported our chief priority was to synthesise a range of diverse quinazolinediones. The application of annulation methodology using functionalised precursors provided a library of N-3 derivatised quinazolinedione analogues. These, along with their N-1 functionalised derivatives provide a wide scope from which to construct a series of pyridinoquinazolinetetrone derivatives while also serving as a unique class of molecules whose biological potential is uncharted. Although the actualisation of the pyridinoquinazolinetetrone was ultimately unsuccessful, our work has led to the development of novel quinoline-5,8-diones which were found to possess excellent anti-cancer activity when assessed by the NCI screen. Of the quinazolinediones synthesised eight compounds were accepted for screening by the NCI. Results from the single-dose tests however indicated that these compounds possessed little cytotoxic activity at 10 μM. The development of this novel template in conjunction with the highly active quinolinediones serves as an excellent rostrum for future synthetic endeavours.

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.

The Quick Mild Cognitive Impairment (Qmci) screen: developing a new screening test for mild cognitive impairment and dementia

Introduction: Identifying mild cognitive impairment (MCI) is challenging. Few short instruments have sufficient sensitivity and specificity for use in busy clinical practice. This thesis explores the development, psychometric evaluation and validation of a new short (3–5 min) cognitive screening instrument, designed to screen for MCI and early dementia, called the Quick Mild Cognitive Impairment (Q