Integrated genetic analysis systems

The overall objective of this thesis is to integrate a number of micro/nanotechnologies into integrated cartridge type systems to implement such biochemical protocols. Instrumentation and systems were developed to interface such cartridge systems: (i) implementing microfluidic handling, (ii) executing thermal control during biochemical protocols and (iii) detection of biomolecules associated with inherited or infectious disease. This system implements biochemical protocols for DNA extraction, amplification and detection. A digital microfluidic chip (ElectroWetting on Dielectric) manipulated droplets of sample and reagent implementing sample preparation protocols. The cartridge system also integrated a planar magnetic microcoil device to generate local magnetic field gradients, manipulating magnetic beads. For hybridisation detection a fluorescence microarray, screening for mutations associated with CFTR gene is printed on a waveguide surface and integrated within the cartridge. A second cartridge system was developed to implement amplification and detection screening for DNA associated with disease-causing pathogens e.g. Escherichia coli. This system incorporates (i) elastomeric pinch valves isolating liquids during biochemical protocols and (ii) a silver nanoparticle microarray for fluorescent signal enhancement, using localized surface plasmon resonance. The microfluidic structures facilitated the sample and reagent to be loaded and moved between chambers with external heaters implementing thermal steps for nucleic acid amplification and detection. In a technique allowing probe DNA to be immobilised within a microfluidic system using (3D) hydrogel structures a prepolymer solution containing probe DNA was formulated and introduced into the microfluidic channel. Photo-polymerisation was undertaken forming 3D hydrogel structures attached to the microfluidic channel surface. The prepolymer material, poly-ethyleneglycol (PEG), was used to form hydrogel structures containing probe DNA. This hydrogel formulation process was fast compared to conventional biomolecule immobilization techniques and was also biocompatible with the immobilised biomolecules, as verified by on-chip hybridisation assays. This process allowed control over hydrogel height growth at the micron scale.

Development of tumour imaging and therapy strategies utilising unengineered bacteria

The ability of systemically administered bacteria to target and replicate to high numbers within solid tumours is well established. Tumour localising bacteria can be exploited as biological vehicles for the delivery of nucleic acid, protein or therapeutic payloads to tumour sites and present researchers with a highly targeted and safe vehicle for tumour imaging and cancer therapy. This work aimed to utilise bacteria to activate imaging probes or prodrugs specifically within target tissue in order to facilitate the development of novel imaging and therapeutic strategies. The vast majority of existing bacterial-mediated cancer therapy strategies rely on the use of bacteria that have been genetically modified (GM) to express genes of interest. While these approaches have been shown to be effective in a preclinical setting, GM presents extra regulatory hurdles in a clinical context. Also, many strains of bacteria are not genetically tractably and hence cannot currently be engineered to express genes of interest. For this reason, the development of imaging and therapeutic systems that utilise unengineered bacteria for the activation of probes or drugs represents a significant improvement on the current gold standard. Endogenously expressed bacterial enzymes that are not found in mammalian cells can be used for the targeted activation of imaging probes or prodrugs whose activation is only achieved in the presence of these enzymes. Exploitation of the intrinsic enzymatic activity of bacteria allows the use of a wider range of bacteria and presents a more clinically relevant system than those that are currently in use. The nitroreductase (NTR) enzymes, found only in bacteria, represent one such option. Chapter 2 introduces the novel concept of utilising native bacterial NTRs for the targeted activation of the fluorophore CytoCy5S. Bacterial-mediated probe activation allowed for non-invasive fluorescence imaging of in vivo bacteria in models of infection and cancer. Chapter 3 extends the concept of using native bacterial enzymes to activate a novel luminescent, NTR activated probe. The use of luminescence based imaging improved the sensitivity of the system and provides researchers with a more accessible modality for preclinical imaging. It also represents an improvement over existing caged luciferin probe systems described to date. Chapter 4 focuses on the employment of endogenous bacterial enzymes for use in a therapeutic setting. Native bacterial enzymatic activity (including NTR enzymes) was shown to be capable of activating multiple prodrugs, in isolation and in combination, and eliciting therapeutic responses in murine models of cancer. Overall, the data presented in this thesis advance the fields of bacterial therapy and imaging and introduce novel strategies for disease diagnosis and treatment. These preclinical studies demonstrate potential for clinical translation in multiple fields of research and medicine.

Ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use

Genetic decoding is not ‘frozen’ as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational ‘correction’ of problem or ‘savior’ indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5′ or 3′ of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3′ from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.

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.

Tropospheric aerosol detection over Southern Ireland using a backscatter Raman lidar

Lidar is an optical remote sensing instrument that can measure atmospheric parameters. A Raman lidar instrument (UCLID) was established at University College Cork to contribute to the European lidar network, EARLINET. System performance tests were carried out to ensure strict data quality assurance for submission to the EARLINET database. Procedures include: overlap correction, telecover test, Rayleigh test and zero bin test. Raman backscatter coefficients, extinction coefficients and lidar ratio were measured from April 2010 to May 2011 and February 2012 to June 2012. Statistical analysis of the profiles over these periods provided new information about the typical atmospheric scenarios over Southern Ireland in terms of aerosol load in the lower troposphere, the planetary boundary layer (PBL) height, aerosol optical density (AOD) at 532 nm and lidar ratio values. The arithmetic average of the PBL height was found to be 608 ± 138 m with a median of 615 m, while average AOD at 532 nm for clean marine air masses was 0.119 ± 0.023 and for polluted air masses was 0.170 ± 0.036. The lidar ratio showed a seasonal dependence with lower values found in winter and autumn (20 ± 5 sr) and higher during spring and winter (30 ± 12 sr). Detection of volcanic particles from the eruption of the volcano Eyjafjallajökull in Iceland was measured between 21 April and 7 May 2010. The backscatter coefficient of the ash layer varied between 2.5 Mm-1sr-1 and 3.5 Mm-1sr-1, and estimation of the AOD at 532 nm was found to be between 0.090 and 0.215. Several aerosol loads due to Saharan dust particles were detected in Spring 2011 and 2012. Lidar ratio of the dust layers were determine to be between 45 and 77 sr and AOD at 532 nm during the dust events range between 0.84 to 0.494.

Learning to drive: from hazard detection to hazard handling

Hazard perception has been found to correlate with crash involvement, and has thus been suggested as the most likely source of any skill gap between novice and experienced drivers. The most commonly used method for measuring hazard perception is to evaluate the perception-reaction time to filmed traffic events. It can be argued that this method lacks ecological validity and may be of limited value in predicting the actions drivers’ will take to hazards encountered. The first two studies of this thesis compare novice and experienced drivers’ performance on a hazard detection test, requiring discrete button press responses, with their behaviour in a more dynamic driving environment, requiring hazard handling ability. Results indicate that the hazard handling test is more successful at identifying experience-related differences in response time to hazards. Hazard detection test scores were strongly related to performance on a driver theory test, implying that traditional hazard perception tests may be focusing more on declarative knowledge of driving than on the procedural knowledge required to successfully avoid hazards while driving. One in five Irish drivers crash within a year of passing their driving test. This suggests that the current driver training system does not fully prepare drivers for the dangers they will encounter. Thus, the third and fourth studies in this thesis focus on the development of two simulator-based training regimes. In the third study participants receive intensive training on the molar elements of driving i.e. speed and distance evaluation. The fourth study focuses on training higher order situation awareness skills, including perception, comprehension and projection. Results indicate significant improvement in aspects of speed, distance and situation awareness across training days. However, neither training programme leads to significant improvements in hazard handling performance, highlighting the difficulties of applying learning to situations not previously encountered.

“Green preservatives” – combating fungi in the food industry by applying antifungal lactic acid bacteria

Fungal spoilage is the most common type of microbial spoilage in food leading to significant economical and health problems throughout the world. Fermentation by lactic acid bacteria (LAB) is one of the oldest and most economical methods of producing and preserving food. Thus, LAB can be seen as an interesting tool in the development of novel bio-preservatives for food industry. The overall objective of this study was to demonstrate, that LAB can be used as a natural way to improve the shelf-life and safety of a wide range of food products. In the first part of the thesis, 116 LAB isolates were screened for their antifungal activity against four Aspergillus and Penicillium spp. commonly found in food. Approximately 83% of them showed antifungal activity, but only 1% showed a broad range antifungal activity against all tested fungi. The second approach was to apply LAB antifungal strains in production of food products with extended shelf-life. L. reuteri R29 strain was identified as having strong antifungal activity in vitro, as well as in sourdough bread against Aspergillus niger, Fusarium culmorum and Penicillium expansum. The ability of the strain to produce bread of good quality was also determined using standard baking tests. Another strain, L. amylovorus DSM19280, was also identified as having strong antifungal activity in vitro and in vivo. The strain was used as an adjunct culture in a Cheddar cheese model system and demonstrated the inhibition of P. expansum. Significantly, its presence had no detectable negative impact on cheese quality as determined by analysis of moisture, salt, pH, and primary and secondary proteolysis. L. brevis PS1 a further strain identified during the screening as very antifungal, showed activity in vitro against common Fusarium spp. and was used in the production of a novel functional wortbased alcohol-free beverage. Challenge tests performed with F. culmorum confirmed the effectiveness of the antifungal strain in vivo. The shelf-life of the beverage was extended significantly when compared to not inoculated wort sample. A range of antifungal compounds were identified for the 4 LAB strains, namely L. reuteri ee1p, L. reuteri R29, L. brevis PS1 and L. amylovorous DSM20531. The identification of the compounds was based on liquid chromatography interfaced to the mass spectrometer and PDA detector

The use of fault detection and diagnostics to reduce air handling unit energy consumption

The contribution of buildings towards total worldwide energy consumption in developed countries is between 20% and 40%. Heating Ventilation and Air Conditioning (HVAC), and more specifically Air Handling Units (AHUs) energy consumption accounts on average for 40% of a typical medical device manufacturing or pharmaceutical facility’s energy consumption. Studies have indicated that 20 – 30% energy savings are achievable by recommissioning HVAC systems, and more specifically AHU operations, to rectify faulty operation. Automated Fault Detection and Diagnosis (AFDD) is a process concerned with potentially partially or fully automating the commissioning process through the detection of faults. An expert system is a knowledge-based system, which employs Artificial Intelligence (AI) methods to replicate the knowledge of a human subject matter expert, in a particular field, such as engineering, medicine, finance and marketing, to name a few. This thesis details the research and development work undertaken in the development and testing of a new AFDD expert system for AHUs which can be installed in minimal set up time on a large cross section of AHU types in a building management system vendor neutral manner. Both simulated and extensive field testing was undertaken against a widely available and industry known expert set of rules known as the Air Handling Unit Performance Assessment Rules (APAR) (and a later more developed version known as APAR_extended) in order to prove its effectiveness. Specifically, in tests against a dataset of 52 simulated faults, this new AFDD expert system identified all 52 derived issues whereas the APAR ruleset identified just 10. In tests using actual field data from 5 operating AHUs in 4 manufacturing facilities, the newly developed AFDD expert system for AHUs was shown to identify four individual fault case categories that the APAR method did not, as well as showing improvements made in the area of fault diagnosis.