Life Sciences Commons^™

Validation Of An Immunomagnetic-Electrochemiluminescence Detection System For The Detection Of Escherichia Coli 0157 In Food And Comparison With An Iso Standard Method, Eimear Carney

Theses

Rapid and sensitive methods for detecting Escherichia coli 0157 in contaminated food, water and other biological samples are important in limiting and preventing the spread of this pathogen. Traditional culture based methods for bacterial identification and detection can be time consuming and labour intensive. Modem alternative methods should be faster whilst maintaining sensitivity. Immunomagnetic separation (IMS) methodology has been investigated and appears promising for rapid bacterial detection in unrelated food and environmental samples. In this study, a commercial sensor which combined IMS with electrochemiluminescence (ECL) was evaluated for the detection of E. coli 0157 in unrelated food matrices. Emitted signals …

Exploring Marine Biotoxins Using Analytical Techniques Based On Mass Spectrometry, Mónica Fernández Amandi

Theses

The impact of phycotoxins on human and animal health is of important concern for worldwide authorities and the scientific community. Domoic acid (DA) is a neurotoxin produced by phytoplankton {Pseudonitzchia spp.). It enters the food chain through the consumption of shellfish that feed on toxic diatoms. DA has been responsible for several outbreaks affecting humans and animals all around the world. Yessotoxin (YTX) is produced by dinoflagellate species {Protoceratiuni reticulatum) which are globally distributed. Its toxicity is low when ingested orally. However, the co-extraction of YTX with diarrhetic shellfish poisoning (DSP) toxins, derives false positives when the mouse bioassay is …

Analytical And Synthetic Methods For Anatoxins., Janet Crowley

Theses

This thesis is focused on the potent cyanobacterial neurotoxins anatoxin-a (AN), its methyl analogue homoanatoxin-a (HMAN) and their non-toxic dihydro- and epoxydegradation products. In commencing the investigation into this area a literature review was performed in order to identify areas where more research and method development was required (Chapter 1). The review highlighted the fact that although a number of analytical methods had been developed for the analysis of AN, there were few methods for the determination of its potent homologue HMAN and their non-toxic degradation products. As these neurotoxins degrade quickly it is important to monitor their degradation products, …

Evaluation Of The Host Range Of Polyvalent Staphylococcal Bacteriophages To Control Mrsa (Staphylococcus Aureus) From Irish Hospitals, Mohamed Farag M. Elbreki

Theses

This study describes firstly, the assembly of a collection of a varied spectrum of staphylococci which can be used to test the efficacy of bacteriophages for the biocontrol of problematic members of this genus. The collection assembled includes a variety of multi-antibiotic resistant MRSA (methicillin resistant S. aureus) strains, hVRSA (hetero vancomycin resistant S. aureus), VRSA and also teicoplenin resistant isolates. It also included a broad range of species of coagulase-negative {non-aureus) staphylococci including S. caprea, S. haemolyticus, S. saprophyticus, S. epidermidis, S. chromogenes, S. capitis, S. hominins and S. hyicus. All strains were characterized by morphology on Baird Parker, …

The Diarrheic Shellfish Poisoning Toxins In Marine Environment Determined Using Liquid Chromatography - Mass Spectrometry Techniques, Patricia Fernández Puente

Theses

A highly specific and sensitive LC-MS/MS analytical method was developed and validated using gradient reversed phase liquid chromatography-tandem mass spectrometry (LC- MS/MS), interfaced using turbo-assisted electrospray ionisation (ESI) in negative mode for the analysis of DSP (okadaic acid (OA), dinophysistoxins (DTXs) and pectenotoxins (PTXs)) toxins in a variety of samples. There were no matrix effects present, proved by the good reproducibility achieved and the linearity of the calibration curves (r" > 0.999) in spiked mussel samples and standards. A DSP toxin profile study was conducted with bulk phytoplankton and mussels samples from the west coast of Ireland. All the samples were …

Analysis Of Phycotoxin And Mycotoxin, Contaminants In Foods, Using Liquid Chromatography - Mass Spectrometry Techniques, Ana Brana Magdalena

Theses

The impact of phycotoxins and mycotoxins on human and animal health are of important concern for worldwide authorities and the scientific community. Liquid chromatography-mass spectrometry (LC-MS) techniques have been developed, optimised and validated for the analysis of these toxins.

Phycotoxins are potentially toxic substances, which are produced by marine phytoplankton and which may accumulate in shellfish or finfish. Azaspiracid poisoning (AZP) is a recently discovered syndrome that has been implicated in several incidents of human intoxications. A study was undertaken to examine the distribution of azaspiracids (AZA) in scallops {Pecten maximiis) and mussels {Mytiliis edulis). Domoic acid (DA) is an …

Development Of An Automated Thiopurine Methyltransferase (Tpmt) Genotyping Assay, Mary Corcoran

Theses

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme that catalyses the S- methylation of aromatic and sulphydryl compounds known as the thiopurines. These therapeutic agents are used in the treatment of acute lymphoblastic leukemia, autoimmune disorders, inflammatory bowel disease and organ transplant recipients.

Erythrocyte TPMT activity is polymorphic and patients with intermediate or deficient TPMT are at significant risk for excessive toxicity after receiving ‘standard’ doses of thiopurine medications. The activity of TPMT is inherited as an autosomal co-dominant trait. Several mutations in the TPMT gene have been described that correlate with a low activity phenotype. Three mutations G238C, G460A and …

An Investigation Of The Effects Of A 12-Week Exercise Programme For People With Diabetes, Olive Fanning

Theses

To determine and examine the effects of a 12-week exercise programme for people with diabetes on their blood glucose control, physiological components and psychological well-being.

The purpose of the study was to examine a wide variety of physiological components including assessment of aerobic capacity, body composition, lung, function, flexibility and blood glucose control (HbAlc levels). Psychological well-being was also assessed which includes sub scales for depression, anxiety, energy and positive well being. This had the purpose of identifying exact levels of these variables so as to compare these to the general public norms. It additionally had the benefit of being …

Development Of A Genomic Approach For The Study Of A Coryneform Bacterial Collection., Carole Feurer

Theses

In the last few years, genomics has profoundly changed research in the biological sciences and in particular in microbiology. Recent studies have demonstrated that comparative genomics is a powerful approach contributing to a deeper understanding of the evolution of microorganisms. Bacterial collections are important resources of microorganisms, however, the potential that genomics has to offer remains to be realised as it is still virtually absent in any study involving strain collections. This feature thus framed the experimental objectives for this study, whose aim was to define the most comprehensive way to apply genomic based methods (high throughput sequencing and bioinformatics) …

Campylobacter Spp: A Molecular Investigation Of Transmission Through The Food Chain In Ireland, Brigid Lucey

Theses

The purpose of this study was to investigate the epidemiology of thermophilic Campylobacter infections in Ireland, and to assess antimicrobial resistance among animal isolates and its relevance to human enteric infection with Campylobacter. The first part of the study focused on 84 isolates of human, poultry and porcine origins, isolated between 1996 and 1998 in the Cork region. These isolates were identified to species level using a combination of biochemical and molecular methods. Campylobacter jejuni (C. jejuni) was the predominant organism among human and poultry isolates, whilst all of the porcine isolates examined were identified as C. coli. DNA amplification …

An Assessment Of Prothrombotic Tendency In Humans Using Functional And Genomic Determinants In The South Eastern Irish Population, Carmel Cullen

Theses

Venous thrombosis occurs in about 1 per 1,000 individuals per year. It is a serious disorder and accounts for a significant number of hospital admissions and deaths annually. Thrombophilia describes the familial or acquired disorders of the haemostatic mechanism that predispose to thrombosis. Inherited thrombophilia can be defined as a genetically determined tendency to venous thromboembolism Mutations in genes that code for proteins involved directly (or indirectly) In blood coagulation have been associated with prothrombotic status. Laboratory investigations for thrombophilia include screening for deficiencies of antithrombin, protein C or protein S and testing for activated protein C resistance. Together these …

An Enzymatic Method For The Measurement Of Magnesium, Joseph Murphy

Theses

Magnesium is quantitatively the most important intracellular divalent cation (4), and has been found to serve in a wide range of enzymatic reactions (2). Studies have shown that up to ten percent of hospital patients may have hypomagnesaemia (6).

Atomic absorption spectrophotometry (AAS) under highly specified conditions is the de facto reference method for magnesium measurement but only about ten per cent of laboratories routinely use this method (4). This is because AAS is time-consuming, requires specialised equipment and a certain level of expertise, and may be hazardous due to the use of inflammable gases. Colourimetric methods e.g. calmagite are …

Β-Lactamase: Evolution Of Extended Spectrum Enzymes, Priscilla Shine

Theses

β-Lactam antibiotics are commonly used antimicrobial agents in clinical medicine. Because of their frequent use it is perhaps not surprising that resistance to these agents has increased over the past few decades, thus compromising the therapeutic advantages offered by these agents. The most common cause of resistance to β-lactam-based antibiotics has been the evolution of a group of enzymes known as β-lactamases, which act by inactivating the drug through structural modification. Plasmid-encoded TEM β-lactamases are examples of these hydrolytic enzymes and are associated with the amoxicillin resistance reported in over 50% of all E. coli isolates.

More recently β-lactamase enzymes …

The Investigation Of Rna Viruses Using The Atomic Force Microscope, Michelle Moloney

Theses

The subject of viral entry into, and subsequent progeny virus egress, from cultured cells, has been extensively studied using numerous scientific techniques in fields ranging from cell culture to biochemistry to microscopy. The atomic force microscope (AFM) is a novel, developing instrument with unprecedented capabilities and this study was undertaken to observe enveloped Semliki Forest virus (SFV) and non-enveloped Theiler's Murine Encephalomyelitis virus (TMEV) entry into and egress from cultured cells using the AFM. Virus particle structure of both enveloped and non-enveloped viruses was also directly examined.

Cytopathic effect studies revealed that TMEV's are unable to produce progeny viruses in …

Improving The Quality Of Foliage Transplants For Supply To The Kerry Foliage Industry, Gwen Lanigan

Theses

The possibility of improving the quality of transplants of ornamental foliage species, Eucalyptus perriniana, E. parvifolia and Pittosporum tenuifolium, for supply to the Kerry Foliage Industry was investigated. At present transplants (seedlings) are propagated from seed under glass and subsequently transplanted into the field. Therefore nursery practices for seed propagation, such as optimum sowing date, pot and medium selection, irrigation and fertilisation regimes, were investigated and optimised. The possibility of eliminating the occurrence of J- roots, a major problem with Eucalyptus species was also investigated. Vegetative propagation methods, such as cutting propagation and in vitro micro-propagation were investigated for comparative …

Applications Of The Atomic Force Microscope Within The Fields Of Haematology And Cytogenetics, Margaret O'Reilly-Crowley

Theses

Atomic force microscopy [AFM] has become a rapidly developing interdisciplinary field of research in recent years. The technique, including instrumentation and specimen preparation methods, is now more sophisticated and its application has increased dramatically. The key advantage of AFM is in providing three-dimensional images of samples imaged. Both size and shape of samples imaged can be obtained allowing their subsequent quantification. Red blood cells [RBCs] and chromosomes were imaged in this work. A brief overview of the current status of biomedical AFM, has been presented in the review.

Measurement of mean cell volume [MCV] of immobilised red blood cells with …

Whole Genome Sequencing Of L.Monocytogenes And L.Innocua : A Basis For Comparative Genomics Of The Genus Listeria, Rachel Purcell

Theses

Listeria monocytogenes is a gram-positive, food-borne human and animal pathogen. L.innocua is a non-pathogenic species the most closely related to L. monocytogenes. Comparative genomics between a pathogenic and a closely related non-pathogenic species is a powerful approach for identifying genes or regions on the chromosome, which might be involved in virulence. Therefore whole genome sequencing of these two organisms was undertaken. The strategy used was based on a random approach combined with end-sequencing of a medium insert library for the finishing phase. Based on the available sequence data for these two species an in silico analysis for the presence of …

Pruning And Nutrition Of Outdoor Cut Foliage Crops, Andy Whelton

Theses

The rapidly expanding cut foliage industry in the South of Ireland aims to satisfy the demands of an exacting market requiring well furnished quality stems for use in the manufacture of foliage and flower bouquets. The studies examined, in detail, aspects of pruning and nutrition of genera currently important to this evolving industry. Recommendations for the optimum time of pruning Eucalyptus pulverulenta and Pittosporum tenujolium are given. Pruning regimes for Eucalyptus parv,folia and E. glaucescens, Pittosporum tenufolium, Ozothamnus rosmarinfolius ‘Silver Jubilee’ and Rhododendron ponticum were examined and guidelines on best practices are outlined. A nutrition experiment to examine the effect …

The Molecular Characterisation Of Selected Epidemic Associated Gram Negative Pathogens In Southern Ireland, Mairéad Daly

Theses

This study was undertaken to evaluate various molecular techniques for the analysis of Gram negative bacterial associated epidemics. The first investigation dealt with bovine mastitis outbreaks, where the causative agent was identified as Pseudomonas aeruginosa. Epidemiological findings, suggested that all herds were infected from teat wipes that were contaminated with this organism. Initial investigations using the polymerase chain reaction (PCR), indicated a possible clonal relationship between all outbreak linked strains, with one exception. This finding was confirmed following pulsed-field gel electrophoresis (PFGE) and ribotype analysis. PCR was again employed to study a rather unusual epidemic, involving Salmonella tel-el-kebir over a …

The Molecular Epidemiology Of Rotavirus In Ireland, Fiona O'Halloran

Theses

Between 1997 and 1998, 3,136 cases of rotavirus diarrhoea were detected in Irish children less than 2 years of age. Hospital inpatients accounted for 80% of these infections, with the remainder being diagnosed in general practice. A large percentage of infections were detected in neonates, suggesting a possible inadequate maternal derived protection from existing indigenous strains. This feature suggested the possible existence of ‘novel’ strains circulating in Ireland. No data describing the epidemiology of rotavirus strains in this country currently exists. Furthermore no assessment of the potential health-economic impact or quantitation of potential disease burden was ever undertaken

Three hundred …

The Atomic Force Microscope In The Elucidation Of Leukocyte Abnormality, Derbrenn O'Connor

Theses

The aim of this project was to investigate the novel use of the atomic force microscope (AFM) as a technique in the elucidation of leukocyte abnormality. To this end, comparative AFM imaging and elasticity studies were performed on normal and myeloid leukaemic leukocytes.

Initially, an evaluation of techniques for the isolation of specific leukocyte (e.g. lymphocytes, granulocytes, etc.) populations from whole blood or leukocyte concentrates, was carried out. It was found that a silanised glass surface failed to select and immobilise a leukocyte population. However, centrifugation of whole blood in an Isopaque-ficoll gradient solution yielded a viable mixed leukocyte population …

New Polyether Toxins From Shellfish And Marine Phytoplankton: Isolation Studies And Development Of Analytical Methods, Alan G. Bishop

Theses

There are three classes of polyether toxins responsible for Diarrhetic Shellfish Poisoning (DSP) world-wide. The causative toxins are okadaic acid (OA) and derivatives (DTXs), pectenotoxins (PTXs) and yessotoxins (YTXs). The origin of these toxins are phytoplankton from Dinophysis or Prorocentrum spp. OA has been demonstrated for most outbreaks in Europe and more recently a new isomer DTX-2 has been reported in Ireland.

Marine phytoplankton biomass samples consisting predominantly of Dinophysis acuta, were acquired using a large double plankton net from waters off the South-west coast of Ireland. Analysis of unialgal samples using high performance liquid chromatography (HPLC) with fluorescence and …

Extraction And Characterisation Of Essential Oils From Waste Eucalyptus Foliage, Hugh O'Reilly

Theses

Extraction procedures such as steam distillation, solvent and soxhlet extraction have been investigated to determine the most effective method of extracting essential oil from the foliage of a number of species of Eucalyptus cultivated in Co. Kerry. Steam distillation has been found to be the most effective method of extraction. The steam distillation extraction method has been thoroughly studied in order to determine the factors affecting yield and composition of the distillate. Alteration of the distillation time has been found to produce oil high in 1,8-cineole content, a requirement of medicinal quality oil. The choice of individual distillation still used …

Methicillin Resistant Staphylococcus Aureus (Mrsa): Molecular Detection And Dna Fingerprinting, Leslie Cotter

Theses

Throughout the 1990s, the incidence of methicillin-resistant Staphylococcus aureus has increased in many health care areas including acute and chronic care facilities, outpatient clinics and in the community. Once introduced into a health care environment, this nosocomial pathogen can spread rapidly and as MRSA are resistant to multiple antibiotics, treatment is often difficult. Therefore effective infection control measures are required to prevent cross-infection and further spread of endemic strains.

In this study, a sensitive and specific triplex-PCR assay was designed for MRSA detection, wherein three genes, the methicillin resistance gene (mecA). femA and the extracellular thermonuclease gene (rmc) were simultaneously …

Development Of An Atomic Force Microscopic Immunosensor For The Ferritin Antigen, Brendan O'Connell

Theses

The adsorption conditions for a range of different immunoreactants to both polystyrene and ultrasmooth silanised silicon surfaces are optimised. The conditions studied were concentration of protein, time and temperature of incubation, pH, molarity and ionic strength of the buffer. The anti-ferritin surface was selected as our model system. This immunosensor was used to develop and evaluate a millispot ELISA for ferritin. The millispot ferritin ELISA developed utilises a sample volume of 6pl and a sample incubation time of 10 minutes. Precision and accuracy studies show the assay to have a dynamic assay range of 7.8-lOOng/ml ferritin. The millispot ELISA detected …

Evaluation Of The Role Of Fructosamine As An Indicator Of Diabetic Control, Katherine Hooley

Theses

Diabetes mellitus, a common metabolic disease, is characterised by excess circulating glucose concentrations. Such a hyperglycaemic status provides the stimulus for irreversible glycation (non-enzymatically post-translational) of structural and functional proteins. Protein glycation, although a cause of complication (neuropathy, micro/macrovascular disease), provides a time-averaged index of diabetes status and control. In particular, glycated plasma proteins analysis, i.e., the fructosamine assay, has received favourable response due to its speed, simplicity, low cost, and ease of automation. However, lack of standardisation and a universally-accepted calibrant has limited technical and subsequent clinical confidence in fructosamine analysis. In response, a comprehensive technical evaluation (including novel …

Development Of A Differential Assay To Selectively Measure Α-Amylase Isoenzymes., Miriam O'Connor

Theses

α-Amylase is a heterogeneous polypeptide, primarily found in the Salivary and Pancreatic glands, giving rise to two isoenzymes, namely Salivary- and Pancreatic-type Amylases. Total α-Amylase activity measurements are routinely carried out in hospital laboratories to aid in the diagnosis of diseases of the pancreas and in the investigation of pancreatic function in patients with severe abdominal pains. With the development and wide availability of modem chromogenic oligosaccharide substrates specific for Amylase, assays have become more sensitive, rapid and easier to use.

Differential Amylase Isoenzyme assays ware developed to distinguish isoenzyme levels. Several methods were developed for isoenzyme determination. Of these. …

Development And Validation Of An Improved Ultrasensitive Assay For Cardiac Troponin I (Ctni), William G. Blake

Theses

Cardiac troponin I (cTnl) has been reported to be a highly specific marker for myocardial injury. In patients suffering from acute coronary syndromes, a sensitive serum marker which is closely related to the extent of myocardial damage which provides prognostic data and which can be measured rapidly is desirable. In this thesis a “sandwich” enzyme immunoassay was developed to measure cTnI. The developed assay was reproducible with the inter assay precision ranging from 4.8-14% and the intra assay precision ranging from 3.1-9.5%. The limit of detection of the assay was calculated as 0.385 ng/ml. The upper reference range (non-parametric 95% …

Development And Validation Of Flow-Injection (Continuous-Flow) Elisa Techniques., Declan Gerard Spillane

Theses

A flow-through enzyme-linked immunosorbent assay was developed based on affinity chromatography using the determination of ferritin in serum as a model system. In this method, samples and standards are introduced to separate columns containing immobilised anti-ferritin antibody, and antigen bound by solid-phase antibody is subsequently detected using an anti-ferritin-alkaline phosphatase conjugate. To detect immobilised label, p-nitrophenyl phosphate is added and product is developed in the column at room temperature. Following elution of product from the column, the absorbance is measured and the columns are regenerated using a low pH elution. The final developed system requires approximately 1.5 h for the …

Molecular Analysis Of The Factor V-Leiden Mutation In A Cardiac Transplant Patient Population, Caroline Maher

Theses

Venous thrombosis is a serious health problem affecting 1 in 1000 individuals annually. Until recently the pathogenic factors underlying thrombosis were associated with genetic defects in protein C, protein S and antithrombin III. However these were recognised in fewer than 5-10% of thrombotic patients. A breakthrough was made with the discovery of activated protein C resistance (APCr) which is associated in 90% of cases v/herein a G-->A transition in the factor V gene results in an abnormal molecule, Factor V Leiden. APCr is a major risk factor for thrombosis being present in 20-60% of thrombotic patients. Recently a G-^A …