Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Epithelial sodium channel in airway epithelium of the newborn infant : Implications for postnatal pulmonary adaptation

The aim of the present thesis was to study the role of the epithelial sodium channel (ENaC) in clearance of fetal lung fluid in the newborn infant by measurement of airway epithelial expression of ENaC, of nasal transepithelial potential difference (N-PD), and of lung compliance (LC). In addition, the effect of postnatal dexamethasone on airway epithelial ENaC expression was measured in preterm infants with bronchopulmonary dysplasia (BPD). The patient population was formed of selected term newborn infants born in the Department of Obstetrics (Studies II-IV) and selected preterm newborn infants treated in the neonatal intensive care unit of the Hospital for Children and Adolescents (Studies I and IV) of the Helsinki University Central Hospital in Finland. A small population of preterm infants suffering from BPD was included in Study I. Studies I, III, and IV included airway epithelial measurement of ENaC and in Studies II and III, measurement of N-PD and LC. In Study I, ENaC expression analyses were performed in the Research Institute of the Hospital for Sick Children in Toronto, Ontario, Canada. In the following studies, analyses were performed in the Scientific Laboratory of the Hospital for Children and Adolescents. N-PD and LC measurements were performed at bedside in these hospitals. In term newborn infants, the percentage of amiloride-sensitive N-PD, a surrogate for ENaC activity, measured during the first 4 postnatal hours correlates positively with LC measured 1 to 2 days postnatally. Preterm infants with BPD had, after a therapeutic dose of dexamethasone, higher airway epithelial ENaC expression than before treatment. These patients were subsequently weaned from mechanical ventilation, probably as a result of the clearance of extra fluid from the alveolar spaces. In addition, we found that in preterm infants ENaC expression increases with gestational age (GA). In preterm infants, ENaC expression in the airway epithelium was lower than in term newborn infants. During the early postnatal period in those born both preterm and term airway epithelial βENaC expression decreased significantly. Term newborn infants delivered vaginally had a significantly smaller airway epithelial expression of αENaC after the first postnatal day than did those delivered by cesarean section. The functional studies showed no difference in N-PD between infants delivered vaginally and by cesarean section. We therefore conclude that the low airway epithelial expression of ENaC in the preterm infant and the correlation of N-PD with LC in the term infant indicate a role for ENaC in the pathogenesis of perinatal pulmonary adaptation and neonatal respiratory distress. Because dexamethasone raised ENaC expression in preterm infants with BPD, and infants were subsequently weaned from ventilator therapy, we suggest that studies on the treatment of respiratory distress in the preterm infant should include the induction of ENaC activity.

Questions to Artificial Nature: A Philosophical Study of Interdisciplinary Models and their Functions in Scientific Practice

This thesis presents an interdisciplinary analysis of how models and simulations function in the production of scientific knowledge. The work is informed by three scholarly traditions: studies on models and simulations in philosophy of science, so-called micro-sociological laboratory studies within science and technology studies, and cultural-historical activity theory. Methodologically, I adopt a naturalist epistemology and combine philosophical analysis with a qualitative, empirical case study of infectious-disease modelling. This study has a dual perspective throughout the analysis: it specifies the modelling practices and examines the models as objects of research. The research questions addressed in this study are: 1) How are models constructed and what functions do they have in the production of scientific knowledge? 2) What is interdisciplinarity in model construction? 3) How do models become a general research tool and why is this process problematic? The core argument is that the mediating models as investigative instruments (cf. Morgan and Morrison 1999) take questions as a starting point, and hence their construction is intentionally guided. This argument applies the interrogative model of inquiry (e.g., Sintonen 2005; Hintikka 1981), which conceives of all knowledge acquisition as process of seeking answers to questions. The first question addresses simulation models as Artificial Nature, which is manipulated in order to answer questions that initiated the model building. This account develops further the "epistemology of simulation" (cf. Winsberg 2003) by showing the interrelatedness of researchers and their objects in the process of modelling. The second question clarifies why interdisciplinary research collaboration is demanding and difficult to maintain. The nature of the impediments to disciplinary interaction are examined by introducing the idea of object-oriented interdisciplinarity, which provides an analytical framework to study the changes in the degree of interdisciplinarity, the tools and research practices developed to support the collaboration, and the mode of collaboration in relation to the historically mutable object of research. As my interest is in the models as interdisciplinary objects, the third research problem seeks to answer my question of how we might characterise these objects, what is typical for them, and what kind of changes happen in the process of modelling. Here I examine the tension between specified, question-oriented models and more general models, and suggest that the specified models form a group of their own. I call these Tailor-made models, in opposition to the process of building a simulation platform that aims at generalisability and utility for health-policy. This tension also underlines the challenge of applying research results (or methods and tools) to discuss and solve problems in decision-making processes.

High-quality polar UV measurements : scientific analyses and transfer of the irradiance scale

The Earth's ecosystems are protected from the dangerous part of the solar ultraviolet (UV) radiation by stratospheric ozone, which absorbs most of the harmful UV wavelengths. Severe depletion of stratospheric ozone has been observed in the Antarctic region, and to a lesser extent in the Arctic and midlatitudes. Concern about the effects of increasing UV radiation on human beings and the natural environment has led to ground based monitoring of UV radiation. In order to achieve high-quality UV time series for scientific analyses, proper quality control (QC) and quality assurance (QA) procedures have to be followed. In this work, practices of QC and QA are developed for Brewer spectroradiometers and NILU-UV multifilter radiometers, which measure in the Arctic and Antarctic regions, respectively. These practices are applicable to other UV instruments as well. The spectral features and the effect of different factors affecting UV radiation were studied for the spectral UV time series at Sodankylä. The QA of the Finnish Meteorological Institute's (FMI) two Brewer spectroradiometers included daily maintenance, laboratory characterizations, the calculation of long-term spectral responsivity, data processing and quality assessment. New methods for the cosine correction, the temperature correction and the calculation of long-term changes of spectral responsivity were developed. Reconstructed UV irradiances were used as a QA tool for spectroradiometer data. The actual cosine correction factor was found to vary between 1.08-1.12 and 1.08-1.13. The temperature characterization showed a linear temperature dependence between the instrument's internal temperature and the photon counts per cycle. Both Brewers have participated in international spectroradiometer comparisons and have shown good stability. The differences between the Brewers and the portable reference spectroradiometer QASUME have been within 5% during 2002-2010. The features of the spectral UV radiation time series at Sodankylä were analysed for the time period 1990-2001. No statistically significant long-term changes in UV irradiances were found, and the results were strongly dependent on the time period studied. Ozone was the dominant factor affecting UV radiation during the springtime, whereas clouds played a more important role during the summertime. During this work, the Antarctic NILU-UV multifilter radiometer network was established by the Instituto Nacional de Meteorogía (INM) as a joint Spanish-Argentinian-Finnish cooperation project. As part of this work, the QC/QA practices of the network were developed. They included training of the operators, daily maintenance, regular lamp tests and solar comparisons with the travelling reference instrument. Drifts of up to 35% in the sensitivity of the channels of the NILU-UV multifilter radiometers were found during the first four years of operation. This work emphasized the importance of proper QC/QA, including regular lamp tests, for the multifilter radiometers also. The effect of the drifts were corrected by a method scaling the site NILU-UV channels to those of the travelling reference NILU-UV. After correction, the mean ratios of erythemally-weighted UV dose rates measured during solar comparisons between the reference NILU-UV and the site NILU-UVs were 1.007±0.011 and 1.012±0.012 for Ushuaia and Marambio, respectively, when the solar zenith angle varied up to 80°. Solar comparisons between the NILU-UVs and spectroradiometers showed a ±5% difference near local noon time, which can be seen as proof of successful QC/QA procedures and transfer of irradiance scales. This work also showed that UV measurements made in the Arctic and Antarctic can be comparable with each other.