Probiotic activity of Lactobacillus delbrueckii subsp. bulgaricus in the oral cavity

Yogurt consumption has been related to longevity of some populations living on the Balkans. Yogurt starter L. delbrueckii subsp. bulgaricus and Str. thermophilus have been recognized as probiotics with verified beneficial health effects. The oral cavity emerges as a arget for probiotic applications. Probiotics have demonstrated promising results in controlling dental diseases and oral yeast infections. However, L. bulgaricus despite its broad availability in dairy products has not been evaluated for probiotic activity in the mouth. These series of studies investigated in vitro properties of L. bulgaricus to outline its potential as an oral probiotic. Prerequisite probiotic properties in the mouth are resistance to oral defense mechanisms, adherence to saliva-coated surfaces, and inhibition of oral pathogens. L. bulgaricus strains showed a strain-dependent inhibition of oral streptococci and Aggregatibacter actinomycetemcomitans, whereas none of the dairy starter strains could affect growth of Porphyromonas gingivalis and Fusobacterium nucleatum. Adhesion is a factor contributing to colonization of the species at the target site. Radiolabeled L. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated surfaces. The effects of lysozyme on adhesion and adhesion of Streptococcus sanguinis after lactobacilli pretreatment were also assessed. Adhesion of L. bulgaricus remained lower in comparison to L. rhamnosus GG. One L. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment significantly increased Lactobacillus adhesion. Low gelatinolytic activity was observed for all strains and no conversion of proMMP-9 to its active form was induced by L. bulgaricus. Safety assessment ruled out deleterious effects of L. bulgaricus on extracellular matrix structures. Cytokine response of oral epithelial cells was assessed by measuring IL-8 and TNF-α in cell culture supernatants. The effect of P. gingivalis on cytokine secretion after lactobacilli pretreatment was also assessed. A strain- and time-dependent induction of IL-8 was observed with live bacteria inducing the highest levels of cytokine secretion. Levels of TNF-α were low and only one of ten L. bulgaricus strains stimulated TNF-α secretion similar to positive control. The addition of P. gingivalis produced immediate reduction of cytokine levels within the first hours of incubation irrespective of lactobacilli strains co-cultured with epithelial cells. According to these studies strains among the L. delbrueckii subsp. bulgaricus species may have beneficial probiotic properties in the mouth. Their potential in prevention and management of common oral infectious diseases needs to be further studied.

Construction of a global map of human gene expression : the process, tools and analysis

This thesis studies human gene expression space using high throughput gene expression data from DNA microarrays. In molecular biology, high throughput techniques allow numerical measurements of expression of tens of thousands of genes simultaneously. In a single study, this data is traditionally obtained from a limited number of sample types with a small number of replicates. For organism-wide analysis, this data has been largely unavailable and the global structure of human transcriptome has remained unknown. This thesis introduces a human transcriptome map of different biological entities and analysis of its general structure. The map is constructed from gene expression data from the two largest public microarray data repositories, GEO and ArrayExpress. The creation of this map contributed to the development of ArrayExpress by identifying and retrofitting the previously unusable and missing data and by improving the access to its data. It also contributed to creation of several new tools for microarray data manipulation and establishment of data exchange between GEO and ArrayExpress. The data integration for the global map required creation of a new large ontology of human cell types, disease states, organism parts and cell lines. The ontology was used in a new text mining and decision tree based method for automatic conversion of human readable free text microarray data annotations into categorised format. The data comparability and minimisation of the systematic measurement errors that are characteristic to each lab- oratory in this large cross-laboratories integrated dataset, was ensured by computation of a range of microarray data quality metrics and exclusion of incomparable data. The structure of a global map of human gene expression was then explored by principal component analysis and hierarchical clustering using heuristics and help from another purpose built sample ontology. A preface and motivation to the construction and analysis of a global map of human gene expression is given by analysis of two microarray datasets of human malignant melanoma. The analysis of these sets incorporate indirect comparison of statistical methods for finding differentially expressed genes and point to the need to study gene expression on a global level.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

The range expansion of the European map butterfly in Finland

Climate change will influence the living conditions of all life on Earth. For some species the change in the environmental conditions that has occurred so far has already increased the risk of extinction, and the extinction risk is predicted to increase for large numbers of species in the future. Some species may have time to adapt to the changing environmental conditions, but the rate and magnitude of the change are too great to allow many species to survive via evolutionary changes. Species responses to climate change have been documented for some decades. Some groups of species, like many insects, respond readily to changes in temperature conditions and have shifted their distributions northwards to new climatically suitable regions. Such range shifts have been well documented especially in temperate zones. In this context, butterflies have been studied more than any other group of species, partly for the reason that their past geographical ranges are well documented, which facilitates species-climate modelling and other analyses. The aim of the modelling studies is to examine to what extent shifts in species distributions can be explained by climatic and other factors. Models can also be used to predict the future distributions of species. In this thesis, I have studied the response to climate change of one species of butterfly within one geographically restricted area. The study species, the European map butterfly (Araschnia levana), has expanded rapidly northwards in Finland during the last two decades. I used statistical and dynamic modelling approaches in combination with field studies to analyse the effects of climate warming and landscape structure on the expansion. I studied possible role of molecular variation in phosphoglucose isomerase (PGI), a glycolytic enzyme affecting flight metabolism and thereby flight performance, in the observed expansion of the map butterfly at two separate expansion fronts in Finland. The expansion rate of the map butterfly was shown to be correlated with the frequency of warmer than average summers during the study period. The result is in line with the greater probability of occurrence of the second generation during warm summers and previous results on this species showing greater mobility of the second than first generation individuals. The results of a field study in this thesis indicated low mobility of the first generation butterflies. Climatic variables alone were not sufficient to explain the observed expansion in Finland. There are also problems in transferring the climate model to new regions from the ones from which data were available to construct the model. The climate model predicted a wider distribution in the south-western part of Finland than what has been observed. Dynamic modelling of the expansion in response to landscape structure suggested that habitat and landscape structure influence the rate of expansion. In southern Finland the landscape structure may have slowed down the expansion rate. The results on PGI suggested that allelic variation in this enzyme may influence flight performance and thereby the rate of expansion. Genetic differences of the populations at the two expansion fronts may explain at least partly the observed differences in the rate of expansion. Individuals with the genotype associated with high flight metabolic rate were most frequent in eastern Finland, where the rate of range expansion has been highest.

Effect of GPS on understanding relationship between mobile map and environment : A field experiment

Objectives: GPS technology enables the visualisation of a map reader s location on a mobile map. Earlier research on the cognitive aspects of map reading identified that searching for map-environment points is an essential element for the process of determining one s location on a mobile map. Map-environment points refer to objects that are visualized on the map and are recognizable in the environment. However, because the GPS usually adds only one point to the map that has a relation to the environment, it does not provide a sufficient amount of information for self-location. The aim of the present thesis was to assess the effect of GPS on the cognitive processes involved in determining one s location on a map. Methods: The effect of GPS on self-location was studied in a field experiment. The subjects were shown a target on a mobile map, and they were asked to point in the direction of the target. In order for the map reader to be able to deduce the direction of the target, he/she has to locate himself/herself on the map. During the pointing tasks, the subjects were asked to think aloud. The data from the experiment were used to analyze the effect of the GPS on the time needed to perform the task. The subjects verbal data was used to assess the effect of the GPS on the number of landmark concepts mentioned during a task (landmark concepts are words referring to objects that can be recognized both on the map and in the environment). Results and conclusions: The results from the experiment indicate that the GPS reduces the time needed to locate oneself on a map. The analysis of the verbal data revealed that the GPS reduces the number of landmark concepts in the protocols. The findings suggest that the GPS guides the subject s search for the map-environment points and narrows the area on the map that must be searched for self-location.