Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0–100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.

Thunderstorm climatology and lightning location applications in northern Europe

Thunderstorm is a dangerous electrical phenomena in the atmosphere. Thundercloud is formed when thermal energy is transported rapidly upwards in convective updraughts. Electrification occurs in the collisions of cloud particles in the strong updraught. When the amount of charge in the cloud is large enough, electrical breakdown, better known as a flash, occurs. Lightning location is nowadays an essential tool for the detection of severe weather. Located flashes indicate in real time the movement of hazardous areas and the intensity of lightning activity. Also, an estimate for the flash peak current can be determined. The observations can be used in damage surveys. The most simple way to represent lightning data is to plot the locations on a map, but the data can be processed in more complex end-products and exploited in data fusion. Lightning data serves as an important tool also in the research of lightning-related phenomena, such as Transient Luminous Events. Most of the global thunderstorms occur in areas with plenty of heat, moisture and tropospheric instability, for example in the tropical land areas. In higher latitudes like in Finland, the thunderstorm season is practically restricted to the summer season. Particular feature of the high-latitude climatology is the large annual variation, which regards also thunderstorms. Knowing the performance of any measuring device is important because it affects the accuracy of the end-products. In lightning location systems, the detection efficiency means the ratio between located and actually occurred flashes. Because in practice it is impossible to know the true number of actually occurred flashes, the detection efficiency has to be esimated with theoretical methods.

Rapsista tuotetun biodieselin ja vehnästä tuotetun bioetanolin energiavirrat ja ympäristövaikutukset Euroopan unionissa

The aim of this thesis was to study the crops currently used for biofuel production from the following aspects: 1. what should be the average yield/ ha to reach an energy balance at least 0 or positive 2. what are the shares of the primary and secondary energy flows in agriculture, transport, processing and usage, and 3. overall effects of biofuel crop cultivation, transport, processing and usage. This thesis concentrated on oilseed rape biodiesel and wheat bioethanol in the European Union, comparing them with competing biofuels, such as corn and sugarcane-based ethanol, and the second generation biofuels. The study was executed by comparing Life Cycle Assessment-studies from the EU-region and by analyzing them thoroughly from the differences viewpoint. The variables were the following: energy ratio, hectare yield (l/ha), impact on greenhouse gas emissions (particularly CO2), energy consumption in crop growing and processing one hectare of a particular crop to biofuel, distribution of energy in processing and effects of the secondary energy flows, like e.g. wheat straw. Processing was found to be the most energy consuming part in the production of biofuels. So if the raw materials will remain the same, the development will happen in processing. First generation biodiesel requires esterification, which consumes approximately one third of the process energy. Around 75% of the energy consumed in manufacturing the first generation wheat-based ethanol is spent in steam and electricity generation. No breakthroughs are in sight in the agricultural sector to achieve significantly higher energy ratios. It was found out that even in ideal conditions the energy ratio of first generation wheat-based ethanol will remain slightly under 2. For oilseed rape-based biodiesel the energy ratios are better, and energy consumption per hectare is lower compared to wheat-based ethanol. But both of these are lower compared to e.g. sugarcane-based ethanol. Also the hectare yield of wheat-based ethanol is significantly lower. Biofuels are in a key position when considering the future of the world’s transport sector. Uncertainties concerning biofuels are, however, several, like the schedule of large scale introduction to consumer markets, technologies used, raw materials and their availability and - maybe the biggest - the real production capacity in relation to the fuel consumption. First generation biofuels have not been the expected answer to environmental problems. Comparisons made show that sugarcane-based ethanol is the most prominent first generation biofuel at the moment, both from energy and environment point of view. Also palmoil-based biodiesel looks promising, although it involves environmental concerns as well. From this point of view the biofuels in this study - wheat-based ethanol and oilseed rape-based biodiesel - are not very competitive options. On the other hand, crops currently used for fuel production in different countries are selected based on several factors, not only based on thier relative general superiority. It is challenging to make long-term forecasts for the biofuel sector, but it can be said that satisfying the world's current and near future traffic fuel consumption with biofuels can only be regarded impossible. This does not mean that biofuels shoud be rejected and their positive aspects ignored, but maybe this reality helps us to put them in perspective. To achieve true environmental benefits through the usage of biofuels there must first be a significant drop both in traffic volumes and overall fuel consumption. Second generation biofuels are coming, but serious questions about their availability and production capacities remain open. Therefore nothing can be taken for granted in this issue, expect the need for development.