Protein cross-linking with oxidative enzymes and transglutaminase : Effects in meat protein systems

The effects of tyrosinase, laccase and transglutaminase (TG) were studied in different meat protein systems. The study was focused on the effects of the enzymes on the gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel Trichoderma reesei tyrosinase was compared to that of the commercial Agaricus bisporus tyrosinase. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule into a complex proteinaceous substrate. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but laccase was also found to cause protein fragmentation. A positive connection between covalent cross-link and gel formation was observed with tyrosinase and TG. Laccase was able to increase the gel formation only slightly. With an excessive laccase dosage the gel formation declined due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase was the only enzyme capable of reducing the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl and phosphate amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt. Increasining the amount of meat, salt and TG contents favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins, i.e. myosin and troponin T. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes.

Bioavailability aspects of hydrophobic contaminant degradation in soils

This thesis concentrates on bioavailability of organic soil contaminants in the context of bioremediation of soil contaminated with volatile or non-volatile hydrophobic pollutants. Bioavailability and biodegradation was studied from four viewpoints: (i) Improvement of bioavailability and biodegradation of volatile hydrocarbons in contained bioremediation systems at laboratory - and pilot-scale. (ii) Improvement of bioavailability of non-volatile, hydrophobic compounds in such systems. (iii) Biodegradation of a non-volatile hydrophobic compound in soil organic matter in microcosms. (iiii) Bioavailability of nitrogen in an open, full-scale bioremediation system. It was demonstrated that volatility of organic compounds can be controlled by amending the soil with adsorbents. The sorbed hydrocarbons were shown to be available to soil microbiota. As the result, biodegradation of the volatile hydrocarbons was greatly favored at the expense of volatilization. PAH compounds were shown to be mobilized and their bioavailability improved by a hydrophobic, non-toxic additive, vegetable oil. Bioavailability of the PAHs was recorded as an increased toxicity of the soil. In spite of the increased bioavailability, biodegradation of the PAHs decreased. In microcosms simulating boreal forest organic surface soil, PAH-compound (pyrene) was shown to be removed from soil biologically. Therefore hydrophobicity of the substrate does not necessarily mean low availability and biodegradation in organic soil. Finally, in this thesis it was demonstrated that an unsuitable source of nitrogen or its overdose resulted in wasteful spending of this nutrient and even harmful effects on soil microbes. Such events may inhibit rather than promote the bioremediation process in soil.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Light scattering in random media with wavelength-scale structures : astronomical and industrial applications

Light scattering, or scattering and absorption of electromagnetic waves, is an important tool in all remote-sensing observations. In astronomy, the light scattered or absorbed by a distant object can be the only source of information. In Solar-system studies, the light-scattering methods are employed when interpreting observations of atmosphereless bodies such as asteroids, atmospheres of planets, and cometary or interplanetary dust. Our Earth is constantly monitored from artificial satellites at different wavelengths. With remote sensing of Earth the light-scattering methods are not the only source of information: there is always the possibility to make in situ measurements. The satellite-based remote sensing is, however, superior in the sense of speed and coverage if only the scattered signal can be reliably interpreted. The optical properties of many industrial products play a key role in their quality. Especially for products such as paint and paper, the ability to obscure the background and to reflect light is of utmost importance. High-grade papers are evaluated based on their brightness, opacity, color, and gloss. In product development, there is a need for computer-based simulation methods that could predict the optical properties and, therefore, could be used in optimizing the quality while reducing the material costs. With paper, for instance, pilot experiments with an actual paper machine can be very time- and resource-consuming. The light-scattering methods presented in this thesis solve rigorously the interaction of light and material with wavelength-scale structures. These methods are computationally demanding, thus the speed and accuracy of the methods play a key role. Different implementations of the discrete-dipole approximation are compared in the thesis and the results provide practical guidelines in choosing a suitable code. In addition, a novel method is presented for the numerical computations of orientation-averaged light-scattering properties of a particle, and the method is compared against existing techniques. Simulation of light scattering for various targets and the possible problems arising from the finite size of the model target are discussed in the thesis. Scattering by single particles and small clusters is considered, as well as scattering in particulate media, and scattering in continuous media with porosity or surface roughness. Various techniques for modeling the scattering media are presented and the results are applied to optimizing the structure of paper. However, the same methods can be applied in light-scattering studies of Solar-system regoliths or cometary dust, or in any remote-sensing problem involving light scattering in random media with wavelength-scale structures.

Inflammation and tissue destruction in arthritides and periodontal disease

The principal aim of this study was to examine diseases characterized by inflammatory injury, especially human arthritides and periodontitis, with specific interest to final effector enzymes of tissue destruction and address the possible future tools to prevent permanent tissue loss. We used biochemical and immunological methods applied to synovial tissue samples, samples of synovial fluid, and samples of peripheral blood. In Study IV, we used established clinical inflammatory injury indicator probing pocket depth and used it to derive a new clinical measure of systemic burden, periodontal inflammatory burden index. In study I, we showed a difference in the effector enzymes of peripheral blood leukocytes and leukocytes from inflamed synovial fluid of rheumatoid arthritis and reactive arthritis patients. The effector enzyme activities were higher in synovial fluid than in peripheral blood. In study II, we showed the presence of collagenase-3 in rheumatoid synovial tissue samples, relative resistance of the enzyme to inhibition in vitro and developed an electrophoretic method for detection of collagenase-3 in presence of collagenase-1. In study III, we carried out an open label study of doxycycline treatment of 12 RA patients. During the treatment period, we observed an improvement in several of the biochemical and psychosocial variables used to assess the status of the patients. In study IV, we showed a clearly lower level of periodontal inflammatory injury in chronic periodontitis patients referred for periodontal treatment. In this cross-sectional pilot study, we showed lower levels of inflammatory injury in periodontitis patients using statin than in those not receiving statin treatment. The difference was of same magnitude in patients using simvastatin or atorvastatin. The weighted index of inflammatory burden, PIBI, which emphasizes the burden imposed by the deepest pathological pockets on the system showed values consistent with a wider scale to ease future studies on the inflammatory burden associated with periodontitis.

The structure of Norway spruce (Picea abies [L.] Karst.) stems in relation to wood properties of sawn timber

An important challenge in forest industry is to get the appropriate raw material out from the forests to the wood processing industry. Growth and stem reconstruction simulators are therefore increasingly integrated in industrial conversion simulators, for linking the properties of wooden products to the three-dimensional structure of stems and their growing conditions. Static simulators predict the wood properties from stem dimensions at the end of a growth simulation period, whereas in dynamic approaches, the structural components, e.g. branches, are incremented along with the growth processes. The dynamic approach can be applied to stem reconstruction by predicting the three-dimensional stem structure from external tree variables (i.e. age, height) as a result of growth to the current state. In this study, a dynamic growth simulator, PipeQual, and a stem reconstruction simulator, RetroSTEM, are adapted to Norway spruce (Picea abies [L.] Karst.) to predict the three-dimensional structure of stems (tapers, branchiness, wood basic density) over time such that both simulators can be integrated in a sawing simulator. The parameterisation of the PipeQual and RetroSTEM simulators for Norway spruce relied on the theoretically based description of tree structure developing in the growth process and following certain conservative structural regularities while allowing for plasticity in the crown development. The crown expressed both regularity and plasticity in its development, as the vertical foliage density peaked regularly at about 5 m from the stem apex, varying below that with tree age and dominance position (Study I). Conservative stem structure was characterized in terms of (1) the pipe ratios between foliage mass and branch and stem cross-sectional areas at crown base, (2) the allometric relationship between foliage mass and crown length, (3) mean branch length relative to crown length and (4) form coefficients in branches and stem (Study II). The pipe ratio between branch and stem cross-sectional area at crown base, and mean branch length relative to the crown length may differ in trees before and after canopy closure, but the variation should be further analysed in stands of different ages and densities with varying site fertilities and climates. The predictions of the PipeQual and RetroSTEM simulators were evaluated by comparing the simulated values to measured ones (Study III, IV). Both simulators predicted stem taper and branch diameter at the individual tree level with a small bias. RetroSTEM predictions of wood density were accurate. For focusing on even more accurate predictions of stem diameters and branchiness along the stem, both simulators should be further improved by revising the following aspects in the simulators: the relationship between foliage and stem sapwood area in the upper stem, the error source in branch sizes, the crown base development and the height growth models in RetroSTEM. In Study V, the RetroSTEM simulator was integrated in the InnoSIM sawing simulator, and according to the pilot simulations, this turned out to be an efficient tool for readily producing stand scale information about stem sizes and structure when approximating the available assortments of wood products.

Management of multi-scale forest resource data over time

During the last decades there has been a global shift in forest management from a focus solely on timber management to ecosystem management that endorses all aspects of forest functions: ecological, economic and social. This has resulted in a shift in paradigm from sustained yield to sustained diversity of values, goods and benefits obtained at the same time, introducing new temporal and spatial scales into forest resource management. The purpose of the present dissertation was to develop methods that would enable spatial and temporal scales to be introduced into the storage, processing, access and utilization of forest resource data. The methods developed are based on a conceptual view of a forest as a hierarchically nested collection of objects that can have a dynamically changing set of attributes. The temporal aspect of the methods consists of lifetime management for the objects and their attributes and of a temporal succession linking the objects together. Development of the forest resource data processing method concentrated on the extensibility and configurability of the data content and model calculations, allowing for a diverse set of processing operations to be executed using the same framework. The contribution of this dissertation to the utilisation of multi-scale forest resource data lies in the development of a reference data generation method to support forest inventory methods in approaching single-tree resolution.

Landscape-scale soil erosion modelling and ecological restoration for a mountainous watershed in Sichuan, China

Sichuanissa Tiibetin ylängön metsäkato on pysähtynyt mutta eroosio-ongelmat jatkuvat Viikin tropiikki-instituutin tutkija Ping ZHOU kartoitti trooppisen metsänhoidon alaan kuuluvassa väitöskirjatyössään maaperän eroosioalttiutta ja sen riippuvuutta metsäkasvillisuudesta Jangtsen tärkeää sivuhaaraa Min-jokea ympäröivällä n. 7400 neliökilometrin suuruisella valuma-alueella Sichuanin Aba-piirikunnassa. Aineistonaan hän käytti muun muassa satelliittikartoitustietoja ja mittaustuloksia yli 600 maastokoealalta. Tutkimuksen nimi suomeksi on "Maaperän eroosion mallinnus ja vuoristoisen valuma-alueen ekologinen ennallistaminen Sichuanissa Kiinassa". Aikaisempien tutkimusten perusteella oli tiedossa että metsien häviäminen tällä alueella pysähtyi jo 1980-luvun alussa. Sen jälkeen on metsien pinta-ala hitaasti kasvanut etupäässä sen vuoksi, että teollinen puunhakkuu luonnonmetsissä kiellettiin kokonaan v. 1998 ja 25 astetta jyrkemmillä rinteillä myös maatalouden harjoittaminen on saatu lopetetuksi viljelijöille tarjottujen taloudellisten houkuttimien avulla. Täten myös pelto- ja laidunmaata on voitu ennallistaa metsäksi. Ping Zhou pystyi jakamaan 5700 metrin korkeuteen saakka kohoavan vuoristoalueen eroosioalttiudeltaan erilaisiin vyöhykkeisiin rinteen kaltevuuden, sademäärän, kasvipeitteen ja maalajin perusteella. Noin 15 prosentilla tutkitun valuma-alueen pinta-alasta, lähinnä Min-joen pääuomaa ympäröivillä jyrkillä rinteillä, eroosioriski oli suuri tai erittäin suuri. Eri tyyppisellä kasvillisuudella oli hyvin erilainen vaikutus eroosioalttiuteen, ja myös alueen sijainti vuoriston eri korkeuksilla vaikutti eroosioon. Säästyneet lähes luonnontilaiset havumetsät, joita on etupäässä vuoriston ylimmissä osissa 2600-4000 metrin korkeudella, edistävät tehokkaasti metsän luontaista uudistumista ja levittäytymistä vaurioituneille alueille. Säilyneiden metsien puulajikoostumus antoi tutkimuksessa mahdollisuuden ennustaa metsien tulevaa kehitystä koko tutkitulla valuma-alueella sen eri korkeusvyöhykkeissä ja eri maaperätyypeillä. Ennallistamisen kannalta ongelmallisimpia olivat alueet joilta metsäpeite oli lähinnä puiden teollisen hakkuun vuoksi kokonaan hävinnyt ja joilla maaperä yleisesti oli eroosion pahoin kuluttama. Näillä alueilla ei ole tehty juuri mitään uudistamis- tai ennallistamistoimenpiteitä. Niillä metsien ennallistaminen vaatii myös puiden tai pensaiden istuttamista. Tähän sopivia ovat erityisesti ilmakehän typpeä sitovat lajit, joista alueella kasvaa luontaisena mm. sama tyrnilaji joka esiintyy myös Suomessa. Työssä tutkittiin yli kahdeksankymmenen paikallisen luontaisen puulajin (joista peräti noin kolmannes on havupuulajeja) ekologisia ominaisuuksia ja soveltuvuutta metsien ennallistamiseen. Avainasemassa työn onnistumisen kannalta ovat nyt paikalliset asukkaat, joiden maankäytön muutokset ovat jo selvästi edistänet luonnonmetsän ennalleen palautumista. Suomen Akatemia rahoitti vuosina 2004-2006 VITRI:n tutkimushanketta, josta Ping Zhou'n väitöskirjatyö muodosti keskeisen osan. Kenttätyö Sichuanissa avasi mahdollisuuden hedelmälliseen monitieteiseen yhteistyöhön ja tutkijavaihtoon Kiinan tiedeakatemian alaisen Chengdun biologiainstituutin (CIB) kanssa; tämä tieteellinen kanssakäyminen jatkuu edelleen.

Plant species diversity of buffer zones in agricultural landscapes: in search of determinants from the local to regional scale

Buffer zones are vegetated strip-edges of agricultural fields along watercourses. As linear habitats in agricultural ecosystems, buffer strips dominate and play a leading ecological role in many areas. This thesis focuses on the plant species diversity of the buffer zones in a Finnish agricultural landscape. The main objective of the present study is to identify the determinants of floral species diversity in arable buffer zones from local to regional levels. This study was conducted in a watershed area of a farmland landscape of southern Finland. The study area, Lepsämänjoki, is situated in the Nurmijärvi commune 30 km to the north of Helsinki, Finland. The biotope mosaics were mapped in GIS. A total of 59 buffer zones were surveyed, of which 29 buffer strips surveyed were also sampled by plot. Firstly, two diversity components (species richness and evenness) were investigated to determine whether the relationship between the two is equal and predictable. I found no correlation between species richness and evenness. The relationship between richness and evenness is unpredictable in a small-scale human-shaped ecosystem. Ordination and correlation analyses show that richness and evenness may result from different ecological processes, and thus should be considered separately. Species richness correlated negatively with phosphorus content, and species evenness correlated negatively with the ratio of organic carbon to total nitrogen in soil. The lack of a consistent pattern in the relationship between these two components may be due to site-specific variation in resource utilization by plant species. Within-habitat configuration (width, length, and area) were investigated to determine which is more effective for predicting species richness. More species per unit area increment could be obtained from widening the buffer strip than from lengthening it. The width of the strips is an effective determinant of plant species richness. The increase in species diversity with an increase in the width of buffer strips may be due to cross-sectional habitat gradients within the linear patches. This result can serve as a reference for policy makers, and has application value in agricultural management. In the framework of metacommunity theory, I found that both mass effect(connectivity) and species sorting (resource heterogeneity) were likely to explain species composition and diversity on a local and regional scale. The local and regional processes were interactively dominated by the degree to which dispersal perturbs local communities. In the lowly and intermediately connected regions, species sorting was of primary importance to explain species diversity, while the mass effect surpassed species sorting in the highly connected region. Increasing connectivity in communities containing high habitat heterogeneity can lead to the homogenization of local communities, and consequently, to lower regional diversity, while local species richness was unrelated to the habitat connectivity. Of all species found, Anthriscus sylvestris, Phalaris arundinacea, and Phleum pretense significantly responded to connectivity, and showed high abundance in the highly connected region. We suggest that these species may play a role in switching the force from local resources to regional connectivity shaping the community structure. On the landscape context level, the different responses of local species richness and evenness to landscape context were investigated. Seven landscape structural parameters served to indicate landscape context on five scales. On all scales but the smallest scales, the Shannon-Wiener diversity of land covers (H') correlated positively with the local richness. The factor (H') showed the highest correlation coefficients in species richness on the second largest scale. The edge density of arable field was the only predictor that correlated with species evenness on all scales, which showed the highest predictive power on the second smallest scale. The different predictive power of the factors on different scales showed a scaledependent relationship between the landscape context and local plant species diversity, and indicated that different ecological processes determine species richness and evenness. The local richness of species depends on a regional process on large scales, which may relate to the regional species pool, while species evenness depends on a fine- or coarse-grained farming system, which may relate to the patch quality of the habitats of field edges near the buffer strips. My results suggested some guidelines of species diversity conservation in the agricultural ecosystem. To maintain a high level of species diversity in the strips, a high level of phosphorus in strip soil should be avoided. Widening the strips is the most effective mean to improve species richness. Habitat connectivity is not always favorable to species diversity because increasing connectivity in communities containing high habitat heterogeneity can lead to the homogenization of local communities (beta diversity) and, consequently, to lower regional diversity. Overall, a synthesis of local and regional factors emerged as the model that best explain variations in plant species diversity. The studies also suggest that the effects of determinants on species diversity have a complex relationship with scale.

Forest inventory-based large-scale forest biomass and carbon budget assessment: new enhanced methods and use of remote sensing for verification

In recent years, concern has arisen over the effects of increasing carbon dioxide (CO2) in the earth's atmosphere due to the burning of fossil fuels. One way to mitigate increase in atmospheric CO2 concentration and climate change is carbon sequestration to forest vegeta-tion through photosynthesis. Comparable regional scale estimates for the carbon balance of forests are therefore needed for scientific and political purposes. The aim of the present dissertation was to improve methods for quantifying and verifying inventory-based carbon pool estimates of the boreal forests in the mineral soils. Ongoing forest inventories provide a data based on statistically sounded sampling for estimating the level of carbon stocks and stock changes, but improved modelling tools and comparison of methods are still needed. In this dissertation, the entire inventory-based large-scale forest carbon stock assessment method was presented together with some separate methods for enhancing and comparing it. The enhancement methods presented here include ways to quantify the biomass of understorey vegetation as well as to estimate the litter production of needles and branches. In addition, the optical remote sensing method illustrated in this dis-sertation can be used to compare with independent data. The forest inventory-based large-scale carbon stock assessment method demonstrated here provided reliable carbon estimates when compared with independent data. Future ac-tivity to improve the accuracy of this method could consist of reducing the uncertainties regarding belowground biomass and litter production as well as the soil compartment. The methods developed will serve the needs for UNFCCC reporting and the reporting under the Kyoto Protocol. This method is principally intended for analysts or planners interested in quantifying carbon over extensive forest areas.

Ecological processes and large-scale climate relationships in northern coniferous forests

Ilmasto vaikuttaa ekologisiin prosesseihin eri tasoilla. Suuren mittakaavan ilmastoprosessit, yhdessä ilmakehän ja valtamerien kanssa, säätelevät paikallisia sääilmiöitä suurilla alueilla (mantereista pallopuoliskoihin). Tämä väistöskirja pyrkii selittämään kuinka suuren mittakaavan ilmasto on vaikuttanut tiettyihin ekologisiin prosesseihin pohjoisella havumetsäalueella. Valitut prosessit olivat puiden vuosilustojen kasvu, metsäpalojen esiintyminen ja vuoristomäntykovakuoriaisen aiheuttamat puukuolemat. Suuren mittakaavan ilmaston löydettiin vaikuttaneen näiden prosessien esiintymistiheyteen, kestoon ja levinneisyyteen keskeisten sään muuttujien välityksellä hyvin laajoilla alueilla. Tutkituilla prosesseilla oli vahva yhteys laajan mittakaavan ilmastoon. Yhteys on kuitenkin ollut hyvin dynaaminen ja muuttunut 1900-luvulla ilmastonmuutoksen aiheuttaessa muutoksia suuren mittakaavan ja alueellisten ilmastoprosessien välisiin sisäisiin suhteisiin.

Computational Methods for Reconstruction and Analysis of Genome-Scale Metabolic Networks

Metabolism is the cellular subsystem responsible for generation of energy from nutrients and production of building blocks for larger macromolecules. Computational and statistical modeling of metabolism is vital to many disciplines including bioengineering, the study of diseases, drug target identification, and understanding the evolution of metabolism. In this thesis, we propose efficient computational methods for metabolic modeling. The techniques presented are targeted particularly at the analysis of large metabolic models encompassing the whole metabolism of one or several organisms. We concentrate on three major themes of metabolic modeling: metabolic pathway analysis, metabolic reconstruction and the study of evolution of metabolism. In the first part of this thesis, we study metabolic pathway analysis. We propose a novel modeling framework called gapless modeling to study biochemically viable metabolic networks and pathways. In addition, we investigate the utilization of atom-level information on metabolism to improve the quality of pathway analyses. We describe efficient algorithms for discovering both gapless and atom-level metabolic pathways, and conduct experiments with large-scale metabolic networks. The presented gapless approach offers a compromise in terms of complexity and feasibility between the previous graph-theoretic and stoichiometric approaches to metabolic modeling. Gapless pathway analysis shows that microbial metabolic networks are not as robust to random damage as suggested by previous studies. Furthermore the amino acid biosynthesis pathways of the fungal species Trichoderma reesei discovered from atom-level data are shown to closely correspond to those of Saccharomyces cerevisiae. In the second part, we propose computational methods for metabolic reconstruction in the gapless modeling framework. We study the task of reconstructing a metabolic network that does not suffer from connectivity problems. Such problems often limit the usability of reconstructed models, and typically require a significant amount of manual postprocessing. We formulate gapless metabolic reconstruction as an optimization problem and propose an efficient divide-and-conquer strategy to solve it with real-world instances. We also describe computational techniques for solving problems stemming from ambiguities in metabolite naming. These techniques have been implemented in a web-based sofware ReMatch intended for reconstruction of models for 13C metabolic flux analysis. In the third part, we extend our scope from single to multiple metabolic networks and propose an algorithm for inferring gapless metabolic networks of ancestral species from phylogenetic data. Experimenting with 16 fungal species, we show that the method is able to generate results that are easily interpretable and that provide hypotheses about the evolution of metabolism.

Computational Methods for Detecting Large-Scale Chromosome Rearrangements in SNP Data

Large-scale chromosome rearrangements such as copy number variants (CNVs) and inversions encompass a considerable proportion of the genetic variation between human individuals. In a number of cases, they have been closely linked with various inheritable diseases. Single-nucleotide polymorphisms (SNPs) are another large part of the genetic variance between individuals. They are also typically abundant and their measuring is straightforward and cheap. This thesis presents computational means of using SNPs to detect the presence of inversions and deletions, a particular variety of CNVs. Technically, the inversion-detection algorithm detects the suppressed recombination rate between inverted and non-inverted haplotype populations whereas the deletion-detection algorithm uses the EM-algorithm to estimate the haplotype frequencies of a window with and without a deletion haplotype. As a contribution to population biology, a coalescent simulator for simulating inversion polymorphisms has been developed. Coalescent simulation is a backward-in-time method of modelling population ancestry. Technically, the simulator also models multiple crossovers by using the Counting model as the chiasma interference model. Finally, this thesis includes an experimental section. The aforementioned methods were tested on synthetic data to evaluate their power and specificity. They were also applied to the HapMap Phase II and Phase III data sets, yielding a number of candidates for previously unknown inversions, deletions and also correctly detecting known such rearrangements.

The Gerbera cDNA Microarray: A Tool for Large-Scale Identification of Genes Involved in Flower Development

During the past ten years, large-scale transcript analysis using microarrays has become a powerful tool to identify and predict functions for new genes. It allows simultaneous monitoring of the expression of thousands of genes and has become a routinely used tool in laboratories worldwide. Microarray analysis will, together with other functional genomics tools, take us closer to understanding the functions of all genes in genomes of living organisms. Flower development is a genetically regulated process which has mostly been studied in the traditional model species Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida. The molecular mechanisms behind flower development in them are partly applicable in other plant systems. However, not all biological phenomena can be approached with just a few model systems. In order to understand and apply the knowledge to ecologically and economically important plants, other species also need to be studied. Sequencing of 17 000 ESTs from nine different cDNA libraries of the ornamental plant Gerbera hybrida made it possible to construct a cDNA microarray with 9000 probes. The probes of the microarray represent all different ESTs in the database. From the gerbera ESTs 20% were unique to gerbera while 373 were specific to the Asteraceae family of flowering plants. Gerbera has composite inflorescences with three different types of flowers that vary from each other morphologically. The marginal ray flowers are large, often pigmented and female, while the central disc flowers are smaller and more radially symmetrical perfect flowers. Intermediate trans flowers are similar to ray flowers but smaller in size. This feature together with the molecular tools applied to gerbera, make gerbera a unique system in comparison to the common model plants with only a single kind of flowers in their inflorescence. In the first part of this thesis, conditions for gerbera microarray analysis were optimised including experimental design, sample preparation and hybridization, as well as data analysis and verification. Moreover, in the first study, the flower and flower organ-specific genes were identified. After the reliability and reproducibility of the method were confirmed, the microarrays were utilized to investigate transcriptional differences between ray and disc flowers. This study revealed novel information about the morphological development as well as the transcriptional regulation of early stages of development in various flower types of gerbera. The most interesting finding was differential expression of MADS-box genes, suggesting the existence of flower type-specific regulatory complexes in the specification of different types of flowers. The gerbera microarray was further used to profile changes in expression during petal development. Gerbera ray flower petals are large, which makes them an ideal model to study organogenesis. Six different stages were compared and specifically analysed. Expression profiles of genes related to cell structure and growth implied that during stage two, cells divide, a process which is marked by expression of histones, cyclins and tubulins. Stage 4 was found to be a transition stage between cell division and expansion and by stage 6 cells had stopped division and instead underwent expansion. Interestingly, at the last analysed stage, stage 9, when cells did not grow any more, the highest number of upregulated genes was detected. The gerbera microarray is a fully-functioning tool for large-scale studies of flower development and correlation with real-time RT-PCR results show that it is also highly sensitive and reliable. Gene expression data presented here will be a source for gene expression mining or marker gene discovery in the future studies that will be performed in the Gerbera Laboratory. The publicly available data will also serve the plant research community world-wide.